Perivascular Clusters Research Articles

Comparison of fluorescent antibody test, immunohistochemistry, and PCR testing for diagnostic confirmation of neurolisteriosis in 25 goats.

Neurolisteriosis, a common disease of small ruminants, is most often caused by Listeria monocytogenes. Here we describe 25 cases of caprine neurolisteriosis diagnosed in our laboratory over a 5-y period and compare our fluorescent antibody test (FAT) results with immunohistochemistry (IHC) and polymerase chain reaction (PCR) testing for diagnostic confirmation. Neurohistologic changes consistent with neurolisteriosis affected the pons in all cases, extending rostrally to the mesencephalon in 6 cases, caudally to the medulla oblongata in 6 cases, and/or dorsally to the cerebellum in 4 cases. Acute inflammatory changes were observed in 17 cases, and included neuroparenchymal microabscesses, neuronal necrosis and neuronophagia, axonal swelling, microgliosis and astrogliosis, and perivascular neutrophils with macrophages, lymphocytes, and plasma cells that occasionally extended to the leptomeninges. Subacute-to-chronic changes (8 cases) consisted of neuroparenchymal and perivascular clusters of macrophages with rare neutrophils, lymphocytes, and plasma cells admixed with glial nodules. Bacterial bacilli were observed within neutrophils or macrophages in H&E-stained tissue sections in 4 cases. Gram stain highlighted gram-positive bacilli in 13 cases. Neurolisteriosis was confirmed by FAT in 2 cases, by IHC in 19 cases, and by PCR in 20 cases.

#1449 Maturation of tertiary lymphoid structures in progressive kidney damage due to chronic phosphate load

Abstract Background and Aims We have recently shown that a high phosphate diet (HPD) in mice leads to an increase in FGF23 levels and causes progressive tubular damage and interstitial fibrosis with increased accumulation of macrophages and concomitant perivascular immune cell aggregates in the corticomedullary zone. Such tertiary lymphoid structures (TLS) can develop as a result of chronic inflammation and have already been described in lupus nephritis and IgA nephropathy, among others. In our model, the question arose to what extent damage to the proximal tubule is temporally associated with the development and maturation of TLS and which factors favor TLS development. Method Wild-type mice were fed HPD for 1, 2, 3, 4 and 6 months and the kidneys of each were compared with those of mice on a normal phosphate diet (NPD). To evaluate the effect of high FGF23 ± phosphate, Fgf23 was overexpressed in mice on NPD and HPD using an adeno-associated virus (AAV-Fgf23), and hypophosphatemic Hyp mice were used. Results An increased tubular damage score was seen after only two months of HPD. In parallel, perivascular immune cell clusters developed in the corticomedullary zone and in the renal cortex, which were characterized by aggregates of CD45R+ B cells and CD3+ T cells, as well as LYVE1+ lymphatic vessels after three months of HPD and were accompanied by increased expression of venous markers (Plvap, Aplnr, EphB4) and cell adhesion molecules (Sell, Vcam1, Madcam1). Further analyses showed a time-dependent induction of the immunofibroblast-derived chemokines Cxcl13 and Ccl19, as well as the lymphotoxins Lta and Ltb, which are important for the differentiation of immunofibroblasts into follicular dendritic cells (FDC) and follicular reticular cells (FRC). Already after four months of HPD, proliferating B-cell clusters with FDCs, FRCs located in T-cell clusters, podoplanin+ networks, high endothelial venules, plasma cells and increased IgD synthesis indicated fully mature TLS, while tubular damage and fibrosis continued to increase until six months of HPD. Compared to AAV-Fgf23 mice on NPD, only AAV-Fgf23 animals on HPD developed tubular damage with fibrosis and fully mature TLS. Hypophosphatemic Hyp mice with high FGF23 showed neither signs of tubular damage nor the development of TLS. Conclusion The development of renal TLS due to chronic high phosphate load coincides with the development of tubular damage, with earlier maturation of TLS and progression of tubular damage.

Extensive intraocular melanoma with secondary glaucoma in a 15-month-old Thoroughbred filly.

A 15-month-old, grey, Thoroughbred filly presented for investigation of a 6-week history of corneal oedema and blepharospasm on the right eye (OD). The filly was otherwise healthy. Following ophthalmic examination, glaucoma on the OD was diagnosed. A space occupying mass within the anterior chamber was documented on transpalpebral ultrasonographic examination. This mass obliterated most of the anterior intraocular structures on the peripheral nasal side (corneal endothelium and drainage angle), leading to secondary glaucoma. After systemic and topical treatment addressing secondary glaucoma, the corneal oedema reduced. The mass was visualised as an irregularly rounded brown structure associated with the iris on the peripheral nasal side of the anterior chamber. Given the filly's signalment, location and appearance of the mass, a tentative diagnosis of intraocular melanoma was made and enucleation was performed. Histopathological evaluation of the globe revealed solid sheets of heavily pigmented melanocytic cells, disrupting the normal ciliary body architecture and extending into the iris and subretinal. The cells were pleomorphic, polyhedral to round with occasional spindle-shaped cells, and contained moderate to large amounts of granular black-brown pigment (melanin). The iridal component expanded into the anterior chamber, with cells directly opposed to Descemet's membrane, with loss of the endothelium and expanding and occluding the filtration angle in this area. The lesion infiltrated locally into the edge of the sclera, but did not extend through the sclera, though occasional perivascular clusters of melanophages were observed within the scleral stroma adjacent to the optic nerve. Diagnosis of a uveal melanocytic neoplasm was confirmed, with characteristics similar to only one reported case . This is a unique case of a rapidly growing, invasive, uveal melanoma in a young horse. Intraocular melanoma should be considered as a differential diagnoses for glaucoma in grey horses, regardless of the age and absence of melanocytic skin lesions.

Pathologic Features of Anti-Ku Myositis.

Characteristics of myositis with anti-Ku antibodies are poorly understood. The purpose of this study was to elucidate the pathologic features of myositis associated with anti-Ku antibodies, compared with immune-mediated necrotizing myopathy (IMNM) with anti-signal recognition particle (SRP) and anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) antibodies, in muscle biopsy-oriented registration cohorts in Japan and Germany. We performed a retrospective pathology review of patients with anti-Ku myositis samples diagnosed in the Japanese and German cohorts. We evaluated histologic features and performed HLA phenotyping. Fifty biopsied muscle samples in the Japanese cohort and 10 in the German cohort were obtained. After exclusion of myositis-specific autoantibodies or other autoimmune connective tissue diseases, 26 samples (43%) of anti-Ku antibody-positive myositis were analyzed. All the samples shared some common features with IMNM, whereas they showed expression of MHC class II and clusters of perivascular inflammatory cells more frequently than the anti-SRP/HMGCR IMNM samples (71% vs 7%/16%; p < 0.005/<0.005; 64% vs 0%/0%; p < 0.005/<0.005). Anti-Ku myositis biopsies could be divided into 2 subgroups based on the extent of necrosis and regeneration. The group with more abundant necrosis and regeneration showed a higher frequency of MHC class II expression and perivascular inflammatory cell clusters. HLA phenotyping in the 44 available patients showed possible associations of HLA-DRB1*03:01, HLA-DRB1*11:01, and HLA-DQB1*03:01 (p = 0.0045, 0.019, and 0.027; odds ratio [OR] 50.2, 4.6, and 2.8; 95% CI 2.6-2942.1, 1.1-14.5, and 1.0-7.0) in the group with less conspicuous necrosis and regeneration. On the contrary, in the group of more abundant necrosis and regeneration, the allele frequencies of HLA-A*24:02, HLA-B*52:01, HLA-C*12:02, and HLA-DRB1*15:02 were lower than those of healthy controls (p = 0.0036, 0.027, 0.016, and 0.026; OR = 0.27, 0, 0, and 0; 95% CI 0.1-0.7, 0-0.8, 0-0.8, and 0-0.8). However, these HLA associations did not remain significant after statistical correction for multiple testing. While anti-Ku myositis shows necrotizing myopathy features, they can be distinguished from anti-SRP/HMGCR IMNM by their MHC class II expression and clusters of perivascular inflammatory cells. The HLA analyses suggest that anti-Ku myositis may have different subsets associated with myopathologic subgroups.

Changes in the immune landscape of TNBC after neoadjuvant chemotherapy: correlation with relapse.

Patients with high-risk, triple negative breast cancer (TNBC) often receive neoadjuvant chemotherapy (NAC) alone or with immunotherapy. Various single-cell and spatially resolved techniques have demonstrated heterogeneity in the phenotype and distribution of macrophages and T cells in this form of breast cancer. Furthermore, recent studies in mice have implicated immune cells in perivascular (PV) areas of tumors in the regulation of metastasis and anti-tumor immunity. However, little is known of how the latter change during NAC in human TNBC or their impact on subsequent relapse, or the likely efficacy of immunotherapy given with or after NAC. We have used multiplex immunofluorescence and AI-based image analysis to compare the immune landscape in untreated and NAC-treated human TNBCs. We quantified changes in the phenotype, distribution and intercellular contacts of subsets of tumor-associated macrophages (TAMs), CD4+ and CD8+ T cells, and regulatory T cells (Tregs) in PV and non-PV various areas of the stroma and tumor cell islands. These were compared in tumors from patients who had either developed metastases or were disease-free (DF) after a three-year follow up period. In tumors from patients who remained DF after NAC, there was a marked increase in stromal CD163+ TAMs, especially those expressing the negative checkpoint regulator, T-cell immunoglobulin and mucin domain 3 (TIM-3). Whereas CD4+ T cells preferentially located to PV areas in the stroma of both untreated and NAC-treated tumors, specific subsets of TAMs and Tregs only did so only after NAC. Distinct subsets of CD4+ and CD8+ T cells formed PV clusters with CD163+ TAMs and Tregs. These were retained after NAC. Quantification of stromal TIM-3+CD163+ TAMs in tumor residues after NAC may represent a new way of identifying patients at high risk of relapse. PV clustering of immune cells is highly likely to regulate the activation and function of T cells, and thus the efficacy of T cell-based immunotherapies administered with or after NAC.

Neuropathology in COVID-19 autopsies is defined by microglial activation and lesions of the white matter with emphasis in cerebellar and brain stem areas.

This study aimed to investigate microglial and macrophage activation in 17 patients who died in the context of a COVID-19 infection in 2020 and 2021. Through immunohistochemical analysis, the lysosomal marker CD68 was used to detect diffuse parenchymal microglial activity, pronounced perivascular macrophage activation and macrophage clusters. COVID-19 patients were compared to control patients and grouped regarding clinical aspects. Detection of viral proteins was attempted in different regions through multiple commercially available antibodies. Microglial and macrophage activation was most pronounced in the white matter with emphasis in brain stem and cerebellar areas. Analysis of lesion patterns yielded no correlation between disease severity and neuropathological changes. Occurrence of macrophage clusters could not be associated with a severe course of disease or preconditions but represent a more advanced stage of microglial and macrophage activation. Severe neuropathological changes in COVID-19 were comparable to severe Influenza. Hypoxic damage was not a confounder to the described neuropathology. The macrophage/microglia reaction was less pronounced in post COVID-19 patients, but detectable i.e. in the brain stem. Commercially available antibodies for detection of SARS-CoV-2 virus material in immunohistochemistry yielded no specific signal over controls. The presented microglial and macrophage activation might be an explanation for the long COVID syndrome.

O-188 Decoding uterine leiomyoma tumorigenesis using single-cell transcriptomics and single-cell proteomics

Abstract Study question Can single-cell transcriptomics and proteomics contribute to a better understanding uterine of leiomyoma tumorigenesis? Summary answer We demonstrate the significant involvement of the MAPK, PI3K-Akt, and proteoglycan pathways in smooth muscle, endothelial and perivascular cells in leiomyoma tumorigenesis. What is known already Uterine leiomyomas (LM), also known as fibroids, are benign tumors of the uterus that arise from the myometrium. Previous studies have shown cellular heterogeneity in both myometrium and uterine leiomyomas, although cell spatial location within the tissue has not been shown. Further, no data describing single-cell-level proteomic differences and key pathways involved in uterine LM tumorigenesis have yet been reported. Study design, size, duration A prospective, observational, and biomedical study of cohorts was conducted at Hospital La Fe (Valencia, Spain) for one year. Single-cell RNAseq (scRNA-seq; n = 16) and single-cell proteomic (scP; n = 16) analyses were performed on eight sample pairs of LM and matched myometrium (MM), to generate a high-resolution transcriptomic and proteomic map decoupled from cell type, state, and spatial location. Participants/materials, setting, methods After obtaining informed consent, LM and MM samples were collected from eight patients between 35-50 years undergoing hysterectomies. Part of the samples were preserved in paraffin for spatial transcriptomics using VISIUM (10x Genomics). While the remaining tissues were dissociated into single-cell suspensions and subjected to Chromium Controller and Orbitrap Eclipse Tribid mass spectrometry for scRNA-seq and scP, respectively. All data were analyzed using publicly available R/Python tools. Main results and the role of chance After restrictive quality control filtering, we analyzed a total of 52,599 and 5,909 cells by scRNA-seq and scP, respectively. While LM and MM possessed similarities in terms of cellular composition, they displayed differential expression of genes and proteins across all the cell populations studied, particularly in smooth muscle, endothelial and perivascular cells. In LM samples, these cell populations displayed impaired MAPK signaling, which acts as a signal integrator for growth factors, estrogen, and vitamin D. We also observed alterations in the PI3K-Akt and proteoglycan pathways in LM smooth muscle and perivascular clusters, which relate to cell proliferation and tumor growth. Additionally, we encountered a subset of consistently dysregulated genes in all LM populations, which may suggest the existence of a shared tumorigenic pathway independent of cell type. Spatial transcriptomics further demonstrated relationships between cells and their relative locations within the tissue, which we validated by immunofluorescence. Together, our results highlight the relevance of specific cell populations in LM tumorigenesis. Limitations, reasons for caution This study involved a sample cohort limited to Caucasian women; therefore, further studies including more patients, and addressing racial disparities will help to generalize these findings to a broader population. Wider implications of the findings Our work describes an unprecedented transcriptomic and proteomic analysis of LM and MM at single-cell resolution, which supports a novel understanding of myometrial tumorigenesis. Trial registration number NCT04214457

Immune Spatial Differences Between Short and Long-term Surviving Glioblastoma Patients

Abstract Introduction: Immune surveillance, distribution, interactions, and antigen presentation within the tumor microenvironment (TME) may influence anti-tumor reactivity and survival. Methods: Dichotomized newly diagnosed treatment-naïve IDH1 wild-type glioblastoma, negative MGMT promotor methylation (65%), short-term (STS; ≤ 6 months; mean age 66 years; n=10) or long-term (LTS; &amp;gt;2 years; mean age 50 years; n=10) survivors, were profiled using high dimensional fluorescence multiplex staining spatial analysis of the TME using a panel of 26 markers. Results: The amounts of specific immune cells (CD4 +, CD8 +, FOXP3 +, CD11c +, CD20 +, CD68 +, CD205 +, NKG2D +) were similar between cohorts, except STS were enriched with CD163 +expressing macrophages and LTS had more P2RY12 +microglia throughout the TME. There was minimal MHC-I expression and no difference in MHC-II on the myeloid populations (CD68 +, CD11c +, CD163 +, P2RY12 +, and/or in combination) in both cohorts. TIM-3 was the only immune regulator substantially expressed on P2RY12 +microglia and the other myeloid populations, irrespective of survival. Perivascular clustering of CD11c +CD163 +cells was enriched in STS cases. The TME in LTS cases demonstrated adaptive immune activation processes, including CD11c +P2RY12 +CD205 +microglia interacting with CD45RO −naïve, non-exhausted (PD-1 −, LAG3 −, Tim3 −) CD8 +T cells expressing the LCK +immune synaptic marker. Conclusion: Immune activation interactions between CD8+ T cells and microglia in the TME may help determine STS versus LTS in GBM patients.

Fibrinogen Depletion Ameliorates Inflammation and Vision Loss in Mouse Models of Diabetes

Abstract Microglia-mediated inflammation plays a significant role in neuronal and vascular damage in diabetic retinopathy, but the mechanism linking inflammation, neurodegeneration, and impaired vascular integrity is still unclear. Our previous studies from diabetic mouse models showed accumulation of fibrinogen, at vessel lesions surrounded by perivascular microglial clusters. In this study, we evaluated whether the pathological hallmarks of gliosis and vascular aberrations characterized in diabetic animal models are consistent with those in diabetic human retinas. Postmortem human retinas were analyzed by immunohistochemistry for markers of gliosis, vascular integrity, and fibrinogen deposition. Immunohistochemical and gene expression analyses of human postmortem retinas revealed evidence of an inflammatory microenvironment, with microgliosis and impaired vasculature. To define the therapeutic potential of reducing fibrinogen in DR, the defibrinogenating agent ancrod was administered in a two-hit inflammatory diabetic mouse model, after which retinal pathology and visual acuity were assessed. Histopathological analyses revealed microglial activation, vascular aberrations, and fibrinogen deposition in the diabetic murine retina. Notably, after treatment with ancrod, diabetic mice appeared to improve visual acuity, which was associated with reduced microglia activation and less fibrinogen deposition in the retina. This study shows that fibrinogen-mediated microglial activation, blood-retinal barrier damage, and vision loss, can be ameliorated by reducing fibrinogen levels. Overall, these findings suggest that that fibrinogen contributes to microglia-mediated inflammation in the diabetic retina. Supported by R01 EY029913

Defibrinogenation Ameliorates Retinal Microgliosis and Inflammation in A CX3CR1-Independent Manner.

Microglia-mediated inflammation plays a significant role in neuronal and vascular damage in diabetic retinopathy (DR), but the mechanism linking inflammation, neurodegeneration, and impaired vascular integrity is still unclear. Previous studies from diabetic mouse models showed accumulation of fibrinogen at vessel lesions surrounded by perivascular microglial clusters. The purpose of this study was to evaluate whether the pathological hallmarks of gliosis and vascular aberrations characterized in diabetic animal models are consistent with those in diabetic human retinas, and to assess the effects of the defibrinogenating agent ancrod in retinal pathology and visual acuity in a two-hit inflammatory diabetic mouse model. Post-mortem human eyes were assessed for retinal and inflammatory gene expression by quantitative PCR. Immunohistochemical analyses in human and murine retinas were performed using markers of gliosis, vascular integrity, and fibrinogen deposition. An inflammatory microenvironment, with microgliosis and microaneurysms, was found in the diabetic human eye. Microglial activation, fibrinogen deposition, and axonal loss were also observed in the diabetic murine retina. Ancrod treatment correlated with reduced microgliosis, less fibrinogen deposition, and reduced pro-inflammatory cytokine levels in diseased retinal tissues. Together, these data suggest that fibrinogen contributes to microglia-mediated inflammation in the diabetic retina. Since retinal microgliosis, vascular pathology, and vision deficits manifest in diabetic mice irrespective of CX3CR1 genotype, our results indicate that defibrinogenation can dampen systemic neuroinflammation and vascular insults, thereby improving vision at early stages of diabetes.Summary StatementDiabetic human and murine retinas revealed pronounced microglial morphological activation and vascular abnormalities associated with inflammation. Pharmacological fibrinogen depletion using ancrod dampened microglial morphology alterations, resolved fibrinogen accumulation, rescued axonal integrity, and reduced inflammation in the diabetic murine retina.

T cell activation niches-Optimizing T cell effector function in inflamed and infected tissues.

Successful immunity to infection, malignancy, and tissue damage requires the coordinated recruitment of numerous immune cell subsets to target tissues. Once within the target tissue, effector T cells rely on local chemotactic cues and structural cues from the tissue matrix to navigate the tissue, interact with antigen-presenting cells, and release effector cytokines. This highly dynamic process has been "caught on camera" in situ by intravital multiphoton imaging. Initial studies revealed a surprising randomness to the pattern of T cell migration through inflamed tissues, behavior thought to facilitate chance encounters with rare antigen-bearing cells. Subsequent tissue-wide visualization has uncovered a high degree of spatial preference when it comes to T cell activation. Here, we discuss the basic tenants of a successful effector T cell activation niche, taking cues from the dynamics of Tfh positioning in the lymph node germinal center. In peripheral tissues, steady-state microanatomical organization may direct the location of "pop-up" de novo activation niches, often observed as perivascular clusters, that support early effector T cell activation. These perivascular activation niches appear to be regulated by site-specific chemokines that coordinate the recruitment of dendritic cells and other innate cells for local T cell activation, survival, and optimized effector function.

001 Maintenance of CD4+ tissue-resident memory T cells via perivascular clusters with CD301b+ dermal dendritic cells in a mouse model of allergic dermatitis

Tissue-resident memory T (TRM) cells play a crucial role in local immunity by inducing rapid immune responses upon re-exposure of the antigen. However, how CD4+ TRM cells are retained in the skin after allergic inflammation remains largely unknown. To clarify the mechanism, we used a delayed-type hypersensitivity model, which is mediated by CD4+ T cells. T cell receptor (TCR)-β deficient mice were transferred with CD4+ T cells from GFP-expressing, ovalbumin (OVA)-specific TCR-transgenic (OT-II) mice and sensitized with OVA emulsion, followed by initial challenge with OVA in ear skin (day 0). On day 35, in spite of the resolution of ear swelling, CD4+ T cells remained in the dermis and exhibited CD44+CD69+ TRM cell phenotype. Their residency in the dermis was confirmed using parabiosis and photo-convertible protein (KikGR)-expressing OT-II T cells. In addition, two-photon microscopy revealed that CD4+ T cells were retained in perivascular clusters on day 35. Immunohistochemical analysis revealed that CD301b+ conventional dendritic cell (DC) subset 2 (cDC2) were colocalized with CD4+ T cells in the clusters. We selectively depleted these CD301b+ cells in Mgl2-diphtheria toxin receptor (DTR) mice, which express the DTR under the regulation of the gene encoding CD301b, and found that the number of CD4+ TRM cells and their clusters were reduced after the depletion of CD301b+ cells. Taken together, these results suggest that CD301b+ cDC2 cells are critical in the tissue residency of CD4+ TRM cells after the resolution of allergic inflammation. This mechanism provides a potential new strategy for preventing the recurrence of CD4+ T cell-mediated chronic inflammatory skin diseases.

Perivascular clusters of Th2 cells and M2 macrophages in allergic contact dermatitis to methylchloroisothiazolinone and methylisothiazolinone.

Methylisothiazolinone (MI) and Methylchloroisothiazolinone (MCI) are among the most common skin sensitizers, yet the immunological events that occur during MCI/MI allergic contact dermatitis (ACD) are still poorly understood. To analyse dendrocytes, macrophage subtypes and T cells in skin during the elicitation phase of MCI/MI ACD. Thirteen patients with positive patch test reactions to MCI/MI (ACD group) and 11 individuals with negative patch test results were selected. Skin biopsies were only performed at 48hours of patch testing. Immunohistochemistry was conducted to assess T cells, dendrocytes (Factor XIIIa), M1 (p-Stat1, CD68) and M2 (c-Maf, CD163) macrophages. Transcriptional analyses were performed for cytokines and related factors, and further compared to atopic dermatitis samples (n=4). Immunofluorescence assays addressed T cells location, along with IL-4 or IL-13, within the skin. MCI/MI elicited dermal dendrocytes and macrophages, pronouncedly the M2subtype. T cells, majorly CD4+ T cells, accumulated in the perivascular areas. Similarly, abundant IL-4 protein was detected in these areas. There was an upregulation of IL-4 and IL-13mRNA expression, a mild increase in IFNG mRNA levels and a down-regulation of RORC in the ACD group. Immunofluorescence revealed dermal clusters of T cells co-localized with IL-4. M2macrophages and Th2 cells participate in the immunopathogenesis of MCI/MI ACD. Dermal dendrocytes and M2macrophages may assist the formation of CD4+ T cells perivascular clusters. These findings render a mechanistic insight into the MCI/MI reaction. Further analysis at different timepoints of patch testing is required to fully comprehend this ACD kinetics.

P–744 The features expression of some lymphocyte markers in the pelvic peritoneal adhesions’ tissue at reproductive age women

Abstract Study question To study the expression of CD4, CD8, CD20, CD 138 in the tissue of the pelvic peritoneal adhesions at women of reproductive age. Summary answer Immunohistochemical study of pelvic adhesions revealed the CD8-positive cells is directly involved in the formation of the immune response at the late stages of adhesiogenesis. What is known already One of the reason identifies the high frequency of adhesion formation is the presence of inflammation in the abdominal cavity with different severity and origin. It is known that Insufficiency of the fibrinolytic system, increased levels of a number of cytokines, including transforming growth factor- β1, and tissue hypoxia induce neoangiogenesis and fibrotization of the fibrin matrix, which leads to the formation of adhesions. Data on expression of CD4, CD8, CD20, CD138/syndecan–1 in the pelvic peritoneal adhesions in connection with their prescription, localization and origin is absent at accessible literature. Study design, size, duration Two hundred infertile women (aged 19–49 yrs) with pelvic peritoneal adhesions, who were underwent operative laparoscopy and adhesiolysis. Participants/materials, setting, methods The material for this study was the fragments of surgical material (adhesions and their parts) n = 200, taken from the women of reproductive age who suffered with infertility during operative laparoscopy.The morphological and immunohistochemical study of adhesions were carried out by standard techniques using paraffin blocks, reagents of Dako and monoclonal antibodies to CD4 (Clone 4B12 Ready-to-Use),CD8 (Clone C8/144B Ready-to-Use), CD20 (Clone L26 Ready-to-Use),CD138/syndecan–1 (Clone MI15 Ready-to-Use) of Abcam with automatic coloring Dako Cytomation. Main results and the role of chance To assess the population composition of these cell infiltrates, as well as individual diffusely located inflammatory cells, an immunohistochemical method with the main lymphocytic markers (CD4, CD8, CD20, CD138) was used. First of all, it is necessary to note the complete absence of CD20-positively colored cells in all observations, which indicates that at the final stage of the formation of adhesions, there is no element of the B-lymphocytic immune response. In an immunohistochemical study with syndecan–1 (CD138) antibodies, we identified a small number of positively colored cells that were located mainly perivascular, as part of mononuclear infiltrates. Quantitative analysis showed that the number of such cells is 0.8±0.2. When studying CD4–positive T-lymphocytes, it was found that they are usually located in the form of band-shaped infiltrates and focal perivascular clusters. The number of CD4-positive cells in the spike tissue is 5.6±0.2. CD8-positive cells were located mainly submesothelial, and in the form of perivascular clusters, the number of such cells was 9.2±0.6. Limitations, reasons for caution Age limitation, only women aged 19–49 yrs took part in this study. Exclusion criteria were the following for the groups: acute gynecological diseases, malignant diseases of female genitalia and ovarian tumors. Wider implications of the findings: The absence of B-cells in the “mature” adhesions’ tissue was found.The number of CD8-positive cells in our study was 1.5 times higher than the number of CD4-positive T-lymphocytes.CD4-positive T-lymphocytes play an important role and their number significantly prevails over the number of CD8-positive T-lymphocytes at the initial stages of adhesiogenesis. Trial registration number Case control study

CD40 Agonist Monoclonal Antibody-Mediated Hepatitis in TNF-Receptor 1 Gene Knockout Mice.

Tumor necrosis factor-alpha (TNF-α) plays an important role in liver inflammation. CD40-CD40 ligand (CD40-CD40L) is a key receptor–ligand signaling pair involved in the adaptive immune response and pathogenesis of autoimmune diseases. In mice, CD40 activation leads to sickness behavior syndrome (SBS) comprising weight loss, sleep disruption and depression, which can be blocked by administration of the TNF-inhibitor etanercept. In the present study, we assessed the extent of hepatic inflammation in mice devoid of the TNF-receptor 1 (TNFR1)-mediated signaling pathway. The TNFR1-depleted (TNFR1−/−) adult mice and their wild type littermates were given a single intra-peritoneal injection of CD40 agonist monoclonal antibody (mAb) or rat IgG2a isotope control. As described previously, TNFR1−/− mice were protected from SBS upon CD40 mAb treatment. Cd40, tnf and tnfr1 mRNA and Tnf-α peptide were increased in the liver of CD40 mAb-stimulated wild type mice. Serum alanine aminotransferase was elevated in both CD40-activated wild type and TNFR1−/− mice. TNFR1−/− mice showed much less intra-parenchymal infiltrates, hepatocellular necrosis, and perivascular clusters upon CD40 mAb activation than their wild type littermates. A gene expression microarray detected increased activity of metabolic and detoxification pathways and decreased activity of inflammatory pathways. We conclude that immune activation and development of liver inflammation in CD40L interactions depend on TNFR1-mediated signaling pathways and are counteracted by alterations in metabolic pathways.

Blood Coagulation Factor Fibrinogen in Tumor Pathogenesis of Central Nervous System B-Cell Lymphoma

Central nervous system (CNS) lymphoma is an extranodal non-Hodgkin B-cell lymphoma characterized by malignant lymph tissue arising in the brain or spinal cord, associated with inflammation and blood-brain barrier (BBB) disruption. Although BBB disruption is known to occur in patients with CNS lymphoma, a direct link between these two has not been shown. Herein, abundant deposition of the blood coagulation protein fibrinogen around B-cell lymphoma was detected in CNS lymphoma patients and in the CNS parenchyma in an orthotopic mouse model. Functional enrichment analysis of unbiased cerebrospinal fluid proteomics of CNS B-cell lymphoma patients showed that coagulation protein networks were highly connected with tumor-associated biological signaling pathways. Invivo two-photon imaging demonstrated that lymphoma growth was associated with BBB disruption, and invitro experiments identified a role for fibrinogen in promoting lymphoma cell adhesion. Overall, these results identify perivascular lymphoma clustering at sites of fibrinogen deposition, and suggest that fibrinogen may be a target for pharmacologic intervention in metastatic B-cell lymphoma associated with BBB disruption.

Absence of B Cells in Brainstem and White Matter Lesions Associates With Less Severe Disease and Absence of Oligoclonal Bands in MS

ObjectiveTo determine whether B-cell presence in brainstem and white matter (WM) lesions is associated with poorer pathological and clinical characteristics in advanced MS autopsy cases.MethodsAutopsy tissue of 140 MS and 24 control cases and biopsy tissue of 24 patients with MS were examined for CD20+ B cells and CD138+ plasma cells. The presence of these cells was compared with pathological and clinical characteristics. In corresponding CSF and plasma, immunoglobulin (Ig) G ratio and oligoclonal band (OCB) patterns were determined. In a clinical cohort of 73 patients, the presence of OCBs was determined during follow-up and compared to status at diagnosis.ResultsIn 34% of active and 71% of mixed active/inactive lesions, B cells were absent, which correlated with less pronounced meningeal B-cell infiltration (p < 0.0001). The absence of B cells and plasma cells in brainstem and WM lesions was associated with a longer disease duration (p = 0.001), less frequent secondary progressive MS compared with relapsing and primary progressive MS (p < 0.0001 and p = 0.046, respectively), a lower proportion of mixed active/inactive lesions (p = 0.01), and less often perivascular T-cell clustering (p < 0.0001). Moreover, a lower CSF IgG ratio (p = 0.006) and more frequent absence of OCBs (p < 0.0001) were noted. In a clinical cohort, numbers of patients without OCBs in CSF were increased at follow-up (27.4%).ConclusionsThe absence of B cells is associated with a favorable clinical and pathological profile. This finding may reflect extremes of a continuum of genetic or environmental constitution, but also a regression of WM humoral immunopathology in the natural course of advanced MS.

Deep Spatial Profiling of COVID-19 Brains Reveals Neuroinflammation by Compartmentalized Local Immune Cell Interactions and Targets for Intervention

COVID-19 can cause acute and chronic neurological symptoms. The underlying pathophysiological mechanisms, the involved immune cells, their spatial distribution, cellular interactions and the role of virus tropism remain largely unclear. Here, we deeply interrogated the brain stem and olfactory bulb in COVID-19 patients with imaging mass cytometry to understand the local immune response at a spatially resolved, high-dimensional single-cell level. We observed significant immune activation in the CNS and identified distinct phenotypes of T cells and microglial clusters, their presence in specific anatomical regions and context-specific cellular interactions. Microglial nodules and perivascular immune cell clusters constitute key sites of the local immune response, with viral antigen present in ACE2-expressing cells in the perivascular compartment. Disease-associated neuroinflammation is associated with astrogliosis and severe axonal damage as a structural basis for the neurologic deficits. Finally, we identify compartment- and cluster-specific immune checkpoints that can be targeted for future therapeutic interventions.Funding: This project was supported by grants from the Deutsche Forschungsgemeinschaft (DFG, GermanResearch Foundation) (SFB 992, SFB1160, SFB/TRR167, SFB/TRR179, German Excellency strategyCIBSS - EXC-2189– Project ID390939984) and special research funds from the Ministry for Science, Research and Art of Baden-Wuerttemberg dedicated to “COVID-19 research” and “Neuroinflammation”.B.B. was further supported by DFG grant BE-5496/5-1 and M.P. was further supported by the Sobek Foundation, the Ernst-Jung Foundation, the Reinhart-Koselleck-Grant and Gottfried Wilhelm Leibniz-Prize.H.E.M. was supported by DFG ME-3644/5-1.Ethical Approval: The analyses were performed with the approval of the Institutional Review Boards (Ethic Committee of the Albert-Ludwigs-University, Freiburg: 322/20, 10008/09; Ethics Committee of the Hamburg Chamber of Physicians: WF-051/20, PV7311). The study was performed in agreement with the principles expressed in the Declaration of Helsinki (2013).Conflict of Interest: None to declare.

B and T Lymphocyte Densities Remain Stable With Age in Human Cortex.

One hallmark of human aging is increased brain inflammation represented by glial activation. With age, there is also diminished function of the adaptive immune system, and modest decreases in circulating B- and T-lymphocytes. Lymphocytes traffic through the human brain and reside there in small numbers, but it is unknown how this changes with age. Thus we investigated whether B- and T-lymphocyte numbers change with age in the normal human brain. We examined 16 human subjects in a pilot study and then 40 human subjects from a single brain bank, ranging in age from 44–96 years old, using rigorous criteria for defining neuropathological changes due to age alone. We immunostained post-mortem cortical tissue for B- and T-lymphocytes using antibodies to CD20 and CD3, respectively. We quantified cell density and made a qualitative assessment of cell location in cortical brain sections, and reviewed prior studies. We report that density and location of both B- and T-lymphocytes do not change with age in the normal human cortex. Solitary B-lymphocytes were found equally in intravascular, perivascular, and parenchymal locations, while T-lymphocytes appeared primarily in perivascular clusters. Thus, any change in number or location of lymphocytes in an aging brain may indicate disease rather than normal aging.

Tissue-resident memory T cells invade the brain parenchyma in multiple sclerosis white matter lesions.

Multiple sclerosis is a chronic inflammatory, demyelinating disease, although it has been suggested that in the progressive late phase, inflammatory lesion activity declines. We recently showed in the Netherlands Brain Bank multiple sclerosis-autopsy cohort considerable ongoing inflammatory lesion activity also at the end stage of the disease, based on microglia/macrophage activity. We have now studied the role of T cells in this ongoing inflammatory lesion activity in chronic multiple sclerosis autopsy cases. We quantified T cells and perivascular T-cell cuffing at a standardized location in the medulla oblongata in 146 multiple sclerosis, 20 neurodegenerative control and 20 non-neurological control brain donors. In addition, we quantified CD3+, CD4+, and CD8+ T cells in 140 subcortical white matter lesions. The location of CD8+ T cells in either the perivascular space or the brain parenchyma was determined using CD8/laminin staining and confocal imaging. Finally, we analysed CD8+ T cells, isolated from fresh autopsy tissues from subcortical multiple sclerosis white matter lesions (n = 8), multiple sclerosis normal-appearing white matter (n = 7), and control white matter (n = 10), by flow cytometry. In normal-appearing white matter, the number of T cells was increased compared to control white matter. In active and mixed active/inactive lesions, the number of T cells was further augmented compared to normal-appearing white matter. Active and mixed active/inactive lesions were enriched for both CD4+ and CD8+ T cells, the latter being more abundant in all lesion types. Perivascular clustering of T cells in the medulla oblongata was only found in cases with a progressive disease course and correlated with a higher percentage of mixed active/inactive lesions and a higher lesion load compared to cases without perivascular clusters in the medulla oblongata. In all white matter samples, CD8+ T cells were located mostly in the perivascular space, whereas in mixed active/inactive lesions, 16.3% of the CD8+ T cells were encountered in the brain parenchyma. CD8+ T cells from mixed active/inactive lesions showed a tissue-resident memory phenotype with expression of CD69, CD103, CD44, CD49a, and PD-1 and absence of S1P1. They upregulated markers for homing (CXCR6), reactivation (Ki-67), and cytotoxicity (GPR56), yet lacked the cytolytic enzyme granzyme B. These data show that in chronic progressive multiple sclerosis cases, inflammatory lesion activity and demyelinated lesion load is associated with an increased number of T cells clustering in the perivascular space. Inflammatory active multiple sclerosis lesions are populated by CD8+ tissue-resident memory T cells, which show signs of reactivation and infiltration of the brain parenchyma.