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.     

Positron emission tomography of the thorax. The current clinical status]

The non-invasive diagnostic assessment of thoracic lesions has been greatly enhanced by the use of positron emission tomography with metabolic active radio-compounds such as fluor-18 labeled deoxyglucose. Four clinical fields of use were established: differential diagnostic evaluation of lesions (T-staging); classification of adenopathy (N-staging); therapy response monitoring; diagnostic evaluation of recurrence. The upcoming challenges for clinical use are limited availability and necessary technical and qualitative standardizations.     

High abundances of neurotrophin 3 in atopic dermatitis mast cell

Background  

Neurotrophin 3 (NT-3) is a member of the neurotrophin family, a group of related proteins that are known to regulate neuro-immune interactions in allergic diseases. Their cellular sources and role in the recruitment of mast cell precursors in atopic dermatitis have not been characterized in detail so far.     

Presumed vestibular hemorrhage secondary to warfarin

Hemorrhage secondary to anticoagulant therapy is well documented. We report a patient who presented with acute vertigo and unilateral deafness while on warfarin and was found to have a probable hemorrhage in the labyrinth, identified on MRI.     

Early recognition of postoperative hematoma formation

Summary Postoperative hematoma formation must be treated as a potential infection. One fourth of all postoperative hematomas are already contaminated. Ultrasonographical examination is an effective method for early recognition of such postoperative hematomas. Ultrasonic diagnosis on a routine basis is not necessary, but it should be carried out as soon as clinical symptoms appear. Our postoperative late results with only one early and one late infection after 100 postoperative treatments of the hip joint and femoral shaft emphasize the importance of early diagnosis of hematomas.

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Zusammenfassung Die drohende Infektion im postoperativen Verlauf nach Osteosynthesen bereitet diagnostische Probleme, da keine objektiven Kriterien vorhanden sind. Das postoperative Hämatom ist als eine drohende Infektion zu beurteilen. 1/4 aller postoperativen Hämatome sind bereits kontaminiert. Die Ultraschalluntersuchung ist eine wirksame diagnostische Hilfe zur frühzeitigen Erkennung der drohenden Infektion bei postoperativen Hämatomen. Sie muß sicherlich nicht routinemäßig, bei klinischem Verdacht aber frühzeitig durchgeführt werden. Das postoperative Spätergebnis mit lediglich einem Frühund Spätinfekt bei 100 operativen Eingriffen am Hüftgelenk und Oberschenkelschaft belegen die Bedeutung der frühzeitigen Hämatomdiagnostik.

     

Lipopolysaccharide contamination of beta-lactoglobulin affects the immune response against intraperitoneally and orally administered antigen

BACKGROUND: Microbial components in the environment are potent activators of the immune system with capacity to shift the active immune response towards priming of Th1 and/or Th2 cells. Lipopolysaccharide (LPS), a cell-wall component of Gram-negative bacteria, is extensively present in food products like cow's milk. It is not well established, however, how this presence of LPS affects oral tolerance induction. METHODS: We studied the effect of LPS contamination in a commercial preparation of the cow milk protein beta-lactoglobulin (beta-LG) on antigen-specific immune responses. IgG1/IgG2a production upon intraperitoneal immunization without adjuvant was measured, and oral tolerance induction against beta-LG after administration of either an aqueous solution or water-in-oil (w/o) emulsion of beta-LG was evaluated. RESULTS: LPS contamination of beta-LG provoked a beta-LG-specific IgG2a response, as well as an enhanced beta-LG-specific IgG1 response upon intraperitoneal immunization. Oral tolerance induction to beta-LG was induced by aqueous solutions of beta-LG with and without LPS administration. Conversely, oral administration of w/o-emulsified beta-LG prevented oral tolerance to beta-LG only when the beta-LG was contaminated with LPS. CONCLUSIONS: LPS contamination of an aqueous protein solution does not affect oral tolerance induction, whereas LPS present in emulsion prevents oral tolerance induction towards the food protein.