Comparative Histological Subtyping of Immune Cell Infiltrates in MPO-ANCA and PR3-ANCA Glomerulonephritis

BackgroundAcute kidney injury (AKI) is a common and severe complication of anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV), potentially leading to chronic kidney disease (CKD), end-stage renal disease (ESRD), or death. Pathogenic ANCAs, in particular proteinase 3 (PR3) and myeloperoxidase (MPO), trigger a deleterious immune response with intrarenal immune cell infiltration resulting in a pauci-immune necrotizing and crescentic glomerulonephritis (GN). However, a systematic analysis of intrarenal immune cell subtypes concerning neutrophils, eosinophils, plasma cells, and mononuclear cell infiltrates (macrophages, lymphocytes) in ANCA GN remains elusive. Therefore, we aimed to compare distinct immune cell infiltrates in association with clinicopathological findings in ANCA GN.MethodsA total of 53 kidney biopsies with ANCA GN at the University Medical Center Göttingen were retrospectively analyzed. Histological infiltrates of neutrophils, eosinophils, plasma cells, and mononucleated cells (macrophages, lymphocytes) were quantified as a fraction of the total area of inflammation.ResultsNeutrophilic infiltrates were associated with glomerular necrosis and severe kidney injury in ANCA GN. Among tubulointerstitial lesions, intrarenal neutrophils correlated with interstitial inflammation, tubulitis, and inflammation in areas of interstitial fibrosis/tubular atrophy (IFTA), representing active inflammatory lesions. Concerning eosinophils, infiltrates were associated with severe kidney injury, interstitial inflammation, and cellular casts independent of glomerular lesions, implicating a distinct role in inflammation and damage in ANCA GN. Plasma cell infiltrates correlated with tubulitis and interstitial fibrosis and were associated with renal replacement therapy during the short-term disease course. Finally, mononuclear cell infiltrates correlated with severe kidney injury and active histopathological lesions (glomerular crescents, interstitial inflammation, tubulitis, inflammation, and tubulitis in areas of IFTA) besides chronic lesions (interstitial fibrosis and tubular atrophy) in ANCA GN. Interestingly, intrarenal subtypes of immune cell infiltrates differed in MPO-ANCA versus PR3-ANCA GN and were associated with distinct glomerular and tubulointerstitial lesions, implicating different pathogenic mechanisms of kidney injury in ANCA subtypes.ConclusionOur observations imply distinct pathomechanisms contributing to inflammation and renal injury in MPO vs. PR3-associated ANCA GN and potentially contribute to new therapeutic targets in specific ANCA subtypes.

Mucosa-Associated Lymphoid Tissue and Tertiary Lymphoid Structures of the Eye and Ear in Laboratory Animals

Mucosa-associated lymphoid tissue (MALT) of special senses is poorly described and can be confused with nonspecific mononuclear cell infiltrates and tertiary lymphoid structures (TLS). In the eye, MALT consists mostly of conjunctiva-associated lymphoid tissue (CALT) and lacrimal drainage-associated lymphoid tissue (LDALT). In humans, CALT and LDALT are important components of the normal eye-associated lymphoid tissue (EALT), but EALT is less frequently described in ocular tissues of animals. The EALT are acquired postnatally in preferential mucosal sites, expand with antigenic exposure, form well-developed lymphoid follicles, and are reported to senesce. Lymphoid follicles that are induced concurrently with chronic inflammation are more appropriately considered TLS but must be differentiated from inflammation in MALT. Less understood is the etiology for formation of lymphoid tissue aggregates in the ciliary body, limbus, or choroid of healthy eyes in animals and humans. In the healthy eustachian tube and middle ear of animals and humans, MALT may be present but is infrequently described. Concurrent with otitis media, lymphoid follicles in the eustachian tube are probably expanded MALT, but lymphoid follicles in the middle ear may be TLS. The purpose of this comparative review is to familiarize toxicologic pathologists with MALT in the special senses and to provide considerations for differentiating and reporting eye and ear MALT from immune or inflammatory cell infiltrates or inflammation in nonclinical studies, and the circumstances for reporting TLS in compartments of the eye and ear.

Spontaneous Background and Procedure-Related Microscopic Findings and Common Artifacts in Ocular Tissues of Laboratory Animals in Ocular Studies

Identification of test article–related microscopic findings in ocular toxicology studies requires a working knowledge of the artifacts and procedure-related or background findings commonly encountered in such studies. The objective of this article is to provide a mini-atlas of the artifacts and procedure-related or spontaneous background findings commonly observed in ocular tissues from animals in toxicology studies of ocular drug candidates. Artifacts in the eye are often related to collection or fixation procedures and include swelling and vacuolation of lens fibers, separation of the neuroretina from the retinal pigment epithelium (RPE), and vacuolation of the optic nerve. Common in-life procedure-related findings include intravitreal injection needle tracks in the sclera and ciliary body pars plana and foci of RPE hypertrophy and/or hyperpigmentation at subretinal injection sites. Common background findings include corneal mineralization, uveal mononuclear cell infiltrates, and peripheral displacement of photoreceptor nuclei in the retina. A few uncommon spontaneous background findings that may be confused with test article–related findings, such as bilateral optic atrophy in macaques, are also included.

Diabetic amyotrophy (Bruns-Garland syndrome)

Garland and Taverner first fully described diabetic amyotrophy as a clinical entity in 1950. Its distinctive features were a painful, markedly asymmetrical proximal weakness and wasting of the thighs and legs often with diminished or absent tendon reflexes. Motor signs dominated the picture, but autonomic and sensory nerves could be involved. Characteristically it occurred in poorly controlled diabetics in whom substantial if not always complete recovery was generally observed. A lumbosacral plexus neuropathy, associated with microvasculitis with secondary inflammatory perivascular mononuclear cell infiltrates is the underlying pathology.

Histopathological Findings in Cynomolgus Macaques (Macaca fascicularis) Consistent with Secondary Immunological Reaction to Biotherapeutics with an Emphasis on the CNS and Eye

Biotherapeutics are pharmaceutical products derived from or synthesized by biological systems. Such molecules carry the potential for immunogenicity which may lead to adverse immune responses. The cynomolgus macaque ( Macaca fascicularis) is the species of choice in nonclinical safety assessment of biotherapeutics. The main aim of this study was to confirm whether mononuclear cell infiltrates at specific locations represent a generic effect of biotherapeutics, and therefore the result of their immunogenicity. Following a review of microscopic findings in studies conducted over a 10-year period at one test facility, 15% of biotherapeutics were reported to have such findings. The most commonly affected site was the choroid plexus and less frequently the meninges and ciliary body. The reporting of such findings as test article–related becomes more subjective as the severity and incidence decreases. To assess the accuracy of such associations, a mathematical approach was employed to determine the probability of obtaining the observed results by chance. There was good agreement between this approach and the original findings. In addition to an increased number and size of mononuclear cell infiltrates in the brain, biotherapeutic administration was strongly associated with the presence of plasma cells and eosinophils.

Role of MAdCAM-1-Expressing High Endothelial Venule-Like Vessels in Colitis Induced in Mice Lacking Sulfotransferases Catalyzing L-Selectin Ligand Biosynthesis

Ulcerative colitis (UC) is a chronic inflammatory disease histologically characterized by diffuse mononuclear cell infiltrates in colonic mucosa. These inflammatory cells are considered to be recruited via high endothelial venule (HEV)-like vessels displaying mucosal addressin cell adhesion molecule 1 (MAdCAM-1), the ligand for α4β7 integrin, and/or peripheral lymph node addressin (PNAd), an L-selectin ligand. 6- O-sulfation of N-acetylglucosamine in the carbohydrate moiety of PNAd is catalyzed exclusively by N-acetylglucosamine-6- O-sulfotransferase 1 (GlcNAc6ST-1) and GlcNAc6ST-2. To determine the role of 6- O-sulfation of N-acetylglucosamine on HEV-like vessels in UC, we used a chronic dextran sulfate sodium–induced colitis model using mice deficient in both GlcNAc6ST-1 and GlcNAc6ST-2. We found that more inflammatory cells, with expression of tumor necrosis factor α, were infiltrated in double knockout mouse colitis compared with that in wild-type mice. Moreover, the number of MAdCAM-1-positive vessels was increased in double knockout mouse colitis, and these vessels were bound by E-selectin•IgM chimeras that bind to unsulfated sialyl Lewis X (sLeX). These findings suggest that interactions between MAdCAM-1 and α4β7 integrin and/or unsulfated sLeX and L-selectin may become a dominant mechanism for inflammatory cell recruitment in the absence of 6-sulfo sLeX and contribute to more severe colitis phenotypes seen in double knockout mice.

Eyedrop Inoculation Causes Sublethal Leptospirosis in Mice

ABSTRACT Leptospirosis is potentially a fatal zoonosis acquired by contact of skin and mucosal surfaces with soil and water contaminated with infected urine. We analyzed the outcome of infection of C3H/HeJ mice with Leptospira interrogans serovar Copenhageni using an enzootic mode of transmission, the conjunctival route. Infection led to weight loss and L. interrogans dissemination from blood to urine, and spirochetes were detected in blood and urine simultaneously. The infectious dose that led to consistent dissemination to kidney after conjunctival infection was ∼108 leptospires. Interestingly, a lower number of spirochetes appeared to colonize the kidney, given that we quantified ∼105 and ∼10 leptospires per μl of urine and per μg of kidney, respectively. Leptospira-specific IgM and IgG were detected at 15 days postinfection, and isotyping of the Ig subclass showed that the total IgG response switched from an IgG1 response to an IgG3 response after infection with L. interrogans. Histological periodic acid-Schiff D staining of infected kidney showed interstitial nephritis, mononuclear cell infiltrates, and reduced size of glomeruli. Quantification of proinflammatory immunomediators in kidney showed that keratinocyte-derived chemokine, macrophage inflammatory protein 2, RANTES, tumor necrosis factor alpha, gamma interferon, and interleukin-10 were upregulated in infected mice. We show that the kinetics of disease progression after infection via the ocular conjunctiva is delayed compared with infection via the standard intraperitoneal route. Differences may be related to the number of L. interrogans spirochetes that succeed in overcoming the natural defenses of the ocular conjunctiva and transit through tissue.

Frequency, clinicopathological features and phylogenetic analysis of feline morbillivirus in cats in Istanbul, Turkey

Objectives The aim of the study was to investigate feline morbillivirus (FmoPV) frequency, phylogeny and associated pathology in cats in Istanbul, Turkey. Methods Samples from sick (n = 96) and dead ( n = 15) cats were analysed using reverse transcription PCR. Blood and urine analyses and histopathology were also performed. Results FmoPV RNA was detected in six cats (5.4%), including three sick (in the urine) and three dead cats (tissues). A significantly greater proportion of FmoPV RNA-positive cats had street access compared with non-infected cats. Blood samples from the morbillivirus-positive cats were negative for morbillivirus RNA. Tubular parenchymal cells, lymphoid and plasma cells in kidney and hepatocytes, lymphoid and plasma cells in liver from dead cats were also positive by immunohistochemistry for the viral N protein. Two FmoPV-positive cats were also positive for feline coronavirus RNA and one cat for feline immunodeficiency virus RNA and feline leukaemia virus proviral DNA. Phylogenetic analysis of the six FmoPV-positive cats showed that the strains were grouped into cluster D and had high similarity (98.5–100%) with strains from Japan and Germany. In the three FmoPV RNA-positive sick cats, respiratory, urinary and digestive system signs were observed as well as weight loss, fever and depression in some cats. Similar clinical signs were also seen in the morbillivirus RNA-negative sick cats. FmoPV RNA-positive cats had lower median red blood cell count, haemoglobin, albumin, albumin/globulin and urobilinogen and higher alanine transaminase, alkaline phosphatase and bilirubin compared with non-infected cats. Significant histopathology of FmoPV RNA-positive dead cats included tubulointerstitial nephritis characterised by severe granular and vacuolar degeneration of the epithelial cells of the cortical and medullary tubules as well as mononuclear cell infiltrates. Widespread lymphoid cell infiltrates were detected in the renal cortex and medullary regions of the kidneys. Cellular infiltration, cholangiohepatitis and focal necrosis in the liver were also found. Although virus-infected cells were found in the kidney and liver of FmoRV RNA-positive cats, tubulointerstitial nephritis, cholangiohepatitis and focal necrosis seen in FmoRV RNA-positive cats were similar to those observed in FmoRV RNA-negative cats. Conclusions and relevance This is the first study to show the presence of FmoPV infection in cats in Turkey. Sick cats, particularly those with kidney disease, should be tested for this virus. The genotypes found in this study were similar to previously reported strains, indicating that circulating morbilliviruses in Turkey are conserved.

Murine model of Bacillus cereus gastrointestinal infection

Bacillus cereus is a spore-forming micro-organism responsible for foodborne illness. In this study, we focus on the host response following intragastric challenge with a pathogenic B. cereus strain (B10502) isolated from a foodborne outbreak. C57BL/6J female mice were infected by gavage with strain B10502. Controls were administered with PBS. Infection leads to significant modification in relevant immune cells in the spleen, Peyer's patches (PP) and mesenteric lymph nodes (MLN). These findings correlated with an increase in the size of PP as compared with uninfected controls. Histological studies showed that B. cereus infection increased the ratio of intestinal goblet cells and induces mononuclear cell infiltrates in spleen at 5 days post-infection. Evaluation of cytokine mRNA expression demonstrated a significant increase in IFN-γ in MLN after 2 days of infection. The present work demonstrates that infection of mice with vegetative B. cereus is self-limited. Our findings determined relevant cell populations that were involved in the control of the pathogen through modification of the ratio and/or activation.