Theofilos Poutahidis

Background/Objectives: Ulcerative colitis (UC) is a chronic inflammatory bowel disease in which alterations in the gut microbiota and dietary lipid composition play a central role; this study aimed to evaluate the effects of synbiotics, omega-3 polyunsaturated fatty acids, and their combination on clinical, macroscopic, microbiological, and histopathological outcomes in dextran sodium sulfate (DSS)-induced colitis in Wistar rats. Methods: Seventy-two male Wistar rats were randomly allocated to four groups (n = 18/group) and received 5% DSS in drinking water for eight days to induce colitis. Following DSS withdrawal and histological confirmation of colitis in sentinel animals, groups were treated for 8 days as follows: DSS (control), DSS-S (synbiotics, Ecologic^® 825), DSS-Ω3 (omega-3 fatty acid-enriched diet, ProSure^®), or DSS-S&Ω3 (combined therapy). Eight rats per group were sacrificed on days 4 and 8 post-DSS. Body weight, Disease Activity Index (DAI), distal colon length, hematologic parameters, bacterial translocation to the liver and mesenteric lymph nodes, histological colitis score, and myeloperoxidase (MPO)-positive cell counts were assessed. Results: DSS induced severe colitis characterized by diarrhea, rectal bleeding, and extensive mucosal erosions. After 8 days of treatment, the DSS-S&Ω3 group showed the greatest body-weight recovery (206.1→222.9 g, p < 0.05 vs. other groups), significantly preserved distal colon length, and the largest reduction in DAI (p < 0.05). Both the DSS-S and DSS-S&Ω3 groups demonstrated reduced bacterial translocation compared with DSS. The DSS-Ω3 group demonstrated persistent MPO-positive neutrophil infiltration compared with the DSS-S and DSS-S&Ω3 groups, whereas combined therapy was associated with lower MPO-positive cell counts. Histological colitis scores were significantly improved only in the DSS-S&Ω3 group (p < 0.05). Conclusions: In this DSS colitis model, the DSS-S&Ω3 group demonstrated superior clinical and histological outcomes compared with DSS-S or DSS-Ω3 alone, supporting further evaluation of combined synbiotic and omega-3 therapy as an adjunctive approach in ulcerative colitis.

The thymus, a central organ for T cell development, lacks standardized pathology methods to assess its structure and function. Most studies rely on flow cytometry, which profiles thymic cell populations but offers limited spatial insight into tissue organization. This gap restricts our understanding of how physiological or pathological conditions remodel thymic architecture and impact immune competence. Here, we introduce a digital pathology pipeline that integrates analytical algorithms with rationalized morphometric assessments at tissue-wide, microanatomical, and ultrastructural levels. We develop and validate it using a mouse model of age-dependent thymic involution, characterized by cortical atrophy, medullary expansion, and epithelial disorganization, providing a benchmark for quantifying known lesions and testing reproducibility. As a proof-of-concept, we apply the pipeline to a DMBA-induced carcinogenesis model combined with early-life cholera toxin (CT) exposure, to evaluate whether environmental stimulation (“hygiene hypothesis”) mitigates late-stage thymic pathology. CT normalizes select parameters, particularly in the medulla, including reduced perivascular space and mTEC density, suggesting enhanced thymocyte occupancy. Overall, this versatile and scalable pipeline provides a standardized framework for quantitative evaluation of thymic structure, supporting both basic and translational studies of thymic biology and disease.

The objective of this study was to assess the effect of Sea-buckthorn (SB) on mink productivity and health. The experiment lasted 10 months and was divided into reproductive and growing periods. 100 brown female mink were randomly assigned to two groups (n=50): SB (1%SB) and Con (control). Body weight (BW) of animals during reproduction period was measured. Mating (MR) and whelping rate (WR), litter size (LS) and kit mortality (KM) were recorded. BW of SB group was higher until the end of mating (p0.05), however SB group weaned more kits. Post weaning, 100 offspring from each group (50 males:50 females) were randomly selected and equally allocated to groups: A and B (1%SB) and C and D (control). Groups A and C descended from SB group, while B and D from Con. IL-4, IL-8 and IFN-γ levels in blood serum and Tgf-β1, IL-1B, IFN- γ and IL-6 in spleen samples were determined. Immune cells (B-lymphocytes, granulocytes, T-lymphocytes) in colon, mesenteric lymph node (MLN) and spleen were counted. Blood serum cytokines levels were significantly higher in group B compared to D. Group A had higher IL-4 levels compared to D. The levels of IL-8 and IFN-γ decreased in A and C compared to D. In spleen samples Tgf-β1 was lower in B compared to the rest (p<0.05), while IL1-B in A was higher compared to C and D (p<0.05). Moreover, IL-6 was higher in group A compared to D (p<0.01). A reduction of T-lymphocytes in MLN of A compared to those of B was observed and, in the T-lymphocytes in colon of A compared to the rest groups (p<0.05). In spleen, B-lymphocytes were decreased in group A, compared to B and D, as also in C, compared to B and D (p<0.001). Microbiome analysis revealed enrichment of Lactobacillus in group A and higher abundance of Candidatus Arthromitus, Clostridium sensu stricto 1, and Escherichia-Shigella in D. The findings suggest that 1% SB affected the gut bacterial flora and the immune system tone of mink, while it presented limited effect on their productive traits.

The thymus, a central primary lymphoid organ of the immune system, plays a key role in T cell development. Surprisingly, the thymus is quite neglected with regards to standardized pathology approaches and practices for assessing structure and function. Most studies use multispectral flow cytometry to define the dynamic composition of the thymus at the cell population level, but they are limited by lack of contextual insight. This knowledge gap hinders our understanding of various thymic conditions and pathologies, particularly how they affect thymic architecture, and subsequently, immune competence. Here, we introduce a digital pathology pipeline to address these challenges. Our approach can be coupled to analytical algorithms and utilizes rationalized morphometric assessments of thymic tissue, ranging from tissue-wide down to microanatomical and ultrastructural levels. This pipeline enables the quantitative assessment of putative changes and adaptations of thymic structure to stimuli, offering valuable insights into the pathophysiology of thymic disorders. This versatile pipeline can be applied to a wide range of conditions that may directly or indirectly affect thymic structure, ranging from various cytotoxic stimuli inducing acute thymic involution to autoimmune diseases, such as myasthenia gravis. Here, we demonstrate applicability of the method in a mouse model of age-dependent thymic involution, both by confirming established knowledge, and by providing novel insights on intrathymic remodeling in the aged thymus. Our orthogonal pipeline, with its high versatility and depth of analysis, promises to be a valuable and practical toolset for both basic and translational immunology laboratories investigating thymic function and disease.

Neutrophil and T-cell recruitment contribute to hepatic ischemia/reperfusion injury. The initial inflammatory response is orchestrated by Kupffer cells and liver sinusoid endothelial cells. However, other cell types, including γδ-Τ cells, seem to be key mediators in further inflammatory cell recruitment and proinflammatory cytokine release, including IL17a. In this study, we used an in vivo model of partial hepatic ischemia/reperfusion injury (IRI) to investigate the role of the γδ-Τ-cell receptor (γδTcR) and the role of IL17a in the pathogenesis of liver injury. Forty C57BL6 mice were subjected to 60 min of ischemia followed by 6 h of reperfusion (RN 6339/2/2016). Pretreatment with either anti-γδΤcR antibodies or anti-IL17a antibodies resulted in a reduction in histological and biochemical markers of liver injury as well as neutrophil and T-cell infiltration, inflammatory cytokine production and the downregulation of c-Jun and NF-κΒ. Overall, neutralizing either γδTcR or IL17a seems to have a protective role in liver IRI.

The impact of dietary inclusion of Spirulina platensis on the immune system, intestinal microbiome and skin of mink was investigated. Forty-eight animals were equally separated into four groups. Groups B and D were control animals, while groups A and C had their feed supplemented daily with 100 mg/kg of body weight Spirulina. Mink in groups A and B were descended from dams supplemented with spirulina during their reproductive period, while those in groups C and D were descended from dams fed the control diets. Fur growth rate and quality were graded semi-quantitatively. Fecal microbiome analysis, skin thickness histomorphometry, immunohistochemical labeling and counts of immune cells in the colon, mesenteric lymph nodes and spleen and quantitative gene expression analysis of cytokines in the colon were performed. Skin thickness, fur growth rate and skin quality were similar among groups (p > 0.05). However, differences were observed among groups concerning the relative and differential abundance of bacterial species. Tgf-β expression was lower in group A, whereas IL-β1 was lower in group C compared to group B (p < 0.05). Group D had significantly lower numbers of inflammatory cells in the colon and mesenteric lymph nodes. The results revealed that Spirulina decreased indices of subclinical inflammation in mink gut, while differences in the bacterial communities among groups were observed.

Maternal microbial dysbiosis has been implicated in adverse postnatal health conditions in offspring, such as obesity, cancer, and neurological disorders. We observed that the progeny of mice fed a Westernized diet (WD) with low fiber and extra fat exhibited higher frequencies of stereotypy, hyperactivity, cranial features and lower FMRP protein expression, similar to what is typically observed in Fragile X Syndrome (FXS) in humans. We hypothesized that gut dysbiosis and inflammation during pregnancy influenced the prenatal uterine environment, leading to abnormal phenotypes in offspring. We found that oral in utero supplementation with a beneficial anti-inflammatory probiotic microbe, Lactobacillus reuteri, was sufficient to inhibit FXS-like phenotypes in offspring mice. Cytokine profiles in the pregnant WD females showed that their circulating levels of pro-inflammatory cytokine interleukin (Il)-17 were increased relative to matched gravid mice and to those given supplementary L. reuteri probiotic. To test our hypothesis of prenatal contributions to this neurodevelopmental phenotype, we performed Caesarian (C-section) births using dissimilar foster mothers to eliminate effects of maternal microbiota transferred during vaginal delivery or nursing after birth. We found that foster-reared offspring still displayed a high frequency of these FXS-like features, indicating significant in utero contributions. In contrast, matched foster-reared progeny of L. reuteri-treated mothers did not exhibit the FXS-like typical features, supporting a key role for microbiota during pregnancy. Our findings suggest that diet-induced dysbiosis in the prenatal uterine environment is strongly associated with the incidence of this neurological phenotype in progeny but can be alleviated by addressing gut dysbiosis through probiotic supplementation.

Canine lymphoma is a commonly reported neoplasia and, in most dogs, arises from lymph nodes before spreading to other organs. Renal lymphoma rarely occurs, and kidneys usually are a secondary site of origin. Primary renal lymphoma is infrequently described in the veterinary literature. In this study, we present a rare case of primary renal lymphoma in a dog and a review of similar cases. A 3-year-old male dog was admitted due to anorexia, weakness and vomiting. Clinical examination revealed bilaterally enlarged kidneys. Imaging demonstrated the presence of multiple renal masses. Cytology of abdominal fluid and kidneys led to the diagnosis of large cell lymphoma. Histopathology and immunohistochemistry on tissue samples taken from the kidneys confirmed the cytological diagnosis of lymphoma and categorized it as primary bilateral renal large B-cell lymphoma (LBCL).

SARS-CoV-2 infection outbreaks in minks have serious implications associated with animal health and welfare, and public health. In two naturally infected mink farms (A and B) located in Greece, we investigated the outbreaks and assessed parameters associated with virus transmission, immunity, pathology, and environmental contamination. Symptoms ranged from anorexia and mild depression to respiratory signs of varying intensity. Although the farms were at different breeding stages, mortality was similarly high (8.4% and 10.0%). The viral strains belonged to lineages B.1.1.218 and B.1.1.305, possessing the mink-specific S-Y453F substitution. Lung histopathology identified necrosis of smooth muscle and connective tissue elements of vascular walls, and vasculitis as the main early key events of the acute SARS-CoV-2-induced broncho-interstitial pneumonia. Molecular investigation in two dead minks indicated a consistently higher (0.3–1.3 log₁₀ RNA copies/g) viral load in organs of the male mink compared to the female. In farm A, the infected farmers were responsible for the significant initial infection of 229 out of 1,000 handled minks, suggesting a very efficient human-to-mink transmission. Subsequent infections across the sheds wherein animals were being housed occurred due to airborne transmission. Based on a R₀ of 2.90 and a growth rate equal to 0.293, the generation time was estimated to be 3.6 days, indicative of the massive SARS-CoV-2 dispersal among minks. After the end of the outbreaks, a similar percentage of animals were immune in the two farms (93.0% and 93.3%), preventing further virus transmission whereas, viral RNA was detected in samples collected from shed surfaces and air. Consequently, strict biosecurity is imperative during the occurrence of clinical signs. Environmental viral load monitoring, in conjunction with NGS should be adopted in mink farm surveillance. The minimum proportion of minks that need to be immunized to avoid outbreaks in farms was calculated at 65.5%, which is important for future vaccination campaigns.

The objective of the present study was to investigate the mechanical indices of hoof horn and their association with length measurements and lesion score. The feet of 185 culled sows from three Greek farms (A: 57 sows; B: 64 sows; C: 64 sows) were used. A slice from the dorsal wall of each claw was used to assess by a three-point bending test the Young’s modulus, yield stress and aximum stress values. The available data from a companion study (part 1) on the length measurements and lesion scores of the claws were used to reveal possible relationships. The Young’s modulus values were significantly higher (p < 0.001 or p < 0.01 depending on location of claw) in the sows of farm C compared to those in sows of farms A and B and in sows of farm B compared to those in the sows of farm A. Yield and maximum stress values were significantly higher (p < 0.05 or p < 0.001 depending on the location of the claw) in the sows of farm C compared to those in the sows of farm A and in the sows of farm B compared to those in the sows of farm A. An increase in heel-sole length decreased all mechanical indices. Young’s modulus and yield stress were associated with wall lesion severity while maximum stress with wall and heel lesion severity. Overall, we conclude that mechanical efficiency deteriorates as length and lesion score increases.