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Abstract

Although extensively studied, the particular contribution of the Earth’s orbital parameters to the intensity and periodicity of the Pleistocene glacial–interglacial cycles remains unresolved. Here, I approach the issue from the perspective of hydroclimatic variation by reconsidering the available palaeoclimatic records of the past 1.2 million years. Correlation of various direct and indirect hydroclimatic proxies consistently converges to highlight a hyetal spectrum of hydroclimatic intensity driven by quasi-22-kyr insolation oscillations due to eccentricity-modulated precession of the Earth’s rotational axis. The only and striking exception occurs at ~880 kyr, coinciding with the onset of the quasi-100-kyr glacial periodicity and the extremely cool and arid period known as the “900-kyr event”. The high insolation prevailing during that period suggests that the climate anomaly was not orbitally forced but instead was due to a currently undefined feedback perturbation of the Earth’s internal climate system. Furthermore, lower and higher hyetal periods seem to be closely related to glacial and interglacial cycles, respectively. The real mechanism of that relation is currently not well understood and might constitute a missing link coupling the Earth’s orbital and climatic histories.

Keywords

Early-to-Middle pleistocene transition, Earth’s orbital parameters, Glacial–interglacial cycles, glaciation, Hydroclimate, Pleistocene

Introduction

The onset of the current geologic period, the Quaternary [Holocene and Pleistocene; 2.58 million years (Myr) to present], was characterised by the intensification of Northern Hemisphere glaciation and variation in the intensity, shape, and duration of glacial–interglacial cycles [1]. During those cycles, relatively short interglacial periods with relatively warm climate and duration of a few thousand years were separated by colder periods lasting several tens of thousands of years. The reasons for that climatic variation have been a subject of study for almost two centuries and have been attributed to factors both internal and external to the climate system (ref. [2-4], and references therein). The astronomical hypothesis is one of the oldest explanations but was not formulated as a theory until 1941, when it presented by the Serbian geophysicist and astronomer Milutin Milanković [3]. The Milanković theory claims that the palaeoclimate was driven by perturbations of the Earth’s orbit and rotational axis. That view was largely disputed until the mid-1970s, when critical climatic information was recovered by the Ocean Drilling Programs (ODP) that demonstrated that the pace of the climatic variation matched the variation of the Earth’s orbital parameters [2]. More recent studies revealed, however, that the climate feedback on the Earth is more complicated than a simple linear system. For example, although the Milanković theory claims that obliquity-dominated oscillations in incoming solar radiation (insolation) at latitudes >65°N are the main factor controlling glacial cycles, the Earth is currently going through an interglacial culmination, but the current insolation has the same magnitude as that during the glacial culmination that occurred about 18,000 years ago [5]. Similarly, although the Milanković theory predicts the occurrence of glacial cycles in accordance with the 41-kyr periodicity of the tilt of the Earth’s axis (obliquity), which has a relatively large effect on insolation, most analyses of the last ~900,000 years of climatic history have concluded a quasi-100-kyr periodicity for glacial cycles, which is most compatible with the variation in the eccentricity of the Earth’s orbit, a parameter with a relatively small effect on insolation [2,6]. The inability to demonstrate a linear relationship between the insolation variation and the intensified glacial–interglacial cycles of the last ~900,000 years (from the onset of the middle Pleistocene to today) has led some researchers to introduce a stochastic parameter into their models of the climate system [7], whereas others have concluded that we are currently unable to understand the real mechanism driving glaciation [3,8]. The relationship between the global climate cycles and the Earth’s orbital parameters has been exhaustively investigated through palaeotemperature proxies. I took a different approach by exploring that relationship in terms of the variation in another important climatic factor, the hydroclimatic variation. To that end, I reconsidered the global palaeoclimatic record of the last 1,200,000 years in order to reconstruct the global trends in hydroclimatic intensity during the late Quaternary. The correlation of that variation with the oscillations of orbital parameters and palaeotemperature proxies allows further investigation of whether and how all those parameters are related to one another.

Materials and Methods

The global palaeoclimatic record of the last 1,200,000 years was reconsidered to investigate whether there are direct and indirect palaeohydroclimatic proxies with satisfactory resolution and precise timescale that can reliably reconstruct the global trends in the variation of the late Quaternary hydroclimatic intensity (Figure 1).

Figure 1: Correlation of palaeotemperature (A) and various direct and indirect hydroclimatic proxies of the last 1.2 million years (see the Materials and Methods). A) Stack of 57 globally distributed benthic δ¹⁸O records [6] (palaeotemperature proxy). MS=Marine Stage. B) Atmospheric methane record from the EPICA Dome C ice core, Antarctica [36]. C) LOVECLIM simulation of the annual mean precipitation amount for the Lake Ohrid [63]. D) Palynologic record, Tenagi Philipon, Greece [25]. E) Gamma ray series at ODP Site 1119, east of South Island, New Zealand [64]. F) Composite δ¹⁸Osp from Chinese caves as a proxy of the Asian Monsoon [9]. G) Iron/potassium (Fe/K) ratio, IODP Site U1467, Maldives Archipelago [22]. H) Biogenic silica accumulation, Lake Baikal, Russia [29]. I) Stack of three Southern Ocean carbonate carbon isotope (δ¹³C_carb) records ODP 1090, ODP 1089, and GeoB1211 [65]. J) Stacked and smoothed carbonate carbon isotope (δ¹³C_carb) from benthic foraminifera of the global ocean [66]. K) Organic carbon isotope record (δ¹³C_org) of marine sediments including predominantly terrestrial material from Niger deep-sea fan (GeoB 4901), West Africa [18]. L) Log-ratio of silica to aluminium as a proxy of sediment opal content (ODP Site 658, subtropical North Atlantic [48]. M) Organic carbon isotope record (δ¹³C_org) of marine sediments including equivalently mixed material of terrestrial and marine photosynthesis, ODP Site 1077, Angola Basin [67]. N) Ti/Al ratio, ODP Sites 967 and 968, eastern Mediterranean [17]. O) Mass Accumulation Rate (MAR) of iron deposition, ODP Site 1090, Southern Ocean [30]. The vertical bars in different shades of blue form a hyetal spectrum highlighting periods of various hydroclimatic intensity. The green δ¹³C_org graph from the Niger deep-sea fan and the magenta loess xfd record from China are superimposed on other graphs for comparison. Black arrows show the remarkable values of various proxies during the 900-kyr event.

The correlation of that variation with the oscillations of both orbital parameters and palaeotemperature proxies allows further investigation of whether and how all of those parameters are related to one another (Figure 2). The investigation considered not only direct and well-established proxies of hydroclimatic intensity but also other proxies that might be indirectly related through mechanisms that are not well understood (Figures 1, 3 and 4). Potential covariation of the latter with the direct hydroclimatic proxies is expected to either confirm their physical property as hydroclimatic proxies or to reveal information about the specific mechanism responsible for the observed covariation. Among the well-established hydroclimatic proxies are the oxygen-isotopic composition in speleothems (δ¹⁸O_sp) [9,10] and the loess-paleosol profiles [10,11]. Speleothems are inorganic carbonate deposits (mostly calcite and aragonite) that grow in caves and form from drip water that is supersaturated with CaCO₃. Speleothems are highly suitable for radiometric dating using uranium-series disequilibrium techniques and can provide high temporal resolution ranging from a seasonal scale to a scale of ~100 years, depending on the sampling resolution. The δ¹⁸O_sp records provide information that can be used to reconstruct past changes in precipitation and atmospheric circulation [10,12]. Here, a composite δ¹⁸O_sp record from Chinese caves spanning the past 640 kyr was used (Figure 1F) as proxy of the Asian Monsoon [11]. Loess is a deposit of wind-blown silt that blankets large areas of the continents. Loess accumulated during dry periods when dust fall was high and vegetation was predominantly short grasses. During episodes of warmer and wetter conditions, a reduction in loess deposition and in situ weathering of the loess already on the ground led to accretionary soil formation [10]. Today, the alternating sequence of loess units and paleosols forms the longest and most complete terrestrial record of Quaternary palaeoclimatic conditions on the continents. The magnetic susceptibility and grain size of loess are two well-accepted proxies of summer and winter monsoon climates, respectively [10,13]. Here, loess frequency-dependent magnetic susceptibility (xfd) (Figure 4C) and loess grain-size data expressed as GT32 (>32 μm particle content) (Figure 4D) from the Luochuan loess section, China, have been arrayed in Figure 4 as proxies of the warm/humid southerly East Asian summer monsoon and the dry/cold northerly East Asian winter monsoon, respectively [9]. Continuous archives of terrestrial lithogenic inputs such as concentrations of terrigenous elements (Fe, K, Al, Si, Mg, and Ti) in marine sediments are considered to reflect wet versus dry conditions in the source areas. Moreover, elementary ratios such as the Ti/Al ratio have been used to remove dilution effects of carbonate in order to clarify palaeohydroclimatic information [14-16]. Accordingly, the Ti/Al ratio can also be considered a well-established palaeohydroclimatic proxy. A continuous Ti/Al record from the eastern Mediterranean [17] was arrayed in Figure 1N. In Figure 3E, the Mediterranean record is correlated with another Ti/Al record from the Niger Delta [18]. The hydrogen isotopic composition of sedimentary leaf waxes (δD_wax) has been shown to primarily reflect precipitation (δD_p) and is often taken to reflect the amount of precipitation [19]. The results of such a dataset from the Limpopo catchment, South Africa, were previously analysed [20] through a transient run with the isotope-enabled climate model iLOVECLIM and inferred to the arrayed graph of the mean annual precipitation range (Figure 4E). ¹⁰Be is a long-lived cosmogenic radionuclide produced in the atmosphere, where it attaches to dust and then is deposited mainly through wet precipitation events. Hence, the ¹⁰Be flux from the atmosphere is considered to be a proxy for rainfall (ref. [21] and references therein). Meteoric ¹⁰Be preserved in Pleistocene Chinese loess has been used as a proxy for monsoon palaeo-rainfall [21]. In Figure 4A, such a graph was arrayed for comparison with the other hydroclimatic proxies. Among the not well-established palaeohydroclimatic proxies is the elementary Fe/K ratio, which was recently considered as an alternative to the Ti/Al ratio in marine sediments [22]. A continuous Fe/K record from the Maldives Archipelago [22] was arrayed in Figure 1G. Another Fe/K record from the southwest Indian Ocean [23] was arrayed in Figure 4F. Other potential hydroclimatic proxies include the organic carbon isotope records of sediments containing terrestrial vegetation material (δ¹³C_org-w) and, in a secondary role, the carbon isotope variation in marine carbonates (δ¹³C_carb) [24], the accumulation ratio of biogenic silica in freshwater lakes [10], the continuous arboreal palynologic records [10,25], the dust and iron deposition records in marine sediments [26,27], and the records of atmospheric methane concentration [28]. The variation in the organic carbon isotope fractionation extracted from terrestrial vegetation (δ¹³C_org-w) has long been considered a good proxy of wet/arid climate modes (ref. [24] and references therein). Fossilised wood debris, often stored in near-shore marine sediments, are the best proxies of that variation, given that they provide sedimentary organic material that can safely be regarded as having originated from terrestrial vegetation. The extracted hydroclimatic information can be biased, however, because of the fact that marine-produced organic material is not influenced directly by hydroclimatic variation [24]. Biogenic silica records of lake sediments, such as the one of Lake Baikal (Figure 1H), are considered proxies of diatom productivity [10,29], which is sensitive to changes in the orbital parameters, although the specific climatic factor influencing the records is currently unknown [29]. Two such records, one terrestrial and one marine, were correlated in Figure 1 for comparison (Figure 1H and 1L). Wind-borne mineral aerosol (‘dust’) from marine sediments and ice cores carries considerable information about the aridification history of source areas and atmospheric circulation over different timescales [26]. Eolian dust fluxes in ice ages tend to be greater than those in interglacial periods. That phenomenon can be attributed to a strengthening of dust sources, together with a longer lifetime for atmospheric dust particles in the upper troposphere resulting from a reduced hydrological cycle [27]. Consequently, palaeo-dust records can be a good proxy of the past hydroclimate. In Figure 1O, a dust record derived from the analysis of marine sediments from ODP Site 1090, located in the Atlantic sector of the subantarctic zone [30], was arrayed for correlation together with another record from the marine sediment core MD03-2705 [31]. The latter is located directly adjacent to Ocean Drilling Program Site 659 and includes information of Saharan dust deposition across the subtropical North Atlantic that is considered to reflect the West African monsoon strength (ref. [31] and references therein).

Figure 2: Correlation of the hyetal spectrum with the Earth’s orbital parameters [68, 69]. Blue arrows point out that hyper-hyetal periods always occur during the orbital periods of high eccentricity. Coloured circles indicate the coincidence of the onset of a hyetal period with either the climatic precession and/or the obliquity maxima: green circles indicate exclusive identity; magenta circles indicate coincidence with both orbital phenomena. Red X symbols indicate failures of either climatic precession or obliquity to explain the onset of a hyetal period, that is, whenever their oscillations coincide with decreasing rather than increasing solar insolation trends. The grey arrows note that precession minima correspond to hypo-hyetal and/or arid periods only during periods of eccentricity minima. To emphasise the precessional effect, hyetal cycles are correlated with the insolation oscillations at 65°N high latitude (bottom graph), which is an area mainly dominated by obliquity-dependent insolation. The horizontal magenta dashed lines correlate the insolation power range of the 880-kyr climate anomaly (yellow vertical bar) with those of the hyetal cycles. Note that the astronomical precession minima correspond to climatic precession maxima.

Variation in palynologic records is also known to reflect climate variability [10]. The continuous 1.3 million-years arboreal pollen record from Tenagi Philipon, Makedonia, Greece [25], which is used here as the main proxy (Figure 1D), is expected to have been primarily influenced by palaeohydroclimatic variation rather than by palaeotemperature variation. That is because in palynologic analyses of other locations in Greece [32,33], the hydroclimate was shown to be the dominant factor influencing the vegetation distribution. The records of atmospheric methane (CH₄) concentration have been found to covary closely with the hydrological cycles [34,35], suggesting the CH4 records to be very good hydroclimatic proxies. In Figure 1B, the 800,000 years atmospheric methane record from the EPICA Dome C ice core, Antarctica [36], was arrayed for correlation. All records were arrayed for correlation against their original timescales. All graphs are oriented so that wetter periods are represented by downward oscillations and drier periods are represented by upward oscillations. Hyetal and arid periods were defined on the basis of visual inspection of the graphs and the appearance of covariation among the proxies. Hyetal periods were defined by the occurrence of distinct downward peaks at least in three proxies, whereas arid periods were defined by the presence of zero to two peaks. The classification of the hyetal periods into categories of intensity was based on the amplifications of the oscillation peaks. Although the resulting hyetal spectrum is somewhat generalised, the resolution is satisfactory and in line with the purpose of the analysis. The separation of the hydroclimatic intensity into hyetal subcategories was done in order to cover a gap in the hydrological terminology by offering a term for the rainfall strength based not on the amount of rain water (precipitation) but on the amount of continental weathering caused by the rainfall. The concluded hyetal spectrum is thus mainly a contribution of proxies from weathering on land.

Results

Figure 1 shows how the best direct and indirect hydroclimatic proxies (see the Materials and Methods) correlate with the palaeotemperature and CO2 records of the last 1,200,000 years. The covariation of hydroclimatic proxies converges to highlight 50 hyper-hyetal, hemi-hyetal, hypo-hyetal, and arid periods characterised by high, semi-high, low, and very low levels of hydroclimatic intensity and continental weathering, respectively. The result is the synthesis of the spectrum of hydroclimatic intensity affecting Eurasia and tropical and northern Africa or, approximately, the Northern Hemisphere. In Figure 2, that spectrum is compared to the Earth’s orbital parameters. Figure 2 shows that hyper-hyetal periods and hypo-hyetal to arid periods largely coincide with the maxima and minima of the quasi-100-kyr eccentricity periods, respectively. Moreover, all of the hyetal periods start during high-insolation peaks, at the culminations of the eccentricity-modulated precession minima. Orbital precession minima correspond to times when the distance between the Earth and the Sun is smallest, resulting in higher insolation and maxima of another term, the climatic precession [37]. Climatic precession also depends on the Earth–Sun distance at the summer solstice. The highest-insolation hyper-hyetal periods occur when the solstice of boreal summer shifts towards the perihelion (e.g., as it was 10,000 years ago). Arid periods, in contrast, occur when the perihelion shifts towards the solstice of boreal winter (e.g., as it is today; see Figure 3). Obliquity plays only an auxiliary role in the configuration of the hyetal spectrum: it amplifies but never triggers hyetal periods. Indeed, there is no visible hyetal period corresponding to a high-insolation peak caused exclusively by an obliquity maximum, although there are plenty caused by climatic precession maxima (green circles in Figure 2). In addition, there are intermediate arid periods caused by precession minima, although they coincide with obliquity maxima (green arrows in Figure 2). As all hyetal periods correspond to insolation highs, this can be explained by the fact that even in the northern high (>65°N) latitudes, where the effect of obliquity on insolation is stronger than that of precession [37], insolation peaks always correspond or are close to precession minima (see the bottom of Figure 2 and Figure 3, where higher resolution data are correlated). Precession minima (climatic precession maxima) correspond to hypo-hyetal and/or arid periods only during periods of eccentricity minima (grey arrows in Figure 2). The only exception to that rule occurs at ~880 kyr (marked by a yellow arrow and a yellow vertical bar in Figures 1 and 2), coinciding with an event that signalled, and probably caused, the onset of the 100-kyr glacial periodicity (see the Discussion). The correlation of the palaeotemperature proxy with the hyetal spectrum in Figure 1 suggests that lower and higher hyetal periods are closely related to glacial and interglacial cycles, respectively. Indeed, in Figure 3 (see also Figure 4 for the Southern Hemisphere), it can be seen that, within the quasi-100-kyr eccentricity cycles, quasi-22-kyr hyetal/arid cycles form ramps of descending hyetal amplitude (from hyper-hyetal to arid) following the eccentricity-modulated precession of the Earth’s rotational axis [centred on a major cycle of ~23,000 years (23,700 and 22,400 years precisely) and a minor cycle of 19,000 years]. Both the CO₂ records and the temperature records seem to be in good agreement with the hyetal cycles (Figure 3), suggesting a relationship that is closer than was previously thought.

Figure 3: Comparison of the Earth’s orbital parameters [68, 69] to hydroclimate proxies supporting the hyetal spectrum of the last 250 thousand years. A) Stable hydrogen isotopic composition of leaf waxes from the Gulf of Aden (core RC09-166) corrected for ice volume contributions [70]. B) Oxygen-isotopic composition of cave calcites (δ¹⁸Osp) from Chinese caves, a proxy of the Asian Monsoon intensity [71]. C) Organic carbon isotope record from the Niger deep-sea fan (core GeoB 4901) [18]. D) Log-ratio of silica to aluminium as a proxy of sediment opal content (ODP Site 658, subtropical North Atlantic [48]. E). Titanium/aluminium (Ti/Al) ratio of sediments from Niger deep-sea fans (core GeoB 4901) [18]. F) Ti/Al ratio of sediments from ODP Sites 967 and 968, eastern Mediterranean [17]. G) Continuous palynologic record, Tenagi Philipon, Greece [25]. H) Pollen composition of cool-temperate deciduous broad leaf trees from lake Nojiri, Japan [72]. I) Atmospheric CO2 record from EPICA Dome C core, Antarctica [73]. J) Stable isotope ratios of oxygen and hydrogen in the Vostok ice core record, Antarctica [74]. Green arrows show the onsets of hyetal events that apparently coincide with climatic precession maxima and obliquity minima (red arrows). Grey vertical bars highlight that even the weakest hypo-hyetal periods coincide with precession maxima: their low hydroclimatic intensity can be justified by the low eccentricity. The “Green Sahara” interval (~11,000 to 5,000 years before present) [75], during which the area of the modern Sahara Desert received high amounts of rainfall, falls within the hyper-hyetal period ht1; however, today we are crossing the ht1 termination and entering into the subsequent arid period. Yellow arrows show the data indicating the current entry into an arid period.

Figure 4: Correlation between the insolation variation and hydroclimatic proxies in the Northern and Southern Hemispheres. A) Meteoric ¹⁰Be record from Pleistocene China as a proxy for monsoon palaeo-rainfall [21]. B) Saharan dust deposition across the subtropical North Atlantic recovered from the marine sediment core MD03-2705 [31] and considered to reflect the West African monsoon strength. C) Loess frequency-dependent magnetic susceptibility (xfd), Luochuan, China [11]. D) Loess grain-size rate (>32 μm particle content), Luochuan, China [11]. E) Hydrogen isotopic composition of sedimentary leaf waxes (δDwax) from the Limpopo catchment, South Africa, as a proxy of the range in the mean annual precipitation [19]. δD values are reported in permille (‰) versus the Vienna Standard of Mean Ocean Water (VSMOW) standard. F) Continuous record of elemental ratios of Fe/K from the marine sediment core CD154-10-06P, southwest Indian Ocean [23].

Discussion

The relationship between variation in orbital precession and eccentricity and the Earth’s hydroclimatic cycles is fundamental in cyclostratigraphy because of the stable 405-kyr period of the eccentricity over hundreds of millions of years [38]. Eccentricity by itself does not influence the variation of annual insolation, but it plays an important role in modulating the amplitude of the precessional cycles [37,38]. Accordingly, precession-driven hydroclimatic cycles have been traced in stratigraphical sequences of the Cenozoic [39,40] and Mesozoic [41,42]. They have also been demonstrated in several climatic models [e.g. 43-45]. On the other hand, the combined influence of precession and obliquity has been found in the configuration of past hydroclimate systems such as that of Mediterranean [46] and the western Pacific Intertropical Convergence Zone [47] (see also Figure 5).

Figure 5: Correlation of the inferred hydroclimatic spectrum with the cyclostratigraphy of the original gamma ray series at ODP Site 1119, east of South Island, New Zealand (modified from ref. [76]: Supplementary Figure 4 with permission). Left: Original gamma ray series from ref. [64]. The original age model [64] has been fine-tuned on the basis of the filtered 40.9-kyr obliquity cycles (Gaussian filter, red line) [76]. Right: 2π power spectrum and evolutionary spectrograms for inspecting stratigraphic frequencies and patterns of the gamma ray series (see methodology in ref. [76]). Notice that all hyper-hyetal periods correspond to gamma ray peaks apart from the ht37 (blue arrow), which follows the 880-Kyr anomaly (yellow arrow and yellow horizontal bar). Although the domination of the 41-kyr obliquity-related cycles seems to have declined since ~930 kyr (red arrow), the transition from 41-kyr obliquity-related cycles to quasi-100-kyr eccentricity-related cycles seems to have been completed at ~870 kyr, succeeding the 880-kyr anomaly.

The correlations of the various palaeoclimatic records in the current study agree with previous results. Furthermore, they open a new window for observations into not only the Earth’s climate history but also the nature of the records, which in one way or another seem to covary in the hyetal spectrum. Specifically, the close matching of the oscillations of well-established proxies of hydroclimatic intensity, such as the oxygen-isotopic composition of speleothems (δ¹⁸O_sp) [9,10] and the loess-paleosol profiles [10,11], and also those of indirect or not-well-established hydroclimatic proxies [e.g., the organic carbon isotope records of sediments containing terrestrial vegetation material (δ¹³C_org_-w) [24], the elemental ratios of iron and potassium (Fe/K) in marine sediments [22], and the records of atmospheric methane concentration [28] (see the Materials and Methods) confirms both the hydroclimatic property of the proxies and the robustness of the concluded hyetal spectrum. In addition, it calls into question the natural processes that were previously inferred from some other records, such as those of the opal precipitation in the Atlantic coast of northwest Africa [48]. Specifically, the opal precipitation peaks during the glacial terminations have been considered to be evidence of deglacial loss of the North Atlantic intermediate water [48]; however, they are understood here as a proxy of hydroclimatic intensity (see Figure 1 and Figure 3). Accordingly, the opal precipitation peaks could be a result of multiple hyper-hyetal “Green Sahara” intervals [49] that caused strong weathering on the Sahara desert, increased riverine runoff, fertilisation of the sea (Saharan dust, rich in iron and phosphorus, fertilises Amazonia even today) [50], growth of diatom blooms, and biogenic opal precipitation through the demise phase of diatom blooms [51]. Indeed, more recent data showed that the ODP Site 658 from which the opal record originated [48] is located on the front of the estuary of the huge palaeoriver Tamanrasett, which had a giant drainage system in the western Sahara [52]. Hydroclimatic intensity and weathering are also suggested to be the previously unrecognised climate factor [29] responsible for diatom productivity in lake sediments such as those of Lake Baikal (Figure 1J). That would also explain why the sedimentary record of biogenic silica from the high-latitude (51.5–56°N) Lake Baikal shows a surprisingly weak obliquity signal, with eccentricity and precessional frequencies dominating the record [10]. Likewise, the δ¹³C_org-wvariation (Figure 1O and 1P) should be seen as a direct hydroclimatic proxy containing information about mean annual precipitation [22]. In contrast, the carbon isotope records of marine carbonates (δ¹³C_carb) seem to contain indirect hydroclimatic information more closely related to continental weathering and carbon sequestration by terrestrial vegetation (Figure 1K and 1L), as previously suggested [22]. The covariation between the temperature oscillations (a proxy of glacial/interglacial climatic cycles) and the hyetal cycles shown in Figure 1 is of particular interest concerning the nature of that relation and its driving mechanism. Is the concluded hyetal spectrum a result of the temperature variation among the glacial/interglacial cycles, or are the latter driven by the eccentricity-modulated precessional hyetal periods? Given that the exact mechanism causing the glacial/interglacial cycles is yet unclear [53], it does not seem unreasonable to ask that question, although today the current consensus is that the physical and biological processes of the oceans are the main climate feedback system responding to orbital oscillations [e.g. 53]. Indeed, the greater precipitation rates characterising the interglacial periods might be considered as a plausible consequence of the prevailing warmer temperatures in the biosphere. As such, interglacial precipitation rates would increase with a rate of approximately 2% per degree of surface warming as a result of an increase in the radiative flux divergence of the atmosphere at a rate of 2% per K [54]. In that way, the change of the global mean annual precipitation would be a slow procedure depending on and following the glacial/interglacial biosphere temperature variation. However, the magnitude of the intense, convectively generated precipitation develops independently of those conditions and increases following the Clausius-Clapeyron law at the same rate as the column moisture, that is, a ~7% increase in specific humidity per K of surface warming [54]. Thus, even with cold glacial mean annual temperatures, strong midday insolation on land (Figure 6) could cause Violent Hydroclimate Perturbation (VHP) and intensified hyetal phenomena, such as the heavy convective rainfalls observed on tropical islands just after the hottest time of day [55].

Figure 6: Simplified schematic explanation of the strong influence of insolation on the creation of convective rainfalls. Increased insolation drives an enhanced land–ocean thermal gradient and moisture convergence over land that strengthens and forces the monsoon to bring rainfall deep into inland areas (A). In contrast, weak insolation moves the cold continental wind masses toward the ocean, keeping the inland arid (B).

Therefore, during the periods of climatic precession maxima, high insolation could maximise the hydroclimatic intensity driven directly by the daily incoming solar radiation and independently of the prevailing mean annual temperatures in the biosphere. Conclusively, it is suggested here as a working hypothesis for future climate simulation models that the missing link coupling the Earth’s orbital and climate histories might be the directly orbitally forced hydroclimatic intensity. Indeed, given that the glacial/interglacial biosphere temperature is driven by atmospheric CO₂ variation, it is worth noting that the Antarctic records of CO₂ and CH₄ concentrations covary [56] in precessional pace [36]. In addition, CH₄ sources have been found that originated in tropical wetlands and seasonally inundated floodplains [57,58], which are known to have been dominated by a precession-driven hydroclimate. On the other hand, the essential contribution of the huge quantities of carbon stored in high-latitude permafrost regions [59] would prove to be more critical in deglaciation only after it was demonstrated that convective hyetal phenomena developed in the Arctic during climatic precession maxima. Such intensified hyetal phenomena might have led to quicker permafrost thawing and carbon release into the atmosphere [60], which otherwise are slow processes that would follow and not precede deglaciation [57]. In addition to the questions about the cause of Pleistocene glaciation, conclusions can be inferred from the hyetal spectrum regarding the mechanism controlling the periodicity of the glacial–interglacial cycles during the last ~900,000 years. The age of 900 kyr (MIS 22) is a threshold in the Pleistocene glaciation because it marks the currently not well understood passing from 41-kyr obliquity-related glacial cycles to quasi-100-kyr eccentricity-related glacial cycles [2,6,7]. That “900-kyr event” [61,62] signals the first long glacial period of the Pleistocene and is characterised by extremely low sea surface temperatures (SSTs) in the North Atlantic and tropical-ocean upwelling regions, increased aridity in Africa and Asia, a δ¹³C_carb minimum (see Figure 1L), sustained decreased carbonate in the subtropical south Atlantic, northward migration of the Antarctic Polar Front, and major change in the deep-water circulation of the oceans [61,62]. The 900-kyr event is believed to have possibly started as early as the MIS24 (~940,000 years ago; see Figure 1E and Figure 5) and was due to orbital changes resulting in minima in the insolation amplitude [7,61,62]. A thorough observation of the hyetal spectrum reveals, however, that the main arid period characterising the 900-kyr event is the only arid period of the hyetal spectrum that falls within a high-insolation interval of both high eccentricity and high climatic precession; in other words, a period that should have resulted in a hyetal period rather than an arid period. That anomaly, dated at ~880,000 years ago (yellow vertical bars in Figures 1 and 2), perfectly coincides with the exact time of transition to the quasi-100-kyr glacial periodicity (see the spectral analysis in Figure 5). Therefore, the 900-kyr event is not a result of any change in insolation. Instead, it should be considered as a currently unexplained feedback perturbation of the Earth’s internal climate system (e.g., a permanent change in the circulation of ocean currents that resulted in large-scale changes in atmospheric circulation).

Declaration of Competing Interest

The author declares no competing interests.

Acknowledgments and Funding Sources

This research was not funded by public, commercial, or not-for-profit grants.

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Anthocyanin Effects in Reducing Platelet Hyperactivity and Thrombotic Risk in Type 2 Diabetes

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DOI: 10.31038/EDMJ.2021513

Abstract

Background: Platelet hyperactivity has a crucial role in initiating vascular thrombosis and subsequent cardiovascular disease (CVD) in type 2 diabetes mellitus (T2DM). This study aims to assess the effect of anthocyanins on several risk markers of thrombosis in T2DM. Twenty-three patients with T2DM consumed 320 mg of AC/day in the form of Medox^® capsules for 28 days. Blood pressure and anthropometric measures were taken before and after the intervention period. Fasting blood samples were collected pre and post-intervention to perform different analyses. Analysis of platelet activation measured the platelet activation measured the expression of platelet surface marker. Surface markers included CD41a and P-selectin in adenosine diphosphate (ADP) stimulated platelets. Platelet aggregation, full blood examination, coagulation and biochemistry profile analyses were also evaluated pre and post-intervention.

Results: Flow cytometric analysis showed no effect of AC on the expression of P-selectin. There were significant reductions in ADP and collagenstimulated platelet aggregation. The hematologic measurements showed no impact of AC. Coagulation analysis demonstrated a non-significant change of prothrombin time, activated partial thromboplastin time, or fibrinogen level in the blood. This study showed a reduction of platelet aggregation and total serum cholesterol. These results suggest that AC positively impacts attenuating platelet function potential improvement in lipid profile, minimising thrombotic risk.

Keywords

anthocyanin, antiplatelet, platelet activation, diabetes mellitus type 2

Introduction

Hyperactivity of platelets, inflammation, and increased oxidative stress have a central role in the pathogenesis of several conditions, including, type 2 diabetes mellitus (T2DM), thrombosis, and cardiovascular disease (CVD) [1]. T2DM is associated with increased macro-vascular complications, which significantly elevate the risk of cardiovascular mortality among these individuals [2]. Platelets are enucleated blood cells that play a vital role in primary haemostasis. Platelet hyperactivity, in the presence of free radicals, can significantly accelerate the progression of atherosclerosis. Free radicals have a significant effect on developing oxidative stress before platelet hyperactivity [3]. For instance, impaired muscle glucose uptake, endothelial dysfunction, and lipid oxidation are predisposed by oxidative stress detected in disorders such as T2DM [4-6]. Platelet activation and coagulation exemplify a biological indicator to predict vascular events in the future [7]. Endothelial damage of the vascular wall or injury of atheromatous plaque is a primary step in platelet–associated thrombogenesis. Platelets stick to the site of endothelial injury and change their shape. Consequently, platelets undergo degranulation and activation process. Activation of platelets leads to fibrinogen binding to platelet receptors and finally, the formation of thrombus.

Aspirin is an antiplatelet drug that reduces platelet hyperactivity. Aspirin target the cyclooxygenase-2 (COX-2) pathway and inhibiting thromboxane A2 (TXA2) production. Although aspirin is still the first-line antiplatelet agent [8] used in the treatment of acute coronary syndromes (ACS), many studies have recently highlighted aspirin resistance [9, 10] and its side effects, especially in individuals with T2DM. With aspirin and clopidogrel, two anti-platelet therapies are the most widely used antiplatelet treatment to treat ACS [8, 11, 12]. A plethora of studies has demonstrated the potential of plantbased antioxidants is not only inhibiting platelet activity but also in alleviating several risk factors that are associated with atherosclerosis and subsequent cardiovascular disease.

Several studies show the positive effects of consuming antioxidantrich diet, especially fruits and vegetables [13-15] in 2004, Hung and colleagues [16] conducted a cohort study recommending to consuming five or more servings of fruits and vegetables to lower CVD risk. Antioxidants reduce or suppress atherosclerotic progression and alleviate CVD development [16-18]. This anti-thrombotic potential of phytochemicals has encouraged nutraceutical industries to explore the use of natural antioxidants as a complementary therapy to the currently used anti-platelet treatment [7]. The effect of natural antioxidants such as anthocyanins to reduce platelet hyperactivity is due to blocking variable platelet receptors and inhibiting free radicals, which initiate platelet activity, thereby eliminates the risk of thrombus [19] [17, 18, 20-23] [7]. Although the effect of polyphenols on overall health is well documented, their actions on function and activity of platelets are changeable [7]. The variability in these findings increases the necessity to conduct a controlled and well-designed human intervention trial. Therefore, this study aims to examine the effect of pure Anthocyanins extracted from bilberries and blackcurrant (Medox^®) on platelet activity and thrombotic risk in patients with type 2 DM.

Materials and methods

Participant recruitment and study design

This study was approved by Griffith University Human Research Ethics Committee, Griffith University, Queensland, Australia (GU Ref No: MSC/07/14/HREC) and is registered with Australia and New Zealand Clinical Trials Registry (ACTRN12615000293561). Twentythree patients with T2DM were recruited from the general population after signing an informed consent before the commencement of the study. All the participants included in the study were carefully screened using health questionnaires and interviews to ensure that they were non-smoking and without bleeding disorders or liver disease. Participants taking an anti-inflammatory, anti-platelet agents or anticoagulants were not included in the study.

Before the commencement of the study, anthropometric measurements and blood pressure were checked. Also, baseline fasting blood samples were collected to determine the presence of any underlying health condition using results from full blood examination, platelet function assays, enzyme-linked immunosorbent assay (ELISA), coagulation and biochemistry profiles. Upon completion of the initial screening, the participants were requested to consume four AC extract caps (80mg per capsule) per day (320mg of AC extract per day) for 28 days. The current study has used this dosage based on previous studies that have demonstrated that AC supplementation at 320 mg per day has significant beneficial effects on reducing risk factors of CVD such as inflammation, lipid profile and thrombosis [24-26]. The four-week intervention was also finalised based on previous clinical trials conducted by our research team, that have shown that four week AC supplementation can significantly reduce platelet aggregation, activation and overall risk of thrombosis in individuals [27-29]. Anthropometric measurements and blood pressure were rechecked. Fasting blood samples were collected after the 28 days supplementation period. Adherence and compliance of AC capsule intake were monitored by checking the capsule strips returned by the participant after the supplementation and by personally interviewing them.

Supplement Information

Patients were assigned to twenty-eight days of AC intervention in capsule form at a daily dose of 320 mg AC. AC supplement (Medox^®) is a hemicellulose capsule, which contains powder of anthocyanins extracted from Bilberries (Vaccinium myrtillus) and Black Currants (Ribes nigrum). Table 2 shows the relative amount of the primary AC components used in the intervention in each capsule. Each capsule contains 80 mg of AC. More details of the relative amount of each AC compounds has been reported in the literature [25]. Patients were asked to consume four capsules per day (two capsules twice daily) after any two main meals of the day, i.e., breakfast, lunch or dinner. Participants were asked to maintain their habitual lifestyle and diet during the study period.

Table 1: Baseline demographic and anthropometric measures of 23 participants with T2DM.

Parameter	participant’s value
Age range (year)	40-78
Gender (male/female)	16/7
Weight (kg) mean	93.1
BMI (kg/m²) mean	31.5

Table 2: Anthocyanins components included in those capsuled which were used in the trial.

Anthocyanin components	Percentage of ingredients
· Delphinidin 3-O-β-glucosides · Delphinidin 3-O-β-galactosides · Delphinidin 3-O-β-arabinosides	59%
· Cyanidin 3-O-β-glucosides · Cyanidin 3-O-β-galactose · Cyanidin 3-O-β-arabinosides	33%
· Malvidin 3-O-β-glucosides · Malvidin 3-O-β-galactose · Malvidin 3-O-β-arabinosides	3%
· Peonidin 3-O-β-glucosides · Peonidin 3-O-β-galactose · Peonidin 3-O-β-arabinosides	2.5%
· Petunidin Petunidin 3-O-β-glucosides · Petunidin 3-O-β-galactose · Petunidin 3-O-β-arabinosides	2.5%
Total	100%

Anthropometric measurements and blood pressure

Weight and body mass index (BMI) were measured before and after the intervention period. Measurements were taken in light clothing, without shoes, watches, or other accessories. Height was determined to the closest 0.1 cm with a rod stadiometer (Surgical & Medical Products, Australia), anybody mass was measured using a BC- 601 digital body composition scale (Tanita Corporation, Australia). Body mass index (BMI) was calculated by dividing the body weight in kilograms by the height in metres and square. Systolic and diastolic blood pressure values were checked before and after the intervention period. The automatic device was used to monitor blood pressure reading. According to the device manual, all instructions were followed carefully during blood pressure measurement.

Blood sample collection and full blood examination

Fasting blood samples pre and post AC supplementation period were collected from the median cubital vein by a trained phlebotomist. The blood was then carefully aliquoted into one Ethylenediaminetetraacetic acid (EDTA; 1.8mg/ml) tube for FBE analysis, three tri-sodium citrate (28.12g/L) tubes for platelet function and coagulation studies and into one serum separation tubes (SST) for biochemical analysis. Beckman Coulter ACT^TM 5Diff CP haematology analyzer (Coulter Corporation, Miami, Florida, USA) was used to perform FBE analysis.

Platelet aggregation assay

Platelet-rich plasma obtained (PRP) from whole blood collected into trisodium citrate anticoagulant tubes was used to perform platelet aggregation studies. PRP was extracted by the spinning of citrated whole blood at 180×g for 10 minutes, followed by which platelet-poor plasma (PPP) was obtained by spinning the same tube at 2000×g for 10 minutes. Platelet agonists stimulated platelet aggregation. The agonists were collagen (2 μg/mL), adenosine diphosphate (ADP; 5 μM), Arachidonic Acid (AA) (200 μg/mL). Recording percentage aggregation was conducted for 6 minutes at a constant temperature of 37^°C. Platelet aggregation studies were performed using Helena AggRam Platelet Aggregometer (Helena laboratory, Beaumont Texas, USA). Platelet aggregation testing was completed within 2 hours of the blood collection.

Evaluation of platelet activation

Trisodium citrate anticoagulated whole blood was used to evaluate platelet activation. Monoclonal antibodies conjugated with specific fluorophores were used to identify and assess platelet activation, degranulation and formation of monocyte-platelet aggregates. CD 41a conjugated with Peridinin-chlorophyll-protein Complex CY5.5 (PerCP-CY5.5) was used to identify platelets. CD62P conjugated with allophycocyanin (APC) was used to quantify platelet degranulation. For analysis, citrated whole blood was diluted in 1:5 ratio with modified Tyrod’s Buffer (MTB). A mixture of monoclonal antibodies was added to the diluted blood and incubated for 15 minutes at room temperature in the dark. ADP (5 μM) was added as an agonist to stimulate platelet activation, followed by which the samples were further incubated for 10 minutes. The samples were then fixed by adding 800 μl of 10% RBC lysing solution (BD Biosciences) and later analyzed on BD LSRFortessa flow cytometer.

Coagulation profile

Platelet-poor plasma (PPP) was used to perform coagulation assays. Coagulation testing was performed on the Stago R-Evolution Coagulation Analyserutilising the Stago STA-R software to run coagulation assays prothrombin time (PT), activated partial thromboplastin time (aPTT) and Fibrinogen concentration as per the manufacturer’s instructions.

Biochemistry profile

Blood collected in serum separation tubes (SST) was centrifuged for 10 minutes at 2000xg at RT to extract serum for biochemical analysis. Serum levels of glucose, cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglyceride (TG), and uric acid (UA) were determined using Integra Cobas 400 Biochemistry Analyser (Roche Diagnostics, Switzerland). Quality controls and calibrators were run before testing to ensure the accuracy of the analyser.

Pro-inflammatory and adhesion markers

Interleukin-8 (IL-8), vascular cell adhesion molecule (VCAM-1) and intercellular adhesion molecule (ICAM-1) were detected using plasma samples collected into EDTA tubes. Human Magnetic Luminex® Assays kit (R&D), and Bio-Plex Analyser 200 (Bio-Rad, Texas, USA) were used to quantify each analyte based on superparamagnetic beads coated with analyte-specific antibodies. Beads recognizing different target analytes are mixed and incubated with the sample. Captured analytes are subsequently detected using a cocktail of biotinylated detection antibodies and a streptavidinphycoerythrin conjugate. A magnet in the analyserattracts and holds the super-paramagnetic microparticles in a monolayer. Two spectrally distinct Light Emitting Diodes (LEDs) illuminate the beads. One LED identifies the parameter that is being spotted and the second LED determines the magnitude of the PE-derived signal, which is in direct proportion to the amount of analyte bound. Each well is imaged with a CCD camera.

Samples were screened for the named pro-inflammatory biomarkers. Individual sets of samples from the same participants were run in the same assay kit. Plasma samples were thawed on ice and spun down at 14000×g for 10 minutes at four^oC, before two-fold dilution and further processing. The assay was conducted according to the manufacturer’s instruction. A further 1/10^th dilution of standard curves was considered to optimise the assay for low-level detection of analytes. As recommended by the manufacturer, a magnetic plate washer was used to guarantee higher yields of analytes.

Statistical analysis

Statistical analysis was performed using a Graph Pad Prism^® version 6 for windows. Paired t-test was used to analyse the data, and the values were expressed as mean ± SME. The p value <0.05 was considered statistically significant.

Results

Full blood examination

Table 3 shows data of ten hematological indices, including differential white blood cells count. There was no change of the blood count under the effect of AC. Most of the hematological indices were similarly affected under both treatments’ conditions (pre and post). There were trends of increased or decreased blood cell counts after AC treatment, but they were non-significant.

Table 3: Descriptive values of FBE parameters in 23 participants pre and post AC supplementation.

Haematological Indices	Pre-AC Mean ± SEM	Post-AC Mean ± SEM	*P value*	Reference Range
WBC (X 10⁹/L)	6.96±0.33	7.16±0.31	0.99	4.0 – 11.0
RBC (X 10¹²/L)	5.22±0.15	5.04±0.11	0.91	3.8 – 6.5
HGB (g/L)	145.63±2.70	143.38±3.27	0.98	120 – 180
HCT (%)	0.44±0.01	0.43±0.01	0.95	0.36 – 0.54
MCV (fL)	85.90±1.65	86.04±1.015	0.99	80 – 100
MCH (pg)	28.07±0.52	28.37±0.38	0.97	27 – 31
MCHC (g/L)	327.063±4.28	330.15±2.99	0.94	320 – 360
RDW (%)	12.88±0.37	12.45±0.29	0.79	11.0 – 15.0
PLT (X 10⁹/L)	250.45±14.52	271.51±15.23	0.68	150 – 400
MPV (fL)	8.92±0.18	8.80±0.14	0.94	6.0 – 10.0

Values are represented as mean± SEM. No significant difference in FBE parameters was observed pre and post AC supplementation. Abbreviations: AC, anthocyanin; WBC, white blood cell; RBC, red blood cell; HGB, Haemoglobin; MCV, mean cell volume; MCH, mean cell haemoglobin; MCHC, mean corpuscular haemoglobin concentration; RDW, red cell distribution width; PLT, platelet; MPV, mean platelet volume.

Anthropometric measurements

The post-intervention measurement did not show any significant changes in the anthropometric data, including BMI and body weight, of participants, as shown in figure 1.

Figure 1: Anthropometric measurements show both body weight and body mass index. Data presented as mean ± SEM.

Blood pressure measurements

Blood pressure measurement showed no change in patients with T2DM after consumption of AC, as illustrated by figure 2.

Figure 2: Blood pressure measurements. were collected before and after ingestion of AC. Data presented as mean ± SEM.

Platelet aggregation study

Mean platelet aggregation was measured by platelet aggregometry. Three different agonists were used, including ADP, collagen, and arachidonic acid (AA). Figure 3 showed three diagrams and each bar chart displays mean platelet aggregation in the presence of a corresponding agonist. This study detected a significant reduction of mean platelet aggregation in the presence of the ADP (p= 0.0198) and collagen (p= 0.0158) agonists respectively, but there is no effect on AA-stimulated platelet aggregation.

Figure 3: Platelet aggregation study. stimulated by different agonists including ADP, Collagen, and AA. Data presented as mean ± SEM.as mean ± SEM.

Immunophenotyping of platelet activation

The flow-cytometry assay demonstrated the cell surface expression of P-selectin (CD62p), which is an activation marker of platelets. The analysis of platelet activation markers showed no effect of AC on platelet activation in patients with T2DM, as shown in figure 4.

Figure 4: Immunophenotyping of platelet activation. Flow-cytometry analysis of expression of surface marker of P-selectin in activated platelets. Data presented as mean ± SEM.

Biochemical analysis

As shown by table 4. The results showed a significant reduction in total cholesterol in response to AC consumption. However, there were trends of insignificantly reduced blood levels of LDL and triglycerides.

Table 4: Biochemical analysis of some parameters under the effect of AC.

Biochemistry Assay	Pre-AC Mean ± SEM	Post-AC Mean ± SEM	*P value*	Reference range
TC (mM)	5.1±0.29	4.6±0.32	0.0051*	<5.5
HDL (mM)	0.94±0.04	0.89±0.04	0.1010	>1.1
TG (mM)	2.4±0.27	1.9±0.22	0.1015	<2.6
FBG (mM)	6.00±0.35	5.9±0.39	0.8211	4.1 – 6.0
UA	312±22	307±19	0.7418	202 – 416
LDL (mM)	3.4±0.23	3.1±0.27	0.1237	2.0 – 3.4

Values are represented as mean± SEM. A significant reduction in total cholesterol levels was observed post AC supplementation. Abbreviations: AC, anthocyanin; TC, total cholesterol; HDL, high-density lipoprotein; TG, triglycerides; FBG, fasting blood glucose; UA, uric acid; LDL, low-density lipoprotein. *P<0.05.

Coagulation analysis

AC supplementation did not influence clotting times for prothrombin time (PT) and activated partial thromboplastin time (aPTT) coagulation assays. Fibrinogen and D-Dimer also showed no change under AC effect observed post AC supplementation, as shown in figure 5.

Figure 5: coagulation analysis. Coagulation assay of samples of T2DM participants before and after consumption of AC. Data presented as mean ± SEM.
Abbreviations:

Cellular adhesion molecules

Analysis of vascular cell adhesion molecule (VCAM-1) and intercellular adhesion molecule (ICAM-1) shows no effect of AC, as shown in figure 6.

Figure 6: cellular adhesion molecules. Soluble adhesion markers under AC effects in patients with diabetes: Abbreviations: vascular-cellular adhesion molecule (VCAM-1) and intercellular adhesion molecule (ICAM-1). The above figures show serum levels of adhesion molecules under 320 mg/ day AC consumption for four weeks intervention. There was no change in their blood levels post-intervention trial. Data presented as mean ± SEM.

Proinflammatory analytes

As illustrated by figure 7, both biomarkers, including high sensitive C-reactive protein (CRP-HS) and IL-8, demonstrate no change in their serum levels under the effect of AC.

Figure 7: proinflammatory analytes. Proinflammatory molecules under AC effects in patients with diabetes: Abbreviations: high sensitivity C reactive protein (HSCRP) and interleukin-8 (IL-8). Data presented as mean ± SEM.

Discussion

The study aim was to investigate the anti-platelet and antithrombotic effects of AC in patients with diabetes. Anthropometric measurements and blood pressure values were measured before and after the treatment period. The aggregation and activation of platelets were assessed by platelet aggregometry and flow cytometry. Coagulation analysis and proinflammatory and adhesion markers were conducted. This study also investigated haematological indices and biochemical blood tests.

Platelet aggregability increases in T2DM due to multifactorial process. Intrinsic platelet factors and high platelet sensitivity to different agonists enhance platelet aggregation [30]. In the present study, three exogenous agonists, including ADP, collagen and AA, were used to stimulate platelet aggregation. These agonists represent three different mechanistic pathways of platelet activation. The P2Y G protein-coupled receptors located on the platelet surface are responsible for ADP induced platelet activation and aggregation that will result in platelet shape change, granule release and thromboxane A2 production. ADP mechanistically has initiated platelet activation by binding to the P2Y1 and P2Y12 receptors to induce internal calcium mobilisation and degranulation. Previously, anti-thrombotic drugs such as clopidogrel have been used to blunt the expression of P2Y1 and P2Y12 receptors and inhibit platelet activation and aggregation. The results from this study have demonstrated that AC supplementation for 28 days can significantly inhibit the ADP-induced platelet aggregation in patients with T2DM. Hence, suggesting that AC extract from bilberries and blackcurrant may exert its anti-platelet effect by blunting the P2Y1 and P2Y12 receptor-mediated platelet activation and aggregation similar to anti-platelet drug clopidogrel.

The observed inhibitory effect of AC supplementation agrees with the finding of several other studies that showed an AC rich diet could inhibit ADP induced platelet aggregation. In a recent study by Thomson K et.al., 28 day AC supplementation inhibited ADP induced platelet aggregation by 29% in the sedentary population [27]. Also, the results from this study have shown that AC supplementation for four weeks can significantly inhibit the collagen-induced platelet aggregation. Anthocyanins are part of other antioxidants family of flavonoids which has an antagonising effect on collagen-stimulated platelet aggregation by mitigating the oxidative burst which is initiated after binding platelets with collagen [31]. There are two primary receptors for collagen on platelets, namely glycoprotein six (GPVI) and the integrin α2β1, which both have a crucial role in the process of haemostasis [32]. Collagen receptors on binding, initiate intracellular signalling pathway and consequently trigger platelet activation and aggregation [32]. The data of this study is parallel with Aviram and colleagues. They detected an 11% reduction of platelet aggregation due to the inhibitory effects of phenolic compounds, including AC, in a dietary intervention study investigating collagen-stimulated platelet aggregation (32).

This study showed no change in AA-stimulated platelet aggregation. This effect is probably due to improved production of thromboxane-A2 as platelets produce more TXA2 in response to different stimuli in T2DM [30, 33]. However, several other in vivo studies have demonstrated that other sources of AC such as strawberries and Queen Garnet plum can inhibit AA-induced platelet aggregation [29, 34].

P-selectin is an adhesion molecule present on the membrane of α-granules expressed to the surface only upon platelet activation by the process of exocytosis [35]. It is believed that the desensitisation of platelet activation-dependent superficial receptors by AC interferes with signal transduction, thus reducing P-selectin release of α-granule contents following platelet activation [36]. Flavonoids, including AC, may reduce platelet production of superoxide anion, and increase platelet nitric oxide production [37], which inhibit platelet adhesion and activation. The inhibitory effect of AC on the expression of P-selectin on activated platelets can reduce platelet hyperactivity in response to various stressors such as oxidative stress and shear stress that lead to thrombotic events and CVD [28, 38].

However, there was no impact of AC on reducing expression of P-selectin in patients with T2DM in the current study. Diminished effect of AC on lowering platelet activity as shown by the expression of CD62P in this study may be due to increased expression of CD62P and upregulation P-selectin receptors on platelets in patients with T2DM [30, 39, 40]. Also, the limited action of AC in T2DM might be due to increased oxidative stress, particularly in uncontrolled patients [30]. Oxidative stress eliminates endothelial nitric oxide synthase activity and lowers the formation of nitric oxide, and augments intracellular signalling of platelet receptors [30]. This action might increase the burden on the current dose and time of AC (320 mg/day)consumption to alleviate the expression of platelet activation marker of CD62p in the current trial.

Other studies have also investigated the effect of AC in reducing P-selectin expression on platelets; however, the source of AC and its concentration, the sample population, or the agonist used for platelet activation were different. Song et al. discovered an inhibitory effect of AC on the level of P-selection in hypercholesteremic patients [41]. Yao et al. found a significant inhibitory effect of cyanidin-3-glucoside on the expression of CD62P [42]. Yang et al. detected a considerable reduction of P-selection in dyslipidaemic rats supplemented with AC extract from black rice [43]. Andreas et al. found an inhibitory effect of AC on the expression of P-selectin of resting and activated platelets [36]. This effect is not consistent with findings of others regarding the impact of AC on the expression of CD62P on the surface of Platelets [27, 28, 37]. However, the sample population in the current trial is different from populations of those studies. The duration of the intervention was short in this study. More extended intervention in future studies may provide more positive results.

There is no effect of AC on levels of adhesion molecules in this study. However, other studies have shown that AC reduces vascular cell adhesion molecules [44, 45]. Cellular adhesion biomarkers have a crucial impact on the pathophysiology of ischemic events and might be used as predictors of high thrombotic risk [46]. It has been shown that increased oxidative stress upregulates adhesion molecules expression [46].

The current study demonstrates no impact of AC on serum levels of pro-inflammatory markers, including HS-CRP and IL-8. Few other studies have also measured the effect of AC in lowering pro-inflammatory biomarkers, but the sample population, type of inflammatory markers, and the source of AC were different. It has been shown by other studies which implemented diverse sample populations and doses and duration of AC treatment, that AC has demonstrated more powerful impact on lowering inflammatory markers [47-52].

Supplementation of AC showed a significant reduction of total cholesterol but no other analytes of the lipid profile nor other biochemical markers in the current trial. It has been hypothesized that AC may improve lipid profile by lowering of HMG-CoA reductase gene activation. It is thus reducing the synthesis of cholesterol in different ways. First, inhibit cholesteryl ester transfer protein (CEPT) which reduces circulating concentrations of LDL [53]. Second, it lowers apolipoprotein B and apolipoprotein C-III–lipoprotein levels in the blood [24, 53].

Additionally, anthocyanin facilitates the excretion of cholesterol through faeces [54]. LDL and triglycerides and fasting blood glucose in patients with T2DM. The inhibitory effect of antioxidants on biochemical parameters has been shown by other researchers [55-57]. The link between dyslipidaemia and inflammation may be attributed by the fact that elevated serum cholesterol is associated with a higher level of pro-inflammatory cytokines. Hence, the protective effect of anthocyanin could also be dual [58, 59]. It has been shown that AC improves glucose tolerance and reduce hyperglycaemia by improving beta-cell function and increase insulin secretion [60].

According to the current study data, there is no impact of AC on different parameters of haematological indices. Few other studies have investigated the effect of AC on variable haematological indices, but they used various sources and concentrations of AC on different sample populations, too [61-63]. Piekarska et al. conducted an animal study to show the impact of AC on increasing different blood cell counts, including RBC, HGB, MCH, MCHC, RDW, and WBC [61]. The most significant strength of this clinical trial is that, to our knowledge, the current clinical trial is one of the few studies that have demonstrated the benefits of AC supplementation in individuals with T2DM. However, compliance with consuming AC capsules and changes to the participant’s diet are self-reported by the participants, which are one of the limitations for the current study.

Conclusion

Anthocyanin (AC)-rich food has been defined to reduce thrombotic susceptibility by attenuating aggregation pathways of platelets potentially. Although many epidemiological studies have shown the effect of anthocyanin-rich food in reducing platelet hyperactivity, those dietary intervention studies have not demonstrated the direct biological action of AC components on the platelet function and activity. Additionally, the bioavailability of AC changes prominently because of other food constituents. Those elements involve micronutrient, macronutrients, and other antioxidants and exist in the ingested foods, altering the absorptive and antioxidant capacities of AC. However, in this study, the effect of AC metabolites has not been identified, and this might be one of the limitations of detecting the direct impact of AC. This study shows that AC applied an inhibitory effect on platelet aggregation, which ADP and collagen have stimulated in patients with T2DM. There was a significant reduction in the blood level of cholesterol under the impact of AC. In summary, AC can potentially alleviate thrombotic risks and probably lessen the risk of cardiovascular events in patients with T2DM. Moreover, further studies are warranted looking at each mechanistic pathway involved in platelet activity.

Acknowledgment and conflict of interest

The corresponding author acknowledges the Iraqi government/Ministry of higher education and scientific research scholarship program and the Australian government, research training program scholarship. The authors declare no conflicts of interest statement.

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Do Pathogenic Chronic Infections Cause Host Senescence?

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DOI: 10.31038/TVI.2021122

Aging and senescence are words used as synonyms, and refer to the progressive and deleterious changes that occur in cells, tissues and organs, which alter their functionality [1]. In complex multicellular organisms such as animals, aging begins as soon as embryonic development reaches its maximum stage of differentiation. Aging cannot solely be explained by telomere shortening optics, but rather a combination of factors,including protein assembly and packaging errors, somatic mutations and errors in DNA repair, free radicals, reactive oxygen species, and epigenetic modifications such as hypermethylation [2]. Through molecular control, cells in their maximum state of differentiation stop dividing or reduce their cell division rates. Even tissues of intense proliferation accumulate mutagenic events, whether by environmental factors, by pathogenic infection, or by the events described above that stimulate senescence. Physiologically, our body uses strategies to eliminate senescent cells, damaged cells, or are able to recycle malformed organelles or proteins without the need for cellular elimination. Through autophagy or the removal of senescent cells by the immune system, our body prevents the accumulation of these cells, thus controlling, for example, the growth of tumors³. This is why there is a higher incidence of tumors in the elderly [3].

Through the action of NK cells and CD8⁺ T cells, the immune system is able to eliminate any cells whose surface protein expression indicates signs of damage and/or malignant transformation. During senescence, there is a significant reduction in the ability of immune cells to fight pathogens, leading to chronic infection [4]. A classic example of senescent cell control and elimination is the red blood cells. Mature red blood cells are anucleated cells whose half-life is approximately 115 days. After this period they become senescent, with the gradual deterioration of their capacity, and they are subsequently eliminated in the spleen. Red blood cells have surface molecules that signal their state of senescence, indicating the right time to eliminate them. The formation of band 3 protein aggregates (one of the most abundant red cell transmembrane proteins), when stabilized by oxidized hemoglobin molecules (hemichromes), are recognized as antigens by autologous IgG antibodies and complement system. With the deposition of a critical density of antibodies and complement molecules, senescent red blood cells are recognized and eliminated [5].

The senescent red blood cells expose phosphatidylserine on the outer portion of their plasma membrane, a sign that indicates that the cell should be phagocytosed. In healthy cells this phospholipid is actively maintained in the cytoplasmic portion of the plasma membrane. Concomitantly, there is down-regulation of the CD47 molecule, a transmembrane protein whose normal expression indicates a non-phagocytic signal. The exposure of phosphatidylserine coupled with the reduction of CD47 expression stimulates phagocytosis and the elimination of these red blood cells [6]. In 2001, Bratosin [7] and colleagues described a process similar to apoptosis occurring in red blood cells, later called erythrosis [8]. Eryptosis has several similarities to apoptosis, regardless of the trigger, induction of an eripotic state usually involves extracellular calcium entry into the cell, caspase and calpain activation, which causes changes in membrane asymmetry, phosphatidylserine exposure and cell shrinkage. and membrane. Erythrosis has been associated with several pathologies, including metabolic syndromes, uremic syndromes, anemias such as sickle cell anemia and thalassemia, and can be triggered by several signs, including osmotic shock and xenobiotics [5].

Infectious processes also induce erythrosis, such as Plasmodium infection that cause malaria [5]. Infection with P. falciparum induces oxidative stress, promoting the opening of calcium channels. Erythrosis also appears to be induced in uninfected red blood cells, both in P. yoelii [9] and P. falciparum infections [10]. That is, chronic infection during malaria induces early red blood cell senescence. Autophagy is a physiological mechanism that allows cells to recognize damaged proteins or organelles and destroy them. In situations of mitochondrial stress, such as the increase of reactive oxygen species, autophagic processes may induce apoptosis cell death [11]. Autophagy also participates in the protection against some intracellular pathogens, although some are able to escape phagolysosome degradation. The relationship between autophagy and senescence is that the latter is characterized precisely by cells resistant to apoptosis and whose autophagic processes do not occur [12].

Like malaria, other chronic infections can also induce host aging. Some bacteria, viruses and protozoa are capable of causing tissue stress leading to molecular and physiological changes in host cells leading to a senescence process. In individuals with cystic fibrosis caused by Pseudomonas aeruginosa, it is believed that the pyocyanin bacterial toxin prevents autophagy. This is due to the increased production of reactive oxygen species, preventing the scaling of the pulmonary epithelium and thus facilitating bacterial colonization [13]. Furthermore, chronic infection with Chlamydia trachomatis, induces increased DNA methylation, and consequently senescence [14].

In infection with Mycobacterium tuberculosis, it is believed that autophagy would function as a protective factor against infection, representing an efficient antimicrobial factor. Although the bacterial toxin ESAT-6 inhibits autophagosome maturation, it is believed that inhibition of autophagy is an activated factor of senescence, so factors that induce autophagosome maturation, such as IFN-gamma, would be inhibitors of senescenia [3]. Coinfection between M. tuberculosis and HIV induces high viremia and functionally altered CD8⁺ T lymphocytes, which are associated with increased expression of cellular markers associated with this characteristic, as well as the absence of other activation factors such as perforins, granzymes and intracellular IFN-gamma [15].

This state of T lymphocytes is compatible with immunosenescence, which is the aging of the immune system that can be caused by chronic infections, such as HIV, Plasmodium spp., or also by tumors [16]. As with M. tuberculosis infection, Trypanosoma cruzi infection is another example of a chronic infection that induces host senescence related to autophagy blockade [17]. In Chagas disease, we observed lymphopenia and signs of T-cell senescence. In patients infected with T. cruzi, CD8⁺ and CD4⁺ T cells display markers of immunosensitivity and show a depleted functional phenotype with decreased production of IFN-gamma and IL-23. Along with evasion of the immune system, T. cruzi can also prevent autophageal intracellular degradation by compromising autophagosome maturation. Autophagy blockade contributes, as the protection of cellular stress, to the activation of senescence [3].

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The Trojan Horse Strategies of Parasites

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DOI: 10.31038/TVI.2021121

The Trojan Horse is a legendary figure of a wooden horse used by the Greeks during the Trojan War to invade and destroy the Trojan city. Inside this seemingly harmless horse, supposedly sent as a sign of truce, were Greek warriors who at dusk, when the Trojan defenses were unarmed, took advantage to attack the city. This legendary figure can be used as an excellent analogy for the mechanisms some pathogens have adopted to invade the host and evade detection by the immune system. The ‘Trojan Horse Theory’ in immunology describes the ability of some microorganisms, using cells of the immune system itself as vectors, to escape the immune response.

The cells commonly used by these pathogens are phagocytes, such as neutrophils and macrophages, which are the first cells to reach the site of infection. These cells have microbicidal mechanisms that are able to eliminate the pathogen and aid in the control of infection. These mechanisms depend on the production of cytokines and chemokines, as well as the production of reactive oxygen and interferon species. However some pathogens are able to circumvent or inhibit some of these mechanisms thus allowing their survival, as well as enabling their dissemination to target sites, where they will multiply protected from the action of the immune system [1].

The neutrophil is the first cell that migrates to infected tissue after inoculation of the promastigote form of the protozoan parasites of the genus Leishmania. Leishmania is capable of inhibiting neutrophil proinflammatory cytokine production, such as TNF-α, increasing the production of anti-inflammatory cytokines, such as TGF-β, as well as preventing neutrophil microbicidal activity by negatively regulating IFN-γ. In addition, the parasite is capable of retarding neutrophil apoptosis, which would naturally go into apoptosis 6-10 hours after activation, to up to two days. Leishmania achieves this by interfering with the caspase pathway, preventing the processing of pre-caspase into caspase 3 [2,3].

The infected and apoptotic neutrophil secretes high levels of the chemokine MIP-1b, which attracts macrophages to the infection site. The moment the infected neutrophil becomes apoptotic and releases MIP-1b coincides with the peak migration of macrophages into infected tissue, so in situ macrophages encounter apoptotic neutrophils containing intracellular parasites rather than free Leishmania promastigotes. Since apoptotic cell phagocytosis is an anti-inflammatory signal, macrophage microbicidal functions are not activated, and the parasite survives [3].

The Trojan Horse Theory can also used to explain how some microorganisms can overcome the blood-brain barrier to reach the brain. The blood-brain barrier is composed of endothelial cells lining the vascular and cerebral system and plays an essential role in the maintenance of hemostasis of neurons and glial cells, and blocking access of endogenous or exogenous toxic substances, including microorganisms [4]. However, some pathogens are able to cross this barrier. The first step is the infection of a phagocyte in the periphery. Once internalized, the pathogen can actively manipulate the phagocyte to promote brain migration; alternatively, it can suppress phagocyte activation and consequent sequestration in the source tissue, allowing the infected cell to circulate normally throughout the body. When an infected phagocyte reaches the brain, it adheres to the luminal side of the brain capillaries (with or without activation of BMECs) and crosses the blood-brain barrier between or through endothelial cells. Upon entry into the brain, the pathogen can exit its Trojan horse to infect other neural structures [5]. This model has been elucidated in more detail for HIV6 and other viruses, but other studies indicate similar strategies are utilized by some bacteria, fungi and protozoa, such as Listeria monocytogenes [6-10], Cryptococcus neoformans [11] and Plasmodium spp [12].

The dendritic cell (DC) can also act as a Trojan horse in some infections. During HIV infection, DCs perform two contradictory functions. On the one hand they are essential for building an efficient response against the virus, and are even the target of several studies for the development of HIV vaccines, whilst on the other hand it carries the virus directly to its target cell, the CD4⁺ T lymphocyte [13]. The CD4 molecule is the HIV-1 receptor, its binding to this receptor and the CCR5 or CXCR4 co-receptors allows the virus to enter the cell and follow its conventional viral cycle, including retrotranscription of its genetic material and integration into the genome. Productive CD infection is a very rare event, less frequent than CD4⁺ T lymphocyte infection. The type-C lectin receptor, DC-SIGN, which is highly expressed in local DCs.

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Invasion Potential, Impact and Population Structure of Non-native Fish Species, Cyprinus carpio (Linnaeus, 1758) from the Tributary of the Ganga River, Central India

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DOI: 10.31038/AFS.2021332

Abstract

Fish population is a subject to natural control processes in respect of resource and a renewable resource if they are exploited in a systematic and planned manner. Random fish samples of Cyprinus carpio were collected. A total of 548 fish specimens in size ranges between 97 to 687 mm and age classes of 0+ to 9+ were used in present study. Invasion potential and population structure of C. carpio was studied during February 2019 to January 2020 from Sirsa fish landing centre at Prayagraj, Uttar Pradesh, Central India. C. carpio is of great socioeconomic importance for the region and keeps active a population of about 100 to 150 fishermen communities in Sirsa at Prayagraj, Uttar Pradesh. The age classes (0+ to 9+) indicated that the C. carpio was powerfully invaded in the Tons river. In total stock, male population comprised 49.63% and female population 50.36% of the total catches. In the pooled samples, 1+ age group was most dominated with 24.09%. Stock of this age group was most abundant in the Tons river. The mesh size of the nets and length of net was also more harvested this age group compared to other age groups. The 7+, 8+ and 9+ age groups were shared minute proportion with 2.19%, 0.91% and 0.55%, respectively. These age groups considered as old age groups. In case of pooled samples, 0+, 2+, 3+, 4+, 5+ and 6+ age groups contributed as 6.35%, 21.35%, 18.61%, 13.87%, 7.85% and 3.83%, respectively.

Keywords

Population structure, Invasion potential, Cyprinus carpio, stock health, Tons river, Ganga basin, Riverine ecosystem

Introduction

The non-native fish impact assessment and native fish stock management in respect of ecosystem function, biodiversity, recruitment pattern and fish stock, presently disputing both environmental executives (e.g. policy maker/government) and scientific communities especially in riverine sector and other large water bodies in developing countries [1-4]. Non-native species may become invasive and are capable of decreasing biodiversity through competition, spreading exotic diseases, predation and habitat degradation, genetic deterioration of wild populations through hybridization and gene introgression in short or long course of time [5-8]. Non-native fish species are also responsible for reduction of fish lenght, damage breeding ground and change food web structure and population structure of indigenous fish species and also earlier introduced fish species [9-13].

Fish stocks are altering, damaging and invading by human activities like as domestic, business, ornamental or trial purposes [14-18]. The Indian riverine fisheries are mostly disturbed by various stressors as like invasion of fishes (example alien species), overexploitation, domestic and industrial effluents [19-22]. Alien or exotic fish species have the great capacity to cause considerable ecological consequences in introducing or receiving ecosystems especially rivers, streams and reservoirs [23-25].

Cyprinus carpio is commonly called as common carp is non-native fish species or an exotic major carp for India. C. carpio forms a capture fishery of great value in the Ganga river system and other major riverine system and large water bodies of the country, apart from being one of the important species in the culture fishery of the country due to high consumer preference and higher production with Indian major carp (Catla catla, Labeo rohita, Cirrhinus mrigala) [3,16,25,26]. It is widely distributed in the inland water in India [27,28]. The present study was thus undertaken to estimate invasion potential and population structure of C. carpio from the Tons river at Prayagraj, Uttar Pradesh, Central India. This study will help in enhancing the productivity of the river and formulating the fishery management policies of C. carpio from the Tons river in respect of Indian major carp.

Material and Methods

The fish samples of Cyprinus carpio were collected from Sirsa fish landing centre at Prayagraj, Uttar Pradesh, India during February 2019 to January 2020 (Map 1). Samples of the key scales from 548 fish specimens in the length ranges between 97 to 687 mm were examined for determination of age group and population structure. The key scales were removed from the row above lateral line and below dorsal fin region [17,29]. The scales were cleaned in 5% KOH solution to remove adhering- tissues and finally washed in distilled water. The scales were then pressed between two glass plates while drying in order to avoid their curling. The total length (mm) from the tip of snout to the end of largest caudal fin rays was measured and key scales were taken from below the dorsal fin (3 or 4 rows) and above the lateral line. The annulus or annuli formation was determined according to the criterion suggested by [30,31]. A percentage frequency table was prepared on the basis of age and to compute in different sexes (male and female). Population structures of male and female fish were determined on the basis of age group.

Map 1: Tons river map with Allahabad district now Prayagraj district. The sampling site Sirsa is confluence of Tons river from the Ganga river at Prayagraj, Uttar Pradesh.

Result and Discussion

Cyprinus carpio is one of the most desirable fish species for food and commercial purposes by majority consumers in this region (Tons river basin). The population structure of the male and female C. carpio was varied from 85 to 472 mm (total length) and 0+ to 9+ age groups. This species is of great socioeconomic importance for the region and keeps active a population of about 100 to 150 fishermen communities at Sirsa at Prayagraj, Uttar Pradesh. In stock, male population comprised 49.63% and female population 50.36% of the total catches (Table 1 and Figure 1). In general, female fishes are more active compared to male in breeding season. Female population was higher due to twice or thrice breeding season.

Table 1: Population structure of Common carp (Cyprinus carpio) from the Tons river at Prayagraj, India.

Age classes	Number of male	Percentage	Number of female	Percentage	Pooled samples	Percentage
0+	20	7.35	17	6.16	37	6.75
1+	64	23.53	68	24.64	132	24.09
2+	57	20.95	60	21.74	117	21.35
3+	50	18.38	52	18.84	102	18.61
4+	37	13.60	39	14.13	76	13.87
5+	20	7.35	23	8.33	43	7.85
6+	12	4.41	9	3.26	21	3.83
7+	7	2.57	5	1.81	12	2.19
8+	3	1.11	2	0.72	5	0.91
9+	2	0.73	1	0.36	3	0.55
Total	272	49.63	276	50.36	548

Figure 1: Population structure of male and female from the Tons river at Prayagraj, Uttar Pradesh, Central India.

In the pooled samples, 1+ age group was most dominated with 24.09%. Stock of this age group was most abundant in the Tons river, Uttar Pradesh, Central India (Figure 2). The mesh size of the nets was also more harvested this age group compared to other age groups. The 7+, 8+ and 9+ age groups were shared small proportion with 2.19%, 0.91% and 0.55%, respectively. These age groups considered as old age groups. In case of pooled samples, 0+, 2+, 3+, 4+, 5+ and 6+ age groups contributed as 6.35%, 21.35%, 18.61%, 13.87%, 7.85% and 3.83%, respectively (Figure 2). The 9+ age group indicated that the ecological condition was most favorable for C. carpio from the Tons river. The age classes also indicated that the C. carpio was powerfully invaded in the Tons river. It is believed that this healthy stock (0+ to 9+ age groups) of C. carpio from the Tons river due to habitat degradation and water quality of the river. Overall, population distribution was systematic in the pooled samples. The 5+ age group probably remain unexploited. Distribution pattern indicated that the population of C. carpio in future will be increased (Figure 2).

Figure 2: Population structure of stock fishes (Pooled samples) from the Tons river at Prayagraj, Uttar Pradesh, Central India.

According to the percentage occurrence 1+ age group was also most dominated with male (23.53%) and female (24.64%) of the total stocks (Table 1). Male population was contributed in 0+, 2+, 3+, 4+, 5+ and 6+ age groups with percentage shared as 7.35%, 20.95%, 18.38%, 13.60%, 7.35% and 4.41%, respectively (Table 1). The old age groups 7+, 8+ and 9+ were contributed minute proportion with 2.57%, 1.11% and 0.73%, respectively.

While female population was contributed in 0+, 2+, 3+, 4+, 5+ and 6+ age groups with percentage as 6.16%, 21.74%, 18.84%, 14.13%, 8.33% and 3.26%, respectively (Table 1). The old age groups 7+, 8+ and 9+ were contributed very minute proportion with 1.81%, 0.72% and 0.36%, respectively (Table 1). In the present study, C. carpio indicates that occurrences of males and females are difference in number; this is possibly caused by the incidence of fish pairs near to the nest area where females take care of their broods. It breeds twice or thrice per year. The frequency of the breeding is more suitable for the stabilization of the stocks in the river.

Present time, the ecological condition of Tons river at Prayagraj is most fitting (example age composition, population structure) for C. carpio. [32] reported that the population structure of Labeo bata, female was greater than male in the Ganga river at Allahabad. The age group 1+ of C. carpio was dominant (21.54%) and constituted nearly one fifth of the total population from the Ganga river at Allahabad [33]. [34] reported that the O. niloticus of males comprised 56.1% and females 43.9% of the catches in Barra reservoir, Brazil. [35] determined the population structure of the Himalayan Mahseer (T. putitora) in the foothill section of the Ganga river and reported that the samples comprised of 1+ to 9+ age groups individuals. Of these 2+ and 4+ age groups constituted 66.01%, while 1+ was nearly 8.07% of the total stock. Most wild fish stocks (example indigenous fish species) in Indian rivers have been over exploited or have reached their maximum sustainable yield but stock of exotic species increasing day by day [26,36,37]. C. carpio with a known capability to adjust to different environmental situation and its high prospective for aquaculture, can now be found in many rivers of India [38-41].

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A Short Note on Climate Change: An Alternative View

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DOI: 10.31038/AFS.2021331

It is the general view of the Media that the cause of climate change is directly related to air pollution from coal burning and vehicle exhaust. Undoubtedly this cause of air pollution is a worrying factor but whether it is the cause of climate change can’t go unchallenged.

In the geological past evidence of glacial and interglacial periods span a minimum of 400 million year and evidence of glaciation in the Lower Paleozoic, 4-500 million years ago, is seen in the Garevellach Islands and Islay, South West Highlands of Scotland. This event is well documented substantiates the concept of worldwide glaciations throughout the past. This geological evidence appears not to concern the media, apparently due to the lack of understanding of geological science.

In geologically recent times, late 7^th to early 19^th century, the River Thames periodically froze over to allow the so-called Frost Fairs to take place. No automobiles or electricity generators existed therefore these events could not have been caused by air pollution from these sources.

The controlling factor is more likely to be changes in solar radiation due to alterations in the Earth’s orbital around the Sun with time, as this would affect the solar radiation falling on the Earth. In which case there is nothing man can do to alter the situation.

Twins with Juvenile Hyaline Fibromatosis

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DOI: 10.31038/PSC.2021111

Abstract

Background: Juvenile hyaline fibromatosis (JAF) is a rare autosomal-recessive disease in which patients progressively develop cutaneous tumoral fibroblastic proliferations, and joint contractures with bone involvement. JAF is caused by aberrant synthesis of glycosaminoglycans by fibroblasts due to a mutation of the capillary morphogenesis factor-2 gene (CMG2). Limited treatment options are available.

Method: We report monozygotic twins who presented with multiple, recurrent, painless cutaneous nodules.

Result: The presence of twins with JAF is extremely rare. A lesion on the head of one boy had ruptured, and pathological analysis indicated benign spindle cells in a periodic acid-Schiff (PAS)-positive hyaline background. One of twins had much more severe clinical presentation than the other, including more frequent diarrhea, larger nodules, more severe joint involvement, and more easily ruptured masses.

Conclusion: Monozygotic twins who present with JAF may have different severity of symptoms despite the presence of identical mutations in CMG2.

Keywords

juvenile hyaline fibromatosis, twins, subcutaneous mass

Introduction

Juvenile hyaline fibromatosis (JHF) is a rare autosomal-recessive disease caused by mutations in capillary morphogenesis gene-2 (CMG2). There have been fewer than 70 cases reported so far in the literature [1–2], and there has only been one report of twins with JHF [3].The clinical onset usually occurs before 5 years of age, and this disorder is slightly more common in boys [4]. JHF is characterized by cutaneous tumoral fibroblastic proliferation, joint contractures, and bone involvement [2].

Materials and Methods

Clinical Features

Two one-and-a-half year-old male monozygotic twins presented with multiple, progressive, painless, variable-sized nodules all over their bodies. The parents had the same family name and lived in the same remote village in Henan province of China, but were not first-degree relatives. In the year prior to presentation, both infants had recurrent diarrhea of unknown etiology, and in the 6 months prior to presentation, both had multiple, painless, variable-sized cutaneous nodules all over their bodies. These nodules were first noticed around the joints, then on the trunk and head, and eventually all over their bodies. The size of nodules increased gradually over time. One year ago, a nodule on one child’s finger was given an incisional biopsy at a local hospital, and the pathological result was “benign”, but there was no definite diagnosis or further investigation. When they were hospitalized, one of masses on the head had decayed spontaneously and the joint contractures had deteriorated gradually, which crippled both of them. They both had very poor appetites. One of them (patient A) had more severe signs and symptoms than the other (patient B) [Table 1]. They both achieved normal mental development milestones.

Table 1. Clinical feature difference between the twins

	patien A	patient B
clinical onset	earlier	later
maximium diameter of nodules	25	15
mass rupture	yes	no
gingival proliferation	svere	moderate
joint constracture	svere	moderate
perianal tissue proliferation	yes	no
nostril narrow	yes	no
diarrhea	conatantly	intermittent

On physical examination, there were multiple, painless, variable-sized nodules all over the bodies, which were soft upon palpation [Figure 1]. The largest nodule was on the head of patient A, whose diameter was 25 cm [Figure 2]. One mass on the head had ruptured and had a terrible odor [Figure 3]. Cutaneous tumoral fibroblastic proliferation around the lips and gingival hyperplasia [Figure 4] were also present. Patient A had a narrow nostril caused by subcutaneous proliferation, but no shortness of breath [Figure 5]. There were many nodules on their hands and fingers [Figure 6], elbows [Figure 7], and abdomens [Figure 8]. There were also flexion contracture deformities of the limbs [Figure 9], scales on the feet [Figure 10], and proliferation in the perianal area [Figure 11], but no significant lymphadenopathy or hepatosplenomegaly.

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Figure 1. Appearance of the twins.

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Figure 2. Mass on the head.

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Figure 3. Decayed mass on the head.

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Figure 4. Cutaneous proliferation around the lips and gingival hyperplasia.

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Figure 5. Patient A had a narrow nostril.

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Figure 6. Nodules on hands and fingers

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Figure 7. Nodules on elbow.

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Figure 8. Nodules on abdomen.

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Figure 9. Flexion contracture deformities of the knee.

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Figure 10. Scales on the foot.

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Figure 11. Proliferation in the perianal area.

The complete blood counts and electrolyte levels were within normal limits. An X-ray indicated osteoporosis in vertebrae of patient A [Figure 12]. Computed tomography (CT) results showed no masses in the abdominal cavities, except for cutaneous nodules in the abdominal wall [Figure 13]. A head CT of patient A showed a large subcutaneous mass but no intracranial abnormalities [Figure 14].

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Figure 12. Osteoporosis in vertebrae.

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Figure 13. Cutaneous nodules in the abdominal wall.

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Figure 14. Subcutaneous mass without intracranial abnormalities.

After consent from the parents, a biopsy of the large mass on patient A’s head was performed. Hyaline subcutaneous tissue was present beneath the skin [Figure 15], which was sent for pathological investigation. The ruptured head mass was covered with dressing and required regular changes.

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Figure 15. Hyaline subcutaneous tissue beneath the skin.

Pathological Findings

Gross examination indicated that the nodules had a gelatinous white and hyaline appearance [Figure 16]. Microscopically, there were poorly circumscribed lesions composed of many uniform spindle cells embedded in an abundant homogenous fibrous matrix. This matrix was eosinophilic and tested positive on periodic acid-Schiff (PAS) staining, findings characteristic of JHF [Figure 17]. The parents did not consent to surgical procedures on the other child.

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Figure 16. Gross pathological examination.

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Figure 17. Microscopical pathological examination.

RNA isolation and cDNA amplification of samples from each patients’ blood were performed. Cloning and sequencing of the CMG2 gene showed that both patients had the same 4 mutations: c.294C→A, c.1069G→C, 1074delT, and c.1153G→C.

Follow up

The twins were followed up for 4 years after the initial presentation. At the last follow-up, the decayed head lesion of patient A was still unhealed, there were more nodules all over their bodies, and the masses on the head of patient A were larger. The parents refused further therapy.

Discussion

JHF is a rare autosomal-recessive disease that was first described by McMurray [1] as molluscum fibrosum, but renamed by Kitano [2]. Patients with JHF present with multiple cutaneous nodules or masses, gingival hypertrophy, joint contractures, and osteolytic lesions [4]. Skin lesions are the most prominent symptoms [8]. Normal motor development is impaired if joint contractures occur during infancy. Eating and delayed dentition can be caused by severe gingival hyperplasia [1].

Histological examination can confirm a diagnosis of JHF [10]. In particular, lesions from the dermis, subcutis, gingivae, bone, and joints contain abundant homogeneous eosinophilic matrix, which is embedded with cords of spindle-shaped cells. The matrix stains positively with PAS and alcian blue, but not with toluideine blue or Congo red [10]. Nodules are calcified occasionally, but have no elastic tissue. The etiology is unknown. The differential diagnosis includes neurofibromatosis, fibromatosis, amyloidosis, infantile systemic hyalinosis, lipoid proteinosis, and Winchester syndrome [2].

The JHF gene is located on gene 4q21 [5], and previous studies have described the effects of mutations in CMG2 [6]. Previous researchers have described JHF as a connective tissue disease that is characterized by aberrant synthesis of glycosaminoglycans. Dermatan sulfate is the predominant glycosaminoglycan in the skin of patients with JHF, which also has chondroitin sulfate and hyaluronan, but hyaluronan is the most abundant glycosaminoglycan in normal skin [7].

There are no specific treatments for JHF, but cosmetic surgery and those that limit orthopedic disability may be employed. This disease has a progressive course, and most patients only survive up to the 4th decade [1–2]. Relapses are common after tumor removal. A previous report indicated good cosmetic results after removal of more than 100 tumors over a period of 19 years [9]. Physiotherapy may be performed to prevent flexion contractures.

The occurrence of twins with JHF is extremely rare, and there has only been one previous case report [3], and the researchers did not report differences between these twins. It is generally accepted that twins will have the same presentations, because JHF is a genetic disease. However, our twins had very different presentations. Patient A had more severe symptoms [Table 1], and a head mass that ruptured and bled, and was refractory to treatment. Patient A also had more frequent diarrhea, larger nodules, and more severe joint involvements.

We do not know the reasons for the different presentations of these twins. They had identical mutations in CMG2, and might be expected to eventually develop the same signs and symptoms. It may be hypothesized that the differences between the normal and genetically mutated somatic cells led to the differences in these twins. It is possible that patient B will eventually develop the same severe symptoms as patient A. A close follow-up of these children is warranted.

Statement

There are no prior publications or submissions with any overlapping information, including studies and patients. The manuscript has not been and will not be submitted to any other journal. There have no financial support or relationships that may pose conflict of interest. Gaoyan, Deng wrote the first draft of the manuscript. No honorarium, grant, or other form of payment was given to anyone to produce the manuscript. Each author listed on the manuscript has seen and approved the submission of this version of the manuscript and takes full responsibility for the manuscript.

References

Nischal KC, Sachdev D, Kharkar V, Mahajan S. (2004) Juvenile hyaline fibromatosis. J Postgrad Med 50: 125–6.
Haleem A, Al-Hindi HN, Juboury MA, Husseini HA, Ajlan AA. (2002) Juvenile hyaline fibromatosis: Morphologic, immuno histochemical and ultrastructural study of three siblings. Am J Dermatopathol 24: 218–24.
Habibeddine S, Khadir K, Azzouzi S, Skalli S, Lakhdar H. (2003) Juvenile hyaline fibromatosis: 2 twin brothers affected. Ann Dermatol Venereol 130: 43–6.
Weiss SW, Goldblum JR. (2001) Fibrous tumors of infancy and childhood. In: Strauss M, editor. Enzinger and Weiss’s Soft Tissue Tumors. 4th ed. St. Louis: Missouri Mosby; 363–7.
Nazneen Rahman, Melanie Dunstan M. Dawn Teare. (2002) The gene for juvenile hyaline fibromatosis maps to chromosome 4q21. Am J Hum Genet 71; 975–980.
Sandra Hanks, Sarah Adams, Jenny Douglas. (2003) Mutations in the gene encoding capillary morphogenesis protein 2 cause juvenile hyaline fibromatosis and infantile systemic hyalinosis. Am J Hum Genet 73; 791–800.
Katagiri K, Takasaki S, Fujiwara S, Kayashima K, Ono T, et al. (1996) Purification and structural analysis of extracellular matrix of a skin tumor from a patient with juvenile hyaline fibromatosis. J Dermatol Sci 13: 37–48.
Sanzalin H, Kiyozuka Y, Uemura Y, Shikata N, Ueda S, et al. (1998) Juvenile hyaline fibromatosis: A report of two unrelated adult sibling cases and a literature review. Pathol Int 48: 230–6.
Woyke S, Domagala W, Markiewicz D. (1984) A 19-year follow-up of multiple juvenile fibromatosis. J Pediatr Surg 19: 302.
Arnold H, Odom RB, James WD. (1990) Andrews’ diseases of the skin, Clinical dermatology. 8th ed. Philadelphia: Saunders, 712.

Physical Activity in Our Modern World: An Evolutionary Necessity

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DOI: 10.31038/CHBT.2021111

In our modern world obesity is a causal factor in numerous metabolic and endocrine disorders including heart disease, non-alcoholic fatty liver disease (NAFLD), type 2 diabetes mellitus (T2DM), bone and joint disorders and some types of cancer [1]. According to the World Health Organization in 2016, globally more than 1.9 billion adults were overweight and of these over 650 million were obese [2]. Childhood obesity is a more disturbing trend; in 2019 thirty eight million children under the age of 5 were overweight or obese and in 2016 over 340 million children and adolescents aged 5-19 were overweight or obese [3]. Obesity is not a cosmetic issue but rather an affliction that places a tremendous burden to quality of life on individuals and health care systems. It is not without exaggeration to say that obesity due to physical inactivity is the biggest public health problem of the 21^stcentury [4]. However, obesity is preventable and reversible and for interpreting the obesity pandemic afflicting modern societies we need to understand our evolutionary history and examine the environmental, physical, and physiological conditions that impelled our ancestral gorilla-chimpanzee lineageto shift from a sedentary, plant and fruit diet existence to a foraging, meat eating, hunter-gatherer existence some 2.5 million years ago [5].

The transition of our ancestral lineage from the gorilla-chimpanzee line to hunter gatherer necessitated changes in diet, metabolism and physiology that are conducive for high levels of physical activity for survival. Although the environmental, physical, and physiological conditions have changed, our physiology remains genetically adapted for a nomadic forager, hunter gatherer life style [6,7]. Our physiology adapted to storing lipids and carbohydrates as fuels to cope with the uncertainties of food availability in our past. This may have been a survival advantage 2.5 million years ago, but in our modern world it is a liability that leads to increased morbidity and mortality due to increased circulating triglycerides, cholesterol, blood pressure, T2D, NAFLD, Alzheimer’s disease, cardiovascular disease (CVD), and several types of cancer [8,9]. For most of our evolutionary history, as conditions changed so did our physiology. Our lungs, brain, muscles, blood vessels and all our organs and physiological organ systems have evolved to support a high activity level demanded for survival and our bodies have retained this adaptation to the present day. However, we no longer have to forage 15-20 kilometers per day, nor do we have to fend off predators or perform exhaustive farming practices to necessitate high energy diets. In evolutionary terms, the modern norms of an inactive lifestyle and a diet based on an excess of energy-dense, nutrient-deficient, foods such animal products, processed foods, alcohol, sugar and sugar derivatives are not conducive for the purpose for which we evolved.

To prevent or reverse the obesity epidemic and minimize our proclivity to obesity and the subsequent metabolic disorders we need to modify our diet and increase our levels of physical activity. We need to exercise. Our physiology is a dynamic product of evolution that adjusts to changes in living conditions. Regular exercise reduces chronic inflammation which constitutes risk factor for CVD, it reduces insulin resistance which is a harbinger for T2D and helps to shuttle glucose into muscle glycogen stores instead of fat, thus helping reduce triglycerides from the circulation [10]. Additionally, it lowers resting levels of testosterone, estrogen and progesterone, which may explain the reduced rate of reproductive cancers among adults who exercise regularly, improves the effectiveness of immune function, which helps stave off infection especially as we age, and may blunt the morning rise of the stress hormone cortisol [11]. Other than maintaining health, physical activity contributes to healthy aging by reducing the accumulation of senescent cells and the of secretion of a complex mixture of different inflammatory cytokines, chemokines, growth factors and proteases such as IL1a, IL1b, IL6, IL8 and metalloproteinases (MP-1, MP-3) that contribute to the aging process [12]. Also, evidence supports an association between exposure to regular exercise and reduced risk for the development of several cancers that might improve clinical outcomes following a diagnosis of primary disease [7,13].

In order to prevent or reverse the obesity pandemic afflicting modern societies the WHO recommends adults undertake at least 150 min moderate or 75 min vigorous intensity physical activity per week, conduct muscle strengthening activities twice a week, and minimize time spent being sedentary [14]. In an effort to counteract the risks of a sedentary lifestyle the “Exercise is Medicine initiative” in collaboration with the American College of Sports Medicine and American Medical Association was introduced in 2007 to advance the implementation of evidence-based strategies and incorporate exercise as part of a standard treatment for conditions related to sedentary lifestyle such as T2D, CVD, and obesity. It calls for the assessment of the every patient’s physical activity program and prescribing a regimen tailored to each individual’s condition and needs. The implementation of this initiative may change patient behaviour, lighten the burden placed on the quality of life and healthcare systems. The benefits of a structured, regular physical activity regimen for health and disease prevention are unequivocal. Natural selection has tailored our bodies for movement and our physiology has retained the metabolic hunter-gatherer characteristics that do not conform to the sedentary way of life of modern societies. Our contemporary lifestyle and dietary habits conflict with our evolutionary outcome. A quote attributed to Hippocrates (460-370 BC) states “If we could give every individual the right amount of nourishment and exercise, neither too much nor too little, we would have found the safest way to health”. A more recent comment by Manini TM (2015) in the JAMA-Internal Medicine testifies that “there is no single medication treatment that can influence as many organ systems in a positive manner as can physical activity” [15].

Author Contributions

Peter Lembessis: Conceptualization of topic, literature search, writing of original draft.

Anastassios Philippou: Review & editing, literature search.

Stamatis Mourtakos: Literature search on nutrition and dietetics, review and editing of draft.

References

Mantovani A, Allavena P, Sica A, Balkwill F (2008) Cancer-related inflammation. Nature 454: 436-444.
WHO (2020) Obesity and overweight.
Saklayen MG (2018) The Global Epidemic of the Metabolic Syndrome. Curr Hypertens Rep 20: 12. [crossref]
Cowan RE (2016) Exercise Is Medicine Initiative: Physical Activity as a Vital Sign and Prescription in Adult Rehabilitation Practice. Arch Phys Med Rehabil 97: S232-237. [crossref]
Sarich VM, Wilson A C (1967) Immunological time scale for hominid evolution. Science 158: 1200-1203.
Hambrecht R, Gielen S (2005) Essay: Hunter-gatherer to sedentary lifestyle. Lancet 366: S60-61. [crossref]
Koelwyn GJ, Quail DF, Zhang X, White RM, Jones LW (2017) Exercise-dependent regulation of the tumour microenvironment. Nat Rev Cancer 17: 620-632. [crossref]
Freese J, Klement RJ, Ruiz-Núñez B, Schwarz S, Lötzerich H (2017) The sedentary (r)evolution: Have we lost our metabolic flexibility? F1000Res 6: 1787. [crossref]
Rezende LFM, Sá TH, Markozannes G, Rey-López JP, Lee IM, et al. (2018) Physical activity and cancer: an umbrella review of the literature including 22 major anatomical sites and 770 000 cancer cases. Br J Sports Med 52: 826-833. [crossref]
Handschin C, Spiegelman BM (2008) The role of exercise and PGC1α in inflammation and chronic disease. Nature 454: 463-469. [crossref]
Kelly RS, Kelly MP, Kelly P (2020) Metabolomics, physical activity, exercise and health: A review of the current evidence. Biochim Biophys Acta Mol Basis Dis 1866: 165936. [crossref]
Schafer MJ, White TA, Evans G, Tonne JM, Verzosa GC, et al. (2016) Exercise Prevents Diet-Induced Cellular Senescence in Adipose Tissue. Diabetes 65: 1606-15. [crossref].
Ruiz-Casado A, Martín-Ruiz A, Pérez LM, Provencio M, Fiuza-Luces C, et al. (2017) Exercise and the Hallmarks of Cancer. Trends Cancer 3: 423-441. [crossref]
Sallis JF, Bull F, Guthold R, Heath GW, Inoue S, et al. (2016) Progress in physical activity over the Olympic quadrennium. Lancet 388: 1325-1336. [crossref]
Manini TM (2015) Using Physical Activity to Gain the Most Public Health Bang for the Buck. JAMA Intern Med 176: 968-9. [crossref]

Viscum album Therapy for Treating Lymphoma in an FIV and FeLV Positive Cat (Felis catus) – Case Report

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DOI: 10.31038/IJVB.2021524

Abstract

Lymphoma is the hematopoietic tumor of higher incidence in domestic cats. Myelosuppression is the most remarkable side effect of the conventional treatments for this disease, especially for patients with debilitating infections such as FIV and FeLV. In this context, alternative therapies could be recommended for treating lymphoma as a sole therapy or even in association with conventional therapies to provide a better quality of life to the patients and fewer side effects.

Material and Methods: A 4-year-old neutered male mixed breed cat was seen at NaturalPet Clinic in Brazil. The cat was positive for FIV and FeLV, diagnosed with lymphoma, and had relative difficulty breathing and swallowing. On the physical examination, a nodule was observed in the right ventral part of the tongue. An incisional biopsy was performed for histopathological analysis, and the lymphoma diagnosis was confirmed. The disease was treated with homeopathic medicines, such as daily applications of Viscum album in the D3, D6, D9, D12, and D30 dilutions. Complete remission of clinical signs was observed after completing the treatment period with the reestablishment of the patient’s health.

Conclusion: The homeopathic treatment used in this case allowed the disease resolution, had no side effects on the patient and increased his life expectancy.

Keywords

Cancer, FeLV, FIV, Viscum album

Introduction

Lymphoma can be defined as neoplasia of lymphoid tissues or lymphosarcoma. It primarily attacks lymph nodes and/or other solid organs, such as the spleen, liver, or thymus. It can also affect no lymphoid tissues [1]. Neoplasms of hematopoietic origin can be considered the most frequent among domestic cats [2,3], and the most frequently diagnosed neoplasms representing about 90% of tumors in this species [4,5].

The possible accepted etiologies for the occurrence of feline lymphoma are viral immunosuppression, and the feline leukemia virus (FeLV) stands out as one of the most significant inducers of this disease [6,7]. This virus was cited in approximately 70% of cats with lymphoma. However, this reality is modifying since only 25% of cats with lymphoma are FeLV-positive [2,8]. Another virus possibly involved with the emergence of lymphomas is the feline immunodeficiency virus (FIV). It is known that approximately 20% of animals with lymphoma in the United States are seropositive for antibodies against FIV [2].

The average age of patients diagnosed with lymphoma is 8 to 10 years [3,9]. However, it varies with the form of the disease, and its etiology can strongly influence the onset of clinical signs [9]. According to [5], there is no evidence for racial or sex predisposition. These authors also claim that neutered males are slightly more affected by lymphoma, especially the alimentary type.

The lymphoma diagnosis is performed by clinical history, physical examination, laboratory and imaging tests, aspiration cytology, biopsy, and immunohistochemistry [8]. The treatment consists of chemotherapy protocols, usually with combined medicines, with no evidence that the associated surgical procedure is more effective than chemotherapy alone [9].

Nevertheless, antineoplastic medicines cause different toxic effects, especially regarding liver parenchyma [10] and animals with viral diseases, such as FIV and FeLV. The hepatotoxicity of chemotherapeutic medicines generally coincides with the increase in serum enzymes, such as alanine aminotransferase (ALT) and alkaline phosphatase (AP) [11]. Therefore, the elevation of the ALT and AP enzymes in the first chemotherapy sessions may indicate the toxic action of these medicines on the liver [11,12]. Myelotoxicity is another frequent and severe limiting factor of chemotherapy, impairing treatment efficacy and increasing the chances of metastases [13]. Additionally, it can lead the animal to death due to septic neutropenia [14]. Neutropenia is dose-limiting and can provoke septicemia with a risk of death for the patient [15].

Within this context, complementary therapies gain ground both as a primary or complementary treatment for cancer patients since they do not present side effects and they stimulate the immune system, improving life quality and, as a consequence, increasing the patient’s survival [16]. Viscum album is the plant most used in the world for the complementary treatment of cancer, with promising results recorded both as a curative or palliative strategy [17-19]. It is used in diverse injectable commercial forms, presenting a bidirectional effect to the treated patient. This medicine presents selective cytotoxicity, being aggressive only against the tumoral cells. It also presents immunomodulatory and anti-inflammatory activities [16].

[20] associated Viscum album with the traditional chemotherapy in dogs and observed a decrease in the total treatment time, reducing the chemotherapy side effects, such as leukopenia.

The objective of this study was to report the case of a domestic feline positive for FIV and FeLV, diagnosed with lymphoma, and successfully treated by injectable homeopathy.

Case Report

A 4-year-old male feline weighing 4.6 Kg, and positive for FIV and FeLV was seen at NaturalPet Clinic in Brasilia, Brazil. The animal has a history of difficulty swallowing concomitant with inappetence and respiratory difficulty. On physical examination, the animal was tachypneic with abdominal breathing, presented pale mucosa, CRT 3″, cardiac auscultation within the normal range considering age and species, mild dehydration, infarcted bilateral submandibular lymph nodes, and absence of pain on abdominal palpation. The examination of the oral cavity showed increased volume on the lower right side of the soft palate, small ulcerated portion, and hyperemic mucosa with apparent edema (Figure 1). Blood was collected for laboratory tests of complete blood count and biochemical measurements of alanine aminotransferase (ALT), alkaline phosphatase (AP), urea, and creatinine. The patient was also referred for a contrast-enhanced computed tomography. After the test results, the animal was referred for punch incisional biopsy (4 mm) and dental treatment, and the material was directed to histopathology. Subsequently, the treatment protocol was established through veterinary homeopathy. The following medicines were also prescribed, subcutaneously: Viscum album D3 (1×10^-3), D6 (1×10^-6), D9 (1×10^-9), D12 (1×12^-12), and D30 (1×10^-30) (Injectcenter^â), a combination of two ampoules, once a day, for 60 days; Arnica montana D4 (1×10^-4) (Injectcenter^â), one ampoule, once a day, for seven days; Apis melifera D4 (1×10^-4) (Injectcenter^â), one ampoule, once a day, for seven days; Mercurius solubilis D18 (1×10^-18) (Injectcenter^â), one ampoule, once a day, for 20 days. In addition, Stomorgyl^â 10, ½ tablet every 24 hours, for seven dias; Omega 3 1000 mg, one capsule, once a day, for 60 days; and Vitamin C 500 mg, one tablet, once a day, for 30 days, were orally prescribed. After five months, the animal returned to the clinic complaining of discomfort in chewing. On the physical examination, the animal presented an ulcerated lesion on the contralateral side of the initial lesion (Figure 2). The same initial treatment was initiated for additional 12 weeks. The day after the evaluation, the animal was submitted to dental treatment to extract molar and premolar teeth and cauterize the injured region.

Figure 1: Presence of slightly ulcerated and contaminated nodule in the oral cavity of the cat.

Figure 2: Presence of ulcerative lesions, represented by the white color arrow, in the initial portion of the soft palate and final portion of the hard palate (left side). On the right side of the image, represented by the green arrow, healed tissue previously affected by lymphoma.

Results

Contrast-enhanced computed tomography images (Figure 3) showed neoformation with an irregular shape, with venous contrast hyper-uptake presenting hypodensity areas in its interior, measuring approximately 3.0 cm in height x 3.5 cm wide x 4.8 cm in length, and located in the right submandibular lymph nodes topography. Signs of invasion of the neoformation mentioned above into the oropharyngeal region were observed, presenting signs of partial obstruction of the nasal choanae. The conclusion of the exam was suggestive of neoformation in the right submandibular lymph nodes topography, followed by the obstruction of the nasal choanae by a mass, as previously described. The results of the blood tests are shown in Table 1.

Figure 3: (A) Image in right longitudinal section (head and neck) – presence of a mass. (B) Ventral cross-sectional image of the animal’s head – presence of a mass on the left side. (C) Cross-sectional image of the sinus of the face – presence of a mass in the left sinus.

Table 1: Blood test data of a 4-year-old male feline, positive for FIV and FeLV, seen at NaturalPet Clinic in Brasilia, Brazil.

	May/2016	July/2016	September/2016	October/2016	January/2017	April/2017
Red blood cells (mL)	7,690,000	8,460,000	8,020,000	6,050,000	7,200,000	6,450,000
Hemoglobin (g/dL)	11	11.5	11.3	9.2	10.3	9.3
Hematocrit (%)	31.3	36.6	35	25.8	31.1	26.2
MCV (g/dL)	40.75	42.25	43.64	42.64	43.19	40.62
MCHC (g/dL)	35.14	31.51	32.29	35.66	33.12	35.5
Leukocytes (mL)	4,500	6,700	9,500	4,600	6,000	7,000
Eosinophils (mL)	585	402	0	506	720	630
Lymphocytes (mL)	1,800	2,278	855	1,794	1,440	1,750
Platelets (mL)	16,000	301,000	230,000	128,000	264,000	368,000
TPP (dL)	11.2	7.0	9.8	8.0	6.4	8.8
ALT (U/L)	55	**	**	**	33	53
Creatinine (mg/dL)	1.5	**	**	**	1.34	1.76
AP (U/L)	13	**	**	**	17	24
Urea (mg/dL)	51	**	**	**	53	31

One week after the biopsy procedure and treatment initiation, the animal returned to the clinic, and the lesion was much smaller (Figure 4A). The clinical improvement can be seen in Figure 4, from the first week after the Viscum album therapy initiation until the 28^th day of treatment, when the initial lesion was healed. The biopsy result was conclusive for lymphoma. The patient was followed up with complete blood count tests. The Viscum album applications should have happened daily, as per the veterinarian’s recommendation. However, the tutor of the animal discontinued the treatment, on her own, from the moment the lesion healed.

Figure 4: Lesion 1 (right side) – Evolution of the treatment and its response. (A) Lesion appearance seven days after treatment initiation. (B) Lesion appearance 14 days after.

After restarting the treatment protocol and extracting the teeth, treatment evolution occurred in three weeks (Figure 5). The tutor continued the treatment for 60 days, and complete lesion healing was observed. Also, the patient remained well for another 36 months and presented normal appetite, water intake, urination, and defecation until he died with severe anemia associated with stage 3 kidney disease.

Figure 5: Lesion 1 (left side) – Evolution of the treatment and its response. (A) Lesion appearance seven days after treatment initiation. (B) Lesion appearance 15 days after treatment initiation. (C) Lesion appearance 28 days after treatment initiation.

Discussion

Lymphoma is the hematopoietic neoplasm most commonly diagnosed in domestic cats, comprising approximately one-third of all tumors in this species. FeLV-positive animals continue to be potential candidates for lymphoma and feline leukemia involvement [21]. Lymphoma is a systemic neoplasm, and the basis of treatment is chemotherapy. Its proper use promotes increased longevity and quality of life of cancer patients, and in some cases, even achieves the complete cure of cancer [22]. The chemotherapeutic treatment aims to induce complete and lasting remission of the neoplasm, and in cases of recurrence, reinduce its remission [23].

Numerous treatment protocols have been reported for feline lymphoma with varied results. The reported literature includes a wide variety of chemotherapy protocols associated or not with corticosteroids that are typically extended for a year or more [21]. Many of these protocols have different side effects, which are limiting factors for FIV and FeLV patients due mainly to the immunosuppression caused by chemotherapy and corticosteroids. In contrast to these authors, the present report describes a case of lymphoma in an FIV and FeLV positive cat treated by the Viscum album therapy with satisfactory results. Furthermore, it is important to emphasize that the therapy used did not have side effects for the patient, nor does it have contraindications.

Also, according to [24-26], overall responses reported for feline lymphoma treated with various chemotherapy protocols vary widely, with median survival times ranging from 50 to 388 days. On the other hand, the present study reports that besides being FIV and FeLV positive, the animal was treated by complementary medicine, basically by the Viscum album therapy. Under this protocol, the patient presented a survival three times greater than the maximum survival recorded for patients treated exclusively by the conventional treatment in FIV and/or FeLV positive or negative animals.

Additionally, our findings are corroborated by [18], who also recorded disease resolution, increased life expectancy, and no side effects when using injectable Viscum album and Magnesia phosphorica for treating feline alimentary lymphoma. Therefore, these are promising data that point to the importance of ongoing studies on the effects of the Viscum album therapy on cancer patients to allow for meaningful decision-making and efficient disease treatment.

Conclusion

This study raises another possibility for treating patients to whom conventional therapies do not contemplate the quality of life associated with survival. Homeopathy is an excellent therapeutic tool for patients with pre-existing diseases, debilitated, or even as a complementary therapy for conventional treatments. Therefore, we propose further studies in the area so that the results obtained in this work are better validated.

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Infant Oral Health: Focus on Caries Prevention during Pregnancy

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DOI: 10.31038/AWHC.2021434

Abstract

Tooth decay or dental caries is the most common chronic disease of childhood. However, caries is also highly preventable through a combination of oral hygiene, the use of fluoride and dietary measures. Prevention of caries should start in the prenatal period because the maternal caries status is strongly associated with the caries status of their children. This short review will address the importance of early risk assessment for identification of parent-infant groups who are at high risk for Early Childhood Caries (ECC) and would benefit significantly from early preventive intervention.

Keywords

Dental caries, Early childhood caries, Pregnancy, Infant oral health

Introduction

Tooth decay or dental caries continues to be a major public health problem worldwide. Caries affects nearly 23% of U.S. children aged 2-5 years and 56% of those aged 6-8 years [1]. Early childhood caries (ECC) can have life-long consequences on the overall health of children due to severe pain, abscesses, chewing difficulty, malnutrition, poor speech articulation, low self-esteem, and increased risk for future caries [2,3]. Importantly, pregnant women are at higher caries risk due to reasons such as increased acidity in the oral cavity, sugary dietary cravings, and limited attention to oral health [4]. The lack of maternal dental care and the transmission of caries pathogens from caries-active mothers to infants lead to increased risk for childhood caries. Appropriate dental care and caries prevention during pregnancy may decrease infant and childhood caries. Therefore, early risk assessment is critical- for identification of parent-infant groups who are at high risk for ECC and would benefit significantly from early preventive intervention.

The Caries Process

The term caries lesions are the scientific term used to describe the signs and symptoms of the disease of dental caries (or tooth decay). A caries lesion is the result of a localized destruction (or demineralization) of the tooth surface caused by acids produced from the fermentation of dietary carbohydrates (mainly free sugars) by dental plaque bacteria. The destruction can affect tooth enamel, dentin and cementum. The first sign of a caries lesion on enamel that can be detected with the naked eye is called a white spot lesion. The caries process can be stopped or reversed at this point because enamel can repair itself by using minerals from saliva, and fluoride from toothpaste or other sources. But if the caries process continues, more minerals will be lost. Over time, the enamel is weakened and destroyed, forming a cavity. A cavity is a permanent damage on the tooth that a dentist has to repair with a filling. Unquestionably, a combination of oral hygiene, the use of fluoride and dietary measures must take place to avoid the onset of white spot lesions and their consequent progression into cavities.

Caries Prevention

Preventing caries and promoting oral health begins with establishing healthy habits in infancy. Dental caries is preventable through a combination of three major behaviors that are proven effective: 1) oral hygiene methods, 2) frequent exposure to fluoride, and 3) a diet that limits the intake of sugary components [5]. Oral Hygiene (OH) is key to remove the acid-producing dental plaque that can demineralize the teeth. The best OH recommendation includes tooth brushing twice a day (especially at bedtime) followed by flossing. In addition, fluoride is a mineral that can prevent, stop the progression, and even reverse early white spot lesions. Fluoride prevents mineral loss in tooth enamel and replaces lost minerals. Sources of fluoride include: drinking fluoridated water from a community water supply (about 74% of Americans served by a community water supply system receive fluoridated water, brushing teeth with a fluoride toothpaste, rinsing your mouth daily with a fluoride mouth rinse, and application of a fluoride gel or varnish to tooth surfaces at dental clinics by dental professionals. Also extremely important in caries prevention is a diet low in foods and drinks containing sugar and starches, which can be metabolized into acids by caries pathogens.

Infant Oral Health

The American Academy of Pediatric Dentistry (AAPD) recognizes infant oral health care as a foundation for offering preventive education and dental care that can enhance the opportunity for a lifetime free from preventable oral diseases [3]. The AAPD recommends that every infant receive an oral health risk assessment from his/her primary health care provider or qualified health care professional by 6 months of age [3], and that parents establish a dental home for infants by 12 months of age [6]. Caries risk assessment for infants allows for the implementation of appropriate prevention and management strategies as the primary dentition begins to erupt [3].

This initial oral health risk assessment should include: 1) evaluation of medical (infant) and dental (parent and infant) histories; 2) a thorough oral examination; 3) an assessment of the patient’s risk of developing oral diseases of soft and hard tissues; 4) education on infant oral health, oral hygiene measures (such as age-appropriate tooth brushing demonstration), nonnutritive sucking habits, teething, injury prevention, and the dietary effects on the dentition; 5) anticipatory guidance regarding dental and oral development; 6) evaluation and optimization of fluoride exposure; 7) development of a preventive treatment plan that includes prophylaxis, fluoride varnish and periodic re-assessment appointments; and 8) referral to the appropriate health professional if specialized intervention is necessary.

The ultimate goal of early assessment is the timely delivery of educational information to populations at high risk for developing caries and prevention of later surgical intervention. Despite the importance of early risk assessment, there is an unfortunate lack of access to dental services among young children in the United States, coupled with a limited workforce to address their oral health needs. In fact, the implementation of Infant Oral Health Programs (IOHP) in dental schools is not only an important community resource for preventive dental care for young children at high risk for caries but should increase the workforce providing care to this population [7].

References

Dye BA, Thornton-Evans G, Li X, Iafolla TJ (2015) Dental caries and sealant prevalence in children and adolescents in the United States, 2011-2012. NCHS Data Brief 1-8. [crossref]
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