HLA-DR residues accessible under the peptide-binding groove contribute to polymorphic antibody epitopes

Many residues involved in polymorphic antibody-binding epitopes on class II molecules are located on the -helix of DRβ chains. Although they have received less attention, residues in the peptide-binding groove and second domain of the DRβ chain may also be critical for polymorphic anti-DR antibody epitopes. In this study, we used transfectants expressing site-directed mutations at positions in the HLA-DR β₁ and β₂ domains and flow cytometry to define the epitopes of several polymorphic anti-DR antibodies. Both DR(β 1^*0403) residues 14 and 25 were shown to be involved in the epitopes of mAbs DA6. 164, HU-20, Q5/6, and 50D6, and DR(β 1^*0701) residue 14 was shown to be critical for the epitopes of two DR7-specific mAbs, SFR16-DR7M and TAL 13. 1. Unlike most other residues shown to be important in antibody-binding epitopes, residue 14 is located in the floor of the peptide-binding groove and residue 25 is in an outer loop, each with their side chains pointing down, such that antibodies may directly contact these residues from below the binding groove. Two residues in the β₂ domain, β180 and β181, were also shown to be involved in the epitopes of three polymorphic anti-DR mAbs, NFLD.D1, NFLD.M1, and LY9. Although these two residues are close to the transmembrane domain in the linear sequence, their solvent accessibility in the DR1 structures is quite impressive. Our data provide new evidence that residues accessible under the peptidebinding groove contribute to polymorphic antibody-binding epitopes.     

Characterization of an inversion duplication of the short arm of chromosome 8 by fluorescent in situ hybridization

A de novo chromosome aberration in a woman with severe mental retardation and minor anomalies has been characterized cytogenetically. The patient's karyotype was described as 46, XX, inv dup (8)(p12 → p23.1). Previous Southern blot dosage studies with the marker locus D8S7 demonstrated that the patient was monosomic for this locus, suggesting that the rearrangement generated a duplication-deficiency chromosome. We have reinvestigated this patient using fluorescent in situ hybridization with chromosome 8 cosmids and an Alu-PCR product specific for 8p. These studies have confirmed directly that the duplicated chromosome also has undergone deletion. © 1992 Wiley-Liss, Inc.     

Real-time PCR method for the quantitative analysis of human T-cell receptor gamma and beta gene rearrangements

Analyzing the status of T-cell receptor (TCR) gene rearrangements has been an essential part of deciphering the stages of thymocyte development, understanding the β vs. γδ lineage decision, and characterizing T-cell leukemias. Methods such as PCR and quantitative Southern blotting provide useful information, but also have significant shortcomings such as lack of quantitation in the case of PCR and technical challenges in the case of Southern blotting. Here we describe a real-time PCR method that overcomes many of these shortcomings. This new method shows comparable results for the fraction of unrearranged TCRγ and TCRβ genes in human thymocytes and peripheral blood T cells as Southern blotting, and has the advantages of being simple to perform, highly quantitative, and requiring nanogram quantities of DNA. We also describe a real-time PCR method to quantitate T-cell receptor excision circles formed during TCRβ rearrangements.     

Establishment and characterization of cell line TNMY1 derived from human malignant fibrous histiocytoma

Although malignant fibrous histiocytoma (MFH) is one of the most common soft tissue sarcomas, its pathogenesis remains unclear. In this study, a cell line derived from human MFH, TNMY1, was established from a metastatic chest-wall lesion of a 60-year-old woman with MFH. The TNMY1 cell line was passaged 95 times, and it still retained the biological characteristics of the original tumor. TNMY1 consists of spindle-shaped cells and pleomorphic cells associated with multinucleated giant cells. Immunohistochemical studies showed that the spindle-shaped and pleomorphic cells were positive for vimentin, CD68 and alpha-smooth muscle actin, but negative for epithelial membrane antigen, desmin, muscle actin, alpha-sarcomeric actin, myoglobin, lysozyme and S-100 protein. The cells expressed collagen types I, III and V. These results indicate that MFH may originate from mesenchymal stem cells with the potential to differentiate into either fibroblasts or histiocytes. An elevated level of collagen type V mRNA expression is considered to support a diagnosis of MFH.     

音猬因子的功能受体斑片在培养神经干细胞中的表达

目的　观察在培养的神经干细胞内是否有发育调控分子———音猬因子 (sonichedgehog)功能受体———斑片 (patched)表达。　方法　神经干细胞克隆在体外培养传代后 ,用patched的特异性引物对培养的神经干细胞进行RT PCR分析 ,PCR产物经克隆测序后 ,用地高辛标记克隆的探针 ,对神经干细胞进行原位杂交分析。　结果　神经干细胞克隆内大量的细胞均可表达sonichedgehog的功能受体patched ,patched阳性细胞间未见明显差别 ,克隆边缘与中央的patched分布也未见明显差别。　结论　sonichedgehog信号传导路可能在神经干细胞的增殖与分化过程中起重要作用。     

PCR-based assays for the detection of monoclonality in non-Hodgkin's lymphoma: application to formalin-fixed,paraffin-embedded tissue and decalcified bone marrow samples

In crucial cases the diagnosis of non-Hodgkin's lymphoma (NHL) still represents a challenge to the pathologist since morphological criteria do not always help to distinguish between reactive and malignant lymphoproliferations. Clonality assays are a useful supplement since monoclonal cell proliferation is strong evidence for malignancy. The polymerase chain reaction (PCR) can be utilized to establish the clonal origin of B-or T-cell lymphocyte populations by amplification of rearranged immunoglobulin and T-cell receptor (TCR) genes. In the present study DNA was isolated from a variety of neoplastic and nonneoplastic formalin-fixed, paraffin-embedded lymph nodes (n=62), cutaneous tissue (n=9), samples of miscellaneous origin (n=11), and, reported here for the first time, decalcified bone marrow samples (n=35). These samples were submitted to PCR-based assays directed against the immunoglobulin heavy-chain (IgH), immunoglobulin light-chain (IgL), and TCR chain genes. The impact of various decalcifying agents on the ability to amplify DNA was investigated by PCR-based amplification of a single copy gene. Buffered and nonbuffered EDTA was found not to impede amplification of DNA fragments up to 300 bp in length. In lymph node and cutaneous specimens monoclonality was detected in 83% of B-NHL cases using a seminested PCR approach for the amplification of IgH, whereas the same approach gave rise to monoclonal bands in 80% of bone marrow samples. The subsequent amplification of IgL helped to raise the sensitivity of detection to 94%. Monoclonality was detected in seven of nine T-cell NHLs by amplification of TCR. Most of the false-negative results were related to DNA extracted from centroblastic-centrocytic lymphoma and lymphoplasmocytic immunocytoma (37% negative each). PCR-based rearrangement analysis of immunoglobulin and TCR chain genes should be used in diagnostic pathology for cases which are histopathologically and immunohistochemically questionable. The application of clonality assays to bone marrow samples previously decalcified with EDTA provides a new tool for the detection of minimal residual disease.Abbreviations BALT bronchus-associated lymphoid tissue - dNTP deoxynucleoside triphosphate - Ig immunoglobulin - IgH immunoglobulin heavy chain - IgL immunoglobulin light chain - MALT mucosa-associated lymphoid tissue - NHL non-Hodgkin's lymphoma - PCR polymerase chain reaction - TCR T-cell receptor     

Screening for the two most frequent mutations in Leber's hereditary optic neuropathy by duplex PCR based on allele-specific amplification

This report describes a rapid and inexpensive assay, which allows detection, in whole blood and by PCR alone, of the two most frequent mitochondrial DNA mutations causing Leber's hereditary optic neuropathy. The assay is based on allele-specific amplification, using primers with the mutation-specific base in the 3′ position, and a deliberately introduced G→C Substitution of base no. four from the 3′ end, which prevents amplification of the wild-type allele. © 1993 Wiley-Liss, Inc.