Granular Cytoplasmic Staining Research Articles

A new algorithm for histopathological diagnosis of periprosthetic infection using CD15 focus score and computer program CD15 Quantifier

Introduction. A simple microscopic diagnostic quantification system for neutrophile granulocytes (NG) was developed evaluating a single focal point (CD15 focus score) which enables the detection of bacterial infection in SLIM (synoviallike interface membrane) Additionally a diagnostic algorithm is proposed how to use the CD15 focus score and the quantification software (CD15 Quantifier). Methods. 91 SLIM removed during revision surgery for histopathological diagnosis (hip; n=59 and knee; n=32) underwent histopathological classification according to the SLIM-consensus classification. NG where identified immunohistochemically by means of a CD15-specific monoclonal antibody exhibiting an intense granular cytoplasmic staining pattern. This pattern is different from CD15 expression in macrophages showing a pale and homogenous expression in mononuclear cells. The quantitative evaluation of CD15-positive neutrophils granulocytes (CD15NG) used the principle of maximum focal infiltration (focus) together with an assessment of a single focal point (approximately 0.3 mm2). This immunohistochemical data made it possible to develop CD15 Quantifier software which automatically quantifies CD15NG. Results. SLIM-cases with positive microbiological diagnosis (n=47) have significantly (p

The clinical and biological significance of MICA in clear cell renal cell carcinoma patients.

Major histocompatibility complex class I-related chains A (MICA), a ligand of Natural killer group 2, member D (NKG2D) receptor, is broadly upregulated in epithelial originated tumor cells. MICA plays a critical role in the immune surveillance against tumor cells and is associated with the prognosis of several malignancies. The aim of this study is to evaluate the clinical and biological significance of MICA in clear cell renal cell carcinoma (ccRCC). The expression of MICA was analyzed by quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). Both MICA mRNA and protein levels were upregulated in ccRCC tissues, compared with normal tissues. IHC staining revealed a homogenous pattern of MICA staining within each tumor, which combined both membrane staining and granular cytoplasmic staining. Furthermore, high MICA expression was associated with lymph node metastasis and advanced clinical stage and predicted poor prognosis in patients with ccRCC. Gene set enrichment analysis (GSEA) was performed using RNA-sequencing data from The Cancer Genome Atlas Research Network (TCGA) to elucidate the biological role of MICA in ccRCC and revealed that MICA was significantly associated with the epithelial-to-mesenchymal transition (EMT) gene set, which was further confirmed by qRT-PCR. Our findings contribute to the studies on biomarkers of kidney cancers and the mechanism of renal cancer progression driven by EMT pathway.

Is DOG1 really useful in the diagnosis of salivary gland acinic cell carcinoma? - A DOG1 (clone K9) analysis in fine needle aspiration cell blocks and the review of the literature.

Introduction:DOG1 is a transmembrane protein originally “discovered on gastrointestinal stromal tumors,” works as a calcium-activated chloride channel protein. There is a limited number of studies on the potential usage of this antibody in the diagnosis of salivary gland tumors on routine practice in cell blocks. The aim of this study was to search for the usefulness of K9 clone in oncocytic type tumors and review of the literature.Materials and Methods:Sixty-nine fine needle aspiration (FNA) cytologic materials of predominantly oncocytic morphology salivary gland tumors; acinic cell carcinoma (AciCC) (n = 8), adenoid cystic carcinoma (n = 2), pleomorphic adenoma (PA) (n = 22), Warthin tumor (WT) (n = 20), myoepithelioma (ME) (n = 5), benign oncocytoma (BeO) (n = 3), mucoepidermoid carcinoma (MEC) (n = 7), mammary analog salivary gland carcinoma (n = 2) were immunostained with DOG1 (clone K9) stain.Results:Of the 8 AciCCs, 7 were observed apical-luminal positive staining, demonstrating 1–3 + intensity, and involving 40–70% of the tumor cells. One MEC of 7 (14%), 1 ME of 5 (20%), and 4 PA of 22 (18%) showed weak (1+) cytoplasmic granular staining in 5–10% of the tumor cells. Pure oncocytic neoplasms (WT, BeO) showed no expression with DOG1-K9.Conclusions:FNA is a common tool in the diagnosis and management of salivary gland tumors. DOG1-K9 clone was very useful with a unique staining pattern of apical-luminal positivity in the differential diagnosis of AciCC from other oncocytic salivary gland tumors.

Abstract 4950: Defective expression of partition-defective 3 (PAR-3) is associated with adverse prognostic factors in esophageal squamous cell carcinoma

Abstract Esophageal cancer is the sixth leading cause of cancer mortality and squamous cell carcinoma is a prevalent histological subtype worldwide. The partition-defective 3 (PAR-3) protein belongs to a partition defective complex that controls polarity, which is mainly composed of PAR-3, PAR-6, and atypical protein kinase C (aPKC). PAR-3 contains one self-oligomerization domain in the N-terminus, three PDZ protein interaction domains and one aPKC-binding domain. Owing to these domains, PAR-3 is able to form a conserved protein complex with PAR-6 and aPKC. In mammalian epithelial cells, PAR complex assembles at tight junctions, and plays a role in controlling apical-basal polarity, asymmetric cell division, and directional cell migration. It is well known that disrupted cell polarity is a feature of epithelial cancers. Therefore, it is believed that PAR proteins may be involved in multiple aspects of oncogenesis and tumor-promoting function. We previously detected a homozygous deletion of PAR-3 gene in esophageal squamous cell carcinoma (ESCC) cell lines by a high-density oligonucleotide microarray approach. (Oncogene 28:2910-2918, 2009). A copy number loss of PAR-3 gene was observed in 15% of primary ESCC tumors. The expression of PAR-3 mRNA was significantly reduced in 70% of ESCC tumors when compared with their nontumorous tissue counterparts. Exogenous expression of PAR-3 gene in ESCC cells lacking this gene (KYSE30 cells) enhanced the recruitment of Zonula occludens 1 (ZO-1), a marker of tight junctions, to sites of cell-cell contact. Conversely, knockdown of PAR-3 gene in ESCC cells expressing this gene disrupted ZO-1 localization at cell-cell borders. Although our previous findings suggested that deletion and reduced expression of PAR-3 gene may be a novel mechanism that drives the progression of ESCC, the clinical relevance of PAR-3 in ESCC tumor progression was not known. In the present study, we aimed to investigate the expression pattern of PAR-3 protein in primary ESCC tumors and to clarify any potential relationship between the PAR-3 expression and clinicopathological parameters. Immunohistochemical analyses showed that PAR-3 protein was expressed at the basal layer of the normal squamous epithelium of the esophagus, esophageal glands proper and the duct of esophageal glands. Both normal esophageal cells and ESCC tumor cells showed granular cytoplasmic staining and particularly strong membranous staining of PAR-3 protein. PAR-3 protein expression was lost in 20 (37%) of 74 primary ESCC tumors. Loss of PAR-3 protein expression was significantly associated with invasion depth (P = 0.0065) and lymph node metastasis (P = 0.03). Our results suggest that loss of PAR-3 protein expression may lead to tumor progression and subsequent lymph node metastasis in ESCC. Citation Format: Tomoko Kitaichi, Koichiroh Yasui, Akira Tomie, Yasuyuki Gen, Osamu Dohi, Yuji Naito, Yoshito Itoh. Defective expression of partition-defective 3 (PAR-3) is associated with adverse prognostic factors in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4950. doi:10.1158/1538-7445.AM2015-4950

Autoantibodies in systemic vasculitis.

Autoantibodies in systemic vasculitis.

Immunohistochemical expression of Bcl-2 in oral epithelial dysplasia and oral squamous cell carcinoma.

The B cell lymphoma-2 gene is a proto-oncogene whose protein product inhibits apoptosis. Its role is associated with keeping cells alive, but not by stimulating them to proliferation, as other proto-oncogenes do. Increased expression of protein product of Bcl-2 gene appears in the early phase of carcinogenesis leading to apoptosis impairment and in consequence to the progression of neoplastic changes. To evaluate and compare the expression of Bcl-2 protein in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC). Sixty cases of formalin-fixed paraffin-embedded archival specimens comprising of 30 cases of leukoplakia with oral epithelial dysplasia and 30 cases of OSCC were taken for immunohistochemical analysis using monoclonal antibody against anti-human Bcl-2 oncoprotein. Immunostaining for Bcl-2 protein was identified in basal and parabasal layers as granular cytoplasmic staining in oral epithelial dysplasia. In OSCC, Bcl-2 immunoreactivity was most prominent in the peripheral cells of the infiltrating tumor islands which diminished toward the center in well-differentiated and moderately differentiated OSCC, whereas stronger and more diffuse expression of Bcl-2 oncoprotein was seen in poorly differentiated OSCC. Overall positivity of 26.7% (8/30) was observed in oral epithelial dysplasia and 30% (9/30) in OSCC in this study. Altered expression of Bcl-2 oncoprotein may be an early molecular event which leads to prolonged cell survival, increased chances of accumulation of genetic alterations, and subsequent increase in malignant transformation potential.

34. BRAF V600E mutation detection in cell blocks of thyroid fine needle aspiration cytology samples

Introduction BRAF mutation c.1799T>A, p.Val600Glu (or V600E) in thyroid neoplasms is exclusively associated with papillary carcinoma (PTC) or anaplastic thyroid carcinoma. Demonstration of BRAF mutation has been suggested as a supplement to confirmation of PTC in cytology material. Recently a V600E mutation specific antibody has been developed. We aim to assess the feasibility of testing for BRAF V600E mutational status in cytology (cell block) material. Methods 39 adequate cell blocks (CBs) and 18 available follow-up histology cases were tested by immunohistochemistry (IHC) (clone VE1, 1:400, Spring Biosciences, Pleasanton, California, using Optiview amplification kit) and molecular methods (castPCR: Competitive Allele Specific Taqman PCR, Life Technologies, CA, USA), followed by single-strand confirmation polymorphism (SSCP) if required. All cell blocks tested included H+E sections to confirm adequacy of abnormal cells. Any fine granular cytoplasmic staining in abnormal cells was considered as a positive IHC result. DNA content on CBs ranged from 2.4–54.1 ng/μL and was considered adequate for both molecular methods. Results Tabled 1 CYTOLOGY (n=39) IHC POSNEG MolecularPOS172 sensitivity =89.5% NEG416 specificity =80.0% HISTOLOGY (n=18) IHC POSNEG MolecularPOS130sensitivity =100% NEG14specificity =80.0% CYTOLOGY AND HISTOLOGY CORRELATION (n=18) Cytology BRAF(any method) POSNEG Histology BRAF (any method) POS104sensitivity=71.4% NEG04specificity =100% Open table in a new tab Conclusions 1.BRAF V600E mutation assessment in thyroid cell block cytology material is feasible although the role in diagnostic algorithm requires further investigation. 2.Specificity of CB BRAF positivity by both IHC and molecular methods was 100% (no false positive findings). 3.Discrepancies noted between IHC and molecular methods requires further investigation. BRAF mutation c.1799T>A, p.Val600Glu (or V600E) in thyroid neoplasms is exclusively associated with papillary carcinoma (PTC) or anaplastic thyroid carcinoma. Demonstration of BRAF mutation has been suggested as a supplement to confirmation of PTC in cytology material. Recently a V600E mutation specific antibody has been developed. We aim to assess the feasibility of testing for BRAF V600E mutational status in cytology (cell block) material. 39 adequate cell blocks (CBs) and 18 available follow-up histology cases were tested by immunohistochemistry (IHC) (clone VE1, 1:400, Spring Biosciences, Pleasanton, California, using Optiview amplification kit) and molecular methods (castPCR: Competitive Allele Specific Taqman PCR, Life Technologies, CA, USA), followed by single-strand confirmation polymorphism (SSCP) if required. All cell blocks tested included H+E sections to confirm adequacy of abnormal cells. Any fine granular cytoplasmic staining in abnormal cells was considered as a positive IHC result. DNA content on CBs ranged from 2.4–54.1 ng/μL and was considered adequate for both molecular methods. 1.BRAF V600E mutation assessment in thyroid cell block cytology material is feasible although the role in diagnostic algorithm requires further investigation. 2.Specificity of CB BRAF positivity by both IHC and molecular methods was 100% (no false positive findings). 3.Discrepancies noted between IHC and molecular methods requires further investigation.

An International Interpretation Study Using the ALK IHC Antibody D5F3 and a Sensitive Detection Kit Demonstrates High Concordance between ALK IHC and ALK FISH and between Evaluators

The goal of personalized medicine is to treat patients with a therapy predicted to be efficacious based on the molecular characteristics of the tumor, thereby sparing the patient futile or toxic therapy. Anaplastic lymphoma kinase (ALK) inhibitors are effective against ALK-positive non-small-cell lung cancer (NSCLC) tumors, but to date the only approved companion diagnostic is a break-apart fluorescence in situ hybridization (FISH) assay. Immunohistochemistry (IHC) is a clinically applicable cost-effective test that is sensitive and specific for ALK protein expression. The purpose of this study was to assemble an international team of expert pathologists to evaluate a new automated standardized ALK IHC assay. Archival NSCLC tumor specimens (n =103) previously tested for ALK rearrangement by FISH were provided by the international collaborators. These specimens were stained by IHC with the anti-ALK (D5F3) primary antibody combined with OptiView DAB IHC detection and OptiView amplification (Ventana Medical Systems, Inc., Tucson, AZ). Specimens were scored binarily as positive if strong granular cytoplasmic brown staining was present in tumor cells. IHC results were compared with the FISH results and interevaluator comparisons made. Overall for the 100 evaluable cases the ALK IHC assay was highly sensitive (90%), specific (95%), and accurate relative (93%) to the ALK FISH results. Similar results were observed using a majority score. IHC negativity was scored by seven of seven and six of seven evaluators on three and two FISH-positive cases, respectively. IHC positivity was scored on two FISH-negative cases by seven of seven readers. There was agreement among seven of seven and six of seven readers on 88% and 96% of the cases before review, respectively, and after review there was agreement among seven of seven and six of seven on 95% and 97% of the cases, respectively. On the basis of expert evaluation the ALK IHC test is sensitive, specific, and accurate, and a majority score of multiple readers does not improve these results over an individual reader's score. Excellent inter-reader agreement was observed. These data support the algorithmic use of ALK IHC in the evaluation of NSCLC.

Characterization of TgPuf1, a member of the Puf family RNA-binding proteins from Toxoplasma gondii

BackgroundPuf proteins act as translational regulators and affect many cellular processes in a wide range of eukaryotic organisms. Although Puf proteins have been well characterized in many model systems, little is known about the structural and functional characteristics of Puf proteins in the parasite Toxoplasma gondii.MethodsUsing a combination of conventional molecular approaches, we generated endogenous TgPuf1 tagged with hemagglutinin (HA) epitope and investigated the TgPuf1 expression levels and localization in the tachyzoites and bradyzoites. We used RNA Electrophoretic Mobility Shfit Assay (EMSA) to determine whether the recombination TgPuf1 has conserverd RNA binding activity and specificity.ResultsTgPuf1 was expressed at a significantly higher level in bradyzoites than in tachyzoites. TgPuf1 protein was predominantly localized within the cytoplasm and showed a much more granular cytoplasmic staining pattern in bradyzoites. The recombinant Puf domain of TgPuf1 showed strong binding affinity to two RNA fragments containing Puf-binding motifs from other organisms as artificial target sequences. However, two point mutations in the core Puf-binding motif resulted in a significant reduction in binding affinity, indicating that TgPuf1 also binds to conserved Puf-binding motif.ConclusionsTgPuf1 appears to exhibit different expression levels in the tachyzoites and bradyzoites, suggesting that TgPuf1 may function in regulating the proliferation or/and differentiation that are important in providing parasites with the ability to respond rapidly to changes in environmental conditions. This study provides a starting point for elucidating the function of TgPuf1 during parasite development.

Basophils are elevated in nasal polyps of patients with chronic rhinosinusitis without aspirin sensitivity

Basophils are elevated in nasal polyps of patients with chronic rhinosinusitis without aspirin sensitivity

A cross sectional study of p504s, CD133, and Twist expression in the esophageal metaplasia dysplasia adenocarcinoma sequence

The incidence of esophageal adenocarcinoma has increased dramatically over recent years and Barrett's esophagus is considered the most established risk factor for its development. Endoscopic surveillance of Barrett's esophagus is therefore recommended but hinges on histological interpretation of randomly taken biopsies which is poorly reproducible. The use of biomarkers presents an opportunity to improve our ability to risk-stratify these patients.We examined three biomarkers namely p504s, CD133, and Twist in the setting of Barrett's esophagus, low-grade dysplasia, and esophageal adenocarcinoma to evaluate differential expression between benign, dysplastic, and malignant Barrett's tissue in an exploratory cross-sectional study. Twenty-five cases each of Barrett's esophagus, low-grade dysplasia, and esophageal adenocarcinoma were included along-with 25 cases of esophagectomy resections for Barrett's adenocarcinoma. The biomarkers were immunostained on automated Ventana(®) immunostainer. The biopsies were assessed for biomarker expression by two independent observers. Granular cytoplasmic staining of p504s was observed in dysplastic Barrett's biopsies and esophageal adenocarcinoma but not in Barrett's esophagus. Apical and membranous CD133 expression was also observed in dysplastic Barrett's and esophageal adenocarcinoma. Nuclear Twist expression was seen predominantly in stromal cells. There was increased p504s expression in dysplastic Barrett's esophagus and esophageal adenocarcinoma compared with controls. CD133 expression was detected for the first time in esophageal adenocarcinoma and dysplastic Barrett's esophagus. Twist expression was not convincing enough to be labeled as Barrett's biomarker. p504s and CD133 have the potential to differentiate benign from malignant Barrett's tissue in this exploratory study. Their validity should be established in prospective longitudinal studies.

Expression of Protein Gene Product 9.5 and Sal-like Protein 4 in Canine Seminomas

The aim of the present study was to characterize canine classical seminoma (SE) and spermatocytic seminoma (SS) by immunohistochemical expression of gonocytic and spermatogonial cellular markers (c-Kit, placental alkaline phosphatase [PLAP], protein gene product 9.5 [PGP9.5]and Sal-like protein 4 [Sall4]) and histochemically by the periodic acid-Schiff (PAS) reaction. Twenty-five cases of SE and 23 cases of SS were investigated. Two cases of dysgerminoma were also examined. c-Kit was expressed on the cell membrane of 13 of 25 cases of SE (52%) and four of 23 cases of SS (16%). This marker was not expressed in dysgerminoma. PLAP immunoreactivity was observed in the cytoplasm of neoplastic cells of six of 25 cases of SE (24%). PLAP was not expressed in cases of SS and dysgerminoma. All samples of SE, SS and dysgerminoma showed cytoplasmic expression of PGP9.5 and nuclear immunoreactivity for Sall4. There was fine granular cytoplasmic PAS staining in neoplastic cells in five of 25 cases of SE (20%), while all samples of SS and dysgerminoma cases were PAS negative. These findings suggest that it is not possible to differentiate canine SE and SS using these markers. This may be because canine SS may be derived from spermatogonia that can differentiate to spermatocytesand also because cases of canine SE might consist of neoplastic cells that have lost their gonocytic nature. This study was the first to show positive immunoreactivity for Sall4 in canine seminomas and dysgerminomas and expression of PGP9.5 in canine dysgerminomas.

Pheochromocytoma-paraganglioma due to germline SDHB mutation in children are common

Introduction Pheochromoctyoma-paraganglioma syndrome (PHEO-PGL) due to SDH germline mutation is easily identified by loss of SDHB by immunohistochemistry. Pham et al . showed a 16% (5/30) prevalence of SDH mutation in a retrospective review of the Mayo clinic involving thirty patients (1975-2005). Aim Ascertain the prevalence of PHEO-PGL due to SDHB germline mutations in a local paediatric population by using IHC. Method 14 patients were ascertained from the pathology database with pheochromocytoma and or paraganglioma, SDHA and SDHB IHC was performed on FFPE blocks. Results Three patients had SDHB germline mutations, three had VHL mutation and eight had no genetic studies performed. Of the 14 patients, four had multiple tumours and of these, three showed loss of SDHB staining. All three patients with loss of SDHB by IHC had genetically confirmed germline mutations of SDHB. A novel observation was that three patients with VHL syndrome showed a characteristic weak granular cytoplasmic staining, a pattern distinct from SDHB loss and normal controls. Discussion (1) We found the prevalence of SDH germline mutations was 28% in children with PHEO-PGL, which is higher than historically identified. (2) In patients with multiple tumours the prevalence was 75% (3/4 patients). (3) We found a distinctive weak granular positive cytoplasmic SDHB staining pattern in tumours with VHL germline mutations.

Pathological features of FTLD-FUS in a Japanese population: Analyses of nine cases

We investigated the pathological features of frontotemporal lobar degeneration (FTLD) with fused in sarcoma protein (FUS) accumulation (FTLD-FUS) in the Japanese population. Only one out of nine FTLD-FUS cases showed pathology that corresponds to atypical FTLD with ubiquitin-positive inclusions (aFTLD-U). Five were basophilic inclusion body disease (BIBD) and two were neuronal intermediate filament inclusion disease. The last case was unclassifiable and was associated with dystrophic neurites (DNs) as the predominant FUS pathology. The results of this study indicate an ethnic difference from western countries. In Japan, BIBD is the most common subtype of FTLD-FUS and aFTLD-U is rare, a finding which contrasts with aFTLD-U being the most common form in western countries. Immunohistochemical analyses of these FTLD-FUS cases reveal that FUS abnormally accumulated in neuronal cytoplasmic inclusions (NCIs) and DNs has an immunohistochemical profile distinct from that of normal, nuclear FUS. NCIs and DNs are more readily stained than the nuclei by antibodies to the middle portion of FUS. Antibodies to the carboxyl terminal portion, on the other hand, stain the nuclei more readily than NCIs and DNs. Such an immunohistochemical profile of NCIs and DNs was similar to that of cytoplasmic granular FUS staining which we previously reported to be associated with dendrites and synapses. Redistribution of FUS from the nucleus to the cytoplasm could be associated with the formation of abnormal FUS aggregates in FTLD-FUS.

Anaplastic lymphoma kinase-positive diffuse large B cell lymphoma: immunohistochemical and FISH analysis of a rare tumor with unusual clinical presentation

Anaplastic lymphoma kinase-positive diffuse large B cell lymphoma represents a distinct subtype of diffuse large B cell lymphoma with a characteristic morphology, a distinct immunophenotypic profile, and recurrent cytogenetic/molecular genetic abnormalities. We report a case of anaplastic lymphoma diagnosed in the bone marrow trephine of a 24-year-old male. Histologically, the tumor cells exhibited plasmablastic morphology with expression of CD45, CD138, EMA, MUM1, and kappa immunoglobulin light chain, but negative for CD20, CD30, CD79a, PAX-5, CD10, and lambda light chain. In addition, the neoplastic cells showed positive immunoreactivity for anaplastic lymphoma kinase (ALK) with exclusive cytoplasmic granular staining pattern. Furthermore, interphase fluorescence in situ hybridization analysis, revealed ALK gene rearrangement in the lymphoid cells. The diagnosis of this rare entity in unusual extranodal sites such as the bone marrow is challenging, and it is based on detailed morphological and immunohistochemical analysis. Fluorescence in situ hybridization provides also a helpful tool in order to establish the diagnosis.

No correlation between somatostatine 2 receptor expression analyzed by RNAin-situhybridization and real-time qRT-PCR in clinically non-functioning pituitary adenomas

ISH for SSTR2 mRNA was performed using the RNAscope 2.0 FFPE Assay (Advanced Cell Diagnostics, Inc., Hayward, CA, USA) on 4-μm formalin fixed and paraffin embedded tissue sections. After deparaffinization they were pretreated with heat and protease prior to hybridization with a target probe to the SSTR2. A horseradish peroxidase-based signal amplification system was then hybridized to the target probes followed by color development with 3,3 -diaminobenzidine. Control probes for the bacterial gene DapB (negative control) and for the POLR2A gene (positive control – evidence of adequate RNA) were also included in each case. The study slides (and corresponding controls) were read independently by two study pathologists (A.K. and J.L) and classified in a binary manner as either positive or negative. Positive cases had to have granular cytoplasmic and/or nuclear brown staining that was higher than the signal on the DapB negative control slide. Positive cases were subsequently semi-quantitatively assessed according to the manufacturer s scoring guideline as follows: Methods

Enteroviruses and the pathogenesis of type 1 diabetes revisited: cross‐reactivity of enterovirus capsid protein (VP1) antibodies with human mitochondrial proteins

Current or recent enteroviral infections show an association with type 1 diabetes. However, evidence for this has mainly been generated using a particular mouse monoclonal antibody (clone 5-D8/1) which binds the viral capsid protein VP1. Difficulty in confirming these findings using other independent methods has led to the concern that this might be artefactual. To address this, we examined the potential cross-reactivity of clone 5-D8/1 with normal islet proteins. Western blotting, two-dimensional gel electrophoresis, and mass spectrometry were used to identify human islet proteins bound by the clone 5-D8/1. We found a distinct reactivity with two mitochondrial proteins, creatine kinase B-type and ATP synthase beta subunit. Immunohistochemistry using the clone 5-D8/1 revealed a granular cytoplasmic staining pattern in mitochondria-rich cells, ie hepatocytes, ductal epithelial cells, vascular endothelial cells, skeletal muscle cells, and the neoplastic salivary gland oncocytoma cells, whereas connective tissue and infiltrating immune cells were negative. Staining on islets of Langerhans from subjects with recent-onset type 1 diabetes, but not on isolated human islets infected in vitro with enteroviruses, could be blocked after mixing the clone 5-D8/1 with the mitochondrial proteins. Collectively, our data show that the clone 5-D8/1 detects two human mitochondrial enzymes in addition to enteroviral VP1. The notion that the previously reported VP1 positivity in islets of recent-onset type 1 diabetes patients could reflect cross-reactivity to native islet proteins and not the presence of EV is supported by difficulties in demonstrating EV infection by independent techniques such as PCR or in situ hybridization. These findings call for revisiting the presence of enteroviruses in pancreatic islets of patients with type 1 diabetes.

The Process of Apoptosis in a Holocrine Gland as Shown by the Avian Uropygial Gland

This study was designed to elucidate the presence of apoptosis and the localization of apoptosis-related Bax and survivin proteins and proliferating cell nuclear antigen (PCNA) within the chicken uropygial gland, a specialized holocrine secretory gland. In day-old chicks, survivin and Bax immunoreactivities were observed in the cell cytoplasm of the germinative and secretory layers of the luminal epithelium and tubules. During this period, the TUNEL reaction, an indication of apoptosis, was only sporadically positive in the tubules. From the 7th day to the 150th day of posthatching, survivin was detected in the cytoplasm of cells in the germinative layer and in the nuclei of some cells in the secretory layers of the gland. The germinative layer cells showed weak homogeneous cytoplasmic staining for Bax, whereas the cells of the secretory and intermediate layers of luminal epithelium and tubules exhibited granular cytoplasmic staining. After day 7, TUNEL-positive cells were observed in the secretory and degenerative layers of the luminal epithelium and central tubules. After day 12, some TUNEL-positive cells were also seen in the peripheral tubules. At all posthatch ages, the cytoplasm and nucleus of the germinative layers of luminal epithelium and tubules reacted with PCNA, whereas only a small number of cell nuclei in the secretory layers were immunopositive. These results support the theory that specific PCNA/Bax/survivin expression patterns could reflect particular cell differentiation states in the uropygial gland and that holocrine secretion in the gland is realized mainly by way of apoptosis.

Cellular protein quality control and the evolution of aggregates in spinocerebellar ataxia type 3 (SCA3)

A characteristic of polyglutamine diseases is the increased propensity of disease proteins to aggregate, which is thought to be a major contributing factor to the underlying neurodegeneration. Healthy cells contain mechanisms for handling protein damage, the protein quality control, which must be impaired or inefficient to permit proteotoxicity under pathological conditions. We used a quantitative analysis of immunohistochemistry of the pons of eight patients with the polyglutamine disorder spinocerebellar ataxia type 3. We employed the anti-polyglutamine antibody 1C2, antibodies against p62 that is involved in delivering ubiquitinated protein aggregates to autophagosomes, antibodies against the chaperones HSPA1A and DNAJB1 and the proteasomal stress marker UBB⁺¹. The 1C2 antibody stained neuronal nuclear inclusions (NNIs), diffuse nuclear staining (DNS), granular cytoplasmic staining (GCS) and combinations, with reproducible distribution. P62 always co-localized with 1C2 in NNI. DNS and GCS co-stained with a lower frequency. UBB⁺¹ was present in a subset of neurones with NNI. A subset of UBB⁺¹-containing neurones displayed increased levels of HSPA1A, while DNAJB1 was sequestered into the NNI. Based on our results, we propose a model for the aggregation-associated pathology of spinocerebellar ataxia type 3: GCS and DNS aggregation likely represents early stages of pathology, which progresses towards formation of p62-positive NNI. A fraction of NNI exhibits UBB⁺¹ staining, implying proteasomal overload at a later stage. Subsequently, the stress-inducible HSPA1A is elevated while DNAJB1 is recruited into NNIs. This indicates that the stress response is only induced late when all endogenous protein quality control systems have failed.

Endobronchial ultrasound fine‐needle aspiration biopsy of pulmonary non–small cell carcinoma with subclassification by immunohistochemistry panel

With the introduction of new treatment modalities and guidelines, it is important to subclassify primary pulmonary non-small cell carcinoma (NSCCA). Subsequent treatment and testing is dependent on accurate subclassification. Endobronchial ultrasound fine-needle aspiration (EBUS FNA) is used for primary evaluation and diagnosis, and can provide a cell block for ancillary testing. EBUS FNA cases from primary pulmonary NSCCA with an immunohistochemical (IHC) panel performed on a cell block with concomitant surgical pathology biopsy were analyzed. Cell block preparations underwent an IHC panel including monoclonal antibodies for napsin A (nap-A), thyroid transcription factor (TTF-1), p63, and cytokeratin 5/6 (CK5/6). A total of 81 cases from 81 patients were identified. Of these, 69 cases (85%) were provided a specific diagnosis of adenocarcinoma (ADCA) or squamous cell carcinoma (SCCA) on the EBUS FNA. In 12 cases (15%), a diagnosis of NSCCA, not otherwise specified was provided. For specific subclassifications, there were 35 ADCA cases, 34 SCCA cases, and 12 NSCCA, not otherwise specified cases. For ADCA, nap-A showed granular cytoplasmic staining and TTF-1 nuclear staining. For SCCA, CK5/6 showed cytoplasmic staining and p63 nuclear staining. Surgical pathology concomitant material was present in 29 of 81 cases with 18 correlations and 11 noncorrelations. With new treatment guidelines for patients with primary pulmonary NSCCA, specific diagnosis is increasingly important. EBUS FNA with cell block provided a specific subclassification of NSCCA in 85% of cases when used in conjunction with a specific IHC panel including nap-A, TTF-1, CK5/6, and p63.