Iron-cofactored superoxide dismutase inhibits host responses to Mycobacterium tuberculosis.

Superoxide dismutase (SOD) is a ubiquitous metalloenzyme in aerobic organisms that catalyzes the conversion of superoxide anion to hydrogen peroxide. Mycobacterium tuberculosis is unusual in that it secretes large quantities of iron-cofactored SOD. To determine the role of SOD in pathogenesis, we constructed mutants of M. tuberculosis H37Rv with reduced SOD production. Compared with controls, SOD-diminished isolates were more susceptible to killing by hydrogen peroxide. The isolates were markedly attenuated, exhibiting nearly 100,000-fold fewer bacilli than virulent control strains in the lungs and spleens of C57BL/6 mice 4 wk after intravenous inoculation. In the lung, SOD-attenuated M. tuberculosis induced robust interstitial mononuclear cell infiltration within 24 h and many cells were apoptotic by TUNEL staining, whereas virulent H37Rv exhibited minimal early inflammatory response and only rare interstitial mononuclear cell apoptosis. During prolonged infections, C57BL/6 mice tolerated SOD-attenuated M. tuberculosis better than BCG, exhibiting 68% greater weight gain, quicker eradication of bacilli from the spleen, and less alveolar lung infiltration. These results establish the importance of SOD in the pathogenesis of tuberculosis. Its effect appears to be mediated in part by inhibiting innate host immune responses, including early mononuclear cell infiltration of infected tissues and apoptosis.

Helicobacter pylori strain-specific differences in genetic content, identified by microarray, influence host inflammatory responses.

Helicobacter pylori enhances the risk for ulcer disease and gastric cancer, yet only a minority of H. pylori-colonized individuals develop disease. We examined the ability of two H. pylori isolates to induce differential host responses in vivo or in vitro, and then used an H. pylori whole genome microarray to identify bacterial determinants related to pathogenesis. Gastric ulcer strain B128 induced more severe gastritis, proliferation, and apoptosis in gerbil mucosa than did duodenal ulcer strain G1.1, and gastric ulceration and atrophy occurred only in B128+ gerbils. In vitro, gerbil-passaged B128 derivatives significantly increased IL-8 secretion and apoptosis compared with G1.1 strains. DNA hybridization to the microarray identified several strain-specific differences in gene composition including a large deletion of the cag pathogenicity island in strain G1.1. Partial and complete disruption of the cag island in strain B128 attenuated induction of IL-8 in vitro and significantly decreased gastric inflammation in vivo. These results indicate that the ability of H. pylori to regulate epithelial cell responses related to inflammation depends on the presence of an intact cag pathogenicity island. Use of an H pylori whole genome microarray is an effective method to identify differences in gene content between H. pylori strains that induce distinct pathological outcomes in a rodent model of H. pylori infection.

Helicobacter pylori genotypes, host factors, and gastric mucosal histopathology in peptic ulcer disease.

From 183 patients undergoing upper gastrointestinal endoscopy, we used antral and corpus gastric biopsies for bacterial culture and histopathologic examination, blood samples to detect immunoglobulin G antibodies against Helicobacter pylori, and H pylori genomic DNA to analyze cytotoxin-associated gene A (cagA) and vacuolating cytotoxin (vacA) genotypes. As expected, among H pylori biopsy-positive patients, those with duodenal ulcer (DU) (n = 34) had significantly more severe chronic and acute inflammation (P <.001) and epithelial degeneration (P =.004) in the gastric antrum than in the gastric corpus. Each of those 3 parameters and H pylori density were significantly higher in the antrum of patients with DU than in patients with gastric ulcer (GU) or no ulcer. Colonization with vacA s1/cagA-positive strains of H pylori was associated with inflammation and epithelial degeneration in gastric mucosa and increased risk for peptic ulcer disease (PUD), whereas colonization with vacA s2m2/cagA-negative strains was associated with mild gastric histopathology and was not associated with any significant risk for PUD. The predominant H pylori strains in African Americans were vacA s1bm1/cagA-positive, whereas all genotypes were well represented in non-Hispanic-Caucasians. By multivariate analysis, H pylori colonization was significantly associated with DU (Adjusted odds ratio [AdjOR] = 3.2 [1.4-7.2]) and nonsteroidal anti-inflammatory drugs (NSAID) use was inversely associated (AdjOR = 0.3 [0.2-0.7]). NSAID use (AdjOR = 4.3 [1.02-18.5]) and African-American ethnicity (AdjOR = 10.9 [2.6-50]) were significantly associated with GU. Smoking and age were not significantly associated with either DU or GU. These data indicate that DU is associated with an antral-dominant gastritis, and H pylori genotype and NSAID use independently contribute to the pathogenesis of PUD. HUM PATHOL 32:264-273. This is a US Government work. There are no restrictions on its use.

Quantitative detection of Helicobacter pylori gene expression in vivo and relationship to gastric pathology.

The iceA locus of Helicobacter pylori includes one of two mutually exclusive gene families, iceA1 and iceA2. Colonization with iceA1 strains is associated with enhanced acute mucosal inflammation, and adherence to gastric epithelial cells in vitro induces expression of iceA1 but not iceA2 mRNA; however, both transcripts can be detected in vivo. The aim of this study was to determine whether differing levels of iceA transcription in vivo may contribute to disease pathogenesis. RNA from 41 H. pylori-positive gastric biopsy specimens was reverse transcribed to cDNA. Quantitative PCR was performed using biotinylated iceA1, iceA2, and 16S rRNA primers, and binding of biotinylated products to streptavidin-coated plates was detected by hybridization with a fluorescein-labeled probe. iceA genotypes were determined by PCR and sequence analysis. All 41 samples contained detectable H. pylori 16S rRNA, with similar levels in iceA1- (n = 10) and iceA2 (n = 31)-colonized patients (P = 0.34). Biopsy specimens from four (40%) and 19 (61%) persons colonized with iceA1 or iceA2 strains, respectively, had detectable iceA RNA. Acute inflammatory scores were significantly higher in iceA1 RNA-positive patients than in iceA1 RNA-negative, iceA2 RNA-positive, or iceA2 RNA-negative subjects (P

Helicobacter pylori alters gastric epithelial cell cycle events and gastrin secretion in Mongolian gerbils.

BACKGROUND & AIMS: Human colonization with Helicobacter pylori increases the risk for distal gastric adenocarcinoma, possibly by altering gastric epithelial cell cycle events and/or gastrin secretion. This study aimed to determine whether H. pylori virulence-related characteristics affect apoptosis, proliferation, and gastrin levels in a rodent model of gastric adenocarcinoma. METHODS: Mongolian gerbils were challenged with H. pylori wild-type or isogenic cagA(-) and vacA(-) mutants, and apoptotic and proliferating cells were identified by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and proliferating cell nuclear antigen immunohistochemistry, respectively. Serum gastrin levels were determined by radioimmunoassay. RESULTS: Gastric epithelial cell turnover was no different after infection with the wild-type, cagA(-), or vacA(-) strains. H. pylori infection significantly increased antral apoptosis 2-4 weeks after challenge, before apoptotic indices decreased to baseline. In contrast, antral proliferation rates were significantly higher 16-20 weeks after inoculation, but then decreased by 40 weeks. Antral proliferation was significantly related to serum gastrin levels, whereas antral apoptosis was inversely related to acute inflammation and lymphoid follicles. CONCLUSIONS: In H. pylori-infected gerbils, enhanced antral apoptosis is an early and transient cell cycle event. Epithelial cell proliferation peaks later and is significantly related to increased gastrin levels, suggesting that epithelial cell growth in H. pylori-colonized mucosa may be mediated by gastrin-dependent mechanisms.

Adherence to gastric epithelial cells induces expression of a Helicobacter pylori gene, iceA, that is associated with clinical outcome.

Most persons infected with Helicobacter pylori strains that produce vacuolating cytotoxin and possess cytotoxin-associated gene A (cagA) genotype nonetheless remain asymptomatic, suggesting that additional genes are important in virulence. We hypothesized that adherence to gastric epithelium provides stimuli that induce expression of some virulence genes. Our aims were to identify expression of H. pylori genes induced by adherence and to determine if such genes were correlated with peptic ulceration, mucosal interleukin-8 (IL-8) levels, and gastric inflammation. RNA was isolated from an ulcer-derived strain and a gastritis-derived strain that were exposed or not exposed to gastric epithelial cells. These RNAs were used for random arbitrarily primed reverse transcription polymerase chain reaction to identify newly expressed transcripts unique to the ulcer-derived strain following adherence. Clinical isolates of H. pylori were characterized for presence of the newly identified gene, and mucosal IL-8 and inflammation were examined in gastric biopsies from the source patients. A novel H. pylori gene, iceA (induced by contact with epithelium), was identified. DNA sequences revealed two families, iceA1 and iceA2. iceA1 strains were significantly associated with peptic ulceration and increased mucosal concentrations of IL-8. Both iceA1 and iceA2 were expressed in vivo by respective H. pylori strains in gastric biopsies. Adherence to gastric epithelial cells in vitro stimulates the transcription of iceA1, an H. pylori gene that is highly correlated with pathological outcome.

Experimental infection of Mongolian gerbils with wild-type and mutant Helicobacter pylori strains.

Experimental Helicobacter pylori infection was studied in Mongolian gerbils with fresh human isolates that carry or do not carry cagA (cagA-positive or cagA-negative, respectively), multiply passaged laboratory strains, wild-type strain G1.1, or isogenic ureA, cagA, or vacA mutants of G1.1. Animals were sacrificed 1 to 32 weeks after challenge, the stomach was removed from each animal for quantitative culture, urease test, and histologic testing, and blood was collected for antibody determinations. No colonization occurred after >/=20 in vitro passages of wild-type strain G1.1 or with the ureA mutant of G1.1. In contrast, infection occurred in animals challenged with wild-type G1.1 (99 of 101 animals) or the cagA (25 of 25) or vacA (25 of 29) mutant of G1.1. Infection with G1.1 persisted for at least 8 months. All 15 animals challenged with any of three fresh human cagA-positive isolates became infected, in contrast to only 6 (23%) of 26 animals challenged with one of four fresh human cagA-negative isolates (P < 0.001). Similar to infection in humans, H. pylori colonization of gerbils induced gastric inflammation and a systemic antibody response to H. pylori antigens. These data confirm the utility of gerbils as an animal model of H. pylori infection and indicate the importance of bacterial strain characteristics for successful infection.

Optimal screening and diagnosis of microsporida in tissue sections: a comparison of polarization, special stains, and molecular techniques.

With improving therapeutic protocols, confirmation of microsporidiosis has become increasingly important. We designed a study to determine the best screening method(s) for microsporidian detection in biopsy specimens. Forty-two small intestinal biopsy specimens from 31 immunocompromised patients (68% AIDS) were stained (hematoxylin-eosin [H & E], modified trichrome, Warthin-Starry, and Brown-Brenn) and polarized. Polymerase chain reaction and Southern blot assays were performed on all positive cases. Microsporida were detected in nine cases (21%) by modified trichrome (all patients with AIDS). Of these, seven were Brown-Brenn positive, and five Warthin-Starry positive. Two cases polarized on H & E and three on special stains. Four of nine positive cases were confirmed by molecular studies. We found polarization to be the least sensitive screening method. The modified trichrome was the most sensitive when screening for microsporidiosis in paraffin-embedded biopsy specimens. Furthermore, combining Brown-Brenn or Warthin-Starry with the trichrome stain helps exclude false-positive results due to granular artifacts (eg, eosinophils, Paneth cells).

Helicobacter pylori Lewis expression is related to the host Lewis phenotype.

BACKGROUND & AIMS: Lewis antigens occur in human gastric epithelium and in Helicobacter pylori lipopolysaccharide; their expression is polymorphic in both. Autoimmune mechanisms induced by bacterial Lewis expression have been proposed to cause gastritis. The aim of this study was to examine the relationship between bacterial and host gastric Lewis expression, as determined by the erythrocyte Lewis(a/b) phenotype, and between gastric histopathology and bacterial Lewis expression. METHODS: H. pylori Lewis expression was determined by enzyme immunoassays, erythrocyte Lewis phenotype was assessed by agglutination tests, and gastric histopathology was scored blindly. RESULTS: The host Lewis phenotype was (a+b-) in 15, (a-b+) in 34, and (a-b-) in 17 patients, therefore expressing Lewis x, y, or neither as their major gastric epithelial Lewis type 2 antigen. H. pylori from patients with Lewis(a+b-) expressed Lewis x more than y (1147 +/- 143 vs. 467 +/- 128 optical density units [ODU]; P = 0.006), isolates from patients with Lewis(a-b+) expressed Lewis x less than y (359 +/- 81 vs. 838 +/- 96 ODU; P = 0.0001), and isolates from Lewis(a-b-) patients expressed Lewis x and y approximately equally. Gastritis was unrelated to H. pylori Lewis expression. CONCLUSIONS: In mimicking host gastric epithelium, H. pylori cells not only express Lewis x and y, but the relative proportion of expression corresponds to the host Lewis phenotype, suggesting selection for host-adapted organisms.

Helicobacter pylori cagA+ strains and dissociation of gastric epithelial cell proliferation from apoptosis.

BACKGROUND: Infection with Helicobacter pylori induces chronic gastritis in virtually all infected persons, and such gastritis has been associated with an increased risk of developing gastric cancer. This risk is further enhanced with cagA+ (positive for cytotoxin-associated gene A) H. pylori strains and may be a consequence of induced gastric cell proliferation and/or alteration in apoptosis (programmed cell death) in the gastric epithelium. PURPOSE: To determine whether the H. pylori cagA genotype and another virulence-related characteristic, the vacA (vacuolating cytotoxin A) s1a genotype, differentially affect epithelial cell proliferation, apoptosis, and the histologic parameters of inflammation and injury, we quantitated these characteristics in infected and uninfected persons. METHODS: Fifty patients underwent upper gastrointestinal endoscopy, and biopsy specimens were taken. Apoptotic cells in the specimens were quantitated after terminal deoxynucleotidyl transferase labeling of DNA fragments with digoxigenin-deoxyuridine triphosphate; epithelial cell proliferation was scored by immunohistochemical analysis of the proliferation-associated antigen Ki-67. Antibodies directed against H. pylori and CagA protein were measured in the serum of patients by means of enzyme-linked immunosorbent assays. Analysis of H. pylori genomic DNA, by use of the polymerase chain reaction, was performed to determine the cagA and vacA genotypes. Acute and chronic inflammation, epithelial cell degeneration, mucin depletion, intestinal metaplasia, glandular atrophy, and vacuolation were each scored in a blinded manner. Reported P values are two-sided. RESULTS: Persons harboring cagA+ strains (n = 20) had significantly higher gastric epithelial proliferation scores than persons infected with cagA-strains (n = 9) or uninfected persons (n = 21) (P = .025 and P<.001, respectively), but the difference in cell proliferation between the latter two groups was not statistically significant. The number of apoptotic cells per 100 epithelial cells (apoptotic index) in persons infected with cagA+ strains was lower than in persons infected with cagA-strains (P = .05). Apoptotic indices in the cagA+ group were similar to those in the uninfected group (P = .2). Epithelial cell proliferation was significantly correlated with acute gastric inflammation, but only in the cagA+ group (r = .44; P = .006). The cagA+ and vacA s1a genotypes were found to be concordant, confirming the close relationship between these virulence-related genotypes. CONCLUSIONS: Gastric mucosal proliferation was significantly correlated with the severity of acute gastritis in persons infected with cagA+ vacA s1a strains of H. pylori. This increased proliferation was not accompanied by a parallel increase in apoptosis. IMPLICATIONS: Increased cell proliferation in the absence of a corresponding increase in apoptosis may explain the heightened risk for gastric carcinoma that is associated with infection by cagA+ vacA s1a strains of H. pylori.

Clinical and pathological importance of heterogeneity in vacA, the vacuolating cytotoxin gene of Helicobacter pylori.

BACKGROUND & AIMS: vacA encodes the vacuolating cytotoxin of Helicobacter pylori and exhibits marked variation in signal sequence and midgene coding regions. The implications for gastroduodenal pathology are unknown. The aim of this study was to define the association of vacA genotype with gastric inflammation and injury, in vitro cytotoxin activity, and peptic ulceration. METHODS: Sixty-one consecutive dyspeptic patients underwent endoscopy and gastric biopsy. The biopsy specimens were processed for H. pylori culture, and 52 specimens were also processed for histology. H. pylori vacA was typed by polymerase chain reaction and colony hybridization. Cytotoxin activity was assessed by a HeLa cell vacuolation assay. RESULTS: vacA signal sequence type s1a strains were associated with greater antral mucosal neutrophil and lymphocyte infiltration than s1b or s2 strains (P < 0.05). vacA midregion type m1 strains were associated with greater gastric epithelial damage than m2 strains (P < 0.05). Both midregion and signal sequence were associated with cytotoxin activity in vitro. Duodenal ulcer disease occurred in 89% of 18 patients with s1a strains vs. 29% of 14 with s1b strains (P < 0.01), 20% of 10 with s2 strains (P < 0.001), and 16% of 19 uninfected patients (P < 0.001). CONCLUSIONS: H. pylori strains of vacA signal sequence type s1a are associated with enhanced gastric inflammation and duodenal ulceration. vacA s2 strains are associated with less inflammation and lower ulcer prevalence.

Density of Helicobacter pylori infection in vivo as assessed by quantitative culture and histology.

Helicobacter pylori density was assessed by quantitative culture and histologic examination of gastric biopsy specimens from 29 H. pylori-infected dyspeptic patients. Density was correlated with cagA and vacA genotypes (assessed by polymerase chain reaction and colony hybridization), gastric inflammation and epithelial injury (assessed histologically), and peptic ulceration. Quantitative culture was more reproducible than histology, and antral density was more reproducible than corpus density. Mean antral density of cagA+/vacA sl strains was 4-fold higher than that of cagA-/vacA s2 strains (1.9 X 10(6) vs. 4.5 x 10(5) cfu/g, P = .02). Antral density was associated with mucosal neutrophilic and lymphocytic infiltration (P < .01) and with epithelial injury (P < .05). Mean antral bacteria] density was 5-fold higher in duodenal ulcer patients than in others (P = .005). In conclusion, H. pylori density in vivo is easily quantified and is associated with bacterial virulence determinants, gastric inflammation, and duodenal ulceration, suggesting a central role in pathogenesis.

A simple splenic reticuloendothelial function test: counting erythrocytes with argyrophilic inclusions.

The presently accepted methods for evaluation of splenic reticuloendothelial (RE) function include 99mTc sulfur colloid spleen scan, antibody-coated autologous erythrocyte clearance, and pocked erythrocyte count. All methods involve special equipment and/or risk and inconvenience to patients. A simple method of assessing splenic RE function was developed by counting erythrocytes with argyrophilic inclusions using a simple silver stain and an ordinary microscope. To test the validity of this method, blood samples were collected from patients suspected of having hyposplenia or asplenia, including patients with history of splenectomy, sickle cell disease or trait, and newborns. Blood samples were also collected from normal adults and from patients without hyposplenia or asplenia as controls. The samples were tested by this method and compared to the pocked erythrocyte count that served as a gold standard. The results obtained by the two methods were found to be very comparable with little overlap between those from controls and patients with definite hyposplenia or asplenia. With the pocked erythrocyte count as the gold standard, this method has a sensitivity of 88.9% and a specificity of 97.1%. However, this method requires no special equipment. Staining can be applied to fresh blood smears as well as to Wright-stained smears, and the silver-stained smears are permanent.

Heightened inflammatory response and cytokine expression in vivo to cagA+ Helicobacter pylori strains.

BACKGROUND: Helicobacter pylori strains that possess the cytotoxin-associated gene (cagA) are highly associated with peptic ulcer disease, but the role of cagA in pathogenesis is unknown. EXPERIMENTAL DESIGN: To test the hypothesis that cagA+ stains elicit a greater proinflammatory cytokine response in the gastric mucosa than cagA- strains, gastric biopsies were obtained from 52 patients and studied by histology, culture, enzyme-linked immunosorbent assay, and reverse transcription polymerase chain reaction. RESULTS: Of 52 patients, 32 (62%) were infected with H. pylori based upon both serology and histology or culture, 16 (31%) were negative by serology, histology, and culture, and four (7%) were positive by serology only. Of 15 H. pylori-infected patients with peptic ulceration, 14 (92%) were infected with cagA+ strains compared with 8 (50%) of 16 patients with gastritis alone, and those infected with cagA+ strains had significantly higher grades of inflammation in the gastric mucosa. Antral inflammation score was significantly associated with IL-8 production. Antral biopsies from infected patients, compared with uninfected patients, significantly more often demonstrated IL-1 beta, IL-2, and IL-8 expression, and those infected with cagA+ compared with cagA- strains significantly more often expressed IL-1 alpha and IL-1 beta and showed elevated antral IL-8 protein levels. Similarly, patients with ulcer disease significantly more often expressed antral IL-1 alpha and IL-8 than those without ulceration. CONCLUSION: These results indicate that infection with cagA+ H. pylori strains is associated with higher grades of gastric inflammation, correlating with enhanced mucosal levels of IL-8, and increased risk of peptic ulceration.

Ringed sideroblasts: a frequent observation after bone marrow transplantation.

Dyserythropoiesis after bone marrow transplantation is common, but the presence of ringed sideroblasts (RS) has not been evaluated fully. To examine for RS, a combined silver and Perls' Prussian blue stain, shown previously to be more sensitive for detecting RS than Perls' Prussian blue stain alone, was used on post-transplant marrow aspirate sections from 39 patients who received marrow transplants (allogeneic, 28; autologous, 11) for a variety of disorders. Marrow aspirate sections were available for comparison from 11 of these patients before any treatment as well as from five patients with normal marrows and normal peripheral blood cell counts. Aspirates were not performed on donor marrows. By the modified silver stain, RS were present in 34 (87%) patients whose marrows were sampled 0.5 to 39 (median, 1.5) months post-transplant including 10 of 11 patients with autologous transplants (no graft versus host disease prophylaxis). In contrast, seven of 36 (19%) of these marrows contained RS when stained with Perls' reaction alone. Only one of 11 pretransplant marrows and none of five normal marrows contained RS when stained by either method. These results demonstrate that RS are present in most post-transplant marrows even beyond the usual period of reconstitution (28 days), and this finding can be included among the features of dyserythropoiesis seen after transplantation. RS apparently are not related to pretransplant pathology or post-transplant therapy. This study also confirms previous observations that modified silver stains are more sensitive for detecting RS than Perls' Prussian blue stain.

Rhodococcus equi--an increasingly recognized opportunistic pathogen. Report of 12 cases and review of 65 cases in the literature.

Rhodococcus equi, a gram-positive, weakly acid-fast coccobacillus, initially isolated from horses, is becoming increasingly recognized as an important pathogen for immunosuppressed human hosts since the first human case was reported in 1967. A review of the English medical literature yielded 53 cases. During the last 11 years, the microbiology laboratories of the authors isolated the organism from 12 patients. Of the total 65 cases, 60 occurred in immunosuppressed patients with HIV infection, malignant neoplasms, or chronic immunosuppressive therapy. The lung is the most common primary site of infection. Typically, the lesion is densely infiltrated by histiocytes with multiple microabscesses. Intracellular gram-positive coccobacilli are easily demonstrated. R equi grows well on routine non-selective media at 35 degrees C. Previously, many cases may have been missed because the organism resembles oropharyngeal commensal diphtheroids. Clinical information with gram and Kinyoun strains on fresh isolates is helpful in recognizing the possibility of R equi infection.

Detection of Helicobacter pylori gene expression in human gastric mucosa.

Mucosal and systemic immunologic recognition of cagA by Helicobacter pylori-infected individuals is associated with peptic ulcer disease; however, in the laboratory, expression of cagA is subject to artificial conditions which may not accurately reflect the conditions in host tissues. Gastric antral and body biopsy specimens and serum for anti-H. pylori immunoglobulin G serology were obtained from 42 patients. Biopsy specimens were studied by histology, culture, and reverse transcription PCR (RT-PCR). Oligonucleotide primers specific for H. pylori (16S rRNA, ureA, and cagA) were used to detect bacterial mRNA in gastric biopsy specimens. PCR was performed on DNA from corresponding H. pylori isolates to detect genomic 16S rRNA, ureA, and cagA. Of the 42 patients from whom clinical specimens were obtained, 25 were infected with H. pylori on the basis of both serology and histology or culture (i.e., tissue positive); 13 were negative by serology, histology, and culture; and 4 were positive by serology only. RT-PCR with 16S rRNA primers detected 24 of 25 tissue-positive and 0 of 17 tissue-negative patients (P < 0.001). RT-PCR with ureA primers detected 16 of 25 tissue-positive and 0 of 17 tissue-negative patients (P < 0.001). CagA mRNA was detected by RT-PCR in 14 of 25 gastric biopsy specimens in the tissue-positive group and in 0 of 17 gastric biopsy specimens in the tissue-negative group. PCR of genomic DNA for the presence of the cagA gene in the corresponding bacterial isolates correlated absolutely with cagA gene expression in gastric tissue. These results indicate that RT-PCR is a sensitive and specific method for the detection of the presence of H. pylori and the expression of H. pylori genes in human gastric tissue. Detection of H. pylori gene expression in vivo by this approach may contribute to improving the diagnosis and understanding the pathogenesis of H. pylori infections.

Hyphalike pseudofungus in a lymph node.

Mediastinoscopic biopsy specimens of multiple paratracheal lymph nodes in a 66-year-old man with a history of resected pulmonary carcinoma were submitted for histologic evaluation. Structures present in one of these lymph nodes superficially resembled branching septate fungal hyphae. Perls' stain demonstrated that these structures contained iron, and Gomori's methenamine silver stain was negative. The histopathologic features are similar to those of the first and only previous case report of such structures.

Correlation between vacuolating cytotoxin production by Helicobacter pylori isolates in vitro and in vivo.

Approximately 50 to 60% of Helicobacter pylori isolates produce a vacuolating cytotoxin in vitro. To assess cytotoxin production in vivo, we sought to determine whether infection with a Tox+ H. pylori strain is associated with the presence of serum antitoxin antibodies. H. pylori isolates and serum samples were obtained from 30 patients, and serum samples were obtained from 20 uninfected patients as controls. Sera were tested by enzyme-linked immunosorbent assay for reactivity with the purified 87-kDa vacuolating cytotoxin, and the 30 H. pylori isolates were tested for vacuolating cytotoxin production. Supernatants from 14 (47%) of the 30 H. pylori isolates induced vacuolation of HeLa cells. Sera from the 30 H. pylori-infected patients reacted with the purified 87-kDa cytotoxin to a greater extent than sera from the uninfected controls for both immunoglobulin G (IgG) and IgA classes (P = 0.0004 and P < 0.0001, respectively). Serum IgG and IgA responses to the purified 87-kDa cytotoxin were higher among the 14 patients infected with Tox+ strains than among the 16 patients infected with Tox- strains (mean optical densities +/- standard errors of the means of 0.603 +/- 0.11 versus 0.234 +/- 0.07 [P = 0.005] and 0.644 +/- 0.12 versus 0.341 +/- 0.08 [P = 0.04] for IgG and IgA, respectively). Infection with a Tox+ strain compared with a Tox- strain was associated with increased antral polymorphonuclear leukocyte inflammation scores (P = 0.04). These data indicate that cytotoxin production by H. pylori isolates in vitro correlates with cytotoxin production in vivo and that infection with Tox+ H. pylori isolates may be associated with increased antral mucosal polymorphonuclear leukocyte infiltration.