Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins

Abstract:  The identification of tumor-specific proteins located at the plasma membrane is hampered by numerous methodological pitfalls many of which are associated with the post-translational modification of such proteins. Here, we present a new combination of detergent fractionation of cells and of subtractive suppression hybridization (SSH) to gain overexpressed genes coding for membrane-associated or secreted proteins. Fractionation of subcellular components by digitonin allowed sequestering mRNA of the rough Endoplasmatic reticulum and thereby increasing the percentage of sequences coding for membrane-bound proteins. Fractionated mRNAs from the cutaneous T-cell lymphoma (CTCL) cell line HuT78 and from normal peripheral blood monocytes were used for SSH leading to the enrichment of sequences overexpressed in the tumor cells. We identified some 21 overexpressed genes, among them are GPR137B, FAM62A, NOMO1, HSP90, SLIT1, IBP2, CLIF, IRAK and ARC. mRNA expression was tested for selected genes in CTCL cell lines, skin specimens and peripheral blood samples from CTCL patients and healthy donors. Several of the detected sequences are clearly related to cancer, but have not yet been associated with CTCL. qPCR confirmed an enrichment of these mRNAs in the rough endoplasmic reticulum fraction. RT-PCR confirmed the expression of these genes in skin specimens and peripheral blood of CTCL patients. Western blotting verified protein expression of HSP90 and IBP2 in HuT78. GPR137B could be detected by immunohistology in HuT78 and in keratinocytes of dysplastic epidermis, but also in sweat glands of healthy skin. In summary, we developed a new technique, which allows identifying overexpressed genes coding preferentially for membrane-associated proteins.     

Genetics in multiple sclerosis: past and future perspectives

The enormous development in the field of molecular genetics during the last decades has lead to optimism concerning the possibilities for identifying the causes of multiple sclerosis (MS) through genetic studies. However, we have learned that dense mapping of large sample sets is needed, which only can be achieved through large collaborative studies. The contribution from each yet unidentified gene is probably weaker than that of the well established human leukocyte antigen association. The ultimate goal of the search for susceptibility genes in MS is to develop diagnostic tools and better treatments that can prevent or reduce the development of symptoms of this often devastating disease.     

Ex vivo neutrophil function in response to three different doses of glycosylated rHuG-CSF (Lenograstim)

Granulocyte colony-stimulating factor (G-CSF) is being considered as adjuvant treatment for infections in non-neutropenic patients. Normal healthy donors were given rHuG-CSF (Lenograstim) at 2.5, 5.0 and 7.5 μg/kg subcutaneously daily for 5 d. Polymorphonuclear leucocyte (PMN) function tests were carried out on peripheral blood PMN before the first injection and at 24 h and 96 h. Circulating PMN levels were also measured at these time intervals and found to be significantly increased with all doses by 24 and 96 h. Investigation of cell surface antigen expression revealed no changes in β₂ integrin (CD11b/CD18) expression. L-selectin (CD62L) expression was reduced with all doses by 96 h, this being significant with the 7.5 μg/kg dose. FcγRI (CD64) levels were significantly up-regulated with the 7.5 μg/kg dose by 96 h whereas FcγRIII (CD16) expression was found to be reduced during G-CSF treatment. Superoxide anion production was significantly increased in response to N -formylmethionylleucylphenylalanine (FMLP) and opsonized Staphylococcus epidermidis stimulation at 24 h and 96 h with the 5.0 and 7.5 μg/kg doses. The initial rate of phagocytosis (0–2 min) appeared to have increased with the 7.5 and 5.0 μg/kg doses at 96 and 24 h compared with PMN responses pretreatment, although these increases were not statistically significant. These results show that G-CSF enhances the functional responses of PMN stimulated by physiological agonists and may help in the treatment of infections.     

Human Renal Allograft Rejection Despite the Absence of Allogeneic Passenger Leukocytes

Passenger leukocytes have been suggested to be both pro-tolerant and immunogenic. The opportunity to evaluate the role of allogeneic passenger leukocytes in humans was presented by a 47-year-old man who donated bone marrow to his HLA-identical leukemic sister. Eleven years later he developed renal failure. The sister's marrow was noted to be 100% XY karyotype and free of malignancy. She donated a kidney to her brother. Immunosuppression was tapered following transplantation. After 6 months, the recipient was on monotherapy sirolimus, 1 mg every third day. A surveillance biopsy was normal and sirolimus was stopped. Eight weeks later, he presented with severe rejection that reversed with Thymoglobulin. Renal function returned to baseline and has been stable on conventional immunosuppression.     

PCR-SSP法HLA-A、B基因分型与血清学分型的比较

目的比较聚合酶链反应-序列特异性引物(PCR-SSP)进行HLA-Ⅰ类A、B抗原位点分型的准确性,并探讨血清学分型错误发生的原因。方法用PCR-SSP以及单克隆抗体血清学分型技术对HLA-A、B分型并比较。结果34例样本PCR-SSP基因分型无假阳性和假阴性出现。PCR-SSP法与血清学比较,血清学检出错误或漏检率分别为HLA-A位点23.5%,B位点26.5%。血清学发生错误或易混淆的抗原有:A2和A68、A32和A33,B5、B60和61。结论PCR-SSP法进行HLA-A、B抗原等位基因分型具有分辨率高、特异性强、重复性好、实验过程简捷快速、分型结果较血清学更加准确可靠的优点。     

Kawasaki Disease in a Father and Son

The pathogenesis and etiology of Kawasaki disease are unknown, but some studies suggest increased genetic susceptibility. The case is presented of an infant with Kawasaki disease whose father suffered from the same illness 21 years previously. The A, B and C loci of the HLA antigens were examined.     

Identification of Babesia bigemina infected erythrocyte surface antigens containing epitopes conserved among strains

The presence of previously uncharacterized antigens (new antigens) on the surface of intact erythrocytes infected with three strains of Babesia bigemina from Kenya and one each from Puerto Rico, Mexico, St. Croix, and Texcoco-Mexico was demonstrated by indirect immunofluorescent antibody (IFA) reactions. These antigens were not strain specific because antibodies in bovine immune serum to either the Mexico or Kenya isolates reacted with all seven strains tested. Homologous and heterologous immune serum antibodies bound a maximum of 83% and 55%, respectively, of intact erythrocytes infected with the Kenya-Ngong strain but not uninfected erythrocytes. Both sera caused agglutination of only infected erythrocytes. Antibodies eluted from the surface of glutaraldehyde (0.25%) fixed infected erythrocytes had IFA reaction patterns among strains similar to those of immune sera before elation. Eluted antibodies were used to determine if these antigens were protein and encoded by B. bigemina. Eluted antibodies bound seven parasite-encoded proteins of 240, 220, 66, 62, 58, 52 and 38 kDa in an erythrocyte surfacespecific immunoprecipitation reaction of ³⁵-methionine labelled proteins. It was concluded that the surface of B. bigemina infected erythrocytes had parasite-encoded proteins and that these proteins had surface exposed epitopes that were conserved among the seven strains examined which were from two continents.     

Peripheral Blood Lymphocyte Surface Antigen Expression and Prognosis in Myeloma: Australian Leukaemia Study Group Study

The Australian Leukaemia Study Group myeloma study (MM1) aimed to determine the prognostic significance of clinical and immunophenotypic markers in patients with multiple myeloma. All patients were treated with standard dose melphalan and prednisone. Seventy-four patients were entered and the median survival was 27 months. Serum beta 2-microglobulin (βM) and albumin levels were the only significant clinical factors influencing survival (p = 0.007 and p = 0.008, respectively). Patients with raised levels of CD38+ lymphocytes at presentation had a significantly shorter survival than patients with normal levels (p = 0.01, logrank test, median 19 months vs 33 months). CD38 antigen expression was independent of β2M but patients with raised levels of CD38 had significantly lower levels of albumin than patients with normal levels (p = 0.001) which may explain their poorer survival. Salmon and Durie stage was not associated with antigen expression. No other B-cell antigens (CD10, CD19, CD20, CD21, CD22, CD23, FMC1 or FMC7) or plasma cell antigens tested (PCA-1) were found to be associated with prognosis. Patients who achieved plateau phase had a better prognosis than those who did not (p = 0.04 in a landmark analysis). Patients who achieved plateau phase following an objective response appeared to have a better prognosis than those who were in plateau phase at presentation (p = 0.09 in a landmark analysis). Light chain isotype suppression (LCIS) was not associated with a significant survival advantage and did not correlate with any known prognostic indicator. We conclude that phenotypic analysis of peripheral blood lymphocytes for CD38 antigen at diagnosis may be useful as a prognostic indicator in patients with myeloma.     

HLA-DR7 predicts the response to alkylating agents in steroid-sensitive nephrotic syndrome

There is a lack of reliable predictors of the response to alkylating agents in children with idiopathic nephrotic syndrome (NS). HLA-DR7 is strongly associated with the frequency of relapses in steroid-sensitive NS before cytostatic therapy. We therefore examined retrospectively the time to the first relapse and the incidence of subsequent relapses in 54 HLA-typed children with frequently relapsing NS, after treatment with cyclophosphamide (n = 49) or chlorambucil (n = 5) for 8 or 12 weeks; 38 patients were HLA-DR7 positive and 16 negative with 80% in both groups being steroid dependent. HLA typing was performed using serological or DNA typing methods. Renal biopsy showed minimal glomerular changes. A lower proportion of HLA-DR7 positive than negative patients remained in remission after 3 years (36% vs. 81%, P<0.02) and 5 years (36% vs. 72%, P<0.03). In the first 3 years after cytostatic therapy the mean number of prednisone-treated relapses was 1.3/patient per year in HLA-DR7-positive patients compared with 0.4 in negative patients (P<0.025). There was no statistically significant difference in the proportion of relapse-free patients with and without steroid dependency. The HLA status predicts the response of NS patients to alkylating agents better than the rate of previous relapses. Received September 19, 1995; received in revised form and accepted April 16, 1996     

Significance of the BTA Test in Bladder Cancer: A Multicenter Trial

Background :
The BTA test is a latex agglutination assay for the qualitative detection in the urine of analytes that are associated with bladder tumor. We compared the results of the BTA test with those of voided urine cytology (VUC) in patients with bladder cancer.
Methods :
A multicenter trial was performed at 6 institutions. A total of 132 patients with histologically diagnosed bladder cancer were enrolled. Urine samples were split for BTA and VUC testing.
Results :
The sensitivities of the BTA test and VUC were 57.6% and 37.9%, respectively; this difference was significant ( P < 0.001). The BTA test had much higher sensitivity for small, solitary, superficial tumors than did VUC.
Conclusion :
The BTA test is simple to perform, gives rapid results, and is far more sensitive than VUC for detection of bladder cancer. The BTA test has the potential to become an additional tool for detecting bladder cancer.