Cytoplasmic Pattern Research Articles

Distinct Uroplakin II Staining Pattern in Apocrine Breast Carcinoma.

Uroplakin II (UPII) has been shown as a highly specific marker of urothelial carcinoma; however, it can also stain subtypes of apocrine-differentiated breast carcinoma. Given that urothelium and breast epithelium share other common immunohistochemical markers, such as CK7 and GATA3, this can lead to a potential diagnostic pitfall. We stained a cohort of triple-negative breast cancer with UPII. Compared with the diffuse, cytoplasmic staining in urothelial carcinoma, UPII was positive in 38.9% of apocrine carcinoma (7/18) with a course, granular cytoplasmic staining pattern and negative in all nonapocrine triple-negative breast cancer cases. Furthermore, the same staining pattern was present in all apocrine metaplasia of the breast (4/4) and apocrine sweat glands in normal skin (6/6). This distinct subcellular localization of UPII staining in breast carcinoma can offer a potential solution to the above diagnostic pitfall.

P053: The cGAS-STING anti-tumor immune response pathway as a potential therapeutic target in classical Hodgkin Lymphoma (cHL)

Background: Cytosolic DNA of exogenous or endogenous origin triggers activation of cyclic GMP-AMP (cGAMP) synthase (cGAS), a cytosolic DNA sensor, that activates innate immune responses through production of the second messenger cGAMP and subsequently activation of the adaptor protein STING. The latter activates TBK1 and IKK kinases that, in turn, activate IRF3 and NF-κB transcription factors, which induce expression of interferons (IFNs), chemokines and cytokines involved in anti-tumor immune responses. The potential role of cGAS-STING pathway in anti-tumor immune responses in cHL remains unknown to date. Methods: STING expression was immunohistochemically analysed in a pilot study group of 32 untreated patients with cHL and available tissue. The in vitro system included 6 cHL cell lines (MDA-V, L1236, L428, L540, HDLM2, KMH2). Gene expression (RNA level) and protein expression and activation (phosphorylation) of cGAS-STING pathway components at baseline and experimental conditions were analysed by quantitative RT-PCR (RT-qPCR) and Western blot, respectively. The cHL cell lines were treated with a STING agonist and TBK1/IKK inhibitor (Amlexanox) alone or in combination with other agents. Silencing of STING gene was performed using transient transfection with specific STING siRNA. The cGAS-STING-associated anti-tumor immune responses were evaluated by assessing the RNA levels of IFN-β, CXCL10, IFN-γ, and a control gene (GAPDH) with RT-qPCR. Results: Using an arbitrary 10% cutoff, STING was positive in the neoplastic Hodgkin & Reed-Sternberg (HRS) cells of cHL in 20 of 32 (63%) patients with a membranous and cytoplasmic pattern. STING expression at the mRNA and protein level was substantially higher in L1236, L428 and HDLM2 compared to other cHL cell lines. Treatment with STING agonist alone stimulated gene expression of IFN-β and/or CXCL10 at a variable level depending on the cell line indicating functional cGAS-STING anti-tumor immune response pathway in cHL. Knocking down STING gene resulted in decreased CXCL10 and type 1 IFN gene expression by cHL cells. Amlexanox treatment also resulted in downregulation of IFN-β or CXCL10 gene expression in vitro. Conclusion: STING agonists and Amlexanox modulate gene expression of type 1 IFNs in cHL with direct therapeutic implications. A large cohort of cHL patients is currently being analysed for the expression and prognostic significance of STING, and the final results will be available at the time of ISHL 2022.

Expression of the phagocytic receptors αMβ2 and αXβ2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells.

Activation of the integrin phagocytic receptors CR3 (αMβ2, CD11b/CD18) and CR4 (αXβ2, CD11c/CD18) requires Rap1 activation and RIAM function. RIAM controls integrin activation by recruiting Talin to β2 subunits, enabling the Talin-Vinculin interaction, which in term bridges integrins to the actin-cytoskeleton. RIAM also recruits VASP to phagocytic cups and facilitates VASP phosphorylation and function promoting particle internalization. Using a CRISPR-Cas9 knockout approach, we have analyzed the requirement for RIAM, VASP and Vinculin expression in neutrophilic-HL-60 cells. All knockout cells displayed abolished phagocytosis that was accompanied by a significant and specific reduction in ITGAM (αM), ITGAX (αX) and ITGB2 (β2) mRNA, as revealed by RT-qPCR. RIAM, VASP and Vinculin KOs presented reduced cellular F-actin content that correlated with αM expression, as treatment with the actin filament polymerizing and stabilizing drug jasplakinolide, partially restored αM expression. In general, the expression of αX was less responsive to jasplakinolide treatment than αM, indicating that regulatory mechanisms independent of F-actin content may be involved. The Serum Response Factor (SRF) was investigated as the potential transcription factor controlling αMβ2 expression, since its coactivator MRTF-A requires actin polymerization to induce transcription. Immunofluorescent MRTF-A localization in parental cells was primarily nuclear, while in knockouts it exhibited a diffuse cytoplasmic pattern. Localization of FHL-2 (SRF corepressor) was mainly sub-membranous in parental HL-60 cells, but in knockouts the localization was disperse in the cytoplasm and the nucleus, suggesting RIAM, VASP and Vinculin are required to maintain FHL-2 close to cytoplasmic membranes, reducing its nuclear localization and inhibiting its corepressor activity. Finally, reexpression of VASP in the VASP knockout resulted in a complete reversion of the phenotype, as knock-ins restored αM expression. Taken together, our results suggest that RIAM, VASP and Vinculin, are necessary for the correct expression of αMβ2 and αXβ2 during neutrophilic differentiation in the human promyelocytic HL-60 cell line, and strongly point to an involvement of these proteins in the acquisition of a phagocytic phenotype.

Polystyrene nanoparticles enhance the adverse effects of di-(2-ethylhexyl) phthalate on male reproductive system in mice

Coexposure of nanoplastics (NPs) with other pollutants adsorbed from the surroundings has received extensive attention. Currently, the combined effects of NPs and plasticizers remain unclear. Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer that has raised much concern owing to its ubiquitous pollution and endocrine-disrupting potential. This study aimed to investigate the toxic effects on the male reproductive system upon coexposure to NPs and DEHP. The C57BL/6J mice were orally administrated with polystyrene nanoparticles (PSNPs), DEHP or both for 35 days to evaluate their effects on sperm quality, histology of testes and epididymides, testicular transcriptomic characteristics as well as expression of some important genes in the epididymides. The low-dose PSNPs used here did not induce significant changes in sperm quality, while DEHP alone or cotreatment with DEHP and PSNPs caused notable impairment, mainly manifesting as decreased sperm quality and aberrant structure of the testis and epididymis. Moreover, enhanced toxic effects were found in the cotreatment group when compared with the individual DEHP treatment group, as manifested by more obvious alterations in the sperm parameters as well as histological changes in the testis and epididymis. Testicular transcriptomic analysis revealed differential regulation of genes involved in immune response, cytoplasmic pattern recognition receptor signaling pathways, protein ubiquitination, oxidative stress, necrotic cell death, ATP synthesis and the cellular respiratory chain. RT-qPCR verified that the expression patterns of Cenpb, Crisp1 and Mars were changed in testes, and genes relevant to epididymal function including Aqp9 and Octn2 were downregulated in epididymides, particularly in the cotreatment group. Collectively, our results emphasize that DEHP at an environmentally relevant dose can induce male reproductive toxicity, and PSNPs may aggravate the toxic effects.

Comparison of the effect of reporting cytoplasmic patterns as anti-nuclear antibody positive and anti-nuclear antibody negative on reflex test ordering

Abstract Objective Anti-nuclear antibody (ANA) patterns are classified as nuclear, cytoplasmic or mitotic. The International Consensus on ANA patterns (ICAP) suggests three parameters for ANA reporting – assay type, results and advice for reflex testing – but has not yet reached a consensus on the reporting of cytoplasmic/mitotic patterns as ANA-negative or positive. We investigated the effect of ICAP’s two proposals for reporting cytoplasmic patterns as ANA-positive and negative with a view to the recommendation for reflex testing in a country that has no national reimbursement policies for automatic reflex testing. Methods This non-interventional descriptive study included 1241 patients with positive cytoplasmic ANA patterns. 442 patients were reported as ANA-negative and 799 as ANA-positive. Patients were followed up for a two-year period to determine testing recommendations based upon nuclear and cytoplasmic patterns. For statistical analysis, the t-test was used, with a significance threshold of p-value<0.05. Results Appropriate reflex orders were seen more commonly with cytoplasmic patterns reported as ANA-positive (27.30%) than with those reported as ANA-negative (5.51%, p-value<0.05). However, ANA-positive reports led to higher ordering of nuclear pattern reflex tests (12.97%) compared with ANA-negative reports (1.10%, p-value<0.05). A large group of patients (59.73% ANA-positive, 93.39% ANA-negative) did not receive reflex testing. Conclusion Reporting cytoplasmic patterns as ANA-positive was considered more significant, but reading the result report without considering the pattern and recommendation notes could lead to inappropriate reflex testing. Besides reaching a consensus for reporting cytoplasmic patterns as ANA-negative or positive, it is important to consider solutions to reimbursement policies for automatic reflex testing to decrease the impediments in reporting cytoplasmic ANA patterns.

High prevalence of antinuclear antibodies in patients with chronic hepatitis C virus infection

BackgroundHepatitis C virus (HCV) infection is a serious public health concern due to its high prevalence and mortality rate. In chronic infection, HCV may induce autoimmune responses through the production of autoantibodies, including antinuclear antibodies (ANA).MethodsWe assessed the presence of ANA by indirect immunofluorescence using HEp-2 cells in 89 patients with chronic hepatitis C. We also collected data on epidemiological variables; clinical characteristics; and biochemical, hematological, molecular, and histopathological information from the patients to assess the impact of the presence of ANA in those patients.ResultsThe prevalence of ANA in the patients was 20.2%, which was significantly higher than that found in healthy controls (2%). However, there was no association of this marker with epidemiological, clinical-laboratory, molecular or histopathological characteristics of hepatitis C, although a slightly higher prevalence of ANA was detected in women and in patients infected with subgenotype 1a. In a specific analysis, chronic HCV patients with the “rods and rings” cytoplasmic pattern had higher degrees of hepatic fibrosis than did ANA-negative patients.ConclusionsThe results confirm a greater predisposition to the presence of ANA in patients with HCV, which may be associated with a worse prognosis, especially in the presence of the “rods and rings” cytoplasmic pattern.

Comparison of anti-Müllerian hormone and inhibin immunolabeling in canine and equine granulosa cell tumors.

Granulosa cell tumors (GCTs) are common ovarian neoplasms in the mare and bitch that can be challenging to diagnose on histopathology. Inhibin has long been the standard immunohistochemical (IHC) marker for GCTs; however, anti-Müllerian hormone (AMH) has not been evaluated widely as an IHC marker in the bitch and mare. We compared the efficacy of AMH and inhibin as IHC markers in canine and equine GCTs. We selected retrospectively 18 equine and 15 canine cases. All equine tumors were dominated by a cystic pattern; canine tumors often had solid patterns. Both inhibin and AMH had similar punctate cytoplasmic patterns of immunolabeling, although labeling intensity was variable; distribution and intensity of labeling were unrelated to the histomorphologic pattern. Labeling for AMH occurred in 12 of 15 canine and 18 of 18 equine cases. Labeling for inhibin occurred in 15 of 15 canine and 18 of 18 equine cases. AMH in equine GCTs often had stronger immunolabeling than inhibin, and granulosa cells were labeled more extensively. Inhibin and AMH performed comparably in bitches, but AMH had more diffuse immunolabeling than inhibin in mares.

Association of immunohistochemical profiles with histotypes in endometrial carcinomas

ObjectiveAlthough a large number of endometrial cancer patients are cured with surgery alone, there are significant numbers of patients with more aggressive variants of endometrial carcinoma for whom the prognosis remains poor. We investigated the effects of prevalence, histotypes, and immunohistochemical profiles on prognostic value in a hospital-based population. Materials and methodsA retrospective study of surgically resected primary endometrial carcinoma was included. Immunohistochemical stains were performed on formalin-fixed paraffin-embedded tissue microarray sections for β-Catenin, estrogen receptor (ER), progesterone receptor (PR), HER-2, MLH1, MSH2, MSH6, PMS2, and p53. ResultsLoss of mismatch repair expression was detected in 25.4% of samples (29/114, mean age 57 years) of the tumors. The following loss of expression was observed in patients: MLH1/PMS2 in 16.6% of patients, MSH6 in 7.0% of patients, MLH1 in 0.9% of patients, and MSH6/PMS2/MLH1 in 0.9% of patients. Immunohistochemistry of p53 was analyzed for 111 patients. A total of 13 patients (11.7%, mean age 64 years) had p53-abnormal expression (absent, cytoplasmic or diffuse strong positive patterns), and more than half (9/13, 69.2%) had endometrioid histotype. Abnormalities in p53 were significantly associated with histotype (p = 0.001), advanced tumor stage (p = 0.038), death of disease (p = 0.002), PR percentage (p = 0.002), and HER-2 expression (p = 0.018). Immunohistochemical nuclear localization of β-Catenin was detected in 7.1% of the cohort. The combination of p53 and nuclear β-Catenin expressions was not significantly predictive of disease-free or overall survival. ConclusionThe results of this study are useful for management of endometrial cancer in patients with DNA mismatch repair, abnormal p53 expression, or nuclear localization of β-Catenin.

The influence of demography and referral medical specialty on the detection of autoantibodies to HEP-2 cells in a large sample of patients

BackgroundThe prevalence of anti-cell autoantibodies detected by indirect immunofluorescence assay on HEp-2 cells (HEp-2-IIFA) increases with age and is higher in female sex. The number of medical specialties that use HEp-2-IIFA in the investigation of autoimmune diseases has increased lately. This study aimed to determine the prevalence and patterns of autoantibodies on HEp-2-IIFA according to demographics variables and referring medical specialties.MethodsA retrospective analysis of the HEp-2-IIFA carried out between January and June of 2017 was performed. The International Consensus on Antinuclear Antibodies Patterns (ICAP) and the Brazilian Consensus on Autoantibodies were used for patterns definition on visual reading of the slides. Anti-cell (AC) codes from ICAP and Brazilian AC codes (BAC) were used for patterns classification.ResultsFrom 54,990 samples referred for HEp-2-IIF testing, 20.9% were positive at titer ≥ 1/80. HEp-2-IIFA positivity in females and males was 24% and 12%, respectively (p < 0.0001). The proportion of positive results in the 4 age groups analyzed: 0–19, 20–39, 40–59, and ≥ 60 years was 23.3, 20.2, 20.1, and 22.8%, respectively (p < 0.0001). Considering all positive sera (n = 11,478), AC-4 nuclear fine speckled (37.7%), AC-2 nuclear dense fine speckled (21.3%), BAC-3 nuclear quasi-homogeneous (10%) and mixed/composite patterns (8.8%) were the most prevalent patterns. The specialties that most requested HEp-2-IIFA were general practitioner (20.1%), dermatology (15%), gynecology (9.9%), rheumatology (8.5%), and cardiology (5.8%). HEp-2-IIFA positivity was higher in patients referred by rheumatologists (35.7% vs. 19.6%) (p < 0.0001). Moderate (46.4%) and high (10.8%) titers were more observed in patients referred by rheumatologists (p < 0.0001). We observed a high proportion of mixed and cytoplasmic patterns in samples referred by oncologists and a high proportion of BAC-3 (nuclear quasi-homogeneous) pattern in samples referred by pneumologists.ConclusionsOne-fifth of the patients studied were HEp-2-IIFA-positive. The age groups with more positive results were 0–19 and ≥ 60 years. AC-4, AC-2, BAC-3 and mixed/composite patterns were the most frequent patterns observed. Rheumatologists requested only 8.5% of HEp-2-IIFA. Positive results and moderate to high titers of autoantibodies were more frequent in patients referred by rheumatologists.

The Anti-Cytoplasmic and Anti-Mitotic Autoantibodies; Are These Antibodies Associated with Diseases?

Examination of antinuclear antibody (ANA) is used in diagnosis of systemic autoimmune diseases, and the indirect immunofluorescence (IIF) assay using HEp-2 cells is the gold standard method. HEp-2 allows the detection of multiple target antigen-directed autoantibodies. The guide “The International Consensus on ANA Patterns (ICAP)”, characterizes the patterns into three groups: nuclear, cytoplasmic, and mitotic. The majority of these are associated with autoimmune diseases, but some are rarely seen in autoimmune diseases or may be associated with conditions other than autoimmune disease. There is no consensus on how to report cytoplasmic and mitotic patterns-negative or positive. We aimed to examine the characteristics of patients that had cytoplasmic or mitotic staining in ANA evaluation by IIF. In our Medical Microbiology Laboratory, 18985 ANA tests of 16940 patients were studied between 01.01.2015-31.12.2019. Cytoplasmic or mitotic pattern was detected in 393 (2.07%) tests belonging to 385 patients. Cytoplasmic patterns suggestive of anti-mitochondrial antibody (AMA), anti-smooth muscle antibody (ASMA), anti-Jo-1 and anti-ribosomal P-protein were not included. The most common patterns were anti-midbody, anti-spindle fibers, and anti-vimentin patterns. There were 66 rheumatology patients that were negative for ANA but had cytoplasmic or mitotic staining. There was no statistically significant difference between the diagnosis and patterns of these patients. We suggest that the ANA should be reported as “negative” in case of cytoplasmic or mitotic pattern unless the term anti-cell antibody is used. It should be noted in the description part of the report in order to distinguish significant cytoplasmic patterns and give an idea for some specific conditions.

Anti-neutrophil cytoplasmic antibody patterns can predict clinical relapse in ANCA-associated vasculitis: overall population and subgroups.

In anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV), relapses are cause of concern as they are unpredictable and predictors of poor prognosis. We aimed to assess the characteristic and predictors of clinical relapse in AAV. This retrospective study included 132 cases of AAV newly diagnosed from January 2016 through November 2021 in the Affiliated Hospital of Qingdao University. We reviewed clinical data of patients and analysed the risk factors for clinical relapse of overall population and subgroups by univariate and multivariate regression models and the K-M survival curve was plotted. The rate of relapse was highest in the positive conversion group than the others significantly (p<0.001). In overall population, ANCA patterns (p<0.001; persistent positive pattern: HR=3.352, 95%CI 1.463~7.678, p=0.004; positive conversion pattern: HR=4.760, 95%CI 2.094~10.820, p<0.001) and infections (HR =4.684, 95%CI 1.980~11.079, p<0.001) were significantly associated with clinical relapse. In myeloperoxidase (MPO)-AAV patients, ANCA patterns (p=0.001; persistent positive pattern: HR=4.495, 95%CI 1.508~13.396, p=0.007; positive conversion pattern: HR=7.404, 95%CI 2.652~20.671, p<0.001) and infections (HR=3.594, 95%CI 1.511~8.547, p=0.004) were significantly associated with clinical relapse. In renal involvement patients, ANCA patterns (p=0.004; persistent positive pattern: HR=3.618, 95%CI 1.364~9.592, p=0.01; positive conversion pattern: HR=4.492, 95%CI 1.778~11.352, p<0.001) and infections (HR=7.791, 95%CI 2.511~24.174, p<0.001) were significantly associated with clinical relapse, but were not in patients without renal involvement. Persistently positive and re-positive ANCA and infections predict clinical relapse in AAV, especially in patients with MPO-AAV and renal involvement. Regular ANCA monitoring should be carried out in high-risk populations.

Clinical significance of HEp-2 cell cytoplasmic patterns in anti-neutrophil cytoplasmic antibody associated vasculitis.

The study was to investigate the clinical characteristics and significance of antinuclear antibody (ANA) cytoplasmic patterns in ANCA-associated vasculitis (AAV) from Southwest China.A retrospective study including 232 AAV patients from Peoples Hospital of Deyang City was performed. These included 115 patients with ANA cytoplasmic pattern as observation group and 117 patients without ANA cytoplasmic pattern as control group.Chest involvement (60.00 vs 46.15, P = .035), cardiovascular involvement (5.21 vs 29.91, P < .001), and renal involvement (37.39 vs 77.78, P = .001) were different between groups.Total protein (69.55 vs 64.01, P < .001), triglyceride (1.41 vs 1.18, P = .023), mean cell volume (89.76 vs 87.59, P = .040), and estimated glomerular filtration rate (76.67 vs 50.87, P = .035) were higher in ANA cytoplasmic patterns group. Creatinine (73.00 vs 117.50, P = .011), white blood cell (6.93 vs 8.86, P = .001), platelet (196.0 vs 239.0, P = .017), anti-myeloperoxidase (2.44 vs 3.42, P = .042), and anti-proteinase 3 (1.00 vs 4.93, P = .007) were lower in this group. In multivariate analysis, creatinine (odds ratio [OR] = 1.21, 95% confidence interval [CI]: 1.06–1.38), triglyceride (OR = 1.97, 95% CI: 1.10–3.48), and anti-myeloperoxidase (OR = 1.64, 95% CI: 1.37–1.95) were independent risk factors of AAV renal involvement. Total protein (OR = .95, 95% CI: 0.91–0.99) was an independent protective factor of AAV renal involvement. Chi-square test showed that speckled pattern was different among anti-neutrophil cytoplasmic antibody patterns (χ2 = 18.526, P < .001).In summary, HEp-2 cell cytoplasmic patterns have certain clinical significance in AAV, which is a new exploration of the clinical value of ANA.

An exploration in pitfalls in interpreting SDHBimmunohistochemistry.

Mutations and epimutations in genes encoding the succinate dehydrogenase complex (SDHx) are associated with multiple tumour types in which identification of SDH-deficiency has significant management implications. Immunohistochemistry (IHC) for the succinate dehydrogenase B (SDHB) subunit can help to detect SDH-deficiency, which manifests as complete loss of staining in tumour cells. However, a subset of SDH-deficient tumours can show aberrant cytoplasmic SDHB-IHC staining patterns and be misinterpreted as 'retained', a diagnostic pitfall complicating interpretation. Herein, we characterise in detail aberrant SDHB-IHC staining patterns in SDH-deficient tumours. We identified 23 tumours from patients with known germline SDHx and/or molecularly confirmed SDHx pathogenic/likely-pathogenic variants in their tumour. Of these, eight (35%) showed significant SDHB-IHC staining: one SDHA-, one SDHB-, three SDHC- and three SDHD-mutated cases. In all eight cases, closer inspection revealed differences in intensity and intracellular distribution of SDHB-IHC staining in tumour cells compared to adjacent non-neoplastic cells: non-neoplastic cells showed intense cytoplasmic coarse granular staining; tumour cells in seven of eight cases showed weak to focally strong, cytoplasmic blush to fine granular staining, in > 80% of cells. The remaining case in the initial block showed variably strong non-granular cytoplasmic staining with globular perinuclear accentuation throughout, only subtly distinct from the staining pattern of non-neoplastic cells. SDHB-IHC performed on two additional blocks in this latter case revealed significant intratumoral heterogeneity, including convincing areas of complete loss. When evaluating SDHB-IHC, care should be taken to distinguish true retained expression from aberrant cytoplasmic expression, which may be difficult to appreciate. Sometimes this may require additional molecular testing.

Monoclonal antibodies from B cells of patients with anti-MDA5 antibody-positive dermatomyositis directly stimulate interferon gamma production

Anti-melanoma differentiation-associated gene 5 (MDA5) antibody (Ab) positive dermatomyositis (anti-MDA5 DM) is a rare entity associated with poor prognosis and multiple immunologic abnormalities. These include the presence of autoAbs and deleterious interferon (IFN)-gamma production in the severe form of the disease. Here, we show that the autoAbs profile differs between patients, depending on disease severity, and that autoAbs from B cells of patients directly stimulate IFN-gamma production by peripheral blood cells. Serum of 29 anti-MDA5 DM patients were analyzed by indirect immunofluorescence (IIF) on Hep-2 cells, to identify patterns associated with poor outcome. Seventeen (59%) serum gave a specific cytoplasmic MDA5 pattern on Hep-2 cells, while 12 (41%) gave an unspecific pattern. Specific MDA5 pattern was associated with a higher risk to develop interstitial lung disease (p = 0.003). Monoclonal autoAbs were generated from B cell clones of two patients with extreme clinical presentation, one who developed a lethal form of the disease, and the other with a favorable outcome. Supernatants of the autoreactive B cell clones that gave an IIF cytoplasmic pattern were tested for their abilities to stimulate IFN-gamma production by peripheral blood cells. Out of 120,000 B cell clones analyzed, 12 produced monoclonal Abs that triggered direct IFN-gamma secretion by peripheral blood cells, by a monocyte-dependent mechanism. None of them was directed against the MDA5 antigen. Altogether, these findings demonstrated that autoAbs other than the highly specific anti-MDA5 Ab are direct contributors of the IFN-gamma upregulation that is linked to the severity of the disease.

Merkel cell carcinoma on the dorsalis pedis with both dot‐like perinuclear and cytoplasmic patterns of CK20 positivity

Classically known to be a tumor of the head and neck region, primary Merkel cell carcinoma (MCC) has rarely been reported in the lower leg. The present case showed both dot‐like perinuclear and cytoplasmic patterns of CK20 positivity in the primary MCC. We propose that the presence of both dot‐like perinuclear and cytoplasmic patterns of CK20 could reflect the diversity of MCC origin from stem cell to mature Merkel cells in our patient.

S-acylation-dependent membrane microdomain localization of the regulatory Kvβ2.1 subunit

The voltage-dependent potassium (Kv) channel Kvβ family was the first identified group of modulators of Kv channels. Kvβ regulation of the α-subunits, in addition to their aldoketoreductase activity, has been under extensive study. However, scarce information about their specific α-subunit-independent biology is available. The expression of Kvβs is ubiquitous and, similar to Kv channels, is tightly regulated in leukocytes. Although Kvβ subunits exhibit cytosolic distribution, spatial localization, in close contact with plasma membrane Kv channels, is crucial for a proper immune response. Therefore, Kvβ2.1 is located near cell surface Kv1.3 channels within the immunological synapse during lymphocyte activation. The objective of this study was to analyze the structural elements that participate in the cellular distribution of Kvβs. It was demonstrated that Kvβ peptides, in addition to the cytoplasmic pattern, targeted the cell surface in the absence of Kv channels. Furthermore, Kvβ2.1, but not Kvβ1.1, targeted lipid raft microdomains in an S-acylation-dependent manner, which was concomitant with peptide localization within the immunological synapse. A pair of C-terminal cysteines (C301/C311) was mostly responsible for the specific palmitoylation of Kvβ2.1. Several insults altered Kvβ2.1 membrane localization. Therefore, growth factor-dependent proliferation enhanced surface targeting, whereas PKC activation impaired lipid raft expression. However, PSD95 stabilized Kvβ2.1 in these domains. This data shed light on the molecular mechanism by which Kvβ2.1 clusters into immunological synapses during leukocyte activation.

The Use of Pan-Tropomyosin Receptor Kinase Immunohistochemistry as a Screening Tool for the Detection of Neurotrophic Tropomyosin-Related Kinase Fusions: Real-World Data from a National Multicentric Retrospective Study

Introduction: The neurotrophic tropomyosin-related kinase (NTRK) genes encode the tropomyosin receptor kinases (TRKs). Patients with solid tumors harboring an oncogenic NTRK fusion are eligible for treatment with TRK inhibitors. NTRK fusion is often associated with TRK overexpression. Pan-TRK immunohistochemistry (IHC) is used to screen for NTRK fusions, but immunoreactivity patterns are poorly defined. Methods: Data on pan-TRK immunoreactivity patterns in 2,669 solid tumors (comprising carcinomas, sarcomas, and melanocytic lesions) were retrospectively collected by nine laboratories and comprised tumor type, percentage of pan-TRK-positive tumor cells, staining intensity, cytoplasmic, membrane and/or nuclear staining pattern, and the presence or absence of NTRK fusion. Results: Overall, 2,457 tumors (92%) were pan-TRK negative and 212 neoplasms (8%) were pan-TRK positive. Twenty-two pan-TRK-positive tumors (0.8%) harbored an NTRK fusion, representing 10% of all pan-TRK-positive tumors. Cytoplasmic immunoreactivity was most often observed, followed by membrane immunoreactivity. Nuclear pan-TRK positivity was least frequent, but was most often (33%) associated with NTRK fusion. Conclusion: Pan-TRK IHC can be used to screen for NTRK fusions, especially in commonly diagnosed solid tumors with low NTRK fusion prevalence. In case of pan-TRK immunoreactivity, regardless of its intensity and tumor cell percentage, subsequent molecular tests should be performed to formally confirm the presence or absence of NTRK fusions.

Transcriptomics of rhinovirus persistence reveals sustained expression of RIG-I and interferon-stimulated genes in nasal epithelial cells in vitro.

Human rhinoviruses (HRVs) are frequently associated with asthma exacerbations, and have been found in the airways of asthmatic patients. While HRV-induced acute infection is well-documented, it is less clear whether the nasal epithelium sustains prolonged HRV infections along with the associated activation of host immune responses. To investigate sustainably regulated host responses of human nasal epithelial cells (hNECs) during HRV persistence. Using a time-course study, HRV16 persistence and viral replication dynamics were established using an in vitro infection model of hNECs. RNA sequencing was performed on hNECs in the early and late stages of infection at 3 and 14days post-infection (dpi), respectively. The functional enrichment of differentially expressed genes (DEGs) was evaluated using gene ontology (GO) and Ingenuity pathway analysis. HRV RNA and protein expression persisted throughout prolonged infections, even after decreased production of infectious virus progeny. GO analysis of unique DEGs indicated altered regulation of pathways related to ciliary function and airway remodeling at 3 dpi and serine-type endopeptidase activity at 14 dpi. The functional enrichment of shared DEGs between the two time-points was related to interferon (IFN) and cytoplasmic pattern recognition receptor (PRR) signaling pathways. Validation of the sustained regulation of candidate genes confirmed the persistent expression of RIG-I and revealed its close co-regulation with interferon-stimulated genes (ISGs) during HRV persistence. The persistence of HRV RNA does not necessarily indicate an active infection during prolonged infection. The sustained expression of RIG-I and ISGs in response to viral RNA persistence highlights the importance of assessing how immune-activating host factors can change during active HRV infection and the immune regulation that persists thereafter.

124 The Effect of Innate Immune Receptor Activation on Differential Breed Responses to LPS in Sheep

Abstract While studying breed differences in immune response of peripheral blood mononuclear cells (PBMC) to Haemonchus contortus (Hc) antigen, RNA sequencing revealed that St. Croix (STC) PBMC upregulated expression of NLRP3 compared to PBMC from Suffolk (SUF) sheep. Nod-like receptor pyrin containing domain 3 (NLRP3) is a cytoplasmic pattern recognition receptor (PRR) with a wide array of agonists, such as PAMPs, DAMPs, ATP, bacterial product, and viral products. Stimulation of the NLRP3 inflammasome results in proteolytic activation of IL-1b and IL-18, cell pyroptosis, and classically, the induction of proinflammatory responses. Common activation of this pathway can be achieved by stimulation with liposaccharide (LPS) which binds and activates a toll-like receptor-4 (TLR4) pathway, causing production of proinflammatory cytokines. Interestingly, NLRP3 has been recently implicated as an early initiator of IL-4 production in response to helminth antigen. Thus, the objective of this study was to determine differences in breed responses to LPS and the role of NLRP3. To test this hypothesis, PBMC were collected from STC and SUF sheep and cells were incubated with LPS only, LPS in combination with pharmacological inhibitor of NLRP3 (MCC950), or complete media for 3 hrs. Gene expression of PBMC exposed to LPS revealed that Inflammatory linked genes IL-1β (P &amp;lt; 0.001), TLR4 (P &amp;lt; 0.001), TNFα (P &amp;lt; 0.001), NFκB (P &amp;lt; 0.001) were upregulated in STC PBMC. Treatment with MCC950 in combination with LPS, reduced inflammatory gene expression in STC PBMC to the level of SUF PBMC (P &amp;gt; 0.05), thus ablating inflammatory responses in STC PBMC. These data reveal a deficiency in NLPR3 function in SUF sheep and have been confirmed by SNPs found in that gene that have been predicted to alter NLRP3 protein structure.

Human Epidermal Growth Factor Receptor 2 (HER2) Expression in Colorectal Carcinoma: A Potential Area of Focus for Future Diagnostics.

ObjectiveIn this study, we aimed to explore the potential diagnostic utility of human epidermal growth factor receptor 2 (HER2) expression in colorectal carcinoma. We investigated the association of HER2 expression with the type and grade of the tumor along with the pattern, staining intensity, and the percentage of cells stained.MethodsThis was an observational study involving 50 cases of colorectal carcinoma that underwent immunohistochemistry to analyze the HER2 expression.ResultsThe positive expression of HER2 was seen in 16 (32%) cases. The majority of the study population was between the fifth-seventh decades of life. The most commonly diagnosed tumor was conventional adenocarcinoma with grade II, cytoplasmic pattern, +2 positivity, and moderate intensity. The maximum positivity for HER2 was seen in tumors of the rectum in eight (16%) cases.ConclusionA substantial rate of HER2 overexpression paves the way for it to become a potential future target in cancer therapeutics.