Successful triple immunoenzymatic method employing primary antibodies from same species and same immunoglobulin subclass.

Protocols for immunohistochemical (IHC) detection of multiple antigens in the same tissue sections have been developed using primary antibodies directly conjugated to different enzymes or fluorochromes, or ones that have been raised in different species, or from different immunoglobulin (Ig) classes or subclasses. For antibodies lacking such dissimilarities, very few proposals have been published with varying degrees of generalizability. In this report we present a successful triple IHC protocol engaging three unconjugated monoclonal primary antibodies raised in the same species and of the same Ig subclass. Compared to other methods, our results showed that denaturation of the preceding reaction complex by microwave heating, combined with additional suppression of enzyme activity, enabled the detection of all three reactions by using the same detection system, with no cross reaction observed. Moreover, expression patterns of each of the three antigens in the triple stained sections, was found to be similar to the pattern observed when single staining was performed. Unlike previous reports, no damage of targeted antigens or tissues did occur following this protocol. Furthermore, the contrast of the colors employed was investigated by computerized color deconvolution, and the three reactions products were successfully separated into three individual images that could be used for further objective quantification.

Ro52- and Ro60-specific B cell pattern in the salivary glands of patients with primary Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is characterized by the presence of autoantibodies against the ribonucleoprotein (RNP) particles Ro/SSA and La/SSB, and mononuclear cell infiltration of exocrine tissues, especially salivary and lachrymal glands. Low numbers of autoantigen-specific memory B cells and elevated levels of plasma cells have been detected previously in the peripheral blood (PB) of pSS patients compared to controls. As both Ro52 and Ro60-specific cells have been detected in the salivary glands (SG) of pSS patients, we aimed to characterize the SSA-specific B cell pattern in SG biopsies. A series of double immunohistochemical stainings were performed on paraffin-embedded tissue from 10 well-characterized pSS patients for each Ro52 and Ro60 along with CD19, CD5, CD20 or CD27, respectively. Ro52 and Ro60-specific cells detected in SG tissue were found to be CD19(+) B cells located outside the CD19(+)/CD20(+) B cell zones (BCZ) and also interstitially. These SSA-specific cells were also quantified. No SSA-specific cells were CD5(+), indicating that they do not belong to the B-1 B cell subset. Furthermore, no SSA-specific cells were observed within the CD20(+) BCZ. Hence, no SSA-specific memory B cells were detected in these individuals. Contrary to this, SSA-specific cells were found to be CD19(+)/CD27(++), demonstrating that they are differentiating short or long-lived plasma cells. Taken together, our findings suggest that these lower levels of SSA-specific memory B cells in PB and absence of SSA-specific memory B cells in SG of pSS patients could result from activation of these cells into plasma cells at the site of inflammation.

Altered CD40 and E-cadherin expression--putative role in oral lichen planus.

BACKGROUND: Oral lichen planus (OLP) is characterized among other features by apoptosis of basal keratinocytes. To identify potential regulatory mechanisms associated with basal cell apoptosis in OLP, we investigated the expression of CD40, CD40 ligand (CD40L), CD44 and epithelial (E)-cadherin. METHODS: Biopsies from 22 patients with OLP were investigated by immunohistochemistry for detection of CD40, CD40L, E-cadherin, CD44, Laminin-5 and Collagen IV, double-labelling for CD40 and CD3, and in situ mRNA hybridization for CD40 and CD40L. RESULTS: In actively diseased areas of OLP lesions, basal keratinocytes did not express CD40 and were focally E-cadherin-negative, in contrast to non-diseased areas and normal oral mucosa. Demonstration of intraepithelial T cells expressing CD40 and CD40L, indicates a potential role in inflammatory cell responses involved in the disease process of OLP. CONCLUSION: T cells may orchestrate inflammatory cell responses in OLP via CD40-CD40L interactions. As basal keratinocytes downregulate CD40, they may escape CD40-CD40L-induced apoptosis in OLP. On the other hand, loss of E-cadherin expression may contribute to epithelial basal cell destruction and T-cell migration into the epithelial compartment in OLP.

Elevated serum levels of soluble E-cadherin in patients with primary Sjögren's syndrome.

The aim of this study was to investigate serum levels of soluble E-cadherin (sE-cadherin) in relation to lymphocytic organization and to characterize the expression of E-cadherin and integrin alphaEbeta7/CD103 in salivary gland epithelium of patients with Sjögren's syndrome (SS). Serum levels of sE-cadherin were significantly increased in SS compared to non-SS and nonsignificantly in germinal centre (GC)+ compared to GC- patients. Membrane-bound E-cadherin was detected on the majority of acinar and ductal epithelial cells in both SS and non-SS. alphaEbeta7/CD103-positive cells were found scattered in focal infiltrates and GC, and in small clusters close to ductal and acinar epithelium at an increased level in SS compared to non-SS. Interestingly, E-cadherin-positive cells were detected randomly dispersed in focal lymphocytic infiltrates in 10/21 patients. By double-labelling, the cells with the E-cadherin-positive component were identified as CD68(+) macrophages. Elevated serum levels of sE-cadherin indicate an increased epithelial cell turnover and shedding, and sE-cadherin deserves further analysis as a potential diagnostic tool for SS.