Perivascular Areas Research Articles

Cerebrospinal fluid cytokine levels are associated with macrophage infiltration into tumor tissues of glioma patients

BackgroundDiffuse gliomas are the most common malignant tumors of the central nervous system with poor treatment efficacy. Infiltration of immune cells into tumors during immunosurveillance is observed in multiple tumor entities and often associated with a favorable outcome. The aim of this study was to evaluate the infiltration of immune cells in gliomas and their association with cerebrospinal fluid (CSF) cytokine concentrations.MethodsWe applied immunohistochemistry in tumor tissue sections of 18 high-grade glioma (HGG) patients (4 anaplastic astrocytoma, IDH-wildtype WHO-III; 14 glioblastomas (GBM), IDH-wildtype WHO-IV) in order to assess and quantify leucocytes (CD45) and macrophages (CD68, CD163) within the tumor core, infiltration zone and perivascular spaces. In addition, we quantified the concentrations of 30 cytokines in the same patients’ CSF and in 14 non-inflammatory controls.ResultsWe observed a significantly higher percentage of CD68+ macrophages (21–27%) in all examined tumor areas when compared to CD45+ leucocytes (ca. 3–7%); CD163+ cell infiltration was between 5 and 15%. Compared to the tumor core, significantly more macrophages and leucocytes were detectable within the perivascular area. The brain parenchyma showing a lower tumor cell density seems to be less infiltrated by macrophages. Interleukin (IL)-7 was significantly downregulated in CSF of GBM patients compared to controls. Additionally, CD68+ macrophage infiltrates showed significant correlations with the expression of eotaxin, interferon-γ, IL-1β, IL-2, IL-10, IL-13, IL-16 and vascular endothelial growth factor.ConclusionsOur findings suggest that the infiltration of lymphocytes is generally low in HGG, and does not correlate with cytokine concentrations in the CSF. In contrast, macrophage infiltrates in HGG are associated with CSF cytokine changes that possibly shape the tumor microenvironment. Although results point towards an escape from immunosurveillance or even exploitation of immune cells by HGG, further studies are necessary to decipher the exact role of the immune system in these tumors.

Effects of nano-sized titanium dioxide powder and ultraviolet light on superficial veins in a rabbit model.

Titanium dioxide (TiO2) is widely used in many fields such as food, cosmetics, and paper industries. Studies on the photocatalytic properties of TiO2 on living tissue are limited. To examine the histopathological effects of TiO2 solution on the marginal veins of rabbit ears under ultraviolet (UV) light. In this study, 4 groups of rabbits (8 rabbits per group) were used: the 1st group was the control group, the 2nd group received 20% of nano-TiO2 only, the 3rd group received UV light only, and the 4th group received nano-TiO2 and UV light, simultaneously. The study lasted for 14 days and samples were taken from the marginal ear vein on the 15th day. The ear tissues of rabbits in the control and TiO2 groups showed a normal histological appearance. In the UV group, the results showed severe chronic inflammation due to mononuclear cells around the hair follicles and perivascular areas. However, these findings decreased in the UV/nano-TiO2 group. The method applied in this study can be used in the treatment of telangiectasia in the future. However, this study investigating the effects of nano-TiO2 on vascular structures under UV light had a predominantly histological and observational nature. Further studies involving genetic, cytogenetic, biochemical, histochemical, and immunohistochemical analyses need to be performed to test the theories we proposed.

HISTOPATHOLOGICAL FINDINGS OF ACUTE EOSINOPHILIC PNEUMONIA

TOPIC: Lung Pathology TYPE: Original Investigations PURPOSE: It has been reported that AEP histopathologically exhibits diffuse alveolar damage (DAD) with eosinophilia. However, acute respiratory distress syndrome (ARDS), acute interstitial pneumonia (AIP), and acute exacerbation of idiopathic pulmonary fibrosis, also exhibiting DAD histopathologically, are all life-threatening diseases. AFP is rapidly improved by steroids alone without remaining of fibrosis or sometimes even without treatment, whereas these diseases with the same histopathology show greatly different clinical courses, which remains a question to solve. METHODS: Here we investigated the histopathology of AEP in2surgical lung biopsy and 14 TBLB cases. Additionally, we determined the presence or absence of DAD by histopathology in these AEP cases. RESULTS: Characteristic histopathological findings of AEP consist of alveolar edema with infiltration of eosinophils and lymphocytes and edema of perivascular area and interlobular septa. The alveolar spaces showed fibrinous exudates. There were no hyaline membranes or massive intraluminal fibrosis. These histopathological findings of interstitial edema and fluid exudates are consist with radiological findings of lung edema and can explain the rapid and complete improvement. CONCLUSIONS: Because AEP dose not exhibit lung fibrosis histopathologically, it should not to be included in DAD which is associated with lung fibrosis. CLINICAL IMPLICATIONS: We reveal the true histopathology of AEP. DISCLOSURES: No relevant relationships by Hiroshi Mochimaru, source=Web Response

Potential of Gold Nanoparticles for Noninvasive Imaging and Therapy for Vascular Inflammation

PurposeMacrophages contribute to the progression of vascular inflammation, making them useful targets for imaging and treatment of vascular diseases. Gold nanoparticles (GNPs) are useful as computed tomography (CT) contrast agents and light absorbers in photothermal therapy. In this study, we aimed to assess the viability of macrophages incubated with GNPs after near-infrared (NIR) laser light exposure and to evaluate the utility of intravenously injected GNPs for in vivo imaging of vascular inflammation in mice using micro-CT.ProceduresMouse macrophage cells (RAW 264.7) were incubated with GNPs and assessed for GNP cellular uptake and cell viability before and after exposure to NIR laser light. For in vivo imaging, macrophage-rich atherosclerotic lesions were induced by carotid ligation in hyperlipidemic and diabetic FVB mice (n = 9). Abdominal aortic aneurysms (AAAs) were created by angiotensin II infusion in ApoE-deficient mice (n = 9). These mice were scanned with a micro-CT imaging system before and after the intravenous injection of GNPs.ResultsThe CT attenuation values of macrophages incubated with GNPs were significantly higher than those of cells incubated without GNPs (p < 0.04). Macrophages incubated with and without GNPs showed similar viability. The viability of macrophages incubated with GNPs (100 μg/ml or 200 μg/ml) was decreased by high-intensity NIR laser exposure but not by low-intensity NIR laser exposure. In vivo CT images showed higher CT attenuation values in diseased carotid arteries than in non-diseased contralateral arteries, although the difference was not statistically significant. The CT attenuation values of the perivascular area in AAAs of mice injected with GNPs were significantly higher than those of mice without injection (p = 0.0001).ConclusionsMacrophages with GNPs had reduced viability upon NIR laser exposure. GNPs intravenously injected into mice accumulated in sites of vascular inflammation, allowing detection of carotid atherosclerosis and AAAs in CT imaging. Thus, GNPs have potential as multifunctional biologically compatible particles for the detection and therapy of vascular inflammation.

Abstract P380: Common And Distinct Signatures Of Interstitial And Perivascular Fibrosis In Mouse Models Of Hypertensive Heart Disease

Fibrosis is the hallmark of hypertensive heart disease and heart failure with preserved ejection fraction. Perivascular fibrosis impairs vascular function while interstitial fibrosis leads to compromised cardiac contractility. How these fibrosis types are represented in mouse models of hypertensive heart disease and to what extent the transcriptional signatures of cardiac fibrosis are defined by their location is unknown. Mice were dosed over 4 weeks with angiotensin II (AngII) alone or together with α 1 -adrenergic agonist phenylephrine (PE) and were characterized by echocardiography, light sheet imaging and fibrosis histology. While both groups developed systolic and diastolic dysfunction, hypertrophy and perivascular fibrosis, co-administration of PE resulted in a more severe disease phenotype and prevalent interstitial fibrosis, highlighting the benefits of this model in preclinical research. High-precision spatial transcriptomics based on laser capture microdissected perivascular and interstitial fibrotic areas revealed activation of distinct pro-fibrotic as well as cardioprotective pathways in the AngII+PE infusion model. Perivascular and interstitial fibrosis showed remarkable differences in global gene expression signatures, as demonstrated by high expression of osteochondrogenic genes and markers of secretory fibroblasts in perivascular fibrosis. A limited number of upregulated genes is shared between the fibrosis locations. These data collectively show the suitability of mouse models of hypertensive heart disease to study cardiac fibrosis and demonstrate how progression of fibrosis in mice is closely coupled to deteriorating cardiac dysfunction associated with highly distinct molecular signatures of perivascular and interstitial fibrosis.

Immune microenvironment composition in high-grade serous ovarian cancers based on BRCA mutational status.

An in-depth analysis of the tumor microenvironment of ovarian cancer is needed. The purpose of this study was to elucidate the architecture of the immune microenvironment of high-grade serous ovarian cancers (HGSCs) with or without BRCA1 and BRCA2 mutations. A cohort of highly annotated HGSC patients with known germline BRCA1 and BRCA2 status was selected, and pretreatment tumor tissue specimens were analyzed with a multiplexed staining technique aimed at detecting lymphocytes, macrophages, and fibroblasts in the whole tumor area and in specific regions including epithelium, stroma, and perivascular areas. BRCA1- or BRCA2-mutated tumors showed a more immunogenic microenvironment, characterized by a higher abundance of CD8+ and PD-L1+ cells, than did tumors with wild-type BRCA1 and BRCA2. High numbers of PD-L1+ and PD-L1+CD8+ cells were prognostic for event-free survival (hazard ratio [HR]: 0.41, 95% CI 0.21-0.79, p = 0.008 and HR 0.49, 95% CI 0.26-0.91, p = 0.025, respectively), as were high numbers of epithelial PD-L1+ and FAP+PD-L1+ cells (HR 0.52, 95% CI 0.28-0.96, p = 0.037 and HR 0.27, 95% CI 0.08-0.87, p = 0.029) and CD8+ cells (HR 0.51, 95% CI 0.28-0.93, p = 0.027). This study reveals substantial differences between the immune microenvironment composition of germline BRCA-mutated and BRCA wild-type HGSC.

Gastrointestinal AA amyloidosis secondary to chronic pyelonephritis presenting with refractory diarrhea and severe hypoalbuminemia

Secondary amyloidosis is a rare complication of chronic inflammatory diseases, such as collagen diseases, and is often difficult to treat. In addition, the gastrointestinal tract is frequently involved in amyloid deposition that often results in various disorders and symptoms. A 70-year-old woman was admitted to our hospital with refractory diarrhea and hypoalbuminemia. Abdominal computed tomography demonstrated extensive edematous wall thickening of the small intestine and colon. Video capsule endoscopy revealed multiple ulcerations with a white mossy appearance of the ileum. Double-balloon endoscopy showed severe circumferential ulcers in the entire ileum. Histological examination of ileum biopsy samples revealed severe amyloid deposition in the lamina propria and perivascular areas of the submucosa. The patient was diagnosed with gastrointestinal AA amyloidosis. The cause of AA amyloid deposition was presumed to be chronic pyelonephritis due to ureteral stones that had been left untreated for 35years. After treatment with ureteral drainage and antibiotics, the patient's symptoms and serological abnormalities improved dramatically. Here, we describe a case of severe gastrointestinal AA amyloidosis secondary to chronic pyelonephritis. Clinicians should thoroughly investigate the entire gastrointestinal tract in patients with refractory diarrhea and severe hypoalbuminemia considering the possibility of gastrointestinal amyloidosis.

Multi-Antigen Imaging Reveals Inflammatory DC, ADAM17 and Neprilysin as Effectors in Keloid Formation.

Keloid is an aberrant scarring process of the skin, characterized by excessive extracellular matrix synthesis and deposition. The pathogenesis of this prevalent cutaneous disorder is not fully understood; however, a persistent inflammatory process is observed. To obtain more insight into this process, we analyzed lesional, perilesional and healthy tissue using multi-antigen-analysis (MAA) in conjunction with a data mining approach. Here, we demonstrate that monocyte-derived inflammatory dendritic cells (CD1a+, CD11c+, CD14+) and activated CD4+ T lymphocytes (CD45 RO+) dominated the immune infiltration in keloids while associating with fibroblasts. In perilesional tissue, precursor immune cells were dominant in the perivascular area, suggesting that they were attracted by an immune process, potentially in the lesional area. Supporting this hypothesis, only in keloid lesions, high levels of ADAM10/17 and Neprilysin (CD10) were observed in both fibroblasts and leukocytes. The spatial proximity of these two cell types, which could be confirmed by image analysis only in lesional tissue, could be a potential factor leading to the activation of fibroblasts. Our findings provide new insight into the pathogenesis of keloid formation and reveal metalloproteinases as a target for therapeutical intervention.

Comparative Analyses of Clinical Features, Histopathology, and CD123 Immunohistochemistry of Oral Lupus Erythematosus, Lichen Planus, and Other Lichenoid Lesions

Background: Oral lupus erythematosus (OLE) and oral lichen planus (OLP) are among the common causes of oral lichenoid lesions (OLLs). The differential diagnosis among causes of OLLs, particularly between OLE and OLP, is challenging as they have significant clinical and histopathological overlap. Objectives: To compare and summarize the clinical, histopathological, and direct immunofluorescence (DIF) findings between OLE, OLP, and other OLLs and to explore the diagnostic value of CD123 immunohistochemistry. Methods: A retrospective study on patients with OLE, OLP, and other OLLs was performed between January 2014 and December 2019. The baseline characteristics, the clinical, histopathological, and DIF features, as well as CD123 immunohistochemistry for plasmacytoid dendritic cells (PDCs) were statistically analyzed and compared between groups. Results: Of 70 patients, 12 had OLE, 39 had OLP, and 19 had other OLLs. Oral erosions/ulcers were the most common findings in all three groups. Red macules, telangiectases, and discoid plaques were more common in OLE patients, while OLP cases were typified by reticulated patches (p < 0.05). Additionally, white patches were found more often in other OLLs than in both OLE and OLP (p = 0.002). Histologically, mucosal atrophy, basal vacuolization, and perivascular infiltrate were observed in OLE, whereas OLP specimens possessed mucosal hyperplasia, hypergranulosis, and compact orthokeratosis (p < 0.05). Mucosal spongiosis was a histologic feature that favored other OLLs over OLE and OLP (p < 0.001). Data on DIF were nonspecific for all three conditions. For immunohistochemical staining, the median number of total CD123+ PDCs was observed to be higher in OLE than OLP in the mucosal-submucosal junction (MSJ) (p = 0.021), the superficial perivascular area (p = 0.026), and the superficial and deep perivascular areas (p = 0.001). Likewise, PDCs in clusters ≥2+ were seen in significantly higher numbers on OLE than OLP along the MSJ (p = 0.002), the superficial perivascular area (p < 0.001), as well as the superficial and deep perivascular areas (p = 0.011). CD123+ PDCs were found to be significantly more numerous in both OLE and OLP than other OLLs in all of the abovementioned areas (all p < 0.05). Conclusion: While there are some differences in the clinicopathological features between OLE, OLP, as well as other OLLs, a significant overlap remains. The quantity and distribution pattern of CD123 immunohistochemical staining has a diagnostic implication in differentiating OLE from OLP and other OLLs.

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–365 Altered endometrial oestrogen-responsiveness and aberrant expression of cell-fate markers may contribute to the aetiology of recurrent pregnancy loss

Abstract Study question Do women with recurrent pregnancy loss (RPL) have an aberrant expression of oestrogen receptor-β(ERβ) and cell-fate markers during the window of implantation (WOI) endometrium? Summary answer Women with RPL are found to have significantly altered levels of ER βand Ki–67 in the WOI endometrium, possibly resulting in anti-proliferative and anti-angiogenic effects. What is known already RPL affects 1% of all women and has been associated with altered endometrial angiogenesis and proliferation when compared with the endometrium of healthy fertile women. RPL can be subcategorised into recurrent loss of anembryonic pregnancy, fetal loss (following evidence of a fetal heartbeat) and recurrent implantation failure (RIF). ERβis the only oestrogen-receptor (ER) known to be expressed in the vascular endothelium of the endometrium and is the dominant ER during the WOI. It has an important role in endometrial regeneration and is proposed to regulate the angiogenic and vascular changes that occur in embryo implantation. Study design, size, duration: This pilot case-control study took place at the Liverpool Women’s Hospital and included 38 women; 29 who suffered RPL and 9 controls with proven fertility (≥2 healthy pregnancies). Of the RPL group, 9 had recurrent loss of anembryonic pregnancy, 10 had recurrent fetal loss and 10 had RIF. Endometrial samples were collected during the WOI (cycle day 22+/–2). Participants/materials, setting, methods To determine whether markers of endometrial cell proliferation and oestrogen-responsiveness are associated with RPL, we assessed the immuno-staining for ER β, progesterone receptor (PR) and cell-fate marker Ki–67 in endometrial biopsies during the WOI using immunohistochemistry. A semi-quantitative immuno-staining score was used to assess the endometrial glands, stroma, luminal epithelium, perivascular and vascular endothelium compartments separately. Statistical differences between groups were calculated by non-parametric tests and significance level set at p &amp;lt; 0.05. Main results and the role of chance During the WOI, the endometrial epithelium of women with RIF and recurrent anembryonic pregnancy loss showed significantly higher levels of ER βwhen compared with fertile controls (p = 0.01 and p = 0.01, respectively). This may indicate an anti-proliferative process occurring at the site of implantation with very early pregnancy losses. In contrast, with women with recurrent fetal loss, a significantly lower level of ERβwas found within the vascular endothelium when compared with the fertile controls (p &amp;lt; 0.01). This supports the theory that increased oxygen levels may compromise trophoblastic invasion, thereby leading to fetal loss. The presence of Ki–67 (a marker of proliferation) was significantly lower within the vascular endothelium of all types of RPL: recurrent anembryonic loss (p = 0.02), RIF (p = 0.02) and recurrent fetal loss (p &amp;lt; 0.01). These findings suggest ineffective endometrial angiogenesis in RPL, resulting in a suboptimal endometrial microenvironment. PR was found to be significantly reduced (p &amp;lt; 0.01) in the perivascular area of women with RIF versus fertile controls. Since decidualisation and preparation of the endometrium for a successful implantation is controlled by critical target genes downstream of PR, this alteration in PR may be an important feature of their defective endometrial phenotype. Limitations, reasons for caution Samples analysed were taken from the functional endometrium and therefore the results do not reflect the basalis. The WOI was identified using history and histological appearance, rather than timing with ovulation. Although we detected statistical significance, generalisation of the results requires further studies with larger sample size. Wider implications of the findings: This data provides novel insight into the biological correlates of clinical types of RPL and suggests that specific alterations in the regulation of endometrial cell fate and oestrogen- responsiveness are associated with different types of RPL. This highlights possible new therapies for RPL, such as selective oestrogen receptor modulators (SERMs). Trial registration number Not applicable

P-365 Pre-selected for an award: Altered endometrial oestrogen-responsiveness and aberrant expression of cell-fate markers may contribute to the aetiology of recurrent pregnancy loss

Abstract Study question Do women with recurrent pregnancy loss (RPL) have an aberrant expression of oestrogen receptor-β (ERβ) and cell-fate markers during the window of implantation (WOI) endometrium? Summary answer Women with RPL are found to have significantly altered levels of ERβ and Ki-67 in the WOI endometrium, possibly resulting in anti-proliferative and anti-angiogenic effects. What is known already RPL affects 1% of all women and has been associated with altered endometrial angiogenesis and proliferation when compared with the endometrium of healthy fertile women. RPL can be subcategorised into recurrent loss of anembryonic pregnancy, fetal loss (following evidence of a fetal heartbeat) and recurrent implantation failure (RIF). ERβ is the only oestrogen-receptor (ER) known to be expressed in the vascular endothelium of the endometrium and is the dominant ER during the WOI. It has an important role in endometrial regeneration and is proposed to regulate the angiogenic and vascular changes that occur in embryo implantation. Study design, size, duration This pilot case-control study took place at the Liverpool Women’s Hospital and included 38 women; 29 who suffered RPL and 9 controls with proven fertility (≥2 healthy pregnancies). Of the RPL group, 9 had recurrent loss of anembryonic pregnancy, 10 had recurrent fetal loss and 10 had RIF. Endometrial samples were collected during the WOI (cycle day 22+/-2). Participants/materials, setting, methods To determine whether markers of endometrial cell proliferation and oestrogen-responsiveness are associated with RPL, we assessed the immuno-staining for ERβ, progesterone receptor (PR) and cell-fate marker Ki-67 in endometrial biopsies during the WOI using immunohistochemistry. A semi-quantitative immuno-staining score was used to assess the endometrial glands, stroma, luminal epithelium, perivascular and vascular endothelium compartments separately. Statistical differences between groups were calculated by non-parametric tests and significance level set at p &amp;lt; 0.05. Main results and the role of chance During the WOI, the endometrial epithelium of women with RIF and recurrent anembryonic pregnancy loss showed significantly higher levels of ERβ when compared with fertile controls (p = 0.01 and p = 0.01, respectively). This may indicate an anti-proliferative process occurring at the site of implantation with very early pregnancy losses. In contrast, with women with recurrent fetal loss, a significantly lower level of ERβ was found within the vascular endothelium when compared with the fertile controls (p &amp;lt; 0.01). This supports the theory that increased oxygen levels may compromise trophoblastic invasion, thereby leading to fetal loss. The presence of Ki-67 (a marker of proliferation) was significantly lower within the vascular endothelium of all types of RPL recurrent anembryonic loss (p = 0.02), RIF (p = 0.02) and recurrent fetal loss (p &amp;lt; 0.01). These findings suggest ineffective endometrial angiogenesis in RPL, resulting in a suboptimal endometrial microenvironment. PR was found to be significantly reduced (p &amp;lt; 0.01) in the perivascular area of women with RIF versus fertile controls. Since decidualisation and preparation of the endometrium for a successful implantation is controlled by critical target genes downstream of PR, this alteration in PR may be an important feature of their defective endometrial phenotype. Limitations, reasons for caution Samples analysed were taken from the functional endometrium and therefore the results do not reflect the basalis. The WOI was identified using history and histological appearance, rather than timing with ovulation. Although we detected statistical significance, generalisation of the results requires further studies with larger sample size. Wider implications of the findings This data provides novel insight into the biological correlates of clinical types of RPL and suggests that specific alterations in the regulation of endometrial cell fate and oestrogen- responsiveness are associated with different types of RPL. This highlights possible new therapies for RPL, such as selective oestrogen receptor modulators (SERMs). Trial registration number Not applicable

Macrophage-Like Cell Density Is Increased in Proliferative Diabetic Retinopathy Characterized by Optical Coherence Tomography Angiography.

PurposeTo quantitatively characterize macrophage-like cells (MLCs) at the vitreoretinal interface in different severity stages of diabetic retinopathy (DR) using optical coherence tomography angiography (OCTA).MethodsThe study included 72 eyes of 72 subjects: 18 healthy controls, 22 diabetes mellitus (DM) without DR, 17 nonproliferative DR (NPDR), and 15 proliferative DR (PDR). We obtained repeated (average, 6.5; range, 3–10) macular OCTA scans for each eye. We registered and averaged the 3-µm OCT slab above the vitreoretinal interface to visualize MLCs. Using a semiautomated method, we binarized and quantified MLCs and compared MLC densities among groups. We also evaluated MLC distribution relative to underlying superficial capillary plexus vasculature and quantified MLCs overlying blood vessels within the perivascular 30-µm watershed region and within ischemic zones (defined as >30 µm from the nearest vessel).ResultsMLC density was 2.8- to 3.8-fold higher in PDR compared with all other groups (P < 0.05 for all). MLC density in PDR was most increased in perivascular areas (3.3- to 4.2-fold; P < 0.05 vs. all) and on blood vessels (3.0- to 4.0-fold; P < 0.05 vs. all), and elevated to a lesser extent in ischemic areas (2.3- to 3.4-fold; P < 0.05 vs. all). MLCs were more likely to localize on blood vessels in DM without DR, NPDR, and PDR (P < 0.05 for all), but not healthy eyes.ConclusionsMLC density was significantly increased in PDR. MLCs clustered on blood vessels in diabetic but not in healthy eyes. Further studies are needed to confirm the origin, identity, and function of MLCs during DR.

Effect of Sorafenib on Liver Biochemistry Prior to Vitamin B17 Coadministration in Ehrlich Ascites Carcinoma Mice Model: Preliminary Phase Study

Aim: Background: Voluminous of the anti-tumor drugs in practice known to yield adverse side effects for instance hepatotoxicity. Objective: This preliminary was carried out to ascertain the histological change after treatment with Sorafenib (SOR) before execution the B17 coadministration and detection of the histochemical change in Ascites Carcinoma mice model (EAC). Methods: In total 12 Swiss albino mice received intraperitoneal injections of Sorafenib (30 mg/ kg mouse) after tumor cells were injected, semi quantifiable scoring of the architectural disparitiesvalidated by histo-pathological inspection of mice tissues. Results: Upsurge of Liver enzymes, Kidney functions and significant reduction after SOR treatment of Hb, RBCs, and Platelets. Membrane integrity, Separation of muscle fiber, Intrusion of inflammatory cells, Edematous intramuscular space were evaluated in addition, liver tissue revealed large focal area and diffuse pleomorphic darkly basophilic cells in the perivascular area around the dilated central vein (CV) besides the mild dilation of some blood sinusoids. Improvement in the histological pictures of hepatic tissues in comparison to EAC bearing positive control group were enhanced Conclusion: Sorafenib showed alterations in histological mice tissues, however, improvements in biochemical parameters were noted; that’s why the Coadministration of B17 will be applied to alleviate the side effects of Sorafenib on the next perspective study.

Adropin Alleviates Myocardial Fibrosis in Diabetic Cardiomyopathy Rats: A Preliminary Study.

Aim: Adropin (ADR) is a novel regulatory polypeptide and has important effects on energy metabolism in the heart. However, it is still unclear whether ADR can relieve ventricular remodeling in DCM. Therefore, this study was conducted to assess the effect of ADR on myocardial fibrosis in DCM rats.Materials and Methods: Twenty Wistar rats were randomly assigned into four groups: healthy control group (CON), DCM model group (DCM), DCM model treated with ADR group (ADR) and DCM model treated with perindopril group (PER). Collagen volume fraction (CVF) and perivascular collagen area (PVCA) were calculated. Diastolic function was assessed by echocardiography. The mitochondrial membrane potential assay was conducted by Rhodamine 123 staining. The protein expression levels of Col I, Col III, Mitofusin-1, Mitofusin-2 and Drp1 were evaluated using western blot.Results: Compared to CON group, CVF, PVCA and the relative protein expression of Col I, Col III and Drp1 increased in DCM group. And the relative expression of Mitofusin-1 and Mitofusin-2 proteins decreased. During our investigations, CVF, PVCA and the relative protein expression of Col I, Col III and Drp1 decreased in ADR treated rats compared to DCM group. The diastolic function was elevated in ADR group. The fluorescence of Rhodamine 123 and the expression of Mitofusin-1 and Mitofusin-2 also increased in ADR group.Conclusion: Our study demonstrated that ADR could alleviate myocardial fibrosis and improve diastolic function in DCM rats. ADR may be a putative candidate for the treatment of DCM.

Abstract 2720: Perivascular accumulation of immunosuppressive cells in the stroma of human triple negative breast carcinomas: implications for immunotherapy

Abstract A distinct subset of tumor-associated macrophages (TAMs) accumulate around blood vessels in mouse tumors where they stimulate tumor angiogenesis and various steps in the metastatic pathway. They also limit tumor responses to frontline anti-cancer therapies like irradiation and chemotherapy, and dampen anti-tumor immunity by recruiting regulatory T cells and suppressing the proliferation of T cells. However, the presence and phenotype of such perivascular (PV) TAMs have yet to be investigated in human tumors. In this study we have used MultiOmyx multiplex immunofluorescence coupled to advanced analytics to compare the distribution and phenotype of TAMs, CD4+ and CD8+ T cells, and CD4+FOXP3+ regulatory T cells (Tregs) in PV vs non-PV areas in the stroma and tumor cell islands (TCIs) of 40 human triple negative breast carcinomas (TNBCs) - and whether this was altered after neoadjuvant chemotherapy. Tregs along with distinct subsets of TAMs and CD4+ and CD8+ T cells were found to preferentially accumulate in PV areas (ie. within 50um of CD31+ blood vessels), especially in the tumor stroma. CD163+ TIM3+ TAMs often made direct contact with the abluminal surface of blood vessels in these sites, and at increased numbers in in chemotherapy-treated TNBCs. This suggests they may regulate the formation/function of blood vessels in relapsing tumors. In both untreated and chemotherapy-treated tumors, a major subset of CD163+ TAMs lacked PDL1 expression and also accumulated preferentially in stromal PV areas, where many made direct contact with PD1- CD4+ or PD1- CD8+ T cells as well as FOXP3+CD4+ Tregs. We are currently investigating whether such close contact of these subsets of T cells with two immunosuppressive cell types (ie. CD163+ TAMs and Tregs) inhibits their subsequent anti-tumor functions. Taken together, our study shows that, as T cells cross blood vessels into TNBCs, they encounter a high density of at least two immunosuppressive cell types in the perivascular niche. This could lead to the inactivation of T cells before they migrate further into tumors, thereby reducing anti-tumor immunity. It could also limit the efficacy of cancer immunotherapies mediated by activated T cells. Identifying and targeting the mechanism causing these suppressive cells to accumulate and interact in such sites could remove this inhibition. Citation Format: Mohammed Ridha Moamin, Richard J. Allen, Nicholas Stavrou, Mate Nagy, Anna Juncker-Jensen, Claire E. Lewis. Perivascular accumulation of immunosuppressive cells in the stroma of human triple negative breast carcinomas: implications for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2720.

Abstract 2797: Changes in the phenotype of macrophages and CD8+ T Cells in the perivascular niche of prostate tumours following androgen deprivation

Abstract Men with intermediate/high-risk, localized prostate cancer usually receive neoadjuvant androgen deprivation therapy (ADT). Although this markedly reduces tumor burden, it may not be curative and over time cancer cells develop resistance to ADT causing the tumor to regrow and spreads to other sites like the bones. Tumors are then termed ‘castration resistant' (CR) and require more aggressive forms of treatment like radical prostatectomy (RP) and chemotherapy. These can have unpleasant side effects so preventing the development of castration resistance and subsequent tumor regrowth/metastasis, would be highly beneficial for patients. A distinct subset of perivascular (PV) tumor-associated macrophages (TAMs) expressing the cell-surface receptor, MRC1, has been shown to be proangiogenic, immunosuppressive and to stimulate tumor regrowth after chemotherapy, irradiation or vascular targeting drugs. In the current study, we have examined the density and phenotype of TAMs in PV vs non-PV areas of localized, human prostate tumors and orthotopic mouse (Myc-CaP) tumors following neoadjuvant ADT or castration respectively. In human tumors, MRC1+ TAMs accumulated at highest density around stromal blood vessels, and were abundant in these sites following ADT. In mice, castration markedly reduced primary tumor growth and changed the distribution and phenotype of TAMs and cytotoxic CD8+ T lymphocytes (CTLs) just prior to the development of CR. In PV areas of tumors in sham-castrated (i.e. control) mice there were significantly more TAMs expressing MRC1 and the broad-spectrum negative checkpoint regulator, VISTA than in non-PV areas. This was accompanied by the preferential accumulation of CTLs around blood vessels (contrasting with the even distribution of CD4+ (helper) cells and CD4+FOXP3+ Tregs across PV and non-PV areas). Following castration, the PV density of MRC1+ and VISTA+ TAMs was markedly increased, along with that of CTLs expressing the cytotoxic protease, granzyme B - a marker of active CTLs. Although these cells may cross the vasculature into tumors in an active state, passing through a PV niche enriched in VISTA+ TAMs would likely suppress their cytotoxic function before they progressed into the tumor mass. An interesting picture is, therefore, emerging of tumor-promoting TAMs accumulating around blood vessels after various forms of treatment, where they regulate a number of important events including the extravasation and function of various immune effectors. Citation Format: Haider Al-janabi, Xuan Pu, Munitta Muthana, Ning Wang, Mateus Crespo, Bora Gurel, Johann De Bono, Janet E. Brown, Claire E. Lewis. Changes in the phenotype of macrophages and CD8+ T Cells in the perivascular niche of prostate tumours following androgen deprivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2797.

Cardiac changes following arteriovenous fistula creation in a mouse model.

Arteriovenous fistula (AVF) creation may negatively affect cardiac structure and function and impact cardiovascular mortality. The objective of this study was to develop and characterize the cardiac changes following AVF creation in a murine AVF model. AVFs were constructed using the carotid artery and jugular vein in C57BL/6 mice. Sham-operated AVF mice served as the control group. 2D-echocardiography was performed prior to AVF creation (baseline) and at 7 and 21 days after creation in AVF and sham-operated mice. Picrosirius red was used to stain the left ventricle for collagen production. The cardiac output (CO), left ventricular end diastolic (LVEDD) and systolic (LVESD) diameter, and end-diastolic (LVEDV) and systolic (LVESV) volume was significantly increased at 7 and 21 days in AVF compared to sham-operated mice. There was also a significant increase in CO, LVEDD, LVESD, LVEDV, and LVESV from baseline to 21 days within the AVF group, but not the sham-operated mice. There was a significant decrease in ejection fraction and fractional shortening at 21 days in AVF compared to sham-operated mice. Picrosirius red was significantly more prominent around both the perivascular and interstitial areas of the cardiac tissue from AVF mice compared to sham-operated AVF mice at 21 days. The creation of an AVF in our murine model leads to cardiac changes such as increased cardiac output, left ventricular dilation, and cardiac fibrosis, while showing reductions of ejection fraction and fractional shortening.

Histopathological Review on Discoid Lupus Erythematous Mimicking Granuloma Faciale

Discoid lupus erythematosus is the most common forms of chronic cutaneous lupus erythematosus. It is characterized by clinical manifestations of erythematous macules, papules, or plaques with a coin-like shape and the face is the most common predilection site. Clinical features often resemble granuloma faciale. This case report aimed to distinguish discoid lesions on the face based on the histopathological examination. A 71-year-old male with a few reddish lumps appeared on his face since three months ago. Physical examination showed multiple discrete erythematous plaques with overlying squamous. Hematoxylin and eosin staining on the epidermis demonstrated basket weave type orthokeratosis, basal vacuolar cell degeneration, epidermal atrophy with flat rete ridges, and follicular plugging while in the dermis obtained inflammatory cell infiltrates, especially in periadnexal areas. Histopathological features of DLE are hyperkeratosis, pilosebasea gland atrophy, follicular plugging, basal membrane thickening, and cellular infiltrate in periadnexa or perivascular areas more visible than in other types of CLE. In DLE, no subepidermal gren zone and eosinophil infiltrate were found, like histological features of granuloma faciale. Histopathological examination can be used to establish a diagnosis for discoid lesions on the face, although serology examination remains as the gold standart. Keywords: Discoid lupus erythematosus; Granuloma faciale; Histopathology

Membranous nephropathy associated with multicentric Castleman\u2019s disease that was successfully treated with tocilizumab: a case report and review of the literature

BackgroundMulticentric Castleman’s disease is a life-threatening disorder involving a systemic inflammatory response and multiple organ failure caused by the overproduction of interleukin-6. Although renal complications of Castleman’s disease include AA amyloidosis, thrombotic microangiopathy, and membranoproliferative glomerulonephritis, membranous nephropathy is relatively rare. We experienced a case of secondary membranous nephropathy associated with Castleman’s disease.Case presentationThe patient was a 43-year-old Japanese man who had shown a high zinc sulfate value in turbidity test, polyclonal hypergammaglobulinemia, anemia, and proteinuria. A physical examination revealed diffuse lymphadenopathy, an enlarged spleen and papulae of the body trunk. A skin biopsy of a papule on the patient’s back showed plasma cells in the perivascular area and he was diagnosed with multicentric Castleman’s disease, plasma cell variant. Kidney biopsy showed the appearance of bubbling in the glomerular basement membranes in Periodic acid methenamine silver stain and electron microscopy revealed electron dense deposits within and outside the glomerular basement membranes. Since immunofluorescence study showed predominant granular deposition of IgG1 and IgG2, he was diagnosed with secondary membranous nephropathy associated with Castleman’s disease. He was initially treated with prednisolone alone, however his biochemical abnormalities did not improve. After intravenous tocilizumab (700 mg every 2 weeks) was started, his C-reactive protein elevation, anemia, and polyclonal gammopathy improved. Furthermore, his urinary protein level declined from 1.58 g/gCr to 0.13 g/gCr. The prednisolone dose was gradually tapered, then discontinued. He has been stable without a recurrence of proteinuria for more than 6 months.ConclusionsTocilizumab might be a treatment option for secondary membranous nephropathy associated with Castleman’s disease.