Stoffwechseluntersuchungen bei tiefer Hypothermie unter 20° und langdauerndem artefiziellem Kreislaufstillstand

Ohne Zusammenfassung     

The ancillary proteins of HATs: SLC3 family of amino acid transporters

The heteromeric amino acid transporters (HATs) are composed of a light and a heavy subunit linked by a disulfide bridge. The heavy subunits are the SLC3 members (rBAT and 4F2hc), whereas the light subunits are members of the SLC7 family of amino acid transporters. SLC3 proteins are type II membrane glycoproteins (i.e., one single transmembrane domain and the C-terminus located outside the cell) with a bulky extracellular domain that shows homology with alpha-glucosidases. rBAT heterodimerizes with b(0,+)AT (SLC7A9) constituting the amino acid transport b(0,+), the main system responsible for the apical reabsorption of cystine in kidney. The defect in this system causes cystinuria, the most common primary inherited aminoaciduria. 4F2hc subserves various amino acid transport systems by dimerization with different SLC7 proteins. The main role of SLC3 proteins is to help routing of the holotransporter to the plasma membrane. A working model for the biogenesis of HATs based on recent data on the rBAT/b(0,+)AT heterodimeric complex is presented. 4F2hc is a multifunctional protein, and in addition to its role in amino acid transport, it may be involved in other cellular functions. Studies on two SLC7 members (Asc-2 and AGT1) demonstrate heterodimerization with unknown heavy subunits.     

Oral DNA vaccination with a plasmid encoding soluble ICAM-1 modulates cytokine expression profiles in nonobese diabetic mice

Adhesion molecules are important for leukocyte extravasation and for the delivery of costimulatory signals in T cell activation. We therefore interfered in the immune process leading to islet inflammation in diabetes prone NOD mice by oral vaccination with plasmid DNA encoding soluble ICAM-1. Female NOD mice were treated orally with ICAM-1, TGF-beta, or control plasmid DNA and received a single injection of cyclophosphamide for synchronization and acceleration of the disease process in the pancreas. Quantitative RT-PCR analysis of pancreatic mRNA showed that cyclophosphamide induced the expression of Th1 cytokines (IFN-gamma and IL-12p40) in vehicle- or control plasmid-treated mice. Treatment with ICAM-1 and TGF-beta DNA resulted in increased levels of IL-10 mRNA in the pancreas, indicating an anti-inflammatory regulatory immune response. Histological analysis of pancreatic islets showed that the DNA treatment did not alter islet infiltration in response to cyclophosphamide. Hence vaccination with the ICAM-1 plasmid had not suppressed leukocyte migration but rather modulated lymphocyte activity, similarly as seen for the TGF-beta-encoding plasmid. Neither of the three plasmids caused recognizable changes in cytokine expression in the small intestine, Peyer's patches, or mesenteric lymph nodes. We conclude that oral vaccination with DNA encoding immunoregulatory molecules such as ICAM-1 and TGF-beta represents an approach for modulating the ongoing inflammatory process in the pancreas of diabetes prone NOD mice.     

Molecular pathology of well-differentiated thyroid carcinomas

The newly discovered molecular features of well-differentiated thyroid carcinomas derived from follicular cells are reviewed, within the frame of the 2004 WHO classification of thyroid tumours, under the following headings: “Follicular carcinoma”, “Papillary carcinoma”, “Follicular variant of papillary carcinoma” and “Hürthle cell tumours”. A particular emphasis is put on the meaning of PAX8–PPARγ rearrangements, RAS and BRAF mutations, and deletions and mutations of mitochondrial genes and of nuclear genes encoding for mitochondrial enzymes, for thyroid tumorigenesis.     

Biochemical characterization of new strains of<Emphasis Type="Italic"> Trypanosoma cruzi</Emphasis> and<Emphasis Type="Italic"> T. rangeli</Emphasis> isolates from Peru and Mexico

Seven trypanosome stocks isolated have been characterized by lectin agglutination, isoenzyme analysis, and the end products excreted. The stocks were isolated from different geographic areas—one from Mexico (TM5), and six from Peru, four of these isolated from different species of triatoma (TP504, TP702, TP704 and TP706), the other two isolated from the salivary glands of Rhodnius ecuadorensis (TRa605 and TRa606). Additionally, one strain of Trypanosoma cruzi isolated from a human case (strain TC-Maracay) and one strain of T. rangeli (TRa, Cajamarca-Peru strain), characterized and maintained in our laboratory, were used as reference strains. According to statistical study, the stocks were grouped into three clusters: (1) cluster I included the reference strain of T. cruzi (TC-Maracay); (2) cluster II was subdivided into two groups—subcluster IIA for the Mexican isolate (TM5) and subcluster IIB for the Peruvian ones, isolated from the salivary glands of Rhodnius ecuadorensis (TRa 605 and TRa 606) and the reference strain T. rangeli (TRa); these two new isolates were classified as T. rangeli; and (3) cluster III for the rest of the Peruvian isolates, which should be considered at least as a different strain from the T. cruzi strain Maracay. We show that the identification of T. cruzi and T. rangeli in mixed infections is readily achieved by biochemical methods. These findings identified three clusters of Mexican and Peruvian stocks that correlate with geographic origin, although assignment to a T. cruzi linage was not possible.     

Currant allergy and the Rosaceae-grass pollen allergy syndrome: a case report.

BACKGROUND: Despite the increasing use of currants in culinary recipes, currant allergy has rarely been reported. OBJECTIVES: To study a case of currant allergy and to explore cross-reactivity between grass pollen and Rosaceae family fruit allergens. METHODS: Skin prick tests to pollen and skin prick-to-prick tests with currants and peach were performed. Specific IgE levels were determined using the CAP method. We prepared a protein extract of 0.1 mg/mL in phosphate-buffered saline using red currant in the presence of protease inhibitors. Immunoblot inhibition studies were performed to explore cross-reactivity between grass pollen and currant allergens. RESULTS: Skin prick test results were positive to Dactylis, arizonic, and olive pollens. Results of skin prick-to-prick tests with fresh red and black currants were negative and positive, respectively, to peach. The specific IgE level was 5.7 KU/L to red currant and 2.92 KU/L to peach (CAP). Western blot analysis with red currant extract revealed specific IgE protein bands of 37 and 26 kDa. Preincubation of sera with extracts from red currant and peach inhibited both IgE bands, and preincubation with Dactylis pollen inhibited the 37-kDa band only. CONCLUSIONS: We report a case of allergy to grass pollen with an oral allergy syndrome involving several fruits from 2 different families of the Rosidae subclass confirmed by in vitro tests. Inhibition studies demonstrated cross-reactivity between different fruits (currant and raspberry) from the Rosidae subclass and were incomplete with grass pollen allergens.     

Chemokine receptors that mediate B cell homing to secondary lymphoid tissues are highly expressed in B cell chronic lymphocytic leukemia and non-Hodgkin lymphomas with widespread nodular dissemination

B cell neoplasms present heterogeneous patterns of lymphoid organ involvement, which may be a result of the differential expression of chemokine receptors. We found that chemokine receptor (CCR)7, CXC chemokine receptor (CXCR)4, or CXCR5, the main chemokine receptors that mediate B cell entry into secondary lymphoid tissues and their homing to T cell and B cell zones therein, were highly expressed in B malignancies with widespread involvement of lymph nodes. Conversely, those pathologies with little or no nodular dissemination showed no expression to very low levels of CCR7 and CXCR5 and low to moderate levels of CXCR4. These findings provide evidence for the role of CCR7, CXCR4, and CXCR5 in determining the pattern of lymphoid organ involvement of B tumors. Functional studies were performed on B malignancies expressing different levels of CCR7, CXCR5, and CXCR4. Multiple myeloma (MM) cells did not express CCR7 nor CXCR5 and did not migrate in response to their ligands; a moderate expression of CXCR4 on MM cells was accompanied by a migratory response to its ligand, CXCL12. By contrast, cells from B cell chronic lymphocytic leukemia (B-CLL) expressed the highest levels of these chemokine receptors and efficiently migrated in response to all ligands of CCR7, CXCR4, and CXCR5. In addition, the migration index of B-CLL cells in response to both of the CCR7 ligands correlated with the presence of clinical lymphadenopathy, thus indicating that the high expression of functional chemokine receptors justifies the widespread character of B-CLL, representing a clinical target for the control of tumor cell dissemination.     

Microhardness of superficial and deep sound human dentin

Our purpose in this study was to determine the microhardness of superficial and deep dentin by means of two indentation methods (Knoop and Vickers) under two different applied loads. Twelve dentin discs approximately 2-mm thick were obtained from both superficial and deep dentin by transversally sectioning the crowns of sound, extracted human third molars with a diamond blade under water irrigation. Dentin surfaces were sequentially polished, and indentations (n = 20 per surface) were performed with either Vickers indentor at loads of 300 and 500 g, respectively, or Knoop indentor at loads of 50 and 100 g, respectively. Average Vickers hardness number (VHN) and Knoop hardness number (KHN) were calculated and treated with two-way analysis of variance (ANOVA) and Student's t test. Microhardness of dentin was not influenced by the different loads applied for both indentation methods. Knoop hardness was significantly higher for superficial than for deep dentin (p < 0.05). Conversely, Vickers hardness was not significantly different for both substrates (p > 0.05). Differences in dentin hardness as a function of depth exist, but they might not be relevant, and no alteration of the distribution of stresses along the adhesive interface is expected.     

Polarized expression of Na+/H+ exchange activity in LLC-PK1/PKE20 cells: II. Hormonal regulation

LLC-PK₁/PKE₂₀ cells (a continuous epithelial cell line) has two different Na/H exchange activities: Na/H-1 located in the basolateral membrane and Na/H-2 located in the apical membrane [Casavola et al. (1989) Biochem Biophys Res Commun 165:833–837; Haggerty et al. (1988) Proc Natl Acad Sci USA 86:6797–6801]. In the present report we have studied hormone regulation of these exchange activities by measuring Na-dependent recovery of pH_i from an acid load (by using microspectrofluorometry and 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein) in response to activation of regulatory cascades by either pharmacological agents or by vasopressin or calcitonin. Agents leading to activation of protein kinase A (cAMP-dependent), such as forskolin (10 M), 8-Br-cAMP (0.25 mM), and isobutylmethylxanthine (0.5 mM), inhibited Na/H-2 and Na/H-1 by an average of 49%. Stimulation of protein kinase C by a phorbol ester (phorbol 12-myristate 13-acetate, TPA, 100 nM) inhibited Na/H-2 (by an average of 48%) and stimulated Na/H-1 (by an average of 38%); these effects of TPA were also observed in the presence of forskolin (100 M). Addition of either vasopressin (2 M) or calcitonin (0.3 M) onto both sides of the monolayer decreased the activity of Na/H-2 by an average of 26.3% and 27.7% respectively, and stimulated the activity of Na/H-1 by an average of 17.4% and 38.7% respectively; exposure of cells to either hormone stimulated production of cAMP and inositol trisphosphate, respectively. Separate hormone additions to either the apical or basolateral cell surface led to effects similar to those produced by simultaneous hormone additions onto both cell surfaces, although the relative response of Na/H exchangers to either agonist is variable. In summary, these results suggest that in LLC-PK ₁/PKE₂₀ cells, vasopressin and calcitonin can act via receptor systems coupled either to adenylate cyclase or to phospholipase C. Activation of these receptor systems can lead to inhibition of Na/H-2 and stimulation of Na/H-1.     

The Immune Reactivity Role of HCV-Induced Liver Infiltrating Lymphocytes in Hepatocellular Damage

Liver infiltrating lymphocytes (LIL) were isolated from HCV-positive (+) and HCV-negative (–) end-stage livers. Phenotypic analysis and functional studies using proliferative and lymphocytotoxic assays were performed with the isolated LIL. Two CD3+ lymphocyte populations were found in LIL using FITC anti-CD3 monoclonal antibodies (mAb). One was a bright fluorescence intensity population (as in PBL), and the other dim. We calculated the number of FITC-anti-CD3 mAbs bound per lymphocyte on PBL and LIL and found 80,040 ± 4628 and 39,615 ± 3932, respectively. Therefore, HCV+ and HCV– patient PBL contained approximately twice the number of CD3 molecules per cell than patient CD3+ LIL. LIL also contained approximately a threefold higher concentration of TCR+, CD4–CD8–, and CD56,16 (NK) cells than the patient PBL. Thus, a major subset of LIL is phenotypically similar to mouse NK1.1+ intermediate T cells. LIL freshly isolated from HCV+ livers exhibited weak CTL activity against EBV- or Con A-transformed lymphoblast targets infected with vaccinia–HCV recombinant virus (rHCV) or primary hepatocyte cultured cells. However, after in vitro coculture of LIL with rHCV, these cells developed a strong cytotoxicity for the above targets. In contrast, LIL from HCV– livers were not cytotoxic against the same targets. Histochemical studies (in situ) demonstrated that these hepatocytes express CD95, and stains demonstrated apoptosis. The HCV+ hepatocytes also express class I MHC molecules and ICAM-1. The addition of mAb specific for these adhesion molecules inhibited CML activity. Short-term cultured hepatocytes (targets) from HCV+ and HCV– patients produced low levels of cytokines IL-1, IL-2, IL-6, TNF, and IFN- but a high level of IL-8. It is speculated that LIL expressing reduced numbers of CD3 molecules may even function as immune regulators as proposed for intermediate T cells in mice.