Novel Autoantibodies in Idiopathic Small Fiber Neuropathy.

OBJECTIVE: Small fiber neuropathy (SFN) is clinically and etiologically heterogeneous. Although autoimmunity has been postulated to be pathophysiologically important in SFN, few autoantibodies have been described. We aimed to identify autoantibodies associated with idiopathic SFN (iSFN) by a novel high-throughput protein microarray platform that captures autoantibodies expressed in the native conformational state. METHODS: Sera from 58 SFN patients and 20 age- and gender-matched healthy controls (HCs) were screened against >1,600 immune-related antigens. Fluorescent unit readout and postassay imaging were performed, followed by composite data normalization and protein fold change (pFC) analysis. Analysis of an independent validation cohort of 33 SFN patients against the same 20 HCs was conducted to identify reproducible proteins in both cohorts. RESULTS: Nine autoantibodies were screened with statistical significance and pFC criteria in both cohorts, with at least 50% change in serum levels. Three proteins showed consistently high fold changes in main and validation cohorts: MX1 (FC = 2.99 and 3.07, respectively, p = 0.003, q = 0.076), DBNL (FC = 2.11 and 2.16, respectively, p = 0.009, q < 0.003), and KRT8 (FC = 1.65 and 1.70, respectively, p = 0.043, q < 0.003). Further subgroup analysis into iSFN and SFN by secondary causes (secondary SFN) in the main cohort showed that MX1 is higher in iSFN compared to secondary SFN (FC = 1.61 vs 0.106, p = 0.009). INTERPRETATION: Novel autoantibodies MX1, DBNL, and KRT8 are found in iSFN. MX1 may allow diagnostic subtyping of iSFN patients. ANN NEUROL 2022;91:66-77.

Therapeutic effects of lentinan on inflammatory bowel disease and colitis-associated cancer.

In this study, we investigated the therapeutic potential of lentinan in mouse models of inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). Lentinan decreased the disease activity index and macroscopic and microscopic colon tissue damage in dextran sulphate sodium (DSS)-induced or TNBS-induced models of colitis. High-dose lentinan was more effective than salicylazosulfapyridine in the mouse models of colitis. Lentinan decreased the number of tumours, inflammatory cell infiltration, atypical hyperplasia and nuclear atypia in azoxymethane/DSS-induced CAC model. It also decreased the expression of pro-inflammatory cytokines, such as IL-13 and CD30L, in IBD and CAC model mice possibly by inhibiting Toll-like receptor 4 (TLR4)/NF-κB signalling and the expression of colon cancer markers, such as carcinoembryonic antigen, cytokeratin 8, CK18 and p53, in CAC model mice. In addition, lentinan restored the intestinal bacterial microbiotal community structure in IBD model mice. Thus, it shows therapeutic potential in IBD and CAC model mice possibly by inhibiting TLR4/NF-κB signalling-mediated inflammatory responses and disruption of the intestinal microbiotal structure.

Single lipoaminoglycoside promotes efficient intracellular antibody delivery: A comprehensive insight into the mechanism of action.

Delivery of biologically active proteins into cells is emerging as important strategy for many applications. Previous experiments have shown that lipoaminoglycosides were capable of delivery of the anti-cytokeratin8 antibody (anti-K8) but only when formulated with lipid helpers potentially leading to toxicity from excess lipids. Here, we optimized anti-K8 delivery with various lipoaminoglycosides in the absence of a lipid helper. Results led to the identification of the aminoglycoside lipid dioleyl phosphoramido ribostamycin (DOPRI) as a potent intracellular delivery system for anti-K8. Electron microscopy revealed that delivered anti-K8 molecules were bound to intermediate filaments in cells. Anti-K8 was bound to the surface of DOPRI vesicles without perturbing lipid organization. Macropinocytosis and caveolin mediated endocytosis contributed to anti-K8 internalization and to filament labeling with a major contribution being made by the caveolin pathway. The results showed that the unique properties of DOPRI were sufficient for efficient intracellular protein delivery without requiring lipid helpers.

A Comparison of Three Methods for the Detection of Circulating Tumor Cells in Patients with Early and Metastatic Breast Cancer.

BACKGROUND: We directly compared CTC detection rates and prognostic significance, using three different methods in patients with breast cancer (BC). METHODS: Early (n=200) and metastatic (n=164) patients were evaluated before initiating adjuvant or first-line chemotherapy, using the CellSearchTM System, an RT-qPCR for CK-19 mRNA detection and by double immunofluorescence (IF) microscopy using A45-B/B3 and CD45 antibodies. RESULTS: Using the CellSearchTM System, 37% and 16.5% of early BC patients were CTC-positive (at ≥1 and ≥2 CTCs/23 ml of blood), 18.0% by RT-qPCR and 16.9% by IF; no agreement was observed between methods. By the CellSearchTM 34.8% and 53.7% (at≥ 5 and ≥ 2 CTCs/7.5 ml) of metastatic patients were CTC-positive, 37.8% by RT-qPCR and 28.5% by IF. A significant agreement existed only between the CellSearchTM and RT-qPCR. In 60.8% of cases, differential EpCAM and CK-19 expression on CTCs by IF could explain the discrepancies between the CellSearchTM and RT-qPCR. CTC-positivity by either method was associated with decreased overall survival in metastatic patients. CONCLUSION: A significant concordance was observed between the CellSearchTM and RT-qPCR in metastatic but not in early BC. Discordant results could be explained in part by CTC heterogeneity. CTC detection by all methods evaluated had prognostic relevance in metastatic patients.

Absence of keratin 8 or 18 promotes antimitochondrial autoantibody formation in aging male mice.

Human mutations in keratin 8 (K8) and keratin 18 (K18), the intermediate filament proteins of hepatocytes, predispose to several liver diseases. K8-null mice develop chronic liver injury and fragile hepatocytes, dysfunctional mitochondria, and Th2-type colitis. We tested the hypothesis that autoantibody formation accompanies the liver damage that associates with K8/K18 absence. Sera from wild-type control, K8-null, and K18-null mice were analyzed by immunoblotting and immunofluorescence staining of cell and mouse tissue homogenates. Autoantibodies to several antigens were identified in 81% of K8-null male mice 8 mo or older. Similar autoantibodies were detected in aging K18-null male mice that had a related liver phenotype but normal colon compared with K8-null mice, suggesting that the autoantibodies are linked to liver rather than colonic disease. However, these autoantibodies were not observed in nontransgenic mice subjected to 4 chronic injury models. The autoantigens are ubiquitous and partition with mitochondria. Mass spectrometry and purified protein analysis identified, mitochondrial HMG-CoA synthase, aldehyde dehydrogenase, and catalase as the primary autoantigens, and glutamate dehydrogenase and epoxide hydrolase-2 as additional autoantigens. Therefore, absence of the hepatocyte keratins results in production of anti-mitochondrial autoantibodies (AMA) that recognize proteins involved in energy metabolism and oxidative stress, raising the possibility that AMA may be found in patients with keratin mutations that associate with liver and other diseases.

Keratin 8 is a novel autoantigen of rheumatoid arthritis.

OBJECTIVE: This research aims to verify Keratin 8 (K8) as a specific autoantigen in rheumatoid arthritis (RA). METHODS: First, total RNA was extracted from HaCaT cell to obtain cDNA by inverse transcription. Then, PCR was performed to amplify corresponding gene by K8 primers. Next, cloning, expression, and purification technology were used to obtain the recombinant human K8 (rhK8). At last, the purified rhK8, after identified by mass spectrometer, was used to perform further disease-related Western blotting and ELISA test with real clinical samples. RESULTS: Purified rhK8 was successfully obtained and then Western blotting confirmed antigenicity of K8 in rheumatoid arthritis. The reactivity of serum IgG against rhK8 was further detected in 34 of 50 RA patients (68%). The reactivity of RA serum IgG antibodies against K8 was significantly higher than healthy controls and systemic lupus erythematosus (SLE) patients. CONCLUSION: This research confirmed Keratin 8 as a novel autoantigen of RA.

Cytokeratin 8-MHC class I interactions: a potential novel immune escape phenotype by a lymph node metastatic carcinoma cell line.

Defective human leukocyte antigen (HLA) class I expression in malignant cells facilitates their escape from destruction by CD8(+) cytotoxic T lymphocytes. In this study, a post-translational mechanism of HLA class I abnormality that does not involve defects in the HLA subunits and antigen processing machinery components was identified and characterized. The marked HLA class I downregulation phenotype of a metastatic carcinoma cell line can be readily reversed by trypsin, suggesting a masking effect by serine protease-sensitive HLA class I-interacting factors. Co-immunoprecipitation, combined with LC-tandem mass spectrometry and immunoblotting identified these factors as cytokeratin (CK) 8 and its heterodimeric partners CK18 and CK19. Ectopic CK8/18 or CK8/19 expression in HEK293 cells resulted in surface CK8 expression with an HLA class I downregulation phenotype, while redirecting CK8/18 and CK8/19 to the endoplasmic reticulum (ER) had no such effect. This observation and the failure to constrain CK8/18 and CK8/19 membrane trafficking by an ER-Golgi transport inhibitor suggested an ER-independent route for CK8 access to HLA class I molecules. Monoclonal antibody mapping revealed a potential CK8 blockade of HLA class I-CD8 and -TCR contacts. These findings, along with the emerging role of cell surface CK8 in cancer metastasis, may imply a dual strategy for tumor cell survival in the host.

A simple multicolor flow cytometry protocol for detection and molecular characterization of circulating tumor cells in epithelial cancers.

Circulating tumor cells (CTCs) might not only serve as prognostic marker but could also be useful for monitoring treatment efficacy. A multicolor flow cytometry protocol for their detection and molecular characterization in peripheral blood was developed which consisted of erythrocyte lysis followed by staining of cells with fluorochrome-labeled antibodies against CD45 and the epithelial markers EpCam and cytokeratin 7/8. For reducing the number of events acquired by flow cytometry, an electronic threshold for the fluorescent signals from the epithelial markers was applied. After establishment of the protocol by using spiking experiments, its suitability to determine the absolute number of CTCs as well as their expression of epidermal growth factor receptor (EGFR) and its phosphorylated form (phospho-EGFR) in blood samples from patients with squamous cell carcinoma of the head and neck (SCCHN) was validated. Spiking experiments demonstrated an excellent recovery (mean 85%) and a linear performance (R(2) = 0.98) of the protocol. Sensitivity and specificity were comparable to our former protocol using immunomagnetic CTC pre-enrichment. The analysis of 33 SCCHN patient samples revealed the presence of CTCs in 33.3% of cases with a mean ± SD of 1.5 ± 0.5 CTCs per 3.75 ml blood. EGFR was expressed in 100% and phospho-EGFR in 36.4% of the CTC+ cases. We have established a simple and sensitive multicolor flow cytometry protocol for detection of CTCs in patients with epithelial cancers including SCCHN which will allow their detailed molecular characterization.

The role of liver biopsy in the diagnosis and prognosis of patients with acute deterioration of alcoholic cirrhosis.

BACKGROUND & AIMS: The aim of this study was to systematically assess the diagnostic and prognostic value of early liver biopsy in patients who require hospital admission with acute deterioration of alcoholic cirrhosis. METHODS: Sixty-eight patients with acute deterioration of alcoholic cirrhosis underwent a liver biopsy within 7 days and the biopsies were processed using routine stains and K8/18 immunohistochemistry to characterize balloon degeneration. The biopsies were scored by two independent histopathologists using pre-defined criteria. The patients were managed according to institutional protocols and followed until the time of hospital discharge or death. RESULTS: With use of K8/18 immunohistochemistry, very high concordance rate for the diagnosis of balloon degeneration was reached (r = 0.7; p = 0.0001). The presence of a systemic inflammatory response (SIRS) suggestive of acute alcoholic steatohepatitis (ASH), predicts severe ASH histologically in only 50% patients. Moreover, in 41% of SIRS negative patients who were thought not to have ASH, a diagnosis of ASH was subsequently confirmed on histological grading. Patients that have SIRS criteria but no evidence of histological ASH are more likely to develop infection which may be indicated by the severity of canalicular cholestasis. Nineteen patients died during follow up. Patients manifesting ASH on biopsy who were also SIRS positive, had a significantly greater risk of mortality compared to those that were SIRS positive but ASH negative (p < 0.01) and those that were SIRS negative (p < 0.0001). CONCLUSIONS: The use of K8/18 immunostaining allows grading of the severity of alcoholic steatohepatitis. Early liver biopsy in these patients presenting with acute deterioration of cirrhosis is safe and provides important diagnostic and prognostic information.

Optimization of production of the anti-keratin 8 single-chain Fv TS1-218 in Pichia pastoris using design of experiments.

BACKGROUND: Optimization of conditions during recombinant protein production for improved yield is a major goal for protein scientists. Typically this is achieved by changing single crucial factor settings one at a time while other factors are kept fixed through trial-and-error experimentation. This approach may introduce larger bias and fail to identify interactions between the factors resulting in failure of finding the true optimal conditions. RESULTS: In this study we have utilized design of experiments in order to identify optimal culture conditions with the aim to improve the final yield of the anti-keratin 8 scFv TS1-218, during expression in P. pastoris in shake flasks. The effect of: pH, temperature and methanol concentration on the yield of TS1-218 using buffered minimal medium was investigated and a predictive model established. The results demonstrated that higher starting pH and lower temperatures during induction significantly increased the yield of TS1-218. Furthermore, the result demonstrated increased biomass accumulation and cell viability at lower temperatures which suggested that the higher yield of TS1-218 could be attributed to lower protease activity in the culture medium. The optimal conditions (pH 7.1, temperature 11°C and methanol concentration 1.2%) suggested by the predictive model yielded 21.4 mg TS1-218 which is a 21-fold improvement compared to the yield prior to optimization. CONCLUSION: The results demonstrated that design of experiments can be utilized for a rapid optimization of initial culture conditions and that P. pastoris is highly capable of producing and secreting functional single-chain antibody fragments at temperatures as low as 11°C.

Prognostic impact of regional lymph node micrometastasis in patients with node-negative biliary cancer.

OBJECTIVE: To immunohistochemically identify regional lymph node micrometastases in patients with regional node-negative biliary cancer who underwent curative resection, and to evaluate their clinical significance. SUMMARY BACKGROUND DATA: The clinical significance of immunohistochemically detected lymph node micrometastasis has recently been evaluated in various tumors. However, few reports have focused on this issue with regard to biliary cancer. METHODS: A total of 1421 regional lymph nodes from 151 patients with biliary cancer with negative regional nodes (as determined by conventional methods) were immunostained with antibody against cytokeratins 7 and 8 (CAM5.2). Prognostic impact was evaluated among patients with no metastasis, micrometastasis, and obvious metastasis detected by hematoxylin and eosin staining. Immunostained tumor foci were classified as small micrometastasis or large micrometastasis according to size (above or below 0.2 mm). RESULTS: CAM5.2-positive occult carcinoma cells in regional lymph nodes were detected in 33 (22%) of 151 patients and 49 (3%) of 1421 regional lymph nodes. Small micrometastases were detected in 23 patients, whereas large micrometastases were found in 10 patients. Survival for patients with micrometastasis was significantly worse than that for patients without (P = 0.0051), but was significantly better than that for patients with overt metastasis (P = 0.0092). No significant difference in postoperative survival was seen between patients with small and large micrometastases (P = 0.4221). CONCLUSIONS: Occult cancer cells were present in regional lymph nodes of 22% patients with regional node-negative biliary cancer, and were associated with significantly worse survival. Patients with micrometastases should be treated as carefully as node-positive patients.

Effect of Tongxieyaofang decoction on colonic mucosal protein expression profiles in rats with visceral hypersensitivity.

OBJECTIVE: To explore the underlying mechanism of action of Tongxieyaofang decoction in rats with visceral hypersensitivity using proteomics technology. METHODS: Twenty-four female Sprague-Dawley rats were randomly divided into three groups: control group, irritable bowel syndrome (IBS) group and Tongxieyaofang treatment group. An IBS model, characterized as visceral hypersensitivity, was established using the odour of mothballs as conditional stimulation and colorectal distension combined with classic physical restraint as non-conditional stimulation. Rats were intragastrically treated with Tongxieyaofang (2 or 4 mL·kg－1·d－1) for 4 weeks. On the 45th day, the rats were dissected and the colonic mucosal proteins were extracted. Differential protein spots were screened by fluorescent two-dimensional differential gel electrophoresis (2D-DIGE), and identified by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS). Western blotting experiments were performed to verify the changes observed in 2D-DIGE and MALDI-TOF-MS. RESULTS: It was found that the visceral sensitivity of rats in the Tongxieyaofang treatment group (4 mL/kg) was lower than that in the IBS group (P < 0.01). Sixty-one protein spots were differentially expressed between the IBS group and the Tongxieyaofang treatment group. Of these, 23 spots were upregulated in the Tongxieyaofang treatment group, while 38 spots were downregulated. Three specific proteins were successfully identified from the five protein spots with the most obvious changes. The two upregulated proteins were transgelin (TAGLN) and acetaldehyde dehydrogenase 2 (Aldh2) and the downregulated protein was cytokeratin 8 (CK8). CONCLUSION: Tongxieyaofang can dose-dependently ameliorate visceral hypersensitivity in rats and the mechanism of action may involve the upregulation of TAGLN and Aldh2 and the downregulation of CK8.

Keratin 8 is a potential self-antigen in the coronary artery disease immunopeptidome: A translational approach.

BACKGROUND: Inflammation is an important risk factor in atherosclerosis, the underlying cause of coronary artery disease (CAD). Unresolved inflammation may result in maladaptive immune responses and lead to immune reactivity to self-antigens. We hypothesized that inflammation in CAD patients would manifest in immune reactivity to self-antigens detectable in soluble HLA-I/peptide complexes in the plasma. METHODS: Soluble HLA-I/peptide complexes were immuno-precipitated from plasma of male acute coronary syndrome (ACS) patients or age-matched controls and eluted peptides were subjected to mass spectrometry to generate the immunopeptidome. Self-peptides were ranked according to frequency and signal intensity, then mouse homologs of selected peptides were used to test immunologic recall in spleens of male apoE-/- mice fed either normal chow or high fat diet. The peptide detected with highest frequency in patient plasma samples and provoked T cell responses in mouse studies was selected for use as a self-antigen to stimulate CAD patient peripheral blood mononuclear cells (PBMCs). RESULTS: The immunopeptidome profile identified self-peptides unique to the CAD patients. The mouse homologs tested showed immune responses in apoE-/- mice. Keratin 8 was selected for further study in patient PBMCs which elicited T Effector cell responses in CAD patients compared to controls, associated with reduced PD-1 mRNA expression. CONCLUSION: An immunopeptidomic strategy to search for self-antigens potentially involved in CAD identified Keratin 8. Self-reactive immune response to Keratin 8 may be an important factor in the inflammatory response in CAD.

Coordinate expression of cytokeratin 8 and cytokeratin 17 immunohistochemical staining in cervical intraepithelial neoplasia and cervical squamous cell carcinoma: an immunohistochemical analysis and review of the literature.

OBJECTIVE: Several Cytokeratin (CK) isoforms have been analyzed in cervical intraepithelial lesions. However, previously reported numbers of specimens have been too low to evaluate any correlation between CK and CIN. METHODS: We examined the immunohistochemical staining of p16, CK8, and CK17 in 134 cervical tissues obtained by punch biopsy and graded as follows: CIN I (n=39), CIN II (n=31), CIN III (n=43), SCC (n=21). RESULTS: p16 staining was identified in 74.4% of CIN I, 93.6% of CIN II, 97.7% of CIN III, and 100% of SCC cases. CK8 and CK17 staining were identified in 12.8% and 33.3% of CIN I, 22.6% and 58.1% of CIN II, 62.8% and 81.4% of CIN III, and 71.4% and 95.2% of SCC cases, respectively. Interestingly, positivity for CK8 and CK17 correlated with increasing lesion grade of the intraepithelial lesions and also correlated with p16 staining (p16, p=0.0008; CK8, p<0.0001, and CK17, p<0.0001), and a coordinate expression profile of CK8[+]/CK17[+] correlates with increasing CIN grade and carcinoma (likewise, a coordinate expression profile of CK8[-]/CK17[-] correlates with decreasing CIN grade and absence of carcinoma), but expression of just CK8 (CK8[+]/CK17[-]) or just CK17 (CK8[-]/CK17[+]) does not correlate with increasing CIN grade and carcinoma. CONCLUSIONS: Results of the present study showed that p16, CK8, and CK17 immunostaining differed according to the degree of cervical intraepithelial lesions and SCC, and surprisingly, that staining was significantly correlated with increasing lesion grade of CIN and SCC.

Functional mapping and single chain construction of the anti-cytokeratin 8 monoclonal antibody TS1.

The anti-cytokeratin (CK) 8 monoclonal antibody (mab) TS1 has been shown to efficiently bind to CK8 expressed in carcinomas in vivo. The anti-idiotypic antibody of TS1, alphaTS1, can be used to regulate the tumor:non-tumor ratio of TS1 by clearing non-tumor binding TS1 from the circulation. If the interaction of TS1 to CK8 and alphaTS1 is fully understood, mutations can be used to improve the tumor:non-tumor ratio. A scFv was made of the mab TS1 and residues earlier identified by Erlandsson et al. as important for the interaction with both its antigen CK8 and its anti-idiotype alphaTS1, were mutated to alanine or amides and expressed in E. coli. The effects of the mutations were studied by ELISA and residues important for the interactions to both CK8 and alphaTS1 were identified as mainly tyrosines, charged residues, a serine and a tryptophan. Altogether, nine amino acid residues in TS1 were found to be important in the interaction to alphaTS1 and six residues for the interaction to CK8. Important residues, clustered together in the modelled protein, were identified as residues from CDR 3 of the heavy chain and the unexpected participation of a residue in CDR 2 of the light chain. Some of the important residues are likely to be hotspots. Hotspots constitute a few residues in an interaction that contribute most to the binding, energetically. Amino acid residues in hotspots often cluster together in the center of the interaction interface, but can also be spread out to the periphery. The hotspots are often surrounded by hydrophobic patches, which are seen in the modelled TS1 protein used in this study. Amino acid residues that increased the affinity when mutated were also identified for both interactions. These residues are likely to be located outside the interacting interface. It can from this study be concluded that it is wise to precede the mutational procedure with experiments that can give guidelines for the selection of which amino acid residues to mutate. If the guidelines from the chemical modifications from Erlandsson et al. not had been used, this study would have left some residues unmutated and thereby missed important information.

Expression of the O-linked N-acetylglucosamine containing epitope H in normal myometrium and uterine smooth muscle cell tumors.

Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAc) residue in a specific conformation and/or environment recognized by monoclonal antibody H (mAbH). We have previously shown that epitope H is present in more than one polypeptide and in various types of normal and pathological cells. In the present study, we focused on uterine smooth muscle cell tumors and their adjacent normal myometrium to gain further insight into the expression patterns of epitope H in human tissues. The indirect immunoperoxidase method was applied using the mAbH and the monoclonal anti-cytokeratin 8 antibody (AbCK8) in 50 cases of typical uterine leiomyomas and in five cases of uterine leiomyosarcomas, with four cases belonging to Group II A and one to Group III according to Bell et al. [6]. Western immunoblotting was applied using mAbH and AbCK8 in five cases of uterine leiomyomas and their adjacent myometrium. The main results were as follows: (1) epitope H showed intense immunohistochemical expression in 46% (23/50) and moderate expression in 54% (27/50) of uterine leiomyomas, (2) epitope H showed intense immunohistochemical expression in 40% (2/5) and moderate expression in 60% (3/5) of uterine leiomyosarcomas, (3) epitope H showed no difference in the immunohistochemical expression between leiomyomas and their adjacent myometrium and between leiomyosarcomas and their adjacent myometrium, (4) immunohistochemical expression of cytokeratin 8 was not detected in the normal and neoplastic smooth muscle cells, (5) Western immunoblotting showed that in the smooth muscle cells of the myometrium and leiomyomas, epitope H is localized in four polypeptides with molecular weights of 100, 61, 59, and 54 kDa, and (6) Western immunoblotting did not detect cytokeratin 8 in the normal and neoplastic smooth muscle cells. The present results indicate fluctuations of the epitope expression levels in uterine smooth muscle cell tumors and their adjacent myometrium. These fluctuations may be of interest for gaining insight into the pathogenesis of uterine smooth muscle cell tumors, since O-GlcNAc glycosylation is involved in cell cycle and apoptosis pathways and may modify proteins involved in oncogenesis (tumor suppressor proteins and oncoproteins) and proteins with important biological functions such as cytoskeletal proteins, transcription factors, and heat-shock proteins. Furthermore, the present results indicate that cytokeratin 8, without being present in the cells of the myometrium, leiomyomas and leiomyosarcomas, shares its epitope H, which contains its unique sugar O-N-acetylglucosamine residue, with four other unrelated polypeptides produced by the normal and neoplastic smooth muscle cells. This should be considered when using anti-cytokeratin 8 antibodies in immunohistochemistry against smooth muscle cell tumors to avoid false positive immunohistochemical results.

Cytokeratin autoantibodies: useful serologic markers for toluene diisocyanate-induced asthma.

To evaluate the clinical significance of autoantibodies to three major epithelial cytokeratins (CK)--CK8, CK18, and CK19--we compared 66 patients with toluene diisocyanate (TDI)-induced asthma (group I) with three control groups: 169 asymptomatic exposed subjects (group II), 64 patients with allergic asthma (group III), and 123 unexposed healthy subjects (group IV). Serum IgG, specific for human recombinant CKs, were measured by ELISA (enzyme linked immunosorbent assay), and ELISA inhibition tests were performed. The existence of these antibodies was confirmed by IgG immunoblot analysis. Anti-TDI-HSA (human serum albumin) IgE and IgG antibodies were measured by ELISA in the same set of the patients. The prevalence of CK8, CK18, and CK19 auotantibodies in group I was significantly higher than in the other three groups. Results of the ELISA inhibition test showed significant inhibition with the addition of three CKs in a dose-dependent manner. No significant association was found between CK autoantibodies and the prevalence of anti- TDI-HSA IgG and IgE antibodies. These results suggest that autoantibodies to CK18 and CK19 can be used as serologic markers for identifying patients with TDI-induced asthma among exposed workers.