Multiplex Immunofluorescence Analysis of Infiltrating Mononuclear Cell Subsets in Acute T-cell-mediated Rejection and Bk Virus-Associated Nephropathy in The Allograft Kidney

Transplant rejection has been diagnosed by mononuclear cell infiltration patterns light microscopically. Although infiltration involves different types of mononuclear cells, analysis of the rejection-associated variation was difficult by usual immunohistochemistry; one antibody staining on each slide and laborious cell counting. The authors applied the newly developed multiplex immunofluorescence staining(MIF) and image analysis system to overcome those disadvantages. By using formalin-fixed, paraffin-embedded slides of acute T-cell–mediated rejection (TCMR, 9 cases) and BK virus-associated nephropathy (BKVAN, 5 cases), the immunologic analysis was done because both had similar mononuclear cell infiltration patterns histologically. Antibodies to CD4, CD8, CD20, CD68, Foxp3, and cytokeratin were used.The cellular subsets were similar to each other. CD68+ cells were dominant in the cortical interstitium. However, by using this method, we could find differences in cellular distributions in the area by area. In the medullary rays, albeit the location being in the cortex, cellular subsets were similar to those of the medulla. In areas surrounding large vessels thought of as non-diagnostic areas, the CD20+ cells were dominant. This MIF could use as an ancillary method of diagnosis and follow up the cellular changes during rejection or treatment. Further large samples with rejection condition-matched studies should be followed.

Pre-existing thyroiditis ameliorates papillary thyroid cancer: insights from a new mouse model

Papillary Thyroid Cancer (PTC) often co-occurs with Hashimoto’s thyroiditis, an association that has long been reported in clinical studies yet remains controversial. Some studies, in fact, have suggested a protective effect of thyroiditis while others have not. We generated a mouse model where PTC and thyroiditis develop in a predictable manner, combining the oncogenic drive of the BRAF v600E mutation (inducible by tamoxifen) to the thyroiditis susceptibility of the NOD.H2 h4 strain (inducible by iodine). A total of 113 NOD.H2h4_TPO-CRE-ER_BRAFV600E mice (50 followed throughout lifetime and 63 sacrificed at 16 weeks post tamoxifen) were used to determine whether the PTC phenotype differs when thyroiditis precedes or coincides with the onset of PTC. Mice with pre-existing thyroiditis lived longer (median survival of 28.2 weeks post tamoxifen) than those with concomitant (25.6 weeks) or no (24.5 weeks) thyroiditis (p<0.01 by Laplace regression). PTC developed less frequently (33%) in the pre-existing thyroiditis group than the concomitant (100%) or no (100%) thyroiditis groups (p<0.001 by chi-squared) and showed less aggressive histopathological features. The intra-tumoral mononuclear cell infiltration was more prominent in mice with pre-existing thyroiditis (p= 0.002 versus the other groups) and sustained by a significant expansion of effector memory CD8+ T cells and CD19+ B cells. These findings shed light on the controversial PTC-thyroiditis association and emphasize the contribution of intra-tumoral T and B lymphocytes to the evolution of PTC.

IL-17 Upregulates MCP-1 Expression via Act1 / TRAF6 / TAK1 in Experimental Autoimmune Myocarditis

Myocarditis is a myocardial inflammatory infiltration heterogeneous disease. At present, various interventions are not effective in the treatment of myocarditis. IL-17, an important pro‐inflammatory factor secreted mainly by Th17 cells, can promote the expression of multiple cytokines. MCP-1 is an important cytokine that mediates mononuclear cell infiltration. Studies have found that IL-17 could stimulate the expression of MCP-1 to mediate inflammatory infiltration. But the mechanism by which IL-17 induces MCP-1 expression in experimental autoimmune myocarditis (EAM) remains unclear. The purpose of this study is to establish an EAM model to explore the role of Act1/TRAF6/TAK1 cascade in the induction of MCP-1 by IL-17. In the present study, we found that in EAM, IL-17 could stimulate the expression of MCP-1 by activating Act1/TRAF6/TAK1 cascade. After interfering TAK1 with si-TAK1, myocardial tissue inflammation was greatly alleviated, and both MCP-1 mRNA and protein expression were downregulated. In conclusion, IL-17 can activate AP-1, NF-κB via Act1/TRAF6/TAK1 upregulation of MCP-1 expression in EAM.

Resveratrol Butyrate Esters Inhibit BPA-Induced Liver Damage in Male Offspring Rats by Modulating Antioxidant Capacity and Gut Microbiota

Resveratrol can affect the physiology or biochemistry of offspring in the maternal–fetal animal model. However, it exhibits low bioavailability in humans and animals. Fifteen-week SD pregnant female rats were orally administered bisphenol A (BPA) and/or resveratrol butyrate ester (RBE), and the male offspring rats (n = 4–8 per group) were evaluated. The results show that RBE treatment (BPA + R30) compared with the BPA group can reduce the damage caused by BPA (p < 0.05). RBE enhanced the expression of selected genes and induced extramedullary hematopoiesis and mononuclear cell infiltration. RBE increased the abundance of S24-7 and Adlercreutzia in the intestines of the male offspring rats, as well as the concentrations of short-chain fatty acids (SCFAs) in the feces. RBE also increased the antioxidant capacity of the liver by inducing Nrf2, promoting the expression of HO-1, SOD, and CAT. It also increased the concentration of intestinal SCFAs, enhancing the barrier formed by intestinal cells, thereby preventing BPA-induced metabolic disruption in the male offspring rats, and reduced liver inflammation. This study identified a potential mechanism underlying the protective effects of RBE against the liver damage caused by BPA exposure during the peri-pregnancy period, and the influence of the gut microbiota on the gut–liver axis in the offspring.

Lethal infection caused by Tetratrichomonas gallinarum in black swans (Cygnus atratus)

Background Tetratrichomonas gallinarum is parasitic protozoa with a wide host range. However, its lethal infection is rare reported. Case presentation Here, we described the first lethal cases of T. gallinarum infection in black swans in China. Five black swans died within a week in succession without obvious symptoms except mild diarrhea. At necropsy, severe lesions were observed in caeca with thickened caecal walls and hemorrhages in the mucosa. A large number of moving trophozoites were found in the contents of the cecum by microscopic examination. The livers were enlarged with multiple bleeding spots on the surface. Histopathology of the livers showed mononuclear cell infiltration and moderate hyperplasia of fibrous tissue. The histopathology of the cecum showed that the villi of the cecum were edematous. Finally, the presence of T. gallinarum was determined by specific PCR andin-situ hybridization assay. Additionally, common pathogens that can cause similar symptoms were excluded. Conclusions The death of the black swan was caused by T. gallinarum, suggesting that the parasite might be a new threat to the Cygnus birds.

Age-Related Arteriolar Changes With Lipid and Amyloid Deposits in the Gonads of Dogs

Arteriolar lesions with lipid and/or amyloid deposits are frequently detected in canine gonads by routine histopathologic examination; however, they have never been examined in detail. In the present study, a total of 139 testes/epididymides and 200 ovaries from 72 male (4 months to 14 years old) and 105 female (7 months to 16 years old) dogs were examined for arteriolar lesions. Arteriolar lesions were detected in 21 of 72 male dogs (29%) and 54 of 105 female dogs (51%). These lesions were histologically classified into 4 types: “fibromuscular hypertrophy,” characterized by thickening of the tunica intima; “focal vasculitis,” characterized by mononuclear cell infiltration; “vacuolar change,” consisting of lipid accumulation and infiltration of foamy cells; and “hyalinosis,” characterized by irregular thickening with amyloid deposits. In the lesions of vacuolar change and hyalinosis, lipid deposition and infiltration of α-SMA-positive cells and Iba-1-positive cells were also observed. Foamy cells and amyloid deposits were immunopositive for apolipoproteins and oxidized low-density lipoprotein-related proteins. These results indicate that vacuolar change is possibly an early stage of atherosclerosis, and that amyloid may deposit as a consequence of the microenvironment associated with atherogenesis. Logistic regression analysis revealed that arteriolar lesions with lipid deposits were associated with age and interstitial cell tumors in male dogs, and with age in female dogs. Aging is likely an important risk factor of arteriolar lesions with lipid deposits of the canine gonads.

Sublethal toxicity test of mercury (Hg) in the flesh and tissue of Tilapia (Oreochromis niloticus)

This study tested the sublethal toxicity of mercury (Hg) in the flesh and tissue of tilapia (Oreochromis niloticus). This research used an experimental method completely randomized design (CRD) with 3 treatments and 3 replications. The treatments used different Hg concentrations: Treatment A = 0.08 ppm; B = 0.16 ppm; C = 0.24 ppm; and the control group = without the addition of Hg. The parameters observed were the accumulation of mercury (Hg) in the flesh and tissue damage (histopathology) of the gills, kidneys and liver of tilapia. The results showed that the highest Hg accumulation was at the highest Hg concentration (treatment C = 0.24 ppm). Tissue damage was mainly found in the kidneys, liver and gills of fish exposed to Hg with indications of bleeding, tubular necrosis, vacuolization of epithelial cells, and mononuclear cell infiltration. The results showed that the toxicity of mercury to the organs increased with the increase in the concentration of Hg in water. Keywords : flesh, mercury, Oreochromis niloticus, sublethal, tissue

Using Artificial Intelligence to Detect, Classify, and Objectively Score Severity of Rodent Cardiomyopathy

Rodent progressive cardiomyopathy (PCM) encompasses a constellation of microscopic findings commonly seen as a spontaneous background change in rat and mouse hearts. Primary histologic features of PCM include varying degrees of cardiomyocyte degeneration/necrosis, mononuclear cell infiltration, and fibrosis. Mineralization can also occur. Cardiotoxicity may increase the incidence and severity of PCM, and toxicity-related morphologic changes can overlap with those of PCM. Consequently, sensitive and consistent detection and quantification of PCM features are needed to help differentiate spontaneous from test article-related findings. To address this, we developed a computer-assisted image analysis algorithm, facilitated by a fully convolutional network deep learning technique, to detect and quantify the microscopic features of PCM (degeneration/necrosis, fibrosis, mononuclear cell infiltration, mineralization) in rat heart histologic sections. The trained algorithm achieved high values for accuracy, intersection over union, and dice coefficient for each feature. Further, there was a strong positive correlation between the percentage area of the heart predicted to have PCM lesions by the algorithm and the median severity grade assigned by a panel of veterinary toxicologic pathologists following light microscopic evaluation. By providing objective and sensitive quantification of the microscopic features of PCM, deep learning algorithms could assist pathologists in discerning cardiotoxicity-associated changes.

The effect of a sudden temperature decrease on selected physiological indices in the common carp

The objective of the study was to evaluate stress invoked by a temperature decrease in the common carp (n = 56). After acclimatisation to laboratory conditions at a temperature of 27.8 ± 1.0 °C, the fish (n = 28) were transferred to 16.8 ± 1.0 °C water (group T2; temperature difference Δ –11 °C); the rest of the fish were kept at the original water temperature (group T1). The examination of individual fish was made after 6, 12, 24 and 48 hours (h). The impact on the haematological and biochemical indices was evaluated. Furthermore, the effect on the food content passage speed was investigated and a histopathological examination was performed. The changed haematological indices in the T2 group returned to the T1 values 48 h after a temperature decrease, except for the neutrophil-lymphocyte ratio which was found to be higher in all the T2 samplings. Most of the monitored biochemical indices decreased in the T2 group and some of them remained that way at 48 hours. On the contrary, the liver enzymes increased in the T2 group at 48 hours. The passage of food through the digestive tract was mostly finished after 48 h for both groups. However, in the warmer water, the intestine was empty in 71.4% cases after 24 h already, while in the T2 group, the food content passage was only completed in 14.3% of the fish at the same time. Local gill and skin necrosis, and mononuclear cell infiltration were found in the T2 group. A sudden change in the temperature can affect various parameters of the common carp, however, these changes can mostly be eliminated within 48 hours.