Norbert Lehn

Helicobacter heilmannii–associated primary gastric low-grade MALT lymphoma: Complete remission after curing the infection

BACKGROUND & AIMS Cure of Helicobacter pylori infection may lead to complete remission of associated low-grade mucosa-associated lymphoid tissue (MALT) lymphoma in stage EI. This study investigated whether Helicobacter heilmannii infection-associated primary gastric MALT lymphoma will regress after cure of the infection. METHODS H. heilmannii-induced gastritis was diagnosed histologically, by a new specific immunoglobulin G enzyme-linked immunosorbent assay, and with 16S ribosomal RNA amplification and sequencing in 5 consecutive patients with primary gastric MALT lymphoma clinical stage EI. Patients received 40 mg omeprazole and 750 mg amoxicillin 3 times per day for 14 days. Polymerase chain reaction (PCR) was used to detect rearrangement of immunoglobulin heavy-chain genes before treatment and during follow-up. RESULTS Five patients (3 men, 2 women; mean age, 65 years; range, 42-79 years) were studied. H. pylori was not detected by culture, histology, serology, or PCR. Treatment resulted in the cure of H. heilmannii infection in each case and complete histological and endoscopic remission of the tumors. Three of 5 patients showed monoclonal B cells before treatment, 2 of whom remained PCR positive. Within a median follow-up period of 24 months, no relapse of the lymphoma or reinfection with H. heilmannii occurred. CONCLUSIONS These data suggest that gastric MALT lymphoma may arise in patients with H. heilmannii infection. Cure of this infection may lead to complete remission of the MALT lymphoma.

Frequency of rpoB Mutations Inside and Outside the Cluster I Region in Rifampin-Resistant Clinical Mycobacterium tuberculosis Isolates

ABSTRACT The prevalence of recently described mutation V176F, located in the beginning of the rpoB gene and associated with rifampin resistance and the wild-type cluster I sequence, was determined by analyzing the distribution of rpoB mutations among 80 rifampin (RIF)-resistant Mycobacterium tuberculosis strains isolated in Germany during 1997. The most frequent rpoB mutations were changes in codon 456 (52 isolates, 65%), followed by changes in codon 441 (13 isolates, 16%) and codon 451 (11 isolates, 14%). The V176F mutation was detected in one isolate of the study population and in 5 of 18 RIF-resistant strains with no cluster I mutation from six previously published studies. In three isolates, a mixture of resistant and susceptible subpopulations (heteroresistance) prohibited the detection ofrpoB mutations in the initial analysis; however, in these isolates, cluster I mutations could be verified after a passage on RIF-containing medium. IS6110 DNA fingerprinting of 76 strains revealed eight clusters comprising 27 strains with identical restriction fragment length polymorphism patterns that mainly also show identical rpoB mutations and identical or similar drug resistance patterns. In conclusion, our results indicate that the V176F mutation should be included in molecular tests for prediction of RIF resistance in M. tuberculosis. We further demonstrated that heteroresistance caused by a mixture of mycobacterial subpopulations with different susceptibilities to RIF may influence the sensitivity of molecular tests for detection of resistance.

The Helicobacter pylori vacA s1, m1 genotype and cagA is associated with gastric carcinoma in Germany

Background: The H. pylori vacuolating cytotoxin encoded by vacA and the cytotoxin‐associated protein encoded by cagA are considered to be important virulence determinants that have been implicated in the development of peptic ulcers and gastric carcinoma. However, conflicting results regarding the association between these virulence factors and clinical disease have been reported from different geographic regions. Aims: To determine the frequency of vacA genotypes, vacuolating cytotoxin activity, and cagA in H. pylori isolates obtained from patients with gastric cancer in Germany. Methods: H. pylori isolates were obtained from 34 patients with gastric cancer and from 35 subjects with asymptomatic H. pylori gastritis. vacA genotypes and cagA were identified by PCR. Cytotoxic activity was determined by HeLa cell assays. Gastritis was assessed according to the updated Sydney System. Results: The H. pylori vacA s1,m1 genotype was significantly more frequent in patients with gastric cancer (24/34, 70.6%) as compared with controls (12/35, 34.3%) (p = 0.005). Cytotoxic activity was detected in 24 (70.6%) and 15 (42.9%) H. pylori isolates from gastric cancer patients and controls, respectively (p = 0.03). The cagA gene was identified in 30 (88.2%) and 21 (60%) H. pylori isolates in the respective groups (p = 0.01). Conclusions: Our study suggests a significant association between the H. pylori vacA s1,m1 genotype, cytotoxic activity, cagA, and gastric cancer. Detection of H. pylori possessing these virulence determinants may help to identify patients being at an increased risk to develop gastric cancer in our population. Int. J. Cancer 87:322–327, 2000.

Real-time PCR assay targeting IS481 of Bordetella pertussis and molecular basis for detecting Bordetella holmesii.

ABSTRACT Detection of Bordetella holmesii by a real-time PCR assay targeting IS481 of Bordetella pertussis is reported. Sequencing of IS481-specific PCR products from B. pertussis and B. holmesii isolates revealed sequence homology. Restriction fragment length polymorphism demonstrated a low copy number of IS481-like sequences in B. holmesii. These results, and culture of B. holmesii from patients with cough, suggest that the specificity and predictive value of IS481-based PCR assays for pertussis may be compromised.

Real-Time Fluorescence PCR Assays for Detection and Characterization of Shiga Toxin, Intimin, and Enterohemolysin Genes from Shiga Toxin-Producing Escherichia coli

ABSTRACT PCR assays have proved useful for detecting and characterizing Shiga toxin-producing Escherichia coli (STEC). Recent advances in PCR technology have facilitated the development of real-time fluorescence PCR assays with greatly reduced amplification times and improved methods for the detection of amplified target sequences. We developed and evaluated two such assays for the LightCycler instrument: one that simultaneously detects the genes for Shiga toxins 1 and 2 (stx1 and stx2) and another that simultaneously detects the genes for intimin (eae) and enterohemolysin (E-hly). Amplification and sequence-specific detection of the two target genes were completed within 60 min. Findings from the testing of 431 STEC isolates of human and animal origin, 73 isolates of E. coli negative for stx genes, and 118 isolates of other bacterial species with the LightCycler PCR (LC-PCR) assays were compared with those obtained by conventional block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the stx1, eae, and E-hly genes and 96 and 100%, respectively, for the stx2 gene. No stx2 genes were detected from 10 stx2f-positive isolates because of significant nucleotide differences in their primer annealing regions. Melting curve analyses of the amplified Shiga toxin genes revealed sequence variation within each of the tested genes that correlated with described and novel gene variants. The performance characteristics of the LC-PCR assays, such as their speed, detection method, and the potential subtyping information available from melting curve analyses, make them attractive alternatives to block cycler PCR assays for detecting and characterizing STEC strains.

Functional Analysis of the cag Pathogenicity Island in Helicobacter pylori Isolates from Patients with Gastritis, Peptic Ulcer, and Gastric Cancer

ABSTRACT Helicobacter pylori is the causative agent of a variety of gastric diseases, but the clinical relevance of bacterial virulence factors is still controversial. Virulent strains carrying the cag pathogenicity island (cagPAI) are thought to be key players in disease development. Here, we have compared cagPAI-dependent in vitro responses in H. pylori isolates obtained from 75 patients with gastritis, peptic ulcer, and gastric cancer (n = 25 in each group). AGS gastric epithelial cells were infected with each strain and assayed for (i) CagA expression, (ii) translocation and tyrosine phosphorylation of CagA, (iii) c-Src inactivation, (iv) cortactin dephosphorylation, (v) induction of actin cytoskeletal rearrangements associated with cell elongation, (vi) induction of cellular motility, and (vii) secretion of interleukin-8. Interestingly, we found high but similar prevalences of all of these cagPAI-dependent host cell responses (ranging from 56 to 80%) among the various groups of patients. This study revealed CagA proteins with unique features, CagA subspecies of various sizes, and new functional properties for the phenotypic outcomes. We further showed that induction of AGS cell motility and elongation are two independent processes. Our data corroborate epidemiological studies, which indicate a significant association of cagPAI presence and functionality with histopathological findings in gastritis, peptic ulcer, and gastric cancer patients, thus emphasizing the importance of the cagPAI for the pathogenicity of H. pylori. Nevertheless, we found no significant association of the specific H. pylori-induced responses with any particular patient group. This may indicate that the determination of disease development is highly complex and involves multiple bacterial and/or host factors.

Mutations in the beginning of the rpoB gene can induce resistance to rifamycins in both Helicobacter pylori and Mycobacterium tuberculosis.

ABSTRACT A clinical isolate of Helicobacter pylori that developed resistance to rifabutin during therapy carried anrpoB gene that retained a wild-type cluster region sequence but had acquired a novel codon 149 (V149F) mutation. In transformation experiments, the mutation was shown to confer high-level rifabutin resistance. The equivalent mutation (V176F) was present in several resistant isolates of Mycobacterium tuberculosis.

Healthcare-associated outbreaks and community-acquired infections due to MRSA carrying the Panton-Valentine leucocidin gene in southeastern Germany

In response to several isolations of methicillin-resistant Staphylococcus aureus carrying the Panton-Valentine leucocidin gene (PVL-MRSA), the present study was conducted to document the spread of infection in a small region of southeastern Germany. During a 9-month period, two healthcare-associated outbreaks with PVL-MRSA occurred, affecting 83 patients, personnel and contacts of personnel, and 34 additional cases were detected in the community. The clinical spectrum ranged from colonization to skin infection and necrotizing pneumonia. The findings represent the largest number of PVL-MRSA cases detected in Germany so far, and demonstrate the potential of this emerging pathogen to spread within the community and in healthcare institutions.

Tumor Necrosis Factor-Dependent Adhesions as a Major Protective Mechanism Early in Septic Peritonitis in Mice

ABSTRACT The occurrence of peritoneal adhesions in surgical patients is positively correlated with tumor necrosis factor (TNF) levels. In a model of septic peritonitis—cecal ligation and puncture—TNF neutralization prevented formation of peritoneal adhesions and increased mortality, most likely because localization of the septic focus was prevented. To discriminate between the coagulation-independent protective TNF effect and a potential protective procoagulant TNF effect, formation of peritoneal adhesions after CLP was inhibited with heparin, hirudin, or urokinase. Each treatment increased mortality and increased the number of bacteria in the peritoneal lavage fluid, kidney, and liver to various degrees. Under these experimental conditions, antibiotics prevented death. In coagulation-compromised mice, lethality was further enhanced by additional TNF neutralization. These findings demonstrate that peritoneal adhesions early in septic peritonitis are an important mechanism of innate immunity that prevents increased spread of bacteria and reduces mortality.

Induction of Maturation and Cytokine Release of Human Dendritic Cells by Helicobacter pylori

ABSTRACT Helicobacter pylori causes a persistent infection in the human stomach, which can result in chronic gastritis and peptic ulcer disease. Despite an intensive proinflammatory response, the immune system is not able to clear the organism. However, the immune escape mechanisms of this common bacterium are not well understood. We investigated the interaction between H. pylori and human dendritic cells. Dendritic cells (DCs) are potent antigen-presenting cells and important mediators between the innate and acquired immune system. Stimulation of DCs with different concentrations of H. pylori for 8, 24, 48, and 72 h resulted in dose-dependent interleukin-6 (IL-6), IL-8, IL-10 and IL-12 production. Lipopolysaccharide (LPS) from Escherichia coli, a known DC maturation agent, was used as a positive control. The cytokine release after stimulation with LPS was comparable to that induced by H. pylori except for IL-12. After LPS stimulation IL-12 was only moderately released compared to the large amounts of IL-12 induced by H. pylori. We further investigated the potential of H. pylori to induce maturation of DCs. Fluorescence-activated cell sorting analysis of cell surface expression of maturation marker molecules such as CD80, CD83, CD86, and HLA-DR revealed equal upregulation after stimulation with H. pylori or LPS. We found no significant differences between H. pylori seropositive and seronegative donors of DCs with regard to cytokine release and upregulation of surface molecules. These data clearly demonstrate that H. pylori induces a strong activation and maturation of human immature DCs.