Yaser Hosny Ali Elewa

Characterization of mouse mediastinal fat-associated lymphoid clusters

The association between adipose tissue and immunity has been established and fat-associated lymphoid clusters (FALCs) are considered as a source of immune cells. We discovered lymphoid clusters (LCs) in mouse mediastinal fat tissues (MFTs). In Th1-biased C57BL/6N (B6), Th2-biased DBA/2Cr (DBA) and autoimmune-prone MRL/MpJ (MRL) mice strains, LCs without a fibrous capsule and germinal center were observed in white-colored MFTs extending from the diaphragm to the heart. The number and size of the LCs were larger in 12-month-old mice than in 3-month-old mice in all of the examined strains. Moreover, B6 had an especially large number of LCs compared with DBA and MRL. The immune cells in the LCs consisted of mainly T-cells and some B-cells. The majority of T-cells were CD4+ helper T (Th) cells, rather than CD8+ cytotoxic T-cells and no obvious immune cell population difference was present among the strains. Furthermore, high endothelial venules and lymphatic vessels in the LCs were better developed in B6 mice than in the other strains. Interestingly, some CD133+ hematopoietic progenitor cells and some c-Kit+/CD127+ natural helper cells were detected in the LCs. BrdU+ proliferating cells were more abundant in the LCs of B6 mice than in the LCs of the other strains and the number of BrdU+ cells increased with age. This is the first report of LCs in mouse MFTs. We suggest that the mouse genetic background affects LC size and number. We term the LCs “mediastinal fat-associated lymphoid clusters”. These clusters can be considered as niches for Th cell production.

Cytoarchitectural differences of myoepithelial cells among goat major salivary glands

Previously, the distribution of myoepithelial cells (mecs) in the salivary glands was studied by both immunohistochemistry, and transmission electron microscopy; however, little was elucidated concerning their morphological features, especially in goats. This study was performed to investigate the correlation between the cytoarchitecture of the mecs in goat major salivary glands (parotid, mandibular, and sublingual glands) and the nature of the saliva secretion. The cytoarchitectural features of the mecs were examined by scanning and transmission electron microscopy as well as immunohistochemically. The secretory endpieces in the parotid gland are of the pure serous type, but in both the mandibular and sublingual glands they are of the mixed type. In all studied glands, the intercalated ducts were covered by mecs which, unlike the large stellate cells that surrounded the secretory endpieces, were spindle-shaped with few cytoplasmic processes. Interestingly, the mecs were found to bulge on the basal surfaces of the serous acini and intercalated ducts in all glands and to be in close contact to the seromucous tubules surface in the mandibular and sublingual glands forming a continuous network around it. In conclusion, the differences in the degree of development of the mecs as well as the number of their cytoplasmic processes may be correlated with the nature of the secretion and the number of the secretory granules. Thus these observations may have some relevance in the diagnosis of atrophy and pathogenic conditions of these glands.

Comparative analysis of mediastinal fat-associated lymphoid cluster development and lung cellular infiltration in murine autoimmune disease models and the corresponding normal control strains

We previously discovered mediastinal fat‐associated lymphoid clusters (MFALCs) as novel lymphoid clusters associated with mediastinal fat tissue in healthy mice. However, no data about their morphology in immune‐associated disease conditions, and their relationship with lung infiltration, is available to date. In the present study, we compared the morphological features of MFALCs in 4‐month‐old male murine autoimmune disease models (MRL/MpJ‐lpr mice and BXSB/MpJ‐Yaa mice) with those of the corresponding control strains (MRL/MpJ and BXSB/MpJ, respectively). In addition, we analysed their correlation with lung infiltration. Furthermore, immunohistochemistry for CD3, B220, Iba1, Gr1 and BrdU was performed to detect T cells and B cells, macrophages, granulocytes and proliferating cells, respectively. The spleen weight to body weight ratios and anti‐double‐stranded DNA autoantibody titres were found to be significantly higher in the autoimmune models than in the control strains. Furthermore, the autoimmune model presented prominent MFALCs, with a significantly greater ratio of lymphoid cluster area to total mediastinal fat tissue area, and more apparent diffused cellular infiltration into the lung lobes than the other studied strains. Higher numbers of T and B cells, macrophages and proliferating cells, but fewer granulocytes, were observed in the autoimmune models than in the control strains. Interestingly, a significant positive Pearsons correlation between the size of the MFALCs and the density of CD3‐, B220‐ and Iba1‐positive cells in the lung was observed. Therefore, our data suggest a potentially important role for MFALCs in the progression of lung disease. However, further investigation is required to clarify the pathological role of MFALCs in lung disease, especially in inflammatory disorders.

Possible role of Arthrospira platensis in reversing oxidative stress-mediated liver damage in rats exposed to lead

Environmental pollutants, particularly metallic elements, mobilized and released into the environment, eventually accumulate in the food chain and thus pose a serious threat to human and animal health. In the present study, the role of Arthrospira (Spirulina platensis; SP) as a protector against oxidative stress-mediated liver damage induced by an exposure to lead acetate (LA; as a metallic pollutant) was assessed. To achieve this aim, rats were orally administered with 300 mg/kg bw SP for 15 days, before and concurrently with an intraperitoneal injection of 50 mg/kg bw LA (6 injections throughout 15 days). As a result, co-administration of SP with LA reduced the amount of lead that accumulated in both blood and liver tissue of the exposed rats and minimized the increased levels of lipid peroxidation, protein oxidation, DNA oxidative damage, and liver enzyme endpoints. In addition, because of SP administration, the levels of depleted biomarkers of antioxidant status and total antioxidant capacity in LA-exposed rats improved. Moreover, SP protected the liver tissue against the changes caused by LA exposure and also decreased the reactivity of HSP70 in the cytoplasm of hepatocytes. Collectively, our data suggest that SP has a potential use as a food supplement in the regions highly polluted with heavy metals such as lead.

IL-36α Regulates Tubulointerstitial Inflammation in the Mouse Kidney

IL-36α, a member of the IL-1 family, is a crucial mediator of inflammatory responses. We previously found that IL-36α was overexpressed in injured distal tubules (DTs); however, its pathological function remains unclear. Herein, unilateral ureter obstruction (UUO) or folic acid (FA) injection was performed in mouse kidneys to assess the role of IL-36α in kidney injury. IL-36α mRNA and protein expression significantly increased in the kidneys within 24 h after UUO. IL-36α localized to dilated DTs. IL-36α expression significantly correlated with the progression of tubulointerstitial cell infiltration and tubular epithelium cell death in UUO kidneys and with renal dysfunction in FA-induced acute kidney injury mice. At 24 h after UUO, IL-36α+ DT epithelial cells showed loose intercellular digitations. IL-1RL2, an IL-36α receptor protein, localized to podocytes, proximal tubules, and DTs in the healthy kidney. IL-1RL2 was expressed in interstitial cells and platelets or extended primary cilia of DT epithelial cells in UUO kidneys. IL-36α stimulation promoted the production of IL-6 and Prss35, an inflammatory cytokine and collagen remodeling-associated enzyme, respectively, in cultured NIH3T3 fibroblasts. UUO-treated IL-36α-knockout (KO) mice showed milder kidney injury features than wild-type (WT) mice did. In UUO kidneys from IL-36α-KO mice, the expression of genes associated with inflammatory response and sensory perception was significantly different from that in WT mice. Altogether, our data indicate an association between intrarenal IL-36α overexpression and the progression of tubulointerstitial inflammations and morpho-functional alterations of DT epithelial cells. IL-36α may be a novel kidney injury marker useful for evaluating DT damages.

Sex-related differences in autoimmune-induced lung lesions in MRL/MpJ-faslpr mice are mediated by the development of mediastinal fat-associated lymphoid clusters

Abstract MRL/MpJ-Faslpr (lpr) mice are a model for autoimmune diseases such as systemic lupus erythematosus (SLE). These diseases mainly affect women, with a 10:1 female-to-male ratio, and cause pleuropulmonary lesions. We previously revealed a correlation between mediastinal fat-associated lymphoid cluster (MFALC) development and cellular infiltration in the lungs of lpr male mice; however, we did not report on MFALCs in females. The purpose of this investigation was to reveal sex-related differences in MFALCs in lpr mice. We compared the morphological features of MFALCs and lung mononuclear cell aggregates (LMCAs) in 5-month-old male and female lpr mice. The females showed significantly elevated anti-dsDNA autoantibody titers and larger MFALCs, with a higher ratio of lymphatic vessel (LV) and high endothelial venule (HEV) areas to MFALC area, and greater numbers of T- and B-cells, macrophages, and proliferating and dendritic cells in MFALCs and LMCAs than males. Our data indicated that MFALCs were more developed and lung lesions were more severe in female than in male lpr mice, thereby suggesting a potential role for LVs and HEVs in the establishment of MFALCs and lung lesions. Further investigation in female lpr mice will be needed for treatment of human respiratory diseases and autoimmune disorders.

Genetic factors derived from the MRL/MpJ mouse function to maintain the integrity of spermatogenesis after heat exposure

MRL/MpJ mice possess highly heat‐shock‐resistant spermatocytes (HRS) in comparison with C57BL/6 mice. This resistance depends on the MRL/MpJ‐type loci at the 81 cM region of Chromosome (Chr) 1 and the 40 cM region of Chr 11. To evaluate the functions of these loci in detail, we examined the histopathological changes resulting from experimental cryptorchidism or transient scrotal heat stress (SHS) in the testes of C57BL/6‐based congenic strains (B6.MRLc1, B6.MRLc11, and B6.MRLc1c11) carrying the MRL/MpJ‐derived loci responsible for HRS. Among cryptorchid testes from congenic strains, those in B6.MRLc1c11 mice showed the highest heat resistance, indicating that the genetic interactions between MRL/MpJ‐derived HRS loci on Chrs 1 and 11 may be important for maintaining spermatogenesis under continuous testicular hyperthermia. In contrast, immediately after SHS induction, germ cell loss via apoptosis was inhibited in B6.MRLc11 and B6.MRLc1c11 mice, similar to that in MRL/MpJ mice. However, this HRS phenotype was not observed in C57BL/6 or B6.MRLc1 mice after SHS induction. Furthermore, testicular calcification owing to long‐term damage by SHS induction was inhibited in all congenic strains in comparison with that in C57BL/6 mice, indicating that each MRL/MpJ‐derived locus on Chrs 1 and 11 acted independently to facilitate the recovery of heat‐induced testicular damage by inhibiting calcification. B6.MRLc11 and B6.MRLc1c11 mice showed greater recovery in spermatogenesis than B6.MRLc1 mice 60 days after SHS induction. Therefore, the MRL/MpJ‐derived HRS locus on Chr 11 might play an important role in recovery from heat stress damage. On the basis of these results, we concluded that MRL/MpJ‐derived loci on Chrs 1 and 11 cooperatively or independently regulate testicular heat sensitivity depending on the various heat stresses.

Close pathological correlations between chronic kidney disease and reproductive organ-associated abnormalities in female cotton rats

Cotton rat (Sigmodon hispidus) is a useful experimental rodent for the study of human infectious diseases. We previously clarified that cotton rats, particularly females, developed chronic kidney disease characterized by cystic lesions, inflammation, and fibrosis. The present study investigated female-associated factors for chronic kidney disease development in cotton rats. Notably, female cotton rats developed separation of the pelvic symphysis and hypertrophy in the vaginal parts of the cervix with age, which strongly associated with pyometra. The development of pyometra closely associated with the deterioration of renal dysfunction or immunological abnormalities was indicated by blood urea nitrogen and serum creatinine or spleen weight and serum albumin/globulin ratio, respectively. These parameters for renal dysfunction and immunological abnormalities were statistically correlated. These phenotypes found in the female reproductive organs were completely inhibited by ovariectomy. Further, the female cotton rats with pyometra tended to show more severe chronic kidney disease phenotypes and immunological abnormalities than those without pyometra; these changes were inhibited in ovariectomized cotton rats. With regard to renal histopathology, cystic lesions, inflammation, and fibrosis were ameliorated by ovariectomy. Notably, the immunostaining intensity of estrogen receptor α and estrogen receptor β were weak in the healthy kidneys, but both estrogen receptors were strongly induced in the renal tubules showing cystic changes. In conclusion, the close correlations among female reproductive organ-associated abnormalities, immunological abnormalities, and renal dysfunction characterize the chronic kidney disease features of female cotton rats. Thus, the cotton rat is a unique rodent model to elucidate the pathological crosstalk between chronic kidney disease and sex-related factors. Impact statement The increasing number of elderly individuals in the overall population has led to a concomitant age-related increase in chronic kidney disease. Moreover, the global prevalence of patients with chronic kidney disease is gradually increasing, which poses a serious public health problem. The limited number of spontaneous chronic kidney disease animal models, which resemble chronic kidney disease pathogenesis in elderly individuals, is a major limitation in the development of experimental and curative medicines for chronic kidney disease. This pathological study clarified that sex-related factors, including hormones, and abnormalities of the female reproductive system, such as pyometra, are closely associated with chronic kidney disease development by using cotton rats (Sigmodon hispidus). Further, ovariectomy inhibited the phenotypes of the female reproductive system, immunological abnormalities, and chronic kidney disease. Thus, this laboratory rodent serves as a novel and useful spontaneous chronic kidney disease model to elucidate the candidate disease factors and the pathogenesis of chronic kidney disease both in human and experimental medicine.

Hydronephrosis with ureteritis developed in C57BL/6N mice carrying the congenic region derived from MRL/MpJ-type chromosome 11.

Abstract Inbred MRL/MpJ mice show several unique phenotypes in tissue regeneration processes and the urogenital and immune systems. Clarifying the genetic and molecular bases of these phenotypes requires the analysis of their genetic susceptibility locus. Herein, hydronephrosis development was incidentally observed in MRL/MpJ-derived chromosome 11 (D11Mit21-212)-carrying C57BL/6N-based congenic mice, which developed bilateral or unilateral hydronephrosis in both males and females with 23.5% and 12.5% prevalence, respectively. Histopathologically, papillary malformations of the transitional epithelium in the pelvic-ureteric junction seemed to constrict the ureter luminal entrance. Characteristically, eosinophilic crystals were observed in the lumen of diseased ureters. These ureters were surrounded by infiltrating cells mainly composed of numerous CD3+ T-cells and B220+ B-cells. Furthermore, several Iba-1+ macrophages, Gr-1+ granulocytes, mast cells and chitinase 3-like 3/Ym1 (an important inflammatory lectin)-positive cells were detected. Eosinophils also accumulated to these lesions in diseased ureters. Some B6.MRL-(D11Mit21-D11Mit212) mice had duplicated ureters. We determined >100 single nucleotide variants between C57BL/6N- and MRL/MpJ-type chromosome 11 congenic regions, which were associated with nonsynonymous substitution, frameshift or stopgain of coding proteins. In conclusion, B6.MRL-(D11Mit21-D11Mit212) mice spontaneously developed hydronephrosis due to obstructive uropathy with inflammation. Thus, this mouse line would be useful for molecular pathological analysis of obstructive uropathy in experimental medicine.

Morphofunctional analysis of antigen uptake mechanisms following sublingual immunotherapy with beads in mice

Background Recently, sublingual immunotherapy (SLIT) has been used as a safe and efficient method for the treatment of and immunization against asthma and various allergies. However, the routes of antigen uptake through the mucosa of the oral cavity remain incompletely understood, as do the roles of sex and age in the process. For this purpose, to elucidate the mechanism and efficacy of SLIT among different sexes and ages microbeads were dripped into the sublingual region to mimic antigen uptake by the sublingual mucosa. Methods Twenty microliters of either phosphate buffered saline (PBS) or fluorescently labelled microbeads (latex and silica beads) were placed under the tongue of both male and female C57BL/6 mice at young (3 months) and old (6 months) ages. The lower jaw was examined 30 min after administration, and beads were detected with a fluorescence stereomicroscope. Morphological observations of the mucosa of the fluorescent areas were made with scanning electron microscopy (SEM) and an all-in-one light fluorescence microscope (LM). Fluorescence intensity was compared between both sexes and ages. Results Stereomicroscopic observation revealed fluorescent illuminations in three compartments of the sublingual mucosa: the sublingual caruncles (SC), the oral rostral mucosa (OR) and the buccal mucosa (BM). Interestingly, the fluorescence intensity tended to be higher among females than among males in the SC region in particular. However, there were no significant age-related differences. SEM and LM revealed beads in the lumina of both mandibular ducts and sublingual ducts (Sd). Additionally, the apical cytoplasm of some Sd cells contained silica beads. However, there were no specification in the OR mucosa or BM. Conclusions This study reveals the major role Sd play in local immunity via the antigen uptake mechanisms. Furthermore, our data suggest that the efficacy of SLIT in humans could be affected by sex.