The nonfunctional allele TCRBV6S1B is strongly associated with Helicobacter pylori infection

To determine genetic susceptibility factors for Helicobacter pylori infection, polymorphic T-cell receptor gene elements were investigated in 203 H. pylori-infected individuals and 180 uninfected individuals (controls). H. pylori infection is highly associated with individuals homozygous for the nonfunctional TCRBV6S1B element (odds ratio = 5.9; chi(2) = 13; P = 0.00032; P value corrected for multiple comparisons [Bonferroni correction] = 0. 00063).     

Quantitative detection of respiratory Chlamydia pneumoniae infection by real-time PCR

Real-time PCR was evaluated as a quantitative diagnostic method for Chlamydia pneumoniae infection using different respiratory samples. Real-time PCR had efficiency equal to or better than that of nested touchdown PCR. This study confirmed sputum as the best sampling material to detect an ongoing C. pneumoniae infection.     

Tyrosine kinase-dependent and -independent events induced by interleukin-2 stimulation: interleukin-2-mediated NO production required for the induction of lymphokine-activated killer cell activity in rat splenocytes is tyrosine kinase independent.

Nitric oxide (NO) has recently been shown to be an indispensable co-factor in the generation of lymphokine-activated killer (LAK) cells induced by interleukin-2 (IL-2). Upon stimulation with IL-2, cells endowed with specific receptors undergo phosphorylation of substrates mediated by protein tyrosine kinases (PTK). In this work we utilized a well-characterized PTK inhibitor, genistein (GEN), to address the role of PTK on NO-dependent LAK cell generation. The effects of GEN were tested on the expression of the inducible NO synthase (iNOS) gene, proliferation, generation of cytotoxic activity and production of NO upon IL-2 stimulation of rat splenocytes. We report here that GEN displays profound inhibitory effects on recombinant (r)IL-2 induced proliferation and on LAK cell generation, while only marginally affecting NO production, measured as NO2-. In contrast, a specific inhibitor of the NO synthetic pathway (NG-monomethyl-L-arginine; NMMA) blocked generation of LAK cells and NO production without affecting cell proliferation. If added directly to the cytotoxicity tests, GEN exerted minor inhibitory effects, not exceeding 25% of control tests, while NMMA was completely ineffective. Sodium nitroprusside (SNP), a non-enzymatic NO-releasing substance, restored LAK cell generation in cultures performed in the presence of NMMA, but not in those performed in the presence of GEN. These results indicate that IL-2-induced NO production is a PTK-independent event. IL-2-stimulated LAK cell generation obligatorily requires the concurrent activation of PTK dependent and independent signal transduction pathways.     

Feasibility of simultaneous fluorescence immunophenotyping and fluorescence in situ hybridization study for the detection of estrogen receptor expression and deletions of the estrogen receptor gene in breast carcinoma cell lines

For the first time, combined immunophenotyping and fluorescence in situ hybridization (FISH) technique according to the ”fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasms” (FICTION) technique have been successfully applied in solid tumors. Thus, we were able to visualize the antigen expression of cells with chromosomal deletions of a tumor suppressor region directly. In six breast carcinoma cell lines, we investigated the correlation between estrogen receptor (ER) expression status and deletions of the estrogen receptor gene (ESR). To screen for deletions of the ESR gene, dual-color FISH was performed with a YAC (yeast artificial chromosome) probe containing the ESR gene and, as internal control, with a centromeric probe of chromosome 6. Deletions of the ESR gene were detected in four of six cell lines. For direct comparison of ER expression with the copy number of the ESR gene at the single cell level, immunophenotyping with mouse anti-human ER antibody was combined with FISH with the YAC probe containing the ESR gene according to the FICTION technique. There was no correlation between lack of or reduced ER expression and deletions of the ESR gene. One cell line with deletions of the ESR gene did express ER on the protein level, while another cell line without a deletion did not. Cells with deletions of the ESR gene were either ER expression positive or negative. The staining intensity of ER expression was not associated with the copy number of the ESR gene. Thus, this FICTION study unequivocally shows that deletions of the ESR gene are not the major cause of absent or reduced ER expression in breast carcinoma cell lines. Received: 6 September 1999 / Accepted: 14 September 1999     

Functional tyrosine kinase inhibitor profiling: a generally applicable method points to a novel role of platelet-derived growth factor receptor-beta in tuberous sclerosis

A ubiquitous herpesvirus that establishes life-long infection, the Epstein-Barr virus (EBV) has yielded little insight into how a single agent in general accord with its host can produce diverse pathologies ranging from oral hairy leukoplakia to nasopharyngeal carcinoma, from infectious mononucleosis to Hodgkin's disease (HD) and Burkitt's lymphoma. Its pathogenesis is further confounded by the less than total association of virus with histologically similar tumors. In other viral systems, defective (interfering) viral genomes are known to modulate outcome of infection, with either ameliorating or intensifying effects on disease processes initiated by prototype strains. To ascertain whether defective EBV genomes are present in HD, we examined paraffin-embedded tissue from 56 HD cases whose EBV status was first determined by cytohybridization for nonpolyadenylated EBV RNAs (EBERs). Using both standard polymerase chain reaction (PCR) and PCR in situ hybridization, we successfully amplified sequences that span abnormally juxtaposed BamHI W and Z fragments characteristic of defective heterogeneous (het) EBV DNA from 10 of 32 (31%) EBER-positive tumors. Of 24 EBER-negative HD, 8 yielded PCR products indicating presence of het EBV DNA. Two of these contained defective EBV in the apparent absence of the prototype virus. Of the 42 tumors analyzed for defective EBV by both PCR techniques, there was concordance of results in 38 (90%). Detection of defective EBV genomes with the potential to disrupt viral gene regulation suggests one mechanism for pathogenic diversity that may also account for loss of prototypic EBV from individual tumor cells.     

Molecular Manipulations of Extracellular Superoxide Dismutase: Functional Importance for Learning

Extracellular superoxide dismutase (EC-SOD) controls the availability of extracellular superoxide (O ₂ ^- ), which is important for a variety of physiological pathways, including the primary means of inactivating nitric oxide (NO). The role of EC-SOD in neurobehavioral function has been until now unexplored. In the current studies, the phenotypic expression of genotypic alterations of EC-SOD production in mice were characterized for spatial learning and memory. Dramatic impairments in spatial learning in the win-shift 8-arm radial maze were seen in both EC-SOD knockout mice and EC-SOD overexpressing mice. The EC-SOD overexpressing mice were further characterized as having significant deficits in a repeated acquisition task in the radial-arm maze, which permitted the dissociation of long and short-term learning. Long-term learning was significantly impaired by EC-SOD overexpression, whereas short-term learning was not significantly affected by EC-SOD overexpression. NO systems have been shown to be importantly involved in learning and memory. This may be important in the current studies because EC-SOD has primary control over the inactivation of NO. We found that EC-SOD overexpressing mice were resistant to the cognitive effects of L-NAME (N^G-nitro-L-arginine methyl ester hydrochloride), an NO synthase inhibitor. Decreased NO catabolism in these mice may have served to counter the effects of NOS inhibition by L-NAME. The current finding that EC-SOD levels that were either higher or lower than controls impaired learning demonstrates that the proper control of brain extracellular (O ₂ ^- ) may be more vital than merely reduction of brain extracelluar (O ₂ ^- ) in maintaining adequate learning function.     

Platelet interactions with calcium-phosphate-coated surfaces

Many studies have shown that calcium-phosphate (CaP)-coated endosseous implants exhibit more peri-implant bone formation and bone contact at early healing times than uncoated implants. Since the rate of healing is influenced by blood/implant interactions and possibly the degree of blood platelet activation, the aim of this study was to determine whether the topography, microtopography, or the presence of calcium (Ca) and phosphate (PO(4)) ions in the implant surface plays a predominant role in platelet activation. We define the threshold between topography and microtopography as the limit of the scale range of platelets themselves; thus, a microtopographic surface is defined by one which exhibits features 3mum. With the help of four international collaborating laboratories, we prepared 11 titanium and CaP-modified titanium surfaces each with different (micro)topographies and interrogated these surfaces with both platelet adhesion (lactate dehydrogenase activity) and platelet activation (microparticle formation and P-selectin expression) assays. Our results show that: calcium (Ca)- and phosphate (PO(4))-containing surfaces of increasing surface microtopographical complexity exhibit increasing platelet activation; surfaces with similar surface microtopographies show similar levels of platelet activation regardless of the presence of Ca and PO(4) in the surface; and that surface microtopography is responsible for platelet activation rather than the presence of Ca and PO(4) in the surface.     

The effect of immunotherapy on eosinophil accumulation and production of eosinophil chemotactic activity in the lung of subjects with asthma during natural pollen exposure

Two groups of birch pollen--allergic patients with seasonal rhinoconjunctivitis and asthma were followed during two consecutive birch-pollen seasons, one group, N = 10, during a season with high pollen load, and one group, N = 15, during a season of low pollen load. Half the patients were treated with immunotherapy (IT) for 3 and 4 years, respectively. The other half of the patients served as control group (non-IT). Bronchoalveolar lavage (BAL) was performed once before each season and once during the pollen season. Eosinophil (EOS) numbers in BAL were increased (p less than 0.01) during the season with high pollen load but not in the season with a low pollen load, and this increment was absent in the IT-treated group. Also, the EOS cationic protein levels were raised in the non-IT-treated group during the season with a high pollen load. The levels of EOS and neutrophil chemotactic activity were raised in BAL in both seasons in the non-IT-treated group compared with the IT-treated group (p less than 0.02, p less than 0.003, p less than 0.04, and p less than 0.005 in high- and low-load pollen season, respectively). Serum and BAL eosinophil chemotactic activity (ECA) were positively correlated (p less than 0.001). We conclude that there is an influx of active EOSs into the lung of pollen-allergic patients with asthma during a pollen season, which may be abrogated by IT. Furthermore, the generation of ECA appears to be an extremely sensitive marker of antigenic exposure, and the potent inhibition of the generation of ECA by IT may provide a clue as to the mechanism of this treatment.