Phenotypes of streptozotocin-induced gestational diabetes mellitus in mice.

Gestational diabetes mellitus (GDM) in human patients disrupts glucose metabolism post-pregnancy, affecting fetal development. Although obesity and genetic factors increase GDM risk, a lack of suitable models impedes a comprehensive understanding of its pathology. To address this, we administered streptozotocin (STZ, 75 mg/kg) to C57BL/6N mice for two days before pregnancy, establishing a convenient GDM model. Pregnant mice exposed to STZ (STZ-pregnant) were compared with STZ-injected virgin mice (STZ-virgin), citrate buffer-injected virgin mice (CB-virgin), and pregnant mice injected with citrate buffer (CB-pregnant). STZ-pregnant non-obese mice exhibited elevated blood glucose levels on gestational day 15.5 and impaired glucose tolerance. They also showed fewer normal fetuses compared to CB-pregnant mice. Additionally, STZ-pregnant mice had the highest plasma C-peptide levels, with decreased pancreatic islets or increased alpha cells compared to CB-pregnant mice. Kidneys isolated from STZ-pregnant mice did not display histological alterations or changes in gene expression for the principal glucose transporters (GLUT2 and SGLT2) and renal injury-associated markers. Notably, STZ-pregnant mice displayed decreased gene expression of insulin-receiving molecules (ISNR and IGFR1), indicating heightened insulin resistance. Liver histology in STZ-pregnant mice remained unchanged except for a pregnancy-related increase in lipid droplets within hepatocytes. Furthermore, the duodenum of STZ-pregnant mice exhibited increased gene expression of ligand-degradable IGFR2 and decreased expression of GLUT5 and GLUT12 (fructose and glucose transporters, respectively) compared to STZ-virgin mice. Thus, STZ-pregnant mice displayed GDM-like symptoms, including fetal abnormalities, while organs adapted to impaired glucose metabolism by altering glucose transport and insulin reception without histopathological changes. STZ-pregnant mice offer a novel model for studying mild onset non-obese GDM and species-specific differences in GDM features between humans and animals.

Effects of autoimmune abnormalities on fertility and placental morphology in mice.

Autoimmune diseases (AIDs) alter the placental immune environment leading to fetal loss. This study investigated the effects of AIDs on pregnancy and the placenta in AID-prone MRL/MpJ-Faslpr/lpr mice and wild-type MRL/MpJ, which were mated with male MRL/MpJ and MRL/MpJ-Faslpr/lpr at five months and defined as moLpr and moMpJ, respectively. AID indices (spleen weight and serum autoantibody levels) and fertility status (number and size of fetuses, morphology, and comprehensive gene expression of placentas) were evaluated on gestational day 15.5. Both strains showed equivalent fertility, but moLpr showed lighter placentas and fetuses than moMpJ, and decreased fertility with AID severity. moLpr placentas had a higher number of T cells, higher expression of genes associated with T helper 2 and T follicular helper functions, and altered expression of genes (Krt15, Slc7a3, Sprr2a3) that significantly regulate pregnancy or immunity. The gene expression of T cell migration-associated chemokines (Ccl5, Cxcl9) was significantly increased in moLpr placentas, and CCL5 and CXCL9 were detected in moLpr placentas, particularly in T cells and placenta-component cells, respectively. Thus, AID altered placental morphofunction and fertility in mice; however, fertility was maintained at the examined time points. This study enhances our understanding of placental alterations and gestational risk due to AIDs.

Structural Features of Connective Tissue Formed around Resin Implants Subcutaneously Embedded in Dairy Cows.

Foreign body reactions (FBRs) are inadvertently observed in invading or artificially embedded materials, triggering inflammation and subsequent fibrotic processes to occur in situ. Here, we assessed the spatiotemporal formation of connective tissue around implanted materials to establish a technique using connective tissue formed by FBRs as xenografts. An acrylic resin implant, comprising a columnar inner rod and a tubular outer cylinder (OC) with several slits, was embedded in adult dairy cows. Tissues formed in the inner rod and OC groups were histologically analyzed at weeks 2, 4, 8, and 12. Edematous tissues with non-collagenous fibers formed for 2 weeks and showed increased cellularity after 4 weeks. The weight, thickness, amounts of total protein, collagen, DNA, and quantitative scores of α-smooth muscle actin-positive myofibroblasts or elastic fibers notably increased after 8 weeks, with condensed collagen fibers showing orientation. Inflammatory cells were primarily localized in tissues close to the OC, and their numbers increased, with the count of CD204+ cells peaking at 8 weeks and declining at 12 weeks. The count of Ki67+ proliferating cells slightly increased in tissues close to the OC; however, the number and lumen of CD31+ vessels increased. These results may help understand FBR-related tissue remodeling.

Ameliorated Renal Pathological Feature in MRL/MpJ-Faslpr/lpr Background Interleukin-36 Receptor-Deficient Mice.

Systemic autoimmune diseases frequently induce lupus nephritis, causing altered balance and expression of interleukin 36 receptor (IL-36R) ligands, including agonists (IL-36α, ß, γ) and antagonists (IL-36Ra, IL-38), in kidneys. Here, we established and analyzed a mouse model of lupus nephritis, MRL/MpJ-Faslpr/lpr with IL-36R-knockout (KO), compared to wild-type (WT) mice. In both genotypes, indices for immune abnormalities and renal functions were comparable, although female WT mice showed higher serum autoantibody levels than males. IL-36R ligand expression did not differ significantly between genotypes at the mRNA level or in IL-36α and IL-38 scores. However, glomerular lesions, especially mesangial matrix expansion, were significantly ameliorated in both sexes of IL-36R-KO mice compared to WT mice. Cell infiltration into the tubulointerstitium with the development of tertiary lymphoid structures was comparable between genotypes. However, the positive correlation with the IL-36α score in WT mice was not evident in IL-36R-KO mice. Fibrosis was less in female IL-36R-KO mice than in WT mice. Importantly, some IL-36α+ nuclei co-localized with acetylated lysine and GCN5 histone acetyltransferase, in both genotypes. Therefore, IL-36R ligands, especially IL-36α, contribute to the progression of renal pathology in lupus nephritis via IL-36R-dependent and IL-36R-independent pathways.

Effects of autoimmune abnormalities on skeletal muscle regeneration after needle puncture in mice.

Regeneration of injured skeletal muscles is supported by the activation of satellite cells, and excessive traumatic injuries may trigger abnormal processes, such as fibrosis. Because the participation of immune cells is crucial during skeletal muscle repair, systemic autoimmune diseases impair their regeneration. This study focused on a traumatic injury by injection and investigated the effect of autoimmune diseases on skeletal muscle regeneration. Male mice of MRL/MpJ-Faslpr/lpr and MRL/MpJ (6-7 months old) were used for autoimmune disease and healthy groups. The abdominal walls punctured by a needle were histologically analyzed at 1, 3, and 8 days postinjection. In both groups, injured skeletal muscle tissues showed necrosis and inflammatory cell infiltrations on day 1, increased cell density at 3 days, and regenerative myotubes with central nuclei without fibrosis at 8 days. Gr-1+ neutrophils at injured skeletal muscle were abundant at 1 day, and then substantially decreased starting from 3 days in both groups. The number of CD3+ T cells was remarkably higher in MRL/MpJ-Faslpr/lpr than that in MRL/MpJ at 1 day, and a similar tendency was observed in B220+ B cells. The numbers of IBA1+ macrophages and bromodeoxyuridine-incorporating cells tended to be higher at 3 days, and those of the latter, mainly proliferating paired-box-7+ satellite cells, showed significance at other time points and negatively correlated with the autoimmune disease indices, such as spleen weights or serum autoantibody level. Thus, this result suggested that injured skeletal muscle by minor trauma is normally regenerated regardless of the effects of autoimmune diseases, although lymphocyte infiltrations during these processes were more severe in MRL/MpJ-Faslpr/lpr.

Current status and issues with the dosimetric assay of iodine-125 seed sources at medical facilities in Japan: a questionnaire-based survey†.

In conducting dosimetric assays of seed sources containing iodine-125 (125I), several major guidelines require the medical physicist to verify the source strength before patient treatment. Japanese guidelines do not mandate dosimetric assays at medical facilities, but since 2017, three incidents have occurred in Japan wherein seeds with incorrect strengths were delivered to medical facilities. Therefore, this study aimed to survey the current situation and any barriers to conducting the dosimetric assay of iodine-125 seeds at medical facilities in Japan. We conducted a questionnaire-based survey from December 2020 to April 2021, to examine whether seed assay and verification of the number of seeds delivered were being performed. We found that only 9 facilities (16%) performed seed assay and 28 (52%) verified the number of seeds. None of the facilities used an assay method that ensured traceability. The reasons for not performing an assay were divided into two categories: lack of resources and legal issues. Lack of resources included lack of instruments, lack of knowledge of assay methods, shorthand, or all of the above, whereas legal issues included the inability to resterilize iodine-125 seeds distributed in Japan and/or purchase seeds dedicated to the assay. Dosimetric assays, including simple methods, are effective in detecting calibration date errors and non-radioactive seeds. The study findings suggest that familiarization of medical personnel with these assay methods and investigation of the associated costs of labor and equipment should be recommended, as these measures will lead to medical reimbursement for quality assurance.

Relationship between Amino Acid Metabolism and Bovine In Vitro Follicle Activation and Growth.

The amino acid metabolism of bovine follicles during in vitro growth (IVG) was evaluated to identify potential indicators of health during culture. The bovine ovarian cortex was sliced, prepared as strips, and cultured for 6 days. Tissue samples were examined histologically before and after 6 days of culture, and the degree of follicle activation was classified as either high or low based on the number of growing secondary follicles present (high: 7~11; low: 0~1). In a separate experiment, secondary follicles (diameter range: 100~200 µm) were manually isolated and cultured, and their growth was monitored for 6 days. Cultured follicles were classified as growth or degenerate based on diameter change during culture (growth: +60.5~74.1 µm; degenerate: -28~15.2 µm). Free amino acids and their metabolites were measured in the spent culture medium from each group. In cultured ovarian cortical strips, the concentration of α-aminoadipic acid was significantly higher in the low activation group than in the high group (p < 0.05), while those of methionine, lysine, and arginine were higher in the high activation group. In cultured isolated secondary follicles, concentrations of methionine, tyrosine, histidine, and hydroxyproline were higher in the degenerate group (p ≤ 0.05). In conclusion, amino acid metabolism has the potential to serve as an indicator of primordial follicle activation and subsequent growth rate during bovine IVG.

[Technical Report: Quality Assurance for 125I Seed Sources in Permanent Prostate Brachytherapy].

This technical report provides useful information on the current status and issues of quality control in 125I seed source strength measurement for Permanent Prostate Brachytherapy in Japan.With the spread of 125I seed brachytherapy, the traceability of source strength measurements with the single-seed assay was established in Japan. This allows medical facilities to measure reference air kerma rate of 125I seeds with their own well-type of ionization chamber. However, it is difficult to maintain the traceability chain because the 125I reference air kerma rate standards have been hardly utilized by medical facilities so far. Meanwhile, some serious incidents of contamination of the different source strengths and dead seeds were reported in Japan.To address the specific issues in Japan, JASTRO Brachytherapy Subcommittee established a working group (WG) in 2021. The goal of this WG is to investigate the management methods of source strength measurement used in medical facilities, and to discuss the ideal and practicable methods of source management such as verifying the number of seeds and source strength. Initially, a questionnaire survey was conducted to facilities offering 125I seed brachytherapy in Japan. Sixty-seven out of 95 facilities responded (response rate 70.5%). This survey revealed that 41% of facilities did not perform either confirmation of the number of seeds or measurement of source strength. There are several reasons why the source strength was not measured in those facilities. For example, 125I seeds are provided under the sterilized conditions; quality assurance by source suppliers is reliable; and there is not sufficient staff.The single-seed assay is regarded as an internationally standardized and the most reliable measurement method. Therefore, it is an essential measurement technique to ensure traceability of source strength measurements. However, our survey found that most Japanese facilities do not perform single-seed assays. Meanwhile, some facilities have performed batch assay as an alternative method, in which all of the multiple sources in a batch are measured while loaded into sterilized cartridges. Although the measurement by the batch assay is less accurate than the one by the single-seeded assay, the batch assay does not require re-sterilization of the source and can be performed quickly. It might be useful to detect unexpected errors such as differences in the number of sources and abnormalities in source strength.In this report, we will introduce several methods of source strength measurement that have been implemented in medical facilities. The quality assurance of 125I seed sources in prostate interstitial brachytherapy should be provided not only by the source suppliers but also by the medical facilities that use sources to treat patients. We hope that medical facilities will refer to this technical report and use it as an aid to quality assurance in their own facilities.

Pharmacodynamic Model-Based Safety Management of Eribulin-Induced Myelosuppression in Patients With Breast Cancer.

BACKGROUND: Neutropenia is a major dose-limiting toxicity of cancer chemotherapy. Semimechanistic mathematical models have been applied to describe the time course of neutrophil counts. The objectives of this study were to develop a mathematical model describing changes in neutrophil counts during eribulin treatment, to apply the empirical Bayes method to predict the probability of developing neutropenia ≥ grade 3 during eribulin treatment in each patient, and to propose the implementation of this mathematical tool in clinical practice for individual safety management. METHODS: The present model analysis and subsequent external evaluation were performed using the data of 481 patients with breast cancer, previously obtained from a postmarketing surveillance (training set) and a phase 2 clinical study (validation set). The model we previously reported (Kawamura et al 2018) was modified to improve its predictive capability. The individual time course of neutrophil changes during the treatment period was predicted by the empirical Bayes method using the observed neutrophil counts at baseline and the first measurement after the first eribulin dose. To evaluate the predictability of this method, the predicted neutrophil counts were compared with those of the observed values. RESULTS: The developed model provided good individual predictions, as indicated by the goodness-of-fit plots between the predicted and observed neutrophil counts, especially for a lower neutrophil count range. Days required to reach the nadir after the dose were also well-predicted. The sensitivity, specificity, and accuracy of the prediction of neutropenia grade ≥3 were 76%, 53%, and 71%, respectively. CONCLUSIONS: We developed a mathematical method for predicting and managing the risk of neutropenia during eribulin treatment. This method is generally applicable to other cases of chemotherapy-induced neutropenia and can be a new practical tool for individual safety management.

Assessment of using a gamma index analysis for patient-specific quality assurance in Japan.

PURPOSE: The Task Group 218 (TG-218) report was published by the American Association of Physicists in Medicine in 2018, recommending the appropriate use of gamma index analysis for patient-specific quality assurance (PSQA). The paper demonstrates that PSQA for radiotherapy in Japan appropriately applies the gamma index analysis considering TG-218. MATERIALS/METHODS: This survey estimated the acceptance state of radiotherapeutic institutes or facilities in Japan for the guideline using a web-based questionnaire. To investigate an appropriate PSQA of the facility-specific conditions, we researched an optimal tolerance or action level for various clinical situations, including different treatment machines, clinical policies, measurement devices, staff or their skills, and patient conditions. The responded data were analyzed using principal component analysis (PCA) and multidimensional scaling (MDS). The PCA focused on factor loading values of the first contribution over 0.5, whereas the MDS focused on mapped distances among data. RESULTS: Responses were obtained from 148 facilities that use intensity-modulated radiation therapy (IMRT), which accounted for 42.8% of the probable IMRT use in Japan. This survey revealed the appropriate application of the following universal criteria for gamma index analysis from the guideline recommendation despite the facility-specific variations (treatment machines/the number of IMRT cases/facility attributes/responded [representative] expertise or staff): (a) 95% pass rate, (b) 3% dose difference and 2-mm distance-to-agreement, and (c) 10% threshold dose. Conditions (a)-(c) were the principal components of the data by the PCA method and were mapped in a similar distance range, which was easily clustered from other gamma index analytic factors by the MDS method. Conditions (a)-(c) were the universally essential factors for the PSQA in Japan. CONCLUSION: We found that the majority of facilities using IMRT in each region of Japan complied with the guideline and conducted PSQA with deliberation under the individual facility-specific conditions.

Ureteral morphology and pathology during urolithiasis in cats.

Cats exhibit high susceptibility to urinary organ-related diseases. We investigated the healthy ureter morphologies and compared these with ureters that were surgically resected distal to a urolithiasis obstruction in cats. Healthy ureters (total length 9.88 ± 0.38 cm) developed adventitia composed of collagen fibers (ADCF), containing a longitudinal muscular layer, toward the distal segment. The healthy ureter was the smallest in the middle segment (4.71-6.90 cm from the urinary bladder) with significantly decreased luminal and submucosal areas compared to those in the proximal segment. Diseased cats exhibited a high incidence of calcium oxalate urolithiasis with renal dysfunction, regardless of age, sex, and body size. Diseased ureters showed increased perimeters, inflammation, and decreased nerves in ADCF. Collagen fibers were increased in the submucosal area, intermuscular spaces, and ADCF, particularly near the obstructed lesion. The mean resected ureter length was 5.66 ± 0.49 cm, suggesting a high obstruction risk in the middle segment. The middle segment also increased the cross-sectional area of the ureter and ADCF, regardless of the distance from the obstructed lesion. The ureters in several cases either lacked the transitional epithelium, or exhibited transitional epithelial hyperplasia, and some of these formed the mucosal folds. In conclusion, we demonstrated the following characteristics and histopathological features of cat ureters: decreases in the ureter size, lumen area, and submucosa area from proximal to middle segment in healthy; ADCF changes in urolithiasis, including increased connective tissues with inflammation and decreased nerves. These data are important to understand the pathogenesis of feline ureteral obstruction.

Modified foreign body reaction to silicone imbedded in subcutaneous tissues by different mouse systemic immune conditions.

Foreign body reaction (FBR) causes unexpected adverse effects due to implanted materials in humans and animals. Inflammation and subsequent fibrosis during FBR seems to be affected by recipient immunity, such as the balance of T helper (Th) response that has the potential to regulate FBR-related macrophage function. Here, the immunological effects of FBR on subcutaneously imbedded silicone tubes (ST) at 8 weeks were investigated histologically by comparing Th1-biased C57BL/6N, Th2-biased MRL/MpJ, and autoimmune disease-prone MRL/MpJ-Faslpr/lpr . Tissue surrounding ST (TSS) was analyzed at day (D) 7 and 14 (reaction phase) or D35 (stability phase) after surgery. In all strains, the TSS was composed of a thin layer (TL) containing fibrous tissues and loose connective tissues formed outside the TL. Few lymphocytes and mast cells, several neutrophils, and numerous macrophages infiltrated the TSS. Active vascularization was observed at D14 in all strains. For the examined indices, M1-type macrophage density in the TSS of C57BL/6N mice was significantly higher at D14 compared to other strains. No significant strain difference relating to M2-type macrophages was detected, suggesting the effects of Th1-biased immunity on FBR-related inflammation. Collagen fibers in the TSS increased in density and became stable with age in all strains. In particular, MRL/MpJ-Faslpr/lpr showed progressive fibrotic features. Serum autoantibody levels in MRL/MpJ-Faslpr/lpr mice were inversely correlated with M1-type macrophage density. These data from MRL/MpJ-Faslpr/lpr mice suggested modifications of FBR-related inflammation and fibrosis by autoimmune abnormalities. The results provide crucial insights into the pathological modification of FBR by recipient immunity and emphasize its clinicopathological importance in humans and animals.

[Utility of Point Dose Verification Using Measured Ionization Amount Ratio of Reference Irradiation to Volumetric Modulated Arc Therapy].

In the point dose verification of intensity-modulated radiation therapy (IMRT), we compared the commonly used method of measuring absolute dose (absolute method) with the measurement method by the American Association of Physicists in Medicine Task Group 119, which describes the point dose verification for IMRT using the ratio of reference irradiation and measured ionization. The target was 66 plans for head and neck cancer, 46 plans for lung cancer with 6 MV X-ray, and 31 plans for prostate cancer with 10 MV X-ray. They were treated with volumetric-modulated arc therapy (VMAT). Each plan was evaluated by the absolute method and the TG119 method using 3D-array. The average and 2SD of the verification results for head and neck cancer, lung cancer, and prostate cancer were 0.129±2.185%, 0.963±2.125%, and 0.259±2.019% by the absolute method, and 0.952±2.039%, 1.704±2.080%, and 0.524±1.274% by the TG119 method. The ratio between the average of the TG119 method and the absolute method corresponded to the error of reference irradiation dose. Considering that the measurement method is simple, the TG119 method enables more stable point dose verification of VMAT.

A Japanese prospective multi-institutional feasibility study on accelerated partial breast irradiation using multicatheter interstitial brachytherapy: clinical results with a median follow-up of 60 months.

BACKGROUND: We carried out the first multi-institutional prospective study on accelerated partial breast irradiation (APBI) via multicatheter interstitial brachytherapy in a shorter period for early breast cancer in Japan. METHODS: Patient eligibility criteria included positive hormone receptors, tumors ≤ 3 cm and TNM stage pN0M0. After breast-conserving surgery (Japanese cylindrical resection) and histological confirmation of negative surgical margins and the absence of lymph node metastasis, applicator implantation was performed either postoperatively or intraoperatively. High-dose-rate brachytherapy of 36 Gy in 6 fractions was delivered. RESULTS: Forty-six patients from six institutions received this treatment regimen, and the median follow-up time was 60 months (range 57-67 months). The median resected breast tissue volume was 81 cm3 (range 28-260 cm3). No Grade 4 late sequela, local recurrence nor death due to breast cancer were observed. Grade 2-3 sequelae such as rib fracture (2%), soft tissue necrosis (9%), fibrosis (20%), and breast pain (9%) were observed. The resected breast tissue volumes of the patients who had Grade ≥ 2 fibrosis and Grade < 2 fibrosis were 105.9 ± 32.3 cm3 and 76.3 ± 45.6 cm3, respectively, p = 0.02. The overall cosmetic outcome score of Excellent/Good was 74% at 60 months after APBI. Grade ≥ 1 fibrosis was observed in 44% and 92% of patients who scored Excellent/Good and Fair/Poor, respectively, p = 0.004. CONCLUSIONS: This study showed excellent local control and survival results with minimal late sequelae.

Genital organ-associated lymphoid tissues arranged in a ring in the mucosa of cow vaginal vestibules.

Female reproductive tracts are equipped with local and mucosal immune systems; however, structural information remains unclear for farm animals. In this study, the mucosa-associated lymphoid tissue-like structures in cow reproductive tracts were described. Vaginal vestibule (VV) and external parts of the genital organ, including the clitoris and vulva, were morphologically analyzed. Whole-mount specimens revealed several hematoxylin-positive spots arranged in a ring in the mucosa. Histologically, these spots were aggregated immune cells and defined as genital organ-associated lymphoid tissues (GOALTs). GOALTs were composed of lymphatic follicles (LFs) or diffuse lymphoid tissues (DLTs) at different depths of lamina propria. LFs frequently contained germinal centers. Scattered lymphocytes occupied the border area between follicles and epithelium, whereas DLTs had indefinite shapes. GOALTs contained immune cells and high endothelial venules. B cells were dominant both in LFs and DLTs. Abundant collagenous fibers were stretched across VV lamina propria, whereas reticular fibers were primarily observed in the DLT rather than LF. The epithelium covering of GOALTs was partially or fully disrupted by the invasion of immune cells toward the VV lumen. These findings suggest GOALTs function as a "genital lymphoid ring" as in Waldeyer's pharyngeal ring and act as immunological gate systems in cow reproductive tracts.

Pharmacometric analysis of seasonal influenza epidemics and the effect of vaccination using sentinel surveillance data.

The identification of influenza epidemics and assessment of the efficacy of vaccination against this infection are major challenges for the implementation of effective public health strategies, such as vaccination programs. In this study, we developed a new pharmacometric model to evaluate the efficacy of vaccination based on infection surveillance data from the 2010/2011 to 2018/2019 influenza seasons in Japan. A novel susceptible-infected-removed plus vaccination model, based on an indirect response structure with the effect of vaccination, was applied to describe seasonal influenza epidemics using a preseasonal collection of data regarding serological H1 antibody titer positivity and the fraction of virus strains. Using this model, we evaluated Kin (a parameter describing the transmission rate of symptomatic influenza infection) for different age groups. Furthermore, we defined a new parameter (prevention factor) showing the efficacy of vaccination against each viral strain and in different age groups. We found that the prevention factor of vaccination against influenza varied among age groups. Notably, children aged 5-14 years showed the highest Kin value during the 10 influenza seasons and the greatest preventive effect of vaccination (prevention factor = 70.8%). The propagation of influenza epidemics varies in different age groups. Children aged 5-14 years most likely play a leading role in the transmission of influenza. Prioritized vaccination in this age group may be the most effective strategy for reducing the prevalence of influenza in the community.

Close Association between Altered Urine-Urothelium Barrier and Tertiary Lymphoid Structure Formation in the Renal Pelvis during Nephritis.

BACKGROUND: Kidneys with chronic inflammation develop tertiary lymphoid structures (TLSs). Infectious pyelonephritis is characterized by renal pelvis (RP) inflammation. However, the pathologic features of TLSs, including their formation and association with non-infectious nephritis, are unclear. METHODS: RPs from humans and mice that were healthy or had non-infectious chronic nephritis were analyzed for TLS development, and the mechanism of TLS formation investigated using urothelium or lymphoid structure cultures. RESULTS: Regardless of infection, TLSs in the RP, termed urinary tract-associated lymphoid structures (UTALSs), formed in humans and mice with chronic nephritis. Moreover, urine played a unique role in UTALS formation. Specifically, we identified urinary IFN-γ as a candidate factor affecting urothelial barrier integrity because it alters occludin expression. In a nephritis mouse model, urine leaked from the lumen of the RP into the parenchyma. In addition, urine immunologically stimulated UTALS-forming cells via cytokine (IFN-γ, TNF-α) and chemokine (CXCL9, CXCL13) production. CXCL9 and CXCL13 were expressed in UTALS stromal cells and urine stimulation specifically induced CXCL13 in cultured fibroblasts. Characteristically, type XVII collagen (BP180), a candidate autoantigen of bullous pemphigoid, was ectopically localized in the urothelium covering UTALSs and associated with UTALS development by stimulating CXCL9 or IL-22 induction via the TNF-α/FOS/JUN pathway. Notably, UTALS development indices were positively correlated with chronic nephritis development. CONCLUSIONS: TLS formation in the RP is possible and altered urine-urothelium barrier-based UTALS formation may represent a novel mechanism underlying the pathogenesis of chronic nephritis, regardless of urinary tract infection.

Compartmentalization of interleukin 36 subfamily according to inducible and constitutive expression in the kidneys of a murine autoimmune nephritis model.

The interleukin (IL) 36 subfamily belongs to the IL-1 family and is comprised of agonists (IL-36α, IL-36ß, IL-36γ) and antagonists (IL-36Ra, IL-38). We previously reported IL-36α overexpression in renal tubules of chronic nephritis mice. To understand the localization status and biological relationships among each member of the IL-36 subfamily in the kidneys, MRL/MpJ-Faslpr/lpr mice were investigated as autoimmune nephritis models using pathology-based techniques. MRL/MpJ-Faslpr/lpr mice exhibited disease onset from 3 months and severe nephritis at 6-7 months (early and late stages, respectively). Briefly, IL-36γ and IL-36Ra were constitutively expressed in murine kidneys, while the expression of IL-36α, IL-36ß, IL-36Ra, and IL-38 was induced in MRL/MpJ-Faslpr/lpr mice. IL-36α expression was significantly increased and localized to injured tubular epithelial cells (TECs). CD44+-activated parietal epithelial cells (PECs) also exhibited higher IL-36α-positive rates, particularly in males. IL-36ß and IL-38 are expressed in interstitial plasma cells. Quantitative indices for IL-36α and IL-38 positively correlated with nephritis severity. Similar to IL-36α, IL-36Ra localized to TECs and PECs at the late stage; however, MRL/MpJ-Faslpr/lpr and healthy MRL/MpJ mice possessed IL-36Ra+ smooth muscle cells in kidney arterial tunica media at both stages. IL-36γ was constitutively expressed in renal sympathetic axons regardless of strain and stage. IL-36 receptor gene was ubiquitously expressed in the kidneys and was induced proportional to disease severity. MRL/MpJ-Faslpr/lpr mice kidneys possessed significantly upregulated IL-36 downstream candidates, including NF-κB- or MAPK-pathway organizing molecules. Thus, the IL-36 subfamily contributes to homeostasis and inflammation in the kidneys, and especially, an IL-36α-dominant imbalance could strongly impact nephritis deterioration.