The lineage of germ cells (GCs) ensures the perpetuation and diversification of genetic information across generations in most vertebral organisms. As part of their development into functional gametes, PGCs undergo extensive genetic and epigenetic modifications during their specification and development. We aimed to understand the dynamics of pluripotent, germline, and epigenetic markers of porcine PGCs (pPGCs) during the gestational and adult periods in both genders. The results demonstrated morphological differences between embryonic/fetal ages and genders in the expression of pluripotency and germinal markers. Additionally, different patterns of epigenetic markers were found at these embryonic/fetal ages. These findings are significant to the field of germ cells because they exhibit the progression of a variety of markers during pPGCs, and they may allow their use in applied science such as reproductive and regenerative medicine.Primordial germ cells (PGCs) are the precursors of gametes. Due to their importance for the formation and reproduction of an organism, understanding the mechanisms and pathways of PGCs and the differences between males and females is essential. However, there is little research in domestic animals, e.g., swine, regarding the epigenetic and pluripotency profiles of PGCs during development. This study analyzed the expression of epigenetic and various pluripotent and germline markers associated with the development and differentiation of PGCs in porcine (pPGCs), aiming to understand the different gene expression profiles between the genders. The analysis of gonads at different gestational periods (from 24 to 35 days post fertilization (dpf) and in adults) was evaluated by immunofluorescence and RT-qPCR and showed phenotypic differences between the gonads of male and female embryos. In addition, the pPGCs were positive for OCT4 and VASA; some cells were H3k27me3 positive in male embryos and adult testes. In adults, the cells of the testes were positive for germline markers, as confirmed by gene expression analysis. The results may contribute to understanding the pPGC pathways during reproductive development, while also contributing to the knowledge needed to generate mature gametes in vitro. For more than a century, the patterns and mechanisms that control the successful migration of germ cells have been a field of interest in reproductive biology ,2. MammaIn porcine embryos, the first clusters of PGCs were identified 11 to 12 days after fertilization. According to Kobayashi et al. , from E1Several molecular mechanisms and signaling pathways are active during mammalian PGC specification ,8. In miThe PGC cells undergo numerous processes from their emergence to their arrival at the genital ridge and can originate new individuals; hence, germ cells represent a unique cellular type. However, in some mammalian species and humans, when and how germ cells acquire this resource in embryogenesis, or how they carry out the migration process are essential questions in biological development that remain unanswered . In mammIt is known that the genetic, transcriptional, and epigenetic profiles of PGCs in humans and mice have some different characteristics, which may be related to differences in pluripotency status or even post-implantation development . In thisThe porcine, in this context, is a viable and highly advantageous model in regenerative studies and in other fields of reproduction, as it is considered a translational model that presents anatomy, physiology, and metabolism close to humans. The results are highly applicable and can be used in research in human and veterinary medicine, such as xenotransplantation ,22,23.However, knowledge about the in vivo and in vitro profiles of PGCs in porcine is still scarce ,25,26,27Therefore, this study aimed to determine the phenotypic profiles of porcine PGCs during embryonic development and in adulthood and evaluate the dynamics of germinal and pluripotency markers of these cells through immunofluorescence and RT-qPCR techniques. The knowledge acquired in these studies is expected to increase our understanding of the initial mechanisms of reprogramming germ cells during the reproductive process. Furthermore, we aim to understand the dynamics of epigenetic changes in these cells and the differences between such events in females and males. The generation of new knowledge about porcine PGCs in vivo can be transferred to the generation of mature gametes in vitro, which can, in turn, be used both in reproduction biotechnologies in domestic animals and as translational models ,31,32. Sus scrofa) females of a commercial hybrid strain  at the Swine Research Center, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Brazil. Females (n = 5) were submitted to artificial insemination, and embryos (n = 3 per age and sex) from 24 to 35 days dpf were used in this experiment. For all experiments, only females with three or more estrous cycles, aged approximately 220 days, and a body weight of ±130 kg were used. The animals were fed twice a day with commercial Agroceres Multimix® pelleted feed, respecting the requirements of the NRC (1998). Access to water was ad libitum provided by pacifier-type drinkers.All procedures performed in this study were conducted in accordance with the Ethics Committee for the Use of Animals of the Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo  and the Faculty of Animal Science and Food Engineering . The embryos and fetuses used in this experiment were collected from pregnant domestic porcine (The testes (n = 3) and ovaries (n = 3) of adult porcine were obtained from the slaughterhouse of the Faculty of Animal Science and Food Engineering, University of São Paulo, located on the Campus of USP “Fernando Costa”, Pirassununga.SRY gene (forward sequence 5′-TTCTGCAACAGGAGGATCGC-3′ and reverse sequence 5′- CACGGT GAAAAGGCAAGTCG—NM_214452.3) and the endogenous Zfx /Zfy  . The original or transformed data, when this procedure was necessary, were submitted to the Analysis of Variance and Tukey’s test for comparison between the means of the different experimental groups. In all statistical analyses, the significance level considered was 5% , it was possible to visualize the gonadal ridges and, laterally to it, the mesonephros. The tubules of the mesonephros are composed of epithelial cells, which form a simple cubic epithelium and are wrapped around blood vessels and a protective serous layer. In the caudal portion of this organ, it is possible to detect the presence of the metanephros tissue, or primitive kidney B. At 35 days, the gonads separate from the mesonephros, and it is already possible to observe the presence of testicular cords composed of centrally located primordial germ cells and presumably peripherally located supporting cells, or Sertoli cells. Germ cells between 30 and 35 dpf were positive for VASA (green) and the co-localization of OCT4 (red) B,C. Histological analysis of the porcine newborn testes with from 0 to 3 days of life showed some large and rounded cells in the center of the seminiferous tubules that presented morphological characteristics of undifferentiated spermatogonia and some Sertoli cells that were located closer to the basement membrane A. At thiThe morphological analysis of the testicles of adult males A showed The differentiated spermatogonia and primary spermatocytes were VASA-positive (green). DAZL (red) and STRA8 (green) were detected in differentiated spermatogonia close to the tubules’ basal membrane and primary spermatocytes, and some spermatids were STRA8-positive (green). Co-immunolocalization of STRA8 and DAZL and some primary spermatocytes. Furthermore, spermatocytes were c-Kit-positive (red) B. HoweveEmbryos at 24, 29, and 35 dpf in the neonates tests were negative for histone alteration markers (H3k9me2 and H3k27me3) detection, although H3k27me3 was detected in some cells at 25/26 days of gestation and in spermatocytes found in adult porcine testes B. The dyThe PGCs of the female porcine gonads had the following morphological characteristics: large, rounded cells with prominent nucleoli, as observed in germ cells of other species A. Embryocumulus oophorus cells in Graafian or pre-ovulatory follicles , in the primary follicle, and H3k27me3 (green), in interstitial cells, and in the cells that comprise the theca of Graff’s follicle B. OCT4 (POU5F1), SOX2, NANOG) and germline genes , SOX17, TFAP2C, VASA (DDX4), STELLA (Dppa3), DAZL, and STRA8) regarding porcine germ cells from both genders at four different periods, and in most of them, also in neonatal testes and adult ovaries or testes. When germ cells were analyzed at different periods, the expression of OCT4, PRDM14, and DAZL was increased in 35 dpf, regardless of sex . STELLA, DAZL, and VASA expression was observed and visually increased in adult testicles; however, it was not statistically different, possibly due to the high heterogeneity between groups. SOX17, TFAP2C, and PRDM14 were observed during embryonic periods, and TFAP2C and PRDM14 showed similar patterns . SOX2 was visually decreased in females as age increased, as expected. NANOG is a gene that exists in the germline of females at different gestational and adult periods , when gender was analyzed regardless of gestational age, which increased in females. The TFAP2C expression profile, however, was not different and was visually higher in males. Additionally, as previously described, OCT4 was increased at 35 dpf in both genders, SOX2 increased in adults, particularly in males, and DAZL was higher in adults as well; however, other differences may not have been observed due to the high heterogeneity between groups.When comparing the relative gene transcriptional abundance between genders , interesPrevious animal studies have discussed germline markers’ expression during the PGC differentiation process ,37,38,40As observed in dogs , the priAccording to Hyttel et al. , in porcIn porcine, the VASA transcript and protein were detected in embryos and adult germ cells, as described in humans, in both genders ,47. TheyIn male embryos, in all periods analyzed (24–35 dpf), co-expression of the OCT4 and VASA proteins was detected, with the STRA8 protein being detected specifically in embryos of 24 days of gestation, and the transcripts of this gene in the same period and at 28 dpf, similar to that described by Pieri et al.  and Lee DAZL was observed in porcine embryos between 28 and 51 days of gestation, and the marker was present from the 31st day of gestation, in addition to the expression in testes of adult porcine, as well as observed in the gonads of female porcine at 35 days of gestation, and in the testes. DAZL (deleted in azoospermia-like), a germ cell-specific RNA-binding protein, is reported essential for the development of PGCs [DAZL expression results in infertility in both genders, with post migratory germ cell apoptosis and a final block in meiosis [DAZL is a necessary “licensing factor” for the sexual differentiation of PGCs [Pieri et al.  describe of PGCs . In mice meiosis . It is a of PGCs ,51.In male neonates (1–3 days old), cells in the seminiferous tubules presented the markers VASA, DAZL, and STRA8, as evidenced in the gene transcription abundance analysis. Furthermore, PLZF  protein was detected in spermatogonial cells. PLZF is a marker of undifferentiated spermatogonia conserved in large animals ; it playIn mice, the 1-day-old neonatal testes are composed of testicular cords containing Sertoli cells; peripherally located and more centrally located gonocyte cells (pro-spermatogonia) have the PLZF, GFRA1, and CDH1 markers; and a small number of cells have the STRA8 cells or c-KIT markers. After 4 days of birth, it is already possible to identify two spermatogonial populations . In porcAs expected, the porcine adults with more than 150 days of life presented complete spermatogenesis, with spermatogonia, spermatocytes, spermatids, and spermatozoa. We also detected the proteins DAZL, VASA, STRA8, PLZF, and c-Kit, as described in dogs and porcine ,60,61,62In female embryos, at 25/26 dpf, cells positive for OCT4 and VASA were found; at 29 days, in addition to these markers, STELLA and STRA8 were also detected, while at 35 days, VASA was detected in some cells, similar to that described in female dog embryos at 35 days of gestation , The autIn adult females of reproductive age and complete oogenesis, it was possible to histologically observe the follicles in their different stages of maturation, from primordial follicles to tertiary or Graafian follicles, which was expected according to what was described in other species . A revieDAZL transcript. It was confirmed with the detection of this marker in primordial follicles and in the cytoplasm of oocytes, similar to that described in humans, where DAZL was detected in the cytoplasm of oogonia and follicular oocytes in fetal ovaries and adults. This marker, which plays an important role in the differentiation and maturation of germ cells [In the porcine ovaries, we observed the rm cells ,67, was rm cells ,68,69. Orm cells . In micerm cells ,70,71. Irm cells .SOX17, BLIMP1, and TFAP2C, as observed in this study. The pPGCs of both genders were evaluated at various gestational and adult ages, and H3k9me2 was not found. After puberty, H3k27me3+ cells were detected in the testes and ovaries; some of these markers show a progressive reduction in H3K9me2 expression, and global DNA demethylation ensues as pPGCs migrate toward the gonads . FurtherThe global metalation (5 mC) was detected in male embryos of 25–29 dpf. Intense demethylation occurs during germ cell development and at the beginning of embryonic development, compatible with the need to “redefine” the chromatin environment and restore the totipotency and pluripotency of the zygote and several stem cell populations. Such reprogramming steps involve the conversion of 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5 hmC), a likely intermediate in DNA demethylation. In mice, during the beginning of the specification of PGCs, the levels of 5 mC are the same as in other somatic cells ,74. HoweIn mice, PGCs from 6.75 dpc embryos present active markers H3K4me2, H3K4me3, and H3K9ac, along with repressive markers H3K27me2 and H3K27me3, similar to those found in somatic cells. Thus, H3K27me3 is upregulated when PGCs start to migrate around 8.25 dpc; at 9.5 dpc, almost all PGCs show high levels of H3K27me3, and this remains elevated in gonadal mPGCs, suggesting an important role in maintaining genomic integrity during the period of active global demethylation ,80. In cThe H3K27me3 was detected in the testes of adult porcine, in spermatocytes and round spermatids, but not in elongated spermatids, similar to what was described by An et al. , who demOCT4, PRDM14 and DAZL were increased after (35 dpf). In porcine, according to Kobayashi et al. [SOX17 and BLIMP1 in response to BMP signaling. Then, the pPGCs begin the migration process at about E15 days through the hindgut and reach the gonadal ridges at 22 days of gestation, and undergo extensive proliferation between 28 and 42 days of gestation [BLIMP1 gene, which represses somatic mesodermal genes in PGCs [TFPA2C and PRDM14. Herein, the transcripts’ abundance of pluripotency and germ cells related-genes and the development of PGCs and adult life was analyzed in porcine. pPGCs were found in ridge genitals at 24 dpf in male and 25/26 day in female embryos. Pluripotency and early specification genes were expressed in this stage, and estation . In mice in PGCs . At 7.5 SOX17 was increased in females regardless of age. A previous study in humans investigated the potential role of SOX17 involving germ cells maturation after specification, specifically in germ cells from a female fetus and adult gonads. The authors described that they detected SOX17 in both the cytoplasm and nucleus of oogonias at 7 gestational weeks (GW), and oocytes of primordial follicles, primary follicles from 15 to 28 GW, and in the nucleus of oocytes in secondary follicles, although the mechanism and SOX17 function in mature germ cells is unclear. Therefore, currently, there may not be enough information in the literature to form a conclusion about the difference in SOX17 expression between males and females [In embryos at E12.5-13.5, the porcine PGCs exhibit the co-expression of SOX17, BLIMP1, NANOG, OCT4, and TFAP2C proteins [ females . PRDM1 (BLIMP1), TFAP2C, NANOS3, and c-Kit, and the pluripotency genes NANOG and OCT4 were increased, whereas SOX2 was decreased. Similarly, we observed an upregulation of OCT4 in the embryonic gonads at 35 days, regardless of gender; however, this was not found in NANOG or the SOX expression in this same embryonic period. The SOX2 (SRY-Box Transcription Factor 2) belongs to the family of HMG box transcription factors related to SRY, which are considered important in the maintenance of pluripotency in early embryonic cells and in the formation of various epithelial tissues during fetal development [SOX2 + cells (spermatogonia A) was demonstrated in some seminiferous tubules, demonstrating a low transcriptional activity of this gene. The authors mention that SOX2+ adult stem cells originate from fetal Sox2+ tissue progenitors [Zhu et al.  analyzedelopment . In the genitors . OCT4 is expected, regardless of gender. In mice, OCT4 is detected in mPGCs during the specification process, and these remain until sexual differentiation occurs [According to Hyttel et al. , in porcn occurs . In feman occurs ,91, simiVASA and STELLA were present, but not increased at 35 dpf, and STRA8 visually increased at 25–28 and 35dpf, as well as DAZL at 35 dpf, corroborating with our findings in immunofluorescence. During embryonic development, the DAZL transcript abundance in porcine embryos between 28 and 51 days of gestation was analyzed, and transcripts of this marker were detected from the 31st day of gestation, in addition to the transcript abundance in testes of adult porcine, as well as in the gonads of female porcine at 35 days of gestation. The DAZL gene belongs to the gene family of DAZ (Deleted in Azoospermia). The DAZL is conserved in vertebrates and required throughout germ cell development, including in germ line progenitors [In porcine embryonic gonads, genitors ,93. Thisgenitors .DAZL, VASA, STELLA and SOX2 were increased in the adult phase in males, with a decrease in STRA8 during this period, while in female embryos, DAZL and STRA8 were upregulated at 28 and 35 dpf and in the adult phase [STRA8 is expressed in premeiotic germ cells and induces their meiotic entry. In mice, STRA8 gene expression was increased in the ovaries of young females paired with young males or elderly male mice, and the level of gene expression was age-dependent [Interestingly, when comparing male and female embryos, lt phase . STRA8 iependent .OCT4 and VASA transcripts [OCT4 transcript was only detected after 10–20 weeks in differentiated germ cells (spermatocytes and spermatids). Unlike what was observed in porcine by Goel et al. [VASA transcript was detected in porcine evaluated at 3 days of age. However, OCT4 protein was not found in adult testes; although OCT4 was downregulated in porcine, it was also observed in adult mice testes [In newborn porcine with 2 days of life, the qPCR analyses revealed that up until 7 days of life, they did not have nscripts . Furtherl et al. , in thise testes .OCT4, VASA, STELLA, and STRA8 were detected, and the same gene transcripts were quantified in oocytes and oogonial stem cells (OSCs), except for STRA8, which was not detected [STRA8 transcripts during the female’s reproductive cycle, showing overregulation in the estrus stage [Recently, a postnatal mouse ovary study demonstrated the expression of STRA8 in primordial follicles using immunofluorescence and RT-qPCR techniques , as obsedetected . Anotherus stage . In miceus stage . In femaus stage ,91.The present study showed clear morphological differences in the gonads and germ cells after 30 days of gestation, when sexual differentiation occurs in porcine. The formation of the genital ridge can be identified as a protuberance of the mesonephros at 24 days of gestation and continues to develop until 29 days, when differentiation into primitive gonads begins, ending sexual dimorphism at 35 days of gestation. In this period, it was possible to observe apparent histological differences between male and female embryos: in female embryos, at 26 dpf, OCT4 and VASA markers were observed, while at 29 dpf, many cells presented OCT4, VASA, and STELLA markers. STELLA is a maternal protein expressed in PGCs and oocytes. Gene transcription analysis revealed that, regardless of sex, there is a difference in the transcript abundance of the genes OCT4, SOX2, NANOG, STELLA, VASA, PRDM14, and DAZL when different gestational periods or adult tissues are analyzed. The difference in expression could be related to the developmental phase of germ cells in the gonads . In the adult phase, the cells of the spermatogenic lineage presented the markers for VASA, DAZL, STRA8, PLZF, and c-Kit, as observed in other species, and the ovaries and markers NANOG, STELLA, VASA, and DAZL were detected, mainly in the cytoplasm of oocytes and primordial follicles, as confirmed by qPCR analysis and described in other species, such as mice. STELLA is important for development and oocyte competence . OCT4, VFurthermore, although most migration patterns of PGCs in porcine are similar to those described in other species, mainly in comparison to humans, some differences in embryonic development highlight the need for further studies on the peculiarities of each species. Therefore, the results of this study may help in an unprecedented way in understanding the molecular and epigenetic profile of the porcine germ line from embryonic development to adulthood in both genders, even in future studies of in vitro gametogenesis, which can be used in reproductive biotechnology and as a model for applied studies.


Vitamin D is a secosteroid hormone that is highly involved in bone health. Mounting evidence revealed that, in addition to the regulation of mineral metabolism, vitamin D is implicated in cell proliferation and differentiation, vascular and muscular functions, and metabolic health. Since the discovery of vitamin D receptors in T cells, local production of active vitamin D was demonstrated in most immune cells, addressing the interest in the clinical implications of vitamin D status in immune surveillance against infections and autoimmune/inflammatory diseases. T cells, together with B cells, are seen as the main immune cells involved in autoimmune diseases; however, growing interest is currently focused on immune cells of the innate compartment, such as monocytes, macrophages, dendritic cells, and natural killer cells in the initiation phases of autoimmunity. Here we reviewed recent advances in the onset and regulation of Graves’ and Hashimoto’s thyroiditis, vitiligo, and multiple sclerosis in relation to the role of innate immune cells and their crosstalk with vitamin D and acquired immune cells. Vitamin D (VitD) is a secosteroid hormone that is primarily involved in bone health and mineral metabolism . Since iAbout 5–8% of the general population suffers from autoimmune diseases , which dOverall, almost 8 out of 10 patients with autoimmune diseases are women ,16. As aThis review focused on the implications of VitD in the regulation of the immune processes that ultimately result in clinically apparent autoimmune diseases. We chose to better detail AITDs, vitiligo, and multiple sclerosis, which might concur in the context of “Autoimmune Polyendocrine Syndromes” (APS) type III class C (APS3C). Instances of APS3C are relatively frequent and are defined by the combination of an AITD and another organ-specific autoimmune disease  involving, for example, the integumentary system  and nervous system  .Active VitD 2D3) regulates the transcription of more than 300 genes in humans, including some involved in the immune system’s functions, by interacting with the complex vitamin D receptor (VDR)/retinoic acid/promoter. The ability of VitD to boost the innate response and suppress the adaptive ones is supported by preclinical studies. Early clinical evidence comes from the application of cod liver oil supplementation in the prevention/treatment of tuberculosis . CalcitrIn experimental models, calcitriol reduces monocytes’ synthesis of inflammatory cytokines, such as tumor necrosis factor (TNF)-α, IL-1, IL-6, IL-8, and IL-12. Calcitriol also encourages the adoption of a tolerogenic phenotype by dendritic cells (DCs) by increasing inhibitory molecules  ,43,44. TIn vitro, high VitD levels directly suppress T cell proliferation and cell cycle progression from gap (G) 1a to G1b phases and promote the shift from T helper (Th)-1/Th17 to Th2 phenotype by suppressing Th1 , TNF-α) and Th17  cytokines in favour of Th2  cytokines ,50.VitD inhibits B cell activation and differentiation to plasma cells and induces apoptosis of activated B cells and immunoglobulin synthesis .In this context, the relationship between VitD and IL-33 could be of interest because these two molecules share some signaling pathways. For instance, both IL-33 and VitD encourage the activation of Th2 while inhibiting the Th1 response and they both orchestrate immunoregulatory cells . In someIt was hypothesized that VitD status and gender may interact to influence the likelihood of developing autoimmunity . VitD meBased on preclinical evidence, it is expected that VitD acts as a selective immune regulator. Several genetic and observational studies in humans supported the hypothesis that VitD signaling is involved in the prevention of autoimmune disorders . UnfortuAutoimmune thyroid disorders (AITDs), which include Hashimoto’s thyroiditis (HT) and Graves’ disease (GD), represent the most common autoimmune diseases, with a prevalence of 5% in the general population ,56. BothAITDs are caused by the loss of peripheral (defective self-reactive T cell apoptosis and reduction of Treg function) and central (disrupted thymus deletion of autoreactive T cells) immunological self-tolerance . Like ot+ (activated) T cells is higher, while CD8+ T cells are reduced compared with healthy subjects, but only in the active phase [+ T cells predominate in the thyroid infiltrate and many of these are activated [In the initial stage of HT, thyroglobulin (Tg) is presented to antigen-presenting cells (APC) and then processed into peptides that bind to T cell receptors. The interaction between CD80/CD86 molecules (on APC) and CD28 (on T cells) represents the second signal required for T cell activation. Studies on T cell phenotypes in HT revealed that the frequency of circulating HLA-DRve phase . CD4+ T ctivated . Althougctivated . The rolctivated ,61,62.Several genes predisposing to HT emerged, providing insights into the pathogenesis of thyroid diseases, as well as disease co-clustering within families and individuals. It is now known that HT is associated with HLA-DR3 and, to a lesser extent, with HLA-DR4 . PolymorThe link between VitD, inflammation, and HT has aroused interest among researchers worldwide ,73. BaseNotwithstanding these encouraging results made by basic science, only a few ambiguous data are available on the effects of VitD status on thyroid function in humans.Existing data from observational studies point to a higher prevalence of VitD deficiency in patients with HT  and to aMost of the available randomized control trials demonstrated the ability of VitD to decrease the TPOAb titer ,82,83,84GD shares many features with HT and what determines the type of disorder is a critical issue. GD is the most frequent cause of hyperthyroidism in iodine-sufficient areas . In susc+ T cells and an increase in HLA-DR+ T cells in active GD. Tregs largely infiltrate the thyroid gland, but they failed to stop the disorder [+ NKs were inversely related to TSR-Ab levels, while NKG2D+ NK cells were inversely related to serum-free T4 levels [Many studies found a reduction in circulating CD8disorder . From a disorder . Accordidisorder . Similardisorder . Considedisorder , a studydisorder . As prevdisorder . Additio4 levels , support4 levels ,94,95,964 levels ,98, agre4 levels ,100.+CD16+ monocytes were significantly expanded in patients with untreated GD and might participate in the production of TSHR-Ab by secreting relatively higher levels of serum-B-cell-activating factor (BAFF). The presence of monocytes was identified in the thyroid tissues of patients with GD, which might maintain and amplify inflammatory responses in situ, and thus, could serve as an important participant in GD pathogenesis [The pathogenic function of monocytes in GD is poorly understood. Chen and colleagues investigated monocytes in the periphery and thyroid tissue in GD, concluding that CD14ogenesis .The etiopathogenesis of GD is multifactorial with a genetic component. Studies on twins revealed that monozygotic twins are around 20–30% concordant for GD, which is at least 10-fold higher than dizygotic twins . HLA-DR3+ T cells, elocalcitol lowered Th1- and Th17-type cytokines and promoted Th2-type cytokine secretion [Data on the effects of VitD status in GD are scant, but the results of the available preclinical studies supported its protective effect. Elocalcitol (BXL-628) reduced IFN-gamma and TNF-alpha-induced CXCL10 protein secretion in human thyrocytes from GD patients more effectively than a placebo (standard anti-thyroid drug treatment). In CD4ecretion . In addiecretion . As for ecretion .However, the role of VitD in GD is still an unresolved issue, as the association does not necessarily imply a causal relationship. Though carried out on a limited number of GD and HT participants (255 patients), Inoue et al. suggested that several polymorphisms of the VDR gene may be involved in the development of HT and GD severity, thus supporting epidemiological findings . DisappoKawakami-Tami et al. randomly assigned 30 naïve GD patients to standard methimazole treatment alone or associated with calcitriol observing a greater decrease in serum thyroid hormone levels with the combined therapy . A recenThe association of selenium and cholecalciferol to anti-thyroid drug treatment was found to be more effective at improving hyperthyroidism compared with methimazole alone in a group of newly diagnosed GD patients with moderate-to-severe hyperthyroidism . RecentlVitiligo, which is the most common acquired depigmented skin disorder, affects approximately 0.5–2% of the worldwide population; it occurs mainly at a young age without gender predilection. It is characterized by the selective loss of melanocytes, and thus, the patient’s skin presents with milk-white macules and patches ,110. AccAlthough it is known that the vitiligo outcome and progression are due to melanocyte loss, the mechanisms of melanocyte death are not completely clear. Several options were proposed, such as neural, microvascular, degenerative, melanocyte adhesion, genetic, or autoimmunity theories, but none of these can be considered complete and exhaustive ,112. Fol+ T cells, with breaking of the tolerance in which IFN-γ has a fundamental role in the initiation of the disease that leads to the destruction of melanocytes [In vitiligo, innate immunity can be considered a bridge between oxidative stress and adaptative immunity, which is responsible for melanocyte destruction. It seems that human melanocytes, whose deaths are triggered by oxidative stress, release several damage-associated molecular patterns (DAMPs), such as DNA, HMGB1, and HSP70, that activate innate immunity and, consequently, adaptive immunity. Then, DCs are stimulated and they perform uptake, processing, and MHC presentation to CD8anocytes . Keratinanocytes .+ T cells, Th1 cells, NK cells, and other ILCs. In the process of chemokine release, a key role is also played by keratinocytes [Furthermore, IFN-γ can instruct CXCL9 and CXCL10 chemokine production, which reinforces the recruitment of CD8inocytes .+ T cells, and are KLRC1, KLRC2, NKG2D (KLRK1), KLRG1, and others [Interestingly, a transcriptome analysis performed on paired lesional and non-lesional skin biopsies from vitiligo patients and normal skin biopsies from healthy volunteers showed that 17 genes are downregulated (which are mostly related to the melanogenesis process), such as TYRP1, TYR, MLANA, and TRPM1, and 13 genes upregulated, most of which are implicated in innate and acquired immune cells, such as NK and CD8d others .Several reports showed an increase in the number of circulating NK cells in the blood of vitiligo patients ,119,120.+ T cytotoxicity, as shown by several studies in animal models in which NKG2D signaling promoted the development of long-lasting CD8+ T cells with enhanced cytolytic function [Recently, great interest was devoted to the NKGD2 molecule, which is involved in stressed cell recognition by innate immune cells, including NK cells, and the enhancement of TCR signaling. The human ligands for NKG2D consist of distinct stress molecules, such as the major histocompatibility complex (MHC)-class-I-related chain (MIC) A/B and ULBP binding proteins 1–6. Furthermore, NKG2D may have a role in human CD8function ,122,123.+ T cells, and this process is thought to be crucial in the development of vitiligo [+ T cells in lesion areas in the skin of vitiligo patients; these cells moreover present enhanced NKG2D levels and produce increased levels of IFN-γ and TNF-α cytokines [Indeed, melanocyte stress induces MICA and MICB molecules, i.e., NKG2DLs, that interact with NKG2D on CD8vitiligo  because vitiligo . In thisytokines .Vitiligo was associated with the APS, which represent a group of clinical conditions characterized by the failure of immune tolerance, functional deterioration of multiple endocrine glands, and the presence of autoantibodies . APS3C wIndeed, vitiligo  was found in 20% of patients with other autoimmune diseases. Among these, AITDs are the most frequent: up to 34% of vitiligo patients are affected by thyroid disorders . MoreoveThese findings are supported by many studies. A systematic review and meta-analysis by Fan et al. showed that a higher prevalence of anti-thyroperoxidase antibody and anti-thyroglobulin antibody could be observed in vitiligo patients relative to controls, in particular in NSV patients, compared with those with SV . AnotherThe correlation between vitiligo and AITDs can be explained by the presence of common heritable susceptibility genes: genome-wide association and linkage analysis techniques identified nine loci . For insOther examples are a single nucleotide polymorphism (SNP) on chromosome 8 encoding for Tg, which is shared among patients with vitiligo and an AITD, and the region of IL2RA gene on chromosome 10, whose variants are associated with Graves’ disease  .VitD plays an important role in regulating both the innate and adaptive immune systems, but it can also modify the function of other molecules and cells in specific organs, such as the skin. Indeed, VitD  influencLow serum VitD levels characterize many autoimmune disorders, such as AITDs, MS, and vitiligo. The first systematic review about the relationship between VitD levels and vitiligo by Upala et al. showed that vitiligo is significantly associated with a low serum VitD concentration . A case–El-Hanbuli et al. demonstrated that vitiligo melanocytes were characterized by a significant reduction in VDR protein in comparison to normal melanocytes. It was observed that in patients affected by vitiligo, VDR expression was remarkably reduced in the areas of “lesional/perilesional” skin, while re-pigmentation was accompanied by an increase in VDR expression .Vitiligo pathogenesis and etiology are very complex because of the high number of processes and cells that are involved. Moreover, many studies about the association between VDR gene polymorphisms and susceptibility to vitiligo provide interesting results. An analysis by Aydıngöz et al. in 2012 reported that individuals with TaqI polymorphism had a 2.23-fold increased risk of developing vitiligo . In 2015However, the specific molecular mechanism is still not clearly understood, and thus, further studies are needed. An observation conducted on 101 vitiligo patients showed that after six months of follow-up, sufficient 25(OH)D levels were associated with the stability of the disease and re-pigmentation. This evidence suggested a possible beneficial role of VitD supplementation on vitiligo patients . HoweverAutoimmune demyelinating diseases can affect the nervous system at the level of peripheral nerves, ranging from acute inflammatory demyelinating polyneuropathy to chronic forms, and at the level of the central nervous system (CNS), with MS being the most prominent one .MS is an autoimmune disease that affects the CNS and is characterized by chronic inflammation, perivascular infiltrates of mononuclear inflammatory cells, gliosis, demyelination of nerves, and the formation of multiple plaques in the spinal cord and brain, ultimately resulting in nerve damage that leads to various levels of neurological signs and symptoms and disabilities. It is a debilitating condition that presents early in life, with a mean age of onset of 28–31 years of age . The preEpidemiological studies indicated that MS is prevalent in high-latitude countries and this phenomenon is associated with a higher risk of vitamin D deficiency . The occIn the earlier phases of the disease, perivenular inflammatory lesions are markedly present and consist of mononuclear infiltrations. Those inflammatory lesions further lead to the formation of demyelinating plaques, which is the distinguishing pathological hallmark of MS .+ T cells. The presence of B cells and plasma cells was also recorded, although in much lower numbers. Oligodendrocyte damage and demyelination occur as a result of inflammation. Axons are relatively preserved in the early stages of the disease; however, as the disease progresses, irreversible axonal damage develops [The inflammatory infiltrates contain T lymphocytes, which are dominated by MHC class I restricted CD8develops ,163.The traditional view describes MS as the pathology mediated by autoreactive T cells recognizing auto-antigens that are activated in the periphery by mechanisms yet to be unveiled, such as molecular mimicry and the breakdown of immunological tolerance. The characteristic neuronal damage in MS results from a complex and dynamic interplay between the immune system (adaptive and innate), glia , and neurons. It has been confirmed for a few decades now that the CNS is not an immunologically privileged site  since th+ and CD4+), NK cells, dendritic cells, and B cells also infiltrate the CSF and then into CNS tissue, orchestrating local inflammation [During the progression of the disease, in addition to T cells (both CD8ammation .+) and cytotoxic (CD8+) T cells were described in MS lesions: CD4+ T cells are more predominantly present in the perivascular space, while CD8+ T cells are distributed within the parenchyma [Conventionally, MS was considered an autoimmune disease in which the adaptive immune system has a critical role. This is mainly due to the numerous studies done on animals via the usage of experimental autoimmune encephalomyelitis (EAE). In addition, early analysis of humans was done only by analyzing the cerebrospinal fluid and peripheral blood, and thus, the conclusion about the predominant role of the adaptive immune system, in particular T cells, was expected. Both helper  originating from peripheral blood can infiltrate active MS lesions and remove myelin debris and inflammatory derivatives. Microglial cells are the primary endogenous phagocytes of the CNS, and their abundant presence was confirmed in MS lesions. Nevertheless, the exact nature of their predominant role (pathogenic and/or protective) remains ill-defined .bright NK cells work ex vivo as inhibitory cells on CD4+ T cells via adenosine release, whereas in the context of autoimmune diseases, such as in juvenile idiopathic arthritis patients, these innate immune cells failed to perform this effect and, at the same time, CD56brightCD16− NK cells derived from inflammatory pleural effusions had a marked inhibitory function, emphasizing the possible regulatory role played by this subtype of NK cells [At the same time, NK cells are also much studied for their possible role in controlling MS pathogenesis, although the effective role of NK cells in MS remains a subject without conclusive results. In this context, Morandi et al. demonstrated that in healthy subjects, CD56NK cells . AdenosiNK cells .brightCD16− NK cells subset that was able, upon inflammatory cues, to kill autologous CD4+ T cell proliferation via granzyme B release and NKp30 and NKp46 involvement [bright NK cells [More recently, Laroni et al. added new data on the functioning of the NK subset by identifying a CD56olvement , thus reNK cells .Other components of the immune innate system, such as astrocytes or complements, are activated by cytokines and chemokines that are secreted in the early phases of an inflammatory reaction. This activation leads to a direct attack on myelin, axons, and glia, and it is mediated by cytotoxic cells and phagocytosis, proteases, cytokines, complement, glutamate, NO, and other intermediates.VitD effects on immune response in MS patients were confirmed in many in vitro studies, and numerous studies in animal models of MS and EAE strongly suggested the protective role of VitD. Despite this, the results of a few clinical trials that explored the influence of VitD on immune response in patients with MS were contradictory.+ T cells in acute and chronic plaques is well documented, as well as the oligoclonal expansion of these cells in CSF and the peripheral blood of MS patients [The abundant presence of CD8patients ,177.+ T cells have a higher expression of VDR than CD4+ T cells [+ T cells secreted lower quantities of the pro-inflammatory cytokines IFN-γ and TNF-α and more anti-inflammatory cytokines, such as IL-5 and TGF-β [CD8 T cells . Lysandrnd TGF-β .Concerning animal models of MS and EAE, VitD exerts formidable effects on Th1 and Th2 cells and Th1/Th2 cytokine synthesis, thus inhibiting EAE induction ,181.+ T cells through the VDR expressed in the nucleus of these cells, leading to the inhibition of EAE [Pedersen et al. showed that 1,25(OH)2D3 treatment significantly reduces the clinical severity of EAE by decreasing monocyte trafficking and chemokine levels , while Mn of EAE .Conversely, numerous studies carried out on MS patients reported conflicting results.+CD4+ Treg cell function associated with high levels of 25-OHD in serum in MS patients, while this association was not confirmed after vitamin D3 supplementation [Functional deficiencies in Treg cells with markedly decreased production of IL-10 were described in the peripheral blood of MS patients in several studies ,185,186.entation .In an EAE mouse model, it was shown that VitD supplementation can lead to an increase in the differentiation rate of Treg cells while inhibiting the proliferation of Th1 and Th17 cells .The effect of VitD on B cells is still not completely clear. In vitro studies showed that 1,25(OH)2D3 has several effects on B cells that may be beneficial in MS, such as inhibition of B cell proliferation and plasma cell differentiation, induction of apoptosis, augmentation of the generation of B memory cells, enhancement of Breg cell activity, and participation in immunoglobulin production. Moreover, it was shown that VitD promotes IL-10 production in human B cells; however, the inhibitory effects of vitamin D were not confirmed in vivo .Dysregulation of the phenotype and/or function of NK cells was reported in various autoimmune disorders through multiple mechanisms , and we Moreover, in MS, NK cells could have a role in the regulation of the autoimmune process, mainly via cytokine secretion or direct cytotoxic activity on effector cells, such as autoreactive T cells or antigen-presenting cells (APCs) ,193. ActConcerning VitD and NK cells, it was reported that the treatment of EAE-induced SJL mice with VitD3 or with monomethyl fumarate (MMF) contrast can prevent EAE, and this process was associated with enhanced in vitro NK cell lysis of monocyte-derived dendritic cells , corroboOf note, other studies reported that VitD3 ameliorates the clinical score in EAE mice , and othTaken together, these data support the fact that VitD actively modifies the immune response either by acting directly on T, B, and NK cells or by modulating DC function. Henceforward, it is appropriate to presume that a deficiency in VitD could augment the risk and/or progression of MS. However, that might not be the case.Several studies confirmed that 25(OH)D levels are lower in MS patients (20–84%) than in healthy controls ,200,201.Interestingly, lower VitD levels were found in MS patients with progressive forms of the disease with increased disability ,200. MunConversely, two studies demonstrated that the administration of cholecalciferol and/or alfacalcidol to MS patients had no effect or did not lead to significant improvement in clinical symptoms of the disease and the number of relapse episodes ,205. A dA published meta-analysis regarding the beneficial effect of VitD as a treatment for MS gave inconclusive suggestions, although it is worth noting that a higher level of VitD tends toward providing a favorable clinical response ,211,212.Genetic and biochemical data soundly demonstrated that VitD regulates a very large number of genes, most of which are not implicated in bone homeostasis. VitD status has a strong genetic basis, as emerged in twin studies. Large GWAS revealed at least four genes implicated in determining VitD levels and action. VitD’s influence on the immune system is supported by numerous studies in animal models and in vitro. The recent results of the VITAL trial demonstrated a protective role of VitD supplementation on autoimmune disorders. However, there is still no consensus on the extraskeletal effects of VitD on human health and diseases, mainly due to the lack of evidence of the clinical effect of VitD supplementation in the case of deficiency. This is likely due to the poor quality of randomized control trials, which were not homogeneous in VitD treatment , the definition of VitD status , and study population . Still, factors regulating local VitD activity are only partially understood. Standardization of the definition of VitD status, treatment scheme, and VitD assay is warranted to definitively clarify the implication of VitD in autoimmune disorders.


Proisotoma minima are always a dominant species during the study period. Seasonal variations are observed in the abundance, richness and diversity levels. In the severe land-degradation habitats (SLD), the abundance, richness, diversity and community complexity of the Collembolans are aways at the lowest levels. In addition, Proisotoma minima is negatively correlated with a majority of the species of Collembolans in the low levels of the land-degradation habitats, whereas they are positively correlated with most of the other species in the high levels. Epedaphic and euedaphic Collembolans responded to land degradation more obviously. The structural equation model (SEM) displays that soil Collembolan communities respond negatively to land degradation. Overall, our results provide implications that soil Collembolan communities are affected by land degradation, and that different taxa of soil Collembolans respond to degradation in numerous ways.Land degradation in black soil regions has a significant effect on belowground systems, and Collembolans can adequately indicate environmental changes in the soil. However, there is currently a knowledge gap in the literature regarding the responses of soil Collembolans to land degradation. In order to better understand this issue, in this study, a total of 180 soil Collembolan samples were collected from four habitats with varying degrees of land degradation in the Songnen Plain, namely a no land-degradation habitat (NLD), light land-degradation habitat (LLD), moderate land-degradation habitat (MLD) and severe land-degradation habitat (SLD). The results reveal that the different degrees of land degradation caused some differences in the taxonomic composition of the Collembolans; however, the majority of the Collembolan species are distributed relatively evenly.  Land degradation, a global issue affecting billions of people, is a phenomenon caused by a combination of natural forces and artificial activities . ErosionLand degradation can negatively impact the physical, chemical and biological quality of soil, with these effects today considered pervasive and systemic . GeneralCollembolans (springtails), which inhabit soil and litter, belong to the three lineages of modern hexapods, and are the key consumers and decomposers in the terrestrial ecosystem . Having Black soil, or mollisol in the USDA soil taxonomy, is characterized by its dark color and rich texture . Due to In this study, the investigation was conducted in the northern part of the Songnen Plain , where a great deal of black soil is widely distributed. This region is situated in the Xiao Hinggan Mountains and Songnen Plain intersection, and low hills account for a large proportion of its area. It is characterized by a continental monsoon climate, with long cold winters and short cool summers, and has a mean annual average temperature of 1.2 °C and mean annual precipitation of 490 mm. The soil type is mollisol, which is neutral or slightly acidic, with rich humus. Maize is most widely cultivated in the non-conservation tillage system used in the region, and thus land degradation is very common in the area. To evaluate the responses of soil Collembolans to land degradation in a black soil region, habitats with different degrees of land degradation were selected among maize planting fields. Land degradation was divided into four degrees following the classification method of Sun et al. . The hab2) and five replicate sample plots (1 m2) were randomly established. Soil core samples (25 × 25 × 20 cm) were taken in order to collect Collembolans in each plot. A total of 180 soil core samples (4 degrees × 3 replicated stands × 5 replicated plots × 3 sampling periods) were obtained. At the same time, soil samples were collected using soil augers in each plot.In this study, we collected samples in the spring (May), summer (July) and autumn (September) of 2021. Three independent stands  and a Nikon E200 biomicroscope , and Collembolan specimens were identified at a species level ,24,25.2Cr2O7–H2SO4 digestion and FeSO4 titration were applied to determine soil organic matter (SOM); the total nitrogen (TN) and available phosphorus (AP) values of the soil were measured using a SEAL AA3 auto analyzer ; the pipette method was applied to measure contents of soil clay particles and sand grains.After litters, roots and gravels were removed from the soil samples, each sample was air-dried and stored at room temperature. Soil properties were measured using conventional standards . BrieflyA Venn diagram was created to illustrate the differences in soil Collembolan taxonomic composition within the four land-degradation degrees, with endemic species being manually screened and a diagram being created using the “VennDiagram” R package . To anal−2) caused by land degradation, the Tukey’s HSD test was conducted using the “car” R package [To compare the differences in the soil Collembolan abundance  and Isotomiella minor (13.46%), and the common species included Parisotoma dichaeta (8.89%), Proisotoma sp.1 (8.89%), Ceratophysella sp.1 (5.81%), Folsomia candida (5.42%), Bionychiurus sp.1 (5.24%), Protaphorura sp.1 (4.18%), Micronychiurus sp.1 (3.89%), Homidia quadrimaculata (3.47%), Xenylla sp.1 (1.91%), Metaphorura affinis (1.26%) and Tomocerus kinoshitai (1.12%). Additionally, the residual 17 species were rare species, accounting for 6.28% of the total number of individuals.During the study period, 10,341 individual soil Collembolans were collected from the four land-degradation habitats: these belonged to 30 species, 26 genera, 12 families and three orders  . In totaProisotoma minima were always a dominant species during the study period. Isotomiella minor were observed to be a dominant species in the no land-degradation habitat (NLD), light land-degradation habitat (LLD) and moderate land-degradation habitat (MLD), whereas they were a common species (4.32%) in the severe land-degradation habitat (SLD). Parisotoma dichaeta were only a dominant species in the LLD (12.64%) and SLD (10.07%). At the same time, Proisotoma sp.1 were only a dominant species in the NLD (11.15%) and SLD (17.45%). Ceratophysella sp.1 was a dominant species (12.34%) only in the MLD.At the species level, there were both similar dominant species and different taxonomic compositions among the four land-degradation habitats A. ProisoProisotoma minima, Isotomiella minor, Parisotoma dichaeta, Proisotoma sp.1, Ceratophysella sp.1, Folsomia candida, Bionychiurus sp.1, Protaphorura sp.1, Micronychiurus sp.1, Homidia quadrimaculata, Xenylla sp.1, Metaphorura affinis, Desoria spatiosa, Anurida assimilis, Desoria pseudomaritima and Sminthurides sp.1) in common. Pseudosinella sp.1 was only collected in the LLD and MLD, while Friesea sp.1 was only found in the LLD and SLD. Bourletiella sp.1 was the only unique species found in the NLD.Across the different land-degradation habitats, both unique and shared species of soil Collembolans were found, as illustrated in the Venn diagram shown in Proisotoma minima was negatively correlated with a majority of the species of soil Collembolans found in the habitats with low levels of land degradation (NLD and LLD), yet it was positively correlated with most of the other species in the habitats with high levels of land degradation (MLD and SLD).The community structure and key topological features of the soil Collembolans were summarized using network analysis . It was −2 (27 species) Collembolan specimens were collected in the NLD; 333,200 m−2 (29 species) in the LLD; 308,000 m−2 (28 species) in the MLD; and 55,600 m−2 (18 species) in the SLD. Among these habitats, the NLD had the most abundance, and the LLD displayed the most richness. In contrast, the SLD had the least abundance and richness.During the study period, 337,300 mp < 0.05). During spring, significant differences were not found across all the four habitats (p > 0.05), and the highest abundance was observed in the MLD. In summer, there were no significant differences among the NLD, LLD and MLD (p > 0.05), and the MLD again had the greatest abundance. During autumn, the abundance of the NLD was the highest, and was significantly greater than the abundance of the MLD and SLD (p < 0.05).During the different seasons, the diversity characteristics of the soil Collembolans differed across the four habitats . The abuThe rarefaction curves of the soil Collembolan richness and diversity are shown in Proisotoma minima was only able to make up one cluster. Isotomiella minor, Proisotoma sp.1 and Parisotoma dichaeta formed another cluster. Finally, other species were divisible into a third cluster, and this indicated that the majority of Collembolan species were distributed relatively evenly. The heat map shown in Pseudachorutes, Bionychiurus, Micronychiurus, Protaphorura, Metaphorura, Tomocerus, Desoria and Isotomiella were significantly correlated with the majority of the soil environmental variables caused by land degradation (p < 0.05). This revealed that these genera might exhibit relatively sensitive responses to land degradation. At the same time, we found that most of the genera were significantly negatively correlated with the contents of soil sand grains (p < 0.05). This indicated that land sandification might be a major limiting factor for soil Collembolans. In addition, soil available P was found not to be correlated with most of the genera, except Arrhopalites, which had a significantly positive correlation with soil available P (p < 0.05).As illustrated by Pearson’s correlations, different environmental variables were correlated with each genus of soil Collembolans in numerous ways . Pseudac2/df = 2.145, p = 0.365, GFI = 0.907, CFI = 0.919 and RMSEA = 0.017). The SEM found that land degradation significantly negatively affected the soil fertility (regression weight = −0.8877) and soil texture (regression weight = −0.8877). At the same time, the soil Collembolan communities were positively influenced by the soil fertility (regression weight = 0.2521) and soil texture (regression weight = 0.1991). Therefore, this model indicated that land degradation may have impacted the soil Collembolan communities by affecting the soil fertility and soil texture.A structural equation model (SEM) was designed to evaluate the correlations among the land degradation (soil thickness and land productivity), soil fertility , soil texture (clay particles and sand grains) and soil Collembolan communities (abundance and richness) . The finIn this study, we observed that the soil Collembolan specimens from the severe land-degradation habitat (SLD), whether regarding their abundance, richness, diversity or community complexity, were always at the lowest levels compared to the other land-degradation habitats . Further, the community complexity found among the different habitats was consistent with the land degradation. This indicated that land degradation may have limited the soil Collembolan communities in the black soil study region. These findings were in line with our hypothesis that changes in soil Collembolan communities would be consistent with the land degradation present in the black soil study region (H1), and agreed with the previous study of soil bacterial communities . PreviouFurther, in this study, 27 species of Collembolans were collected in the NLD, 29 species in the LLD and 28 species in the MLD. These findings were partially out of line with our first hypothesis (H1). The “intermediate disturbance hypothesis” predicts that maximum levels of biodiversity should be observed under some intermediate disturbance frequency in ecological communities, which permits more species invasions and colonization . ComparePseudachorutes, Bionychiurus, Micronychiurus, Protaphorura, Metaphorura, Tomocerus, Desoria and Isotomiella were significantly correlated with the majority of the soil environmental variables caused by land degradation (p < 0.05) . Previous studies revealed that life histories, feeding guilds, propagation characteristics and adaptability mechanisms differed dramatically across soil Collembolan taxa ,41,42. C < 0.05) . Soil Coembolans . HerbivoProisotoma minima, a dominant species in all the habitats, was negatively correlated with the majority of soil Collembolan species in the habitats with low levels of land degradation (NLD and LLD), yet they were positively correlated with most of the other species in the habitats with high levels of land degradation (MLD and SLD)  . Proisotow niche . In the We found that soil Collembolan communities might have responded negatively to land degradation, because the land degradation changed the soil fertility and soil texture . Land deIn addition, this study still has a lot of room for improvement in the responses of soil Collembolan to land degradation. The morphological characteristics of soil Collembolan have been confirmed to diversify with environmental factors, and thus these characteristics are functional . ConsequProisotoma minima was negatively correlated with a majority of the species of soil Collembolans in the habitats with low levels of land degradation, whereas they were positively correlated with most of the other species in the habitats with high levels of land degradation. Additionally, each genus of soil Collembolans responded to the land degradation in numerous ways, with the epedaphic and euedaphic Collembolans responding more obviously to it. The findings of this study have implications for the study of the relationship between land degradation and soil Collembolans, and can provide some assistance in developing biodiversity guidelines for farmland protection in black soil regions.In summary, in this study, it was found that the soil Collembolan communities may have exhibited negative responses to land degradation in the study region. The Collembolan specimens’ taxonomic composition varied across the different land-degradation habitats, which were located in a black soil region of the Songnen Plain, but the majority of the Collembolan species were distributed relatively evenly across this region. The abundance, richness, diversity and community complexity of the soil Collembolans from the severe land-degradation habitat were always at the lowest levels compared to the other habitats. Further, seasonal variations were observed in the abundance, richness and diversity levels of the soil Collembolans.


GBA gene. Intravenous enzyme replacement therapy (ERT) is recommended for GD patients. In this paper, we report a case of a 2-year and 8-month-old girl with massive splenomegaly and radiological finding of hepatic gaucheroma, in whom a genetic study showed homozygous mutation on the GBA gene at c.1448T>C (p.Leu483Pro) and certified the diagnosis of GD. This patient represents the youngest child reported to have gaucheroma and also the first one presenting with gaucheroma at the diagnosis and not during the follow up, highlighting that GD should be routinely included in the differential diagnosis of children presenting with splenomegaly and hepatomegaly, taking into account that the early start of ERT can change the natural history of the disease-preventing serious complications.Gaucher Disease (GD) is a condition resulting from an autosomal recessive inheritance pattern, characterized by a deficiency of the lysosomal enzyme beta-glucocerebrosidase. This leads to the accumulation of glucocerebroside and other glycolipids in multiple tissues, causing damage to various organ systems. The diagnosis of GD can be challenging due to its heterogeneity, non-specific symptoms, and variability across different geographic regions and age groups. Although GD is suspected based on symptoms or signs, the diagnosis is confirmed through the measurement of deficient b-glucocerebrosidase activity and the identification of biallelic pathogenic variants in the  GBA gene, with more than 400 variants identified to date. The incidence of GD at birth varies in different regions of the world, ranging from approximately 0.39 to 5.80 per 100,000 individuals [Gaucher Disease (GD) is a condition resulting from an autosomal recessive inheritance pattern, characterized by a deficiency of the lysosomal enzyme beta-glucocerebrosidase. This leads to the accumulation of glucocerebroside and other glycolipids in multiple tissues, causing damage to various organ systems ,2,3. TheThe diagnosis of GD can be challenging due to its heterogeneity, non-specific symptoms, and variability across different geographic regions and age groups . GD is dIn the majority of cases, oculomotor neurological implication, usually associated with visceral manifestations, occurs before the age of 20. As GD1, GD3 phenotypes are heterogeneous, especially concerning neurological implications. Furthermore, neurological signs may appear several years after the visceral symptoms, even in patients originally diagnosed and treated as affected by GD1 [GBA gene [Patients affected by GD1 have a life expectancy that is usually around ten years shorter than that of the general population, even though advanced therapies could affect the survival of these patients. On the other hand, patients with GD2 usually do not survive beyond two years of age. Children affected by GD3 experience a slower progression of the disease and may live into adulthood. Often GD is not diagnosed or is diagnosed late; this leads to complications and the persistence of symptoms [In pediatric patients with symptomatic GD1 and GD3, intravenous enzyme replacement therapy (ERT) is recommended. Although ERT does not slow down or stop the progression of a neurological condition, it has been used off-label for palliative care in some cases of GD2 ,5. An orFinally, next-generation SRT, gene therapy, pharmacological chaperone therapy, and histone deacetylase inhibitors represent potential new treatments in development ,5.In this paper, we report a case of GD diagnosed in a child with massive splenomegaly and a radiological finding of hepatic gaucheroma, highlighting that GD should be routinely included in the differential diagnosis of children presenting with splenomegaly and hepatomegaly because the early start of ERT can change the natural history of the disease-preventing serious complications.A 2-year and 8-month-old girl was brought to our attention by her primary care pediatrician. The parents reported persistent abdominal distension during the last 18 months; a series of blood exams performed during the previous year showed anemia, thrombocytopenia, adequate hepato-renal function, and normal hemoglobin chain analysis. At physical examination, significant abdominal distension underlying massive splenomegaly (the splenic pole reached the low hypogastric region) and hepatomegaly was noted without any other significant symptom: general clinical conditions and neurological examination were in order. Development and growth also appeared adequate. Subsequently, the patient was admitted to our Pediatric Clinic Unit for further examination.Blood exams confirmed anemia and thrombocytopenia  with normal white cell count; blood smear showed anysopoikilocytosis without immature forms. Immunoglobulins and immunophenotype on peripheral blood were in order. Infective diseases  were ruled out; an auto-immune panel comprehensive of ANA, LKM1-2, ASMA, and AMA was carried out and resulted normal for age.Radiological assessment of the visceromegaly was performed: computed tomography (CT) and magnetic resonance imaging (MRI) described the massive splenomegaly and highlighted a hepatic focal lesion .GBA gene at c.1448T>C (p.Leu483Pro), and quantitative measurement of lyso-Gb1 was performed, which showed an elevated concentration (732.4 ng/mL). Therefore, the diagnosis of GD was made.At this point, a dried blood spot (DBS) test for GD and Nieman Pick A/B disease was carried out, revealing decreased activity of β-glucocerebrosidase A; genetic study was then conducted, which confirmed a homozygous mutation in the The case was discussed with pediatric radiologists and a pediatric surgeon, and since alfa-fetoprotein and βHCG came out negative, liver biopsy was excluded, and the hepatic lesion was characterized as hepatic gaucheroma.A neurological examination, including an EEG, was performed, which did not reveal any neurological changes, and no gaze palsy was observed.The patient was admitted to the referral center for GD where she began treatment and continued monitoring of the disease. Before starting the treatment, the enzyme glucocerebrosidase was measured again, and it remained deficient (0.3 microMol/L/h). Additionally, the value of lysoGb1 was confirmed to be elevated (782 ng/mL). At the age of 2 years and 10 months, she started ERT with intravenous imiglucerase (Cerezyme) at a dosage of 60 U/kg/dose every 14 days without showing any local or systemic reaction. A lower limb MRI was carried out, which showed marked signs of spinal cord impairment according to the underlying pathology. An echocardiogram and ECG were performed, with normal results. The ophthalmologic examination did not show any pathological changes.3/µL). The CYP2D6 genotype study showed normal activity with the following CYP2D6 genotype 2A/2A, making her a normal metabolizer of CYP2D6 substrates, allowing her to be switched to oral therapy with a substrate inhibitor (Eliglustat-Cerdelga) at the age of 18.Regarding laboratory tests, 3 months after the start of treatment hemoglobin values recovered (11.2 g/dL), while platelets remained stable  in peripheral blood cells and to analyze GBA1 mutations. DBS samples have several advantages, including ease of collection, minimal blood requirement, and straightforward transportation. Nevertheless, DBS has limitations when it comes to measuring GCase activity. Bone-marrow aspiration and biopsies to identify Gaucher cells are no longer considered diagnostic tools for GD and should only be done when assessing another hematologic comorbidity [To diagnose GD in our patient, DBS was performed to evaluate the enzymatic activity of β-glucocerebrosidase (GCase), followed by an analysis of the orbidity . Additioorbidity . Given tRegarding plasma biomarkers, they have been utilized for a significant period of time to diagnose and monitor patients with GD. However, traditional biomarkers, such as ACE, ferritin, alkaline phosphatase, and high-density lipoprotein, are not unique to GD, and more specific biomarkers, such as chitotriosidase and CCL18, have limited usefulness. On the other hand, Glucosylsphingosine (lyso-Gb1), the deacylated form of glucocerebroside, has been identified as a potential biomarker with high sensitivity and specificity for the diagnosis of GD .Pathological macrophages in GD release chitotriosidase. While higher plasma chitotriosidase activity is typically observed in type 1 GD patients compared to those with types 2 and 3, it is important to note that increased enzyme activity is not exclusive to GD. In fact, modest elevations in chitotriosidase activity can also be found in various lysosomal and non-lysosomal diseases . CCL18 is a member of the C-C chemokine family, and like chitotriosidase, it accumulates in the alternatively activated macrophages present in Gaucher cells in GD. An increase in CCL18 can also be found in various lysosomal and non-lysosomal diseases, similar to chitotriosidase .Lyso-Gb1 is involved in GD-associated bone pathology and chronic inflammation, making it a relevant biomarker. During GD treatment, lyso-Gb1 levels decline rapidly, like other biomarkers. Plasma levels of lyso-Gb1 are much higher in untreated GD patients than in healthy controls and correlate with hepatomegaly, splenomegaly, and platelet counts. Patients with neuronopathic GD have notably elevated levels of plasma lyso-Gb1 compared to those with non-neuronopathic GD .We observed hepatomegaly in addition to splenomegaly, and MRI showed a hepatic focal lesion radiologically defined as gaucheroma. Gaucheroma is an uncommon disorder characterized by the formation of a “pseudotumor” consisting of a group of Gaucher cells, typically found in the liver, spleen, bone, and lymph nodes .The prevalence of splenic and hepatic lesions in GD has been reported to range from 19% to 33% and 6% to 20%, respectively, across all age groups . GaucherAccording to our knowledge and the literature available ,11,12,13GBA1 gene: c.1226A>G (N370S), c.1448T>C (L444P), c.84dup, c.115+1G>A (IVS2+1G>A) and RecNciI are the most common [GBA gene variants may have a limited value in predicting organ involvement and disease severity. Patients with the same GBA genotype, and even siblings, may have different clinical manifestations. In addition, the GBA genotype does not seem to determine the response to ERT.As previously mentioned, over 400 mutations have been identified in the 1226A>G N70S, c.14Despite these limitations, the GBA genotype can be useful to distinguish between the classic neuronopathic and non-neuronopathic forms . If the Homozygosity for c.1448T>C p.Leu483Pro) found in our patient has been usually associated with GD3 ,13, whicPro foundSince our patient had no neurologic signs, both clinical and instrumental, it was not possible to define if she was affected by GD1 or GD3, and this will presumably become more evident during the monitoring of the disease.In the diagnostic algorithm of GD, an eventual GD3 definition is of significative importance because ERT does not cross the blood–brain barrier at therapeutic levels, thus potentially having no impact on neurological symptoms and deterioration ,16,17,18Although individuals with the p.Leu483Pr genotype often show neurologic manifestations, cases of adults treated with ERT showing no neurological worsening up to the age of 50 years are reported in the literature . These aRegarding gaucheroma, at the moment there is no clear indication in the literature on treatment regimen . SimilarThis case shows that GD should be routinely included in the differential diagnosis of children presenting with splenomegaly and hepatomegaly, considering that the early start of ERT can change the disease progression, preventing serious complications. There is no clear evidence regarding the efficacy of ERT for gaucheroma, but hopefully, new effective and safe treatments will be available in the next few years.


In Armenia, the presence of nitrofuran residues in food products is unacceptable for both domestic sales and export. However, food may contain nitrofuran metabolites (NMs) due to the illegal use of these drugs in the agrofarming practice. This study aimed to identify NMs as the marker residues for nitrofurans in fish and honey produced in Armenia and assess the potential health risks associated with consuming these foods. The commodities studied were natural honey and three species of farmed fish produced by various regions nationwide. Concentrations of the marker metabolites (3-amino-2-oxazolidinone (AOZ), 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), 1-aminohydantoin (AHD), and semicarbazide (SEM)) were determined through an enzyme-linked immunosorbent assay (ELISA) and verified using liquid chromatography–mass spectrometry (LC-MS/MS). Consumer groups were identified based on their average daily intake of foods. Health risk was assessed by calculating the margin of exposure (MOE). Reference values for health risk assessment were obtained from the European Food Safety Authority (EFSA). Results showed that 33.3% of fish samples and 44.4% of honey samples contained NMs, the mean concentrations ranging from 0.05 μg/kg to 0.52 μg/kg. All MOE values obtained were over 10,000, indicating that the detected concentrations of NMs in fish and honey produced in Armenia pose no health risk to consumers. However, these results highlight the illicit use of highly toxic substances and the need for improved control of farming practices. The presence of residues of veterinary drugs (VD) in food products is a major concern in food safety, leading to strict requirements being set at both the national and international levels ,2,3. TheIn the 1980s, there was a growing global awareness of the threat posed by inadequately regulated veterinary drugs (VDs), such as antimicrobials used in animal husbandry. In response, many countries implemented measures to reduce the use of antimicrobials and prevent their potential adverse effects. Notable examples include Norway, which achieved a significant reduction of antibiotics in aquaculture by tenfold in 1987 through the application of vaccines. This reduction was accompanied by establishing maximum residue levels (MRLs) for substance residues in raw materials and food products and the requirement for veterinary drug sales by prescription from a veterinarian. Numerous countries across the globe supported the fight against antibiotics, with Japan reducing the use of antibiotics in livestock production by nearly 30% from 2000 to 2013 through the reduction of antibiotic tolerance and the prohibition of antimicrobial additives. South Korea also made significant progress, reducing antibiotic use by over a third between 2007 and 2016 by allowing the sale of antimicrobials only with veterinarian prescriptions ,6,7.The European Union has implemented comprehensive regulations that cover the entire food chain, from feed resources (Regulation (EC) No 1831/2003) ,10. DespAmong the VDs, nitrofurans have been widely used as broad-spectrum antimicrobials in livestock farming. However, the use of nitrofurans in food-producing animals is not authorized by the European Union (Council Regulation (EEC) No 2377/90)  as they Robust evidence supports the high toxic hazard posed by nitrofurans. Specifically, nitrofurans have been found to possess carcinogenic, genotoxic/mutagenic, and hepatotoxic potential ,23,24. DIn Armenia, regulatory documents ,27 prohiIn 2018, the export of fish and honey from Armenia was estimated to be USD 23,806.72 and 259.4 thousand, respectively. In 2019, fish exports increased to USD 26,894.40 thousand, while honey exports decreased to USD 158.1 thousand. In 2020, the total export from Armenia accounted for USD 2537 mln, with fish and fish-based products still increasing and accounting for 10,430.49 tons valued at USD 49,684.96 thousand , while the export of natural honey showed a remarkable decrease  . DespiteFood consumption data were obtained through a survey using the food frequency questionnaire (FFQ) method. The FFQ questionnaire featured detailed inquiries about the serving sizes, as well as the frequency of fish and honey consumption. Additionally, the questionnaire included inquiries related to food sources and demographics, including gender, age, employment, education, and income level. To enhance data precision, the portion size was formulated as an open-ended question, offering responses in grams, units, and pieces. Consumption frequency was assessed across different categories from “no consumption” to “2–4 times per week”, also including less frequent options like “2–3 per month”, “once a month”, and “other”. To calculate the average daily intake of foods, we multiplied its daily consumption frequency by the portion size and quantity.In-person interview-based survey was conducted anonymously, ensuring a representation from all districts of Yerevan, the capital of Armenia, with 1040 respondents aged between 18 to 65 years old. Yerevan comprises about 30% of the country’s population, and the sample population is considered as representative of the Armenian adult consumers .For data analysis, the statistical software SPPP (version 22.0) was used. Employing the k-means clustering method, homogeneous clusters of consumers were identified. This approach proved effective, enabling accurate calculation of the risk associated with each consumer group. Indeed, the clusters of fish consumers were identified in a previous paper , and we Salmo Ishkhan), Rainbow trout (Oncorhynchus mykiss), and Sturgeon . The honey and fish analyses were carried out as part of the national monitoring program on residues in animal origin products, performed according to the requirements of the Council Directive 96/23/EC  ; Semicarbazide hydrochloride−13C, 15N2 (WITEGA); Ammonium Acetate (NH4Ac), K2HPO4, and NaOH (Sigma-Aldrich); Methanol  and water . Nitrofuran metabolites were hydrolyzed in an acidic solution and derivatized to nitrobenzyl- (NB) derivatives with 2-nitrobenzyladehyde (2-NBA). The following chemicals/reagents were used: AOZ and SEM•HCl ; 2-NBA (Sigma-Aldrich); DMSO (Sigma-Aldrich); d2 values exceeding 0.995. Rigorous validation of the method’s accuracy and precision was achieved by analyzing fish samples fortified with different concentrations of nitrofuran metabolites. The results showed recovery values ranging from 78% to 112%, along with deviations between 3% and 23%. Importantly, these standard deviations account for variations associated with both sample preparation and analytical instrument usage. The limit of detection (LOD) was equal to 0.05 μg/kg. Calibration curves demonstrated linearity within a range of 0.025 ng/mL to 10 ng/mL, with RThe risk assessment used the margin of exposure (MOE) approach. For genotoxic and carcinogenic agents, the MOE index is not considered a concern from the public health perspective when it is equal to or greater than 10,000 . MOE is selected . The HBGselected . food (Consumption) is the average daily intake of food (kg/day); Cmetabolite (Content)—the mean content of a metabolite in food (mg/kg); BW—a consumer’s body weight assumed as 65 kg on average.The DI is calculated for each consumer cluster using Equation (2):The samples with a value exceeding the limit of detection (LOD) increase the probability of the presence of small quantities of the agent in other samples with no detectable residues; accordingly, values < LOD, “left-censored” are considered as LOD/2 to take into account the uncertainty on the presence of residues below LOD .In risk assessment, the worst-case scenario involves considering the most unfavorable conditions or factors that could potentially occur. In this study, we have adopted a hypothetical worst-case scenario, where we consider the highest detected value per metabolite as the mean content of metabolites. Furthermore, for calculating the daily intake, we took the consumption data of the high consumers, representing the consumer cluster characterized by the highest product consumption.According to survey data, 83% of respondents have fish in their diet; besides, fish is among the special foods commonly served during fest events. The role of occasional high intake during festive events can only be discussed after assessing regular consumption. The share of honey consumers is 70%. Among fish consumers, the statistical data analysis has revealed 3 clusters of daily intake levels. The first cluster is characterized by an average daily intake of 16 g and accounts for 79% of respondents; the second cluster consumes 63 g of fish per day and accounts for 16% of respondents; the daily diet of the third cluster—5% of respondents—includes 156 g of fish.The honey consumers are also grouped into three clusters: 6 g/day, 28 g/day, and 59 g/day , which mNM residues were identified in 33.3% of fish samples and 44.4% of honey samples. Considering the left-censored samples, average NM concentrations in fish samples are presented in Upper bound (UB) values for nitrofuran metabolites in fish and honey samples are presented in The up-to-date reference point for action (RPA) for the four nitrofuran metabolites is 0.0005 mg/kg . Any fooAccording to research data for 2008–2010 in Bangladesh, an unintentional source of NM in shrimps was feed . A collaThe EFSA Scientific Panel on Contaminants in the Food Chain (CONTAM) did not have sufficient data to establish a reference point for adverse effects caused by AMOZ. As a result, no further calculations were conducted for this metabolite. It was determined that AMOZ is not genotoxic in vitro, and there were no available in vivo data on its genotoxicity or carcinogenicity at the time of the Opinion . Due to 10  corresponded to 1.6mg/kg b.w based on bronchial adenocarcinoma in mice. Additionally, a BMDL05(non-neoplastic) value of 0.02 mg/kg b.w. was selected based on adverse changes in alkaline phosphatase in dogs [10 of 1 mg/kg b.w; this value is chosen as HBGV for the risk characterization of noncarcinogenic effects [neoplastic) and noncarcinogenic effects (MOEnon-neoplastic). The calculated MOE values for fish and honey are given in The CONTAM Panel of EFSA has established two reference points for adverse effects related to AOZ: BMDLapproach . For AHDale rats . For AOZ in dogs . Further effects . For eac4 for fish and 6.89 × 104 for honey. Conversely, in the first cluster, AHD has the highest MOE values: 6.31 × 108 for fish and 6.15 × 108 for honey. In the third cluster, AOZ has the lowest MOE values: 3.47 × 10To assess the MOE value for the cumulative consumption of honey and fish in clusters with the highest consumption, we consider the maximum consumption values .It is important to note that the representatives of the third cluster for fish consumption and the third cluster for honey consumption are not the same individuals; we only consider the maximum consumption for the calculation to simulate a potential situation.5 to 1.27 × 108. This range represents the margin of exposure for individuals with the highest cumulative consumption of both fish and honey products. The MOE values at the sum of the maximum portions of fish and honey fall within the range of 1.04 × 10Using the margin of exposure (MOE) approach provides a comprehensive understanding of the potential risks associated with consuming NM-contaminated products. When the calculated MOE values exceed the established safety thresholds , the health concerns related to genotoxicity, carcinogenicity, and other hazards are insignificant.In the current study, the MOE values of all the studied metabolites are greater than 10,000, indicating that none of the indices oversteps the safety margin for carcinogenic as well as noncarcinogenic effects. This is observed, even with the cumulative consumption of the maximum portions of fish and honey, suggesting that occasional festive fish dishes, as well as honey, may not lead to significant health concerns.It is worth noting that, unlike some environmental contaminants, there are no human epidemiological studies on nitrofuran (NF) metabolites. The HBGVs are solely based on animal studies, which may introduce some additional uncertainty. Nonetheless, EFSA has considered the BMDL derived from animal studies as a reliable foundation for the risk characterization of NM.Unlike fish, honey commonly undergoes no culinary treatment before use. For this research, we sampled raw fish that did not undergo any culinary treatment before the laboratory test. NMs are known to remain almost completely preserved in food even after frying, baking, or microwaving . For thifood, representing the largest average daily serving size. The calculation data for this scenario are provided in In the worst-case scenario for fish, the highest concentrations of metabolites are as follows: AHD—1.450 μg/kg, AOZ—2.4 μg/kg, SEM—0.25 μg/kg. The value of the third cluster of consumers is assumed as CIn the scenario for honey, the following concentrations of the metabolites are obtained: AHD—1.85 μg/kg, AOZ—1.93 μg/kg, SEM—0.5 μg/kg. The average daily intake is assumed to be the index of the third cluster of consumers .In this worst-case scenario, the MOE value for the neoplastic effect of AOZ is less than 10,000 for both fish and honey consumers. For the other two NM, the MOE values indicate no concern. In the worst-case scenario, 100% of samples have the maximum concentrations of residues. However, such samples are quite rare; for example, samples with the AOZ concentration of 2.4 μg/kg account for only 6.67% of the total number of fish samples. This indicates that such high concentrations of metabolites are not commonly found in the tested samples, further supporting the overall low risk of adverse effects associated with NM residues in fish or honey consumption in the current scenario of Armenia. Meanwhile, some uncertainties need to be considered: (a) currently, we do not have food consumption data for toddlers and children; this might lead to an underestimation of exposure, as toddlers and infants tend to eat more food per kg body weight ; (b) no The worst-case scenario analysis employed in this study represents a crucial aspect of a risk assessment. By considering the highest detected concentrations of NMs along with maximum consumption rates, the study provides a comprehensive overview of the potential risks even under extreme, yet data-based, conditions. The fact that the MOE values remained comfortably above safety thresholds reinforces the robustness of the study’s conclusions and the safety of these food products.The study showed that exposure to NM residues through consuming fish and honey produced in Armenia does not lead to significant health concerns. Meanwhile, the frequent presence of NM residues in fish flags a problem. The illicit use of nitrofurans in aquaculture does not comply with standards for domestic food marketing, and it hampers the export of food commodities to external markets. The frequent presence of NM residues, while the use of nitrofurans in honeybees is very unlikely, indicates one more concern according to a One Health perspective: the illicit use of nitrofurans leads to the potential transfer of NM into the surrounding environment, with contamination of other food chains and influence on the ecology and resistance of microorganisms. A noteworthy dimension of this study is its alignment with the “One Health” approach. The interconnectedness of human health, animal health, and the environment becomes evident in the context of illicit nitrofuran use. The residues not only affect consumer’s health but also pose risks to other animal species. This underscores the need for collaborative efforts between veterinary, agricultural, public health, and environmental sectors to address these multifaceted challenges.Although we could not definitively identify the cause of NM occurrence in domestic fish and honey in this research, several potential reasons could be suggested: treatments of farmed animals, the illicit use of NFs as feed additives, and the use of NM-contaminated fertilizers in agriculture. Consequently, it is recommended that Armenian fish and honey producers thoroughly analyze the entire food production cycle to identify and address potential sources of contamination. Our findings further support that the presence of residues of prohibited drugs is a real issue  and indiOur data point out that the use of nitrofurans in Armenia, while not of direct public health concern, is of sufficient relevance to call for actions aimed at assessment, prevention, and monitoring; our findings may prompt further studies in other countries in the Caucasus region, that share several socio-economic and environmental features with Armenia. Beyond the identification and quantification of nitrofuran metabolites in food items, the study presents a thorough evaluation of consumer intake and associated risks. Through risk assessment, worst-case scenario examination, and actionable suggestions, the study establishes a model for addressing food safety challenges within Armenia and globally.