[Diagnosis of allergen components in fungi and microbial communities: patient sensitization characteristics and main sensitizing proteins].

The human body, as a highly complex ecosystem, harbors diverse microbial communities, with major factors triggering allergic reactions encompassing the skin microbiome and fungi. The global diversity of fungi is estimated to range from approximately 600 000 to 1 million species, and theoretically, IgE-mediated sensitization may occur to any fungal species. As of now, the World Health Organization/IUIS official database records 113 fungal allergens originating from 30 different fungi species, covering 42 allergen families. Regarding the skin microbiome, 14 distinct Malassezia allergens have been identified, all derived from three different Malassezia fungi species--M. furfur, M. sympodialis, and M. globosa. The conditions of patients with these allergies are exceptionally complex. This article extensively discusses the latest research advancements and clinical applications related to skin microbiome and fungal allergies from the European Academy of Allergy and Clinical Immunology (EAACI) publication, "Molecular Allergology User's Guide 2.0". Additionally, it compiles information on the sources of fungal allergens, characteristics of allergen component protein families, clinical relevance, and management strategies, both domestically and internationally. The aim is to enhance the profound understanding of allergen components among relevant professionals. Through the application of advanced allergen component diagnostic techniques, the goal is to achieve precise diagnosis and treatment of fungal allergy patients and explore the mechanisms underlying fungal sensitization and pathogenesis, laying the foundation for studying the fungal allergen protein sensitization spectrum in the Chinese population.

[Advances of allergen component detection in the prevention and control of allergic diseases].

The incidence of allergic diseases in China is increasing year by year, which has caused heavy public health burden to individuals and society. The detection of specific IgE (sIgE) is an important way to diagnose the etiology of allergic disease. Currently, the diagnosis of allergic rhinitis In vitro mainly focus on the specific IgE of crude extracts in clinical practice, while the detection of sIgE in allergen components is rarely carried out. Clinicians, especially non-allergists, do not have sufficient understanding about the importance of sIgE in allergen component detection. Knowing the related types and clinical significance of allergen components can improve the diagnostic level of allergic diseases. Allergen component detection can distinguish the major components of common allergens, identify cross-sensitization, predict the risk of anaphylaxis, guide allergen immunotherapy and develop precise dietary regimens, so as to provide accurate prevention and control recommendations for patients.

[Correlation analysis of gut microbiota and biochemical indexes in patients with non-alcoholic fatty liver disease].

Objective: To investigate the relationship between gut microbiota structure and biochemical changes in patients with different types of nonalcoholic fatty liver disease (NAFLD), in order to provide evidence for clinical diagnosis and prevention of NAFLD. Methods: Forty-eight NAFLD cases (NAFLD group), 40 NAFLD cases with type 2 diabetes mellitus (NAFLD combined with type 2 diabetes mellitus group) and 30 healthy cases (healthy group) were randomly enrolled, and their body mass index, serum alanine aminotransferase, aspartate aminotransferase, total bilirubin, total cholesterol, triglyceride, high density lipoprotein, low density lipoprotein and uric acid were measured. Serum levels of TNF-alpha and fasting insulin were measured using ELISA, and then insulin resistance index was calculated. The gut microbiota of three groups of subjects was detected using 16S rDNA-based high-throughput sequencing. Lastly, the correlations between the various factors were analyzed. The comparison among groups was conducted by 2 test, and one-way ANOVA was used for comparison among groups with normal distribution and homogeneity of variance. Furthermore, the LSD method was used to compare the two groups. K-W rank sum test was used for comparison among groups without normal distribution or homogeneity of variance. Results: Body mass index, aspartate aminotransferase, triglyceride, total cholesterol, low density lipoprotein, uric acid, tumor necrosis factor-alpha, fasting insulin and insulin resistance index of NAFLD group were higher than healthy group, while the high-density lipoprotein was lower in the healthy group, and the difference was statistically significant (P< 0.05). Compared with NAFLD group, the life expectancy, fasting blood glucose and insulin resistance index of NAFLD combined with type 2 diabetes mellitus group were higher, while the body mass index, aspartic acid aminotransferase, total cholesterol and HDL levels were decreased, and the difference was statistically significant (P< 0.05). NAFLD group (P= 0.016) had decreased abundance of firmicutes than healthy group, and the abundancy of the firmicutes in the NAFLD combined with type 2 diabetes group was significantly lower (P< 0.001). The abundance of bacteroidetes in NAFLD combined with type 2 diabetes group was higher than healthy group, and the difference was statistically significant (P= 0.006). At the "genus level," the abundance of Roseburia and Subdoligranulum in the NAFLD group was decreased, while the Roseburia in the NAFLD group with type 2 diabetes group was significantly lower (P< 0.05). In addition, the abundance of Faecalibacterium, Blautia, Anaerostipes and Fusicatenibacter in NAFLD combined with type 2 diabetes group was lower than healthy group, and the difference was statistically significant (P< 0.001). Fusicatenibacter, Blautia, Anaerostipes, Faecalibacterium, and Roseburia were negatively correlated with fasting blood glucose and insulin resistance index levels (r< 0,P< 0.05), and positively correlated with high-density lipoprotein levels (r> 0,P< 0.05). Fusicatenibacter was negatively correlated with tumor necrosis factor-alpha (r= -0.211,P= 0.044), and Lachnoclostridium was positively correlated with body mass index, alanine aminotransferase, aspartate aminotransferase levels (r> 0,P< 0.05). Fusobacterium was positively correlated with aspartate aminotransferase level (r= 0.245,P= 0.019). Escherichia-shigella was positively correlated with fasting blood glucose, low-density lipoprotein, alanine aminotransferase, aspartate aminotransferase levels (r > 0,P< 0.05). Megamonas was negatively correlated with high-density lipoprotein levels (r= -0.231,P= 0.027). Conclusion: A structural change of gut microbiota had occurred in patients with NAFLD, suggesting changes in some of these bacterial genuses had relation to insulin resistance and inflammatory response, which may become a new target for the treatment of NAFLD.

Vitamin D receptor interacts with NLRP3 to restrict the allergic response.

Vitamin D receptor (VDR) mediates various biochemical activities between the cytoplasm and the nucleus in the cell. The nucleotide-binding, oligomerization domain (NOD)-like receptor family, pyrin domain containing 3 (NLRP3) protein is involved in the T helper type 2 (Th2) response. This study tests a hypothesis that VDR interacts with NLRP3 to restrict the Th2-biased response. In this study, VDR-/- mice and WT (WT) mice were used. Th2 cell differentiation between VDR-/- mice and WT mice was observed. We observed that CD4+ T cell activation was higher in VDR-/- mice. The VDR-/-CD4+ T cells were prone to Th2 polarization. VDR-/- mice produced more immunoglobulin (Ig)E. VDR bound NLRP3 to prevent Th2 differentiation by restricting IL4 gene transcription. Th2 biased inflammation spontaneously developed in the intestine of VDR-/- mice. In conclusion, VDR binds NLRP3 to restrict IL4 gene transcription and prevent biased Th2 polarization.

Manipulation of intestinal dysbiosis by a bacterial mixture ameliorates loperamide-induced constipation in rats.

Constipation has a significant influence on quality of life. Patients with constipation have slow waves in their gastrointestinal smooth muscles and less faecal water contents, which are closely associated with down-regulation of the interstitial cells of Cajal (ICC) in the gastrointestinal muscles and the aquaporin protein AQP3 expressed in colon epithelial cells. Recent studies supported that patients with constipation have altered intestinal microbial structures compared with healthy controls. Intestinal dysbiosis might be one possible pathophysiological mechanism causing constipation. Bacterial strains, such as Lactobacillus spp., have shown many beneficial effects on the amelioration of constipation. However, few studies reported the structural changes of intestinal microbiota post-intervention of probiotics. In this study, a bacterial mixture was administrated to rats with loperamide-induced constipation. Effects of the bacterial mixture on small intestine transit (SIT), faecal water content, and the intestinal microbiome in rats were evaluated. Meanwhile, we investigated several factors involved in signalling pathways that regulate function of ICC and expression of AQP3 to discuss the possible underlying molecular mechanisms. Intervention of the bacterial mixture improved SIT and faecal water content in constipated rats. The up-regulation of C-kit/SP signalling pathways in ICC and AQP3 significantly contributed to improvements. These changes were closely associated with the manipulation of intestinal dysbiosis in constipated rats. Furthermore, our results revealed the important role of intestinal microbiota in affecting gut motility through regulation of serotonin biosynthesis. This monoamine neurotransmitter, secreted from enterochromaffin cells, up-regulated both substance P/neurokinin 1 receptors pathway of ICC and the expression of AQP3 in intestinal epithelial cells. Our study suggested that the disrupted microbiome in patients could be a potential therapeutic target for the improvement of constipation.

Vitamin D contributes to mast cell stabilization.

BACKGROUND AND AIMS: Mast cells are the major effector cells in allergic disorders and many other informatory disorders. The mechanism of mast cell stabilization is not fully understood. Cumulative reports indicate that vitamin D (VitD) contributes to the homeostasis in the body. This study tests a hypothesis that VitD is required in the maintenance of the stability of mast cells. METHODS: The stability of mast cell lines, HMC1 cells, RBL-2H3 cells, p815 cells, and mouse bone marrow-derived mast cells (BMMC) was tested in the presence or absence of VitD3. RESULTS: Mast cells activated automatically in a VitD-deficient environment. Exposure to calcitriol in the culture increased the expression of VitD receptor (VDR) in mast cells. VDR formed complexes with Lyn in mast cells to inhibit the binding of Lyn to the ß chain of FcεRI and MyD88, which decreased the phosphorylation of Syk, decreased the levels of MAPK and NF-κB. VDR bound to the promoter of TNF-α to decrease the acetylation of histone H3/H4, RNA polymerase II and OCT1 (a transcription factor of TNF-α) at the promoter locus and repressed the expression of TNF-α in mast cells. CONCLUSIONS: The data demonstrate that VitD is required to maintain the stability of mast cells. The deficiency of VitD results in mast cell activation.

Dust mite allergen, glutathione S-transferase, induces T cell immunoglobulin mucin domain-4 in dendritic cells to facilitate initiation of airway allergy.

BACKGROUND: Allergens from dust mites play a critical role in the pathogenesis of airway allergy. The mechanism by which dust mite allergens induce allergic diseases is not fully understood yet. OBJECTIVE: This study tests a hypothesis that the eighth subtypes of Dermatophagoides farina allergen (Derf8) play an important role in the induction of airway allergy. METHODS: The protein of Derf8 was synthesized via molecular cloning approach. Dendritic cells (DC) were stimulated with Derf8 in the culture, and then, the expression of T cell immunoglobulin mucin domain 4 (TIM4) in dendritic cells (DC) was analysed. The role of Derf8 in the induction of airway allergy was evaluated with a mouse model. RESULTS: Exposure to Derf8 markedly induced the TIM4 expression in DCs by modulating the chromatin at the TIM4 promoter locus. Derf8 played a critical role in the expansion of the T helper 2 response in the mouse airway via inducing DCs to produce TIM4. Administration with Derf8-depleted dust mite extracts (DME) inhibited the allergic inflammation and induced regulatory T cells in mice with airway allergy. CONCLUSION: Derf8 plays an important role in the initiation of dust mite allergy. Vaccination with Derf8-deficient DME is more efficient to inhibit the dust mite allergic inflammation than using wild DME.

[Effects of Lactobacillus paracasei N1115 combined with fructooligosaccharides on non-alcoholic fatty liver disease induced by high-fat diet in mice].

Objective: To investigate the effects of Lactobacillus paracasei N1115 combined with fructooligosaccharides (FOS) on non-alcoholic fatty liver disease (NAFLD) in mice and its possible mechanism. Methods: A total of 50 male C57 mice were randomly and equally divided into five experimental groups. Group 1 received a normal diet (ND). Other four groups received a high-fat diet (HFD) to establish NAFLD models. In addition to HFD, group 3 received Lactobacillus paracasei N1115 (2.2×10(9) CFU/mL), group 4 received FOS (4 g/kg per day), and group 5 received Lactobacillus paracasei N1115 (2.2×10(9) CFU/mL) and FOS (4 g/kg per day). All groups received continuous intervention for 16 weeks. The following indices were measured for all groups after intervention: general condition, the levels of fasting blood glucose, insulin, and lipopolysaccharide (LPS), and the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and interferon (IFN)-γ in the serum and liver. The mRNA levels of Toll-like receptor (TLR)4, nuclear factor (NF)-κb, insulin receptor (InsR), and insulin receptor substrate (IRS)-1 were measured by real-time RT-PCR. The data were subjected to one-way analysis of variance and comparison between groups was made by Bonferroni method. Results: Compared with group 2, groups 3, 4, and 5 had significantly lower body weight, Lee's index, liver index, and the levels of blood glucose and insulin resistance (P < 0.05). The serum level of LPS in group 2 was significantly higher than that in the other experimental groups (group 1: 8.80 ± 0.85 U/L, group 3: 12.31 ± 1.01 U/L, group 4: 12.27 ± 0.98 U/L, and group 5: 10.17 ± 0.79 U/L vs group 2: 15.45 ± 1.14 U/L, F = 55.117, P < 0.001). The levels of TNF-α, IL-1ß, IL-6, and IFN-γ in the serum and liver in group 2 were also significantly higher than those in the other groups (P < 0.05). Group 2 had significantly higher mRNA levels of TLR4 and NF-κb in the liver than the other groups (F = 82.933, P < 0.001; F = 149.033, P < 0.001); however, it had significantly lower mRNA levels of InsR and IRS-1 in the liver than the other groups (F = 33.347, P < 0.001; F = 70.225, P < 0.001). Conclusion: Lactobacillus paracasei N1115 combined with FOS can reduce the level of LPS in the blood circulation, inhibit activation of the LPS/TLR4 signaling pathway, and reduce the release of inflammatory factor and the body's insulin resistance, so it can relieve NAFLD.

[Jinghuaweikang capsules combined with furazolidone-based triple or quadruple therapy as the rescue treatment for Helicobacter pylori infection: a multicenter randomized controlled clinical trial].

Objective: To explore the efficacy of Jinhuaweikang capsules plus furazolidone-based triple or quadruple therapy as the rescue treatment for Helicobacter pylori (H.pylori) infection. Methods: This is a prospective randomized controlled multicenter clinical trial. Patients with chronic gastritis from H. pylori infection in whom eradication treatment failed were recruited from 6 hospitals. All patients were divided into 4 groups using stratified randomization: group A1 (PAFJ), receiving pantoprazole 40 mg+ amoxicillin 1 000 mg+ furazolidone 100 mg+ Jinghuaweikang 3 capsules, twice a day for 10 d (d1-10); group A2, PAFJ therapy as in group A1, followed by Jinghuaweikang 3 capsules twice a day for 18 d (d11-28); group B1 (PAFB), receiving pantoprazole 40 mg+ amoxicillin 1 000 mg+ furazolidone 100 mg+ bismuth potassium citrate 220 mg, twice a day for 10 d (d1-10); group B2, PAFB therapy as in group B1, followed by Jinghuaweikang 3 capsules twice a day for 18 d (d11-28). At least 28 days after the end of treatment, all patients underwent 13C urea breath test for assessment of H. pylori eradication. Results: A total of 357 patients, 145 males and 212 females, were recruited, including 90 in group A1, 88 in group A2, 89 in group B1, and 90 in group B2. The eradication rates of H. pylori in groups A1 and A2 were 76.1%(67/88)and 79.6%(70/88) in per-protocol (PP) analysis, 74.4%(67/90) and 79.6%(70/88)in intention-to-treat (ITT) analysis; the rates in groups B1 and B2 were as 85.9%(73/85) and 92.1%(81/88) in PP analysis, 82.0%(73/89) and 90.0%(81/90)in ITT analysis. There were statistically significant differences in PP eradication rates among the 4 groups (P=0.020); there was statistically significant difference between groups A1 and B2, and also between groups A2 and B2 (P=0.003, 0.020), but not between groups A1/A2 and B1 (P>0.05), nor between groups B1 and B2 (P>0.05). No statistically significant differences in ITT eradication rates were found among the 4 groups (P>0.05). The improvement of belching and poor appetite for patients in groups A2 and B2 was better than those in groups A1 and B1. Conclusions: The efficacy of Jinghuaweikang capsules plus furazolidone-based quadruple therapy is superior to combination with furazolidone-based triple therapy as the rescue treatment of H. pylori, and superior to bismuth-containing quadruple therapy. Extending administration of Jinghuaweikang capsules to 28 days may better improve symptoms of indigestion.

[The dynamic change of cytokines associated with the speccificimmunotherapy and its clinical significance].

Objective:This project aimed to study the dynamic change of the cytokines associated with specific immunotherapy(SIT) pre- and post-SIT.Searching for immune regulatory indicators would used in SIT.Method:One hundred cases who had accepted SIT were enrolled in the project.Data of serum specific IgE and cytokines were statistically analyzed.In the three periods,pre-SIT,17 weeks post-SIT,57 weeks post-SIT,the levels of the eight kinds of cytokines(IL-4,IL-5,IL-8,IL-10,IL-13,IL-17,IFN-γ and TNF-α)were detected,and the dynamic change of the nasal symptoms score were analyzed.Result:The six kinds of cytokines(IL-5,IL-8,IL-10,IL-13,IL-17 and TNF-α)had no significant difference before and after SIT.The level of house dust mite sIgE level was positively correlated with serum IL-5 when the SIT pre-treatment and 57weeks (P<0.05).Pre-treatment and in 17 weeks after treatment,serum IL-5,IL-17 content difference and reduce the magnitude of nasal symptom scores were positively correlated (P<0.01).In 17 weeks of treatment and 57 weeks of treatment,difference of serum IL-10,IL-13,TNF-α levelsand the difference of nasal symptom scores were negatively correlated(P<0.01).Pre- treatment and 57 weeks,difference of serum IL-13,IL-17,TNF-α and the difference of nasal symptom scores were positively correlated (P<0.05),serum IL-10 levels of difference between the nose ministry of magnitude lower symptom scores were negatively correlated (P<0.01).Conclusion:The cytokines (IL-4,IL-5,IL-8,IL-10,IL-13,IL-17,IFN-γ and TNF-α) associated with the SIT play an important role in allergy and can objectively reflect the immune status during SIT.

Tolerogenic CX3CR1+ B cells suppress food allergy-induced intestinal inflammation in mice.

BACKGROUND: B lymphocytes are an important cell population of the immune regulation; their role in the regulation of food allergy has not been fully understood yet. OBJECTIVE: This study aims to investigate the role of a subpopulation of tolerogenic B cells (TolBC) in the generation of regulatory T cells (Treg) and in the suppression of food allergy-induced intestinal inflammation in mice. METHODS: The intestinal mucosa-derived CD5+ CD19+ CX3CR1+ TolBCs were characterized by flow cytometry; a mouse model of intestinal T helper (Th)2 inflammation was established to assess the immune regulatory role of this subpopulation of TolBCs. RESULTS: A subpopulation of CD5+ CD19+ CX3CR1+ B cells was detected in the mouse intestinal mucosa. The cells also expressed transforming growth factor (TGF)-ß and carried integrin alpha v beta 6 (αvß6). Exposure to recombinant αvß6 and anti-IgM antibody induced naive B cells to differentiate into the TGF-ß-producing TolBCs. Coculturing this subpopulation of TolBCs with Th0 cells generated CD4+ CD25+ Foxp3+ Tregs. Adoptive transfer with the TolBCs markedly suppressed the food allergy-induced intestinal Th2 pattern inflammation in mice. CONCLUSIONS: CD5+ CD19+ CX3CR1+ TolBCs are capable of inducing Tregs in the intestine and suppress food allergy-related Th2 pattern inflammation in mice.

Intestinal epithelial cells express galectin-9 in patients with food allergy that plays a critical role in sustaining allergic status in mouse intestine.

BACKGROUND AND AIMS: Mechanisms in sustaining the allergic hypersensitivity status in the body are unclear. Galectin-9 (Gal-9) has strong immune regulatory capacity. The present study aims to elucidate the role of Gal-9 in sustaining allergic status in the intestine. METHODS: Duodenal biopsies were obtained from 20 patients with peptic ulcer and food allergy (FA). The expression of Gal-9 in intestinal tissue was examined at both protein level and mRNA level. Two coculture systems with intestinal epithelial cells (IEC) and mast cells, or dendritic cells (DC) and T cells were established to investigate the source of Gal-9 in the intestine and the mechanism by which Gal-9 modulated DC's phenotyping and sustained the T helper 2 polarization. RESULTS: Normal IEC showed mild expression of Gal-9 that was markedly enhanced in patients with FA. Mast cells had the capability to induce IEC to produce Gal-9 via releasing tryptase that activated the proteinase-activated receptor 2 on IEC. Gal-9 activated DC to produce TIM4 (T-cell immunoglobulin mucin domain) via ligating TIM3 on DC via activating the cyclic guanosine monophosphate (cGMP) pathway. In a mouse FA model, blocking Gal-9 inhibited the allergic hypersensitivity status and the antigen-specific Th2 response in the intestine. CONCLUSIONS: IEC-derived Gal-9 contributes to sustaining the allergic status in the intestine.

Psychological stress induces eosinophils to produce corticotrophin releasing hormone in the intestine.

BACKGROUND AND AIMS: Psychological stress plays an important role in an array of intestinal disorders. Corticotrophin releasing hormone (CRH) is involved in the pathogenic process induced by psychological stress. The peripheral sources of CRH remain to be further understood. This paper aims to identify the sources of CRH in the intestine. METHODS AND RESULTS: Mice were treated with chronic restraint stress. A double-labelling approach was taken to localise CRH expression in immune cells (including dendritic cells, mast cells, lymphocytes, enterochromaffin cells and eosinophils) in the intestine by confocal microscopy and flow cytometry. As CRH was identified in eosinophils, a cell line of eosinophil, EoL-1 cells were treated with an array of putative stress mediators. The results showed that substance P (SP) induced the expression/release of CRH in eosinophils via neurokinin receptor 1 and 2. Co-culturing SP-primed eosinophils with the mast cell line, HMC-1 cells, we found that HMC-1 cells were activated by eosinophil-derived CRH that further induced T84 monolayer barrier dysfunction, which was further confirmed by a mouse model study. CONCLUSION: Eosinophils express CRH in the jejunum in response to psychological stress. SP and its receptors mediate the effect of stress in the CRH expression in eosinophils. Eosinophil-derived CRH activates mast cells to induce the jejunum epithelial barrier dysfunction.

The status of the cagA gene does not predict Helicobacter pylori-associated peptic ulcer disease in Singapore.

Discrepancies among reports from different geographical regions worldwide on the association between the presence of cagA and peptic ulcer disease prompted this study on the predictive value of the cagA gene in Helicobacter pylori-associated gastroduodenal diseases in the Singapore population. H. pylori strains were obtained from 169 patients with a peptic ulcer, 83 with non-ulcer dyspepsia, and nine with gastric cancer. The presence of the cagA gene was evaluated by polymerase chain reaction (PCR). The expected 400 bp PCR product coding for the cagA gene was present in 232/261 (89%) H. pylori isolates. Of these, 151/169 (89%) strains from patients with peptic ulcer, 73/83 (88%) strains from patients with non-ulcer dyspepsia and 8/9 (89%) strains from cancer patients were positive for the cagA gene. There was no statistically significant difference between the prevalence of cagA-positive strains from patients with distinct clinical outcomes (p > 0.05). The prevalence of cagA-positive strains in the Singapore population is high regardless of clinical disease status. The results suggest that the cagA gene is not a universal virulence marker of H. pylori.

Association of peptic ulcer with increased expression of Lewis antigens but not cagA, iceA, and vacA in Helicobacter pylori isolates in an Asian population.

BACKGROUND: Studies in Western populations suggest that cagA, iceA, and vacA gene status in Helicobacter pylori isolates is associated with increased virulence and peptic ulcer disease. AIM: To investigate the relationship between peptic ulcer and expression of Lewis (Le) antigens as well as cagA, iceA, and vacA in H pylori isolates in Singapore. METHODS: Expression of Le antigens in H pylori isolates obtained from patients with dyspepsia was measured by enzyme linked immunosorbent assay. The cagA, iceA, and vacA status was determined by polymerase chain reaction. RESULTS: Of 108 H pylori isolates, 103 (95.4%) expressed Le(x) and/or Le(y), while Le(a) and Le(b) were expressed in 23 (21.3%) and 47 (43.5%) isolates, respectively. Expression of two or more Le antigens (Le(x), Le(y), Le(a), or Le(b)) was significantly higher in H pylori isolated from ulcer patients than in non-ulcer patients (89.6% v 73.2%, p=0.035). There were no significant differences in the prevalence of cagA or iceA1 in H pylori isolates from peptic ulcer and non-ulcer patients (86.6% v 90.2% for cagA; 70.1% v 68.3% for iceA1), and no association of peptic ulcer with any specific vacA genotype. CONCLUSIONS: The present study indicates that peptic ulcer disease is associated with increased expression of Lewis antigens but not cagA, iceA, or vacA genotype in H pylori isolates in our population. This suggests that cagA, iceA, and vacA are not universal virulence markers, and that host-pathogen interactions are important in determining clinical outcome.

Unchanged characteristics of Helicobacter pylori during its morphological conversion.

Helicobacter pylori strains RH 54 and NCTC 11637 were grown in brain-heart infusion broth up to 56 days, and the coccoid form was obtained during prolonged incubation. Two morphological types of coccoids were observed, one of which was electron-dense and had an intact cellular membrane and flagella, indicating that it was likely to be viable. The other coccoid form was sphaeroblast-like and weakly stained, showing features of degeneration. Catalase activity was positive for aged cultures even up to 160 days. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that most of the protein bands appeared to be similar in both the spiral and coccoid forms. In addition, Lewis blood group antigens were detected in cultures of up to 8 weeks. Furthermore, two sets of primers for the vacA and cagA genes were used in polymerase chain reaction, and these two important genes remained conserved in both the spiral and coccoid forms. The present study shows that the coccoid form of H. pylori retained many important characteristics present in the spiral form despite the morphological conversion, and thus supports the notion that some of the coccoid forms of H. pylori are likely to be viable.

Simultaneous expression of type 1 and type 2 Lewis blood group antigens by Helicobacter pylori lipopolysaccharides. Molecular mimicry between h. pylori lipopolysaccharides and human gastric epithelial cell surface glycoforms.

Previous structural investigations performed on the lipopolysaccharides (LPSs) from the human gastric pathogen Helicobacter pylori have revealed that these cell surface glycan molecules express type 2 partially fucosylated, glucosylated, or galactosylated N-acetyllactosamine O antigen chains (O-chains) of various lengths, which may or may not be terminated at the nonreducing end by Lewis X (Lex) and/or Ley blood group epitopes in mimicry of human cell surface glycoconjugates and glycolipids. Subsequently, serological experiments with commercially available Lewis-specific monoclonal antibodies also have recognized the presence of Lex and Ley blood group antigens in H. pylori but, in addition, have indicated the presence of type 1 chain Lea, Leb, and Led (H-type 1) blood group epitopes in some H. pylori strains. To confirm their presence, structural studies and additional serological experiments were undertaken on H. pylori strains suspected of carrying type 1 chain epitopes. These investigations revealed that the O-chain region of H. pylori strain UA948 carried both Lea (type 1) and Lex (type 2) blood group determinants. The O-chain from H. pylori UA955 LPS expressed the terminal Lewis disaccharide (type 1 chain) and Lex and Ley antigens (type 2). The O-chain of H. pylori J223 LPS carried the type 1 chain precursor Lec, the H-1 epitope (Led, type 1 chain) and an elongated nonfucosylated type 2 N-acetyllactosamine chain (i antigen). Thus, O-chains from H. pylori LPSs can also express fucosylated type 1 sequences, and the LPS from a single H. pylori strain may carry O-chains with type 1 and 2 Lewis blood groups simultaneously. That monoclonal antibodies putatively specific for the Leb determinant can detect glycan substructures (Le disaccharide, Lec, and Led) of Leb indicates their nonspecificity. The expression of both type 1 and 2 Lewis antigens by H. pylori LPSs mimics the cell surface glycomolecules present in both the gastric superficial (which expresses mainly type 1 determinants) and the superficial and glandular epithelium regions (both of which express predominantly type 2 determinants). Therefore, each H. pylori strain may have a different niche within the gastric mucosa, and each individual LPS blood group antigen may have a dissimilar role in H. pylori adaptation.

Phase variation in Helicobacter pylori lipopolysaccharide.

Helicobacter pylori NCTC 11637 lipopolysaccharide (LPS) expresses the human blood group antigen Lewis x (Le(x)) in a polymeric form. Le(x) is beta-D-galactose-(1-4)-[alpha-L-fucose-(1-3)]-beta-D-acetylglucosamine. Schematically the LPS structure is (Le(x))n-core-lipid A. In this report, we show that Le(x) expression is not a stable trait but that LPS displays a high frequency (0.2 to 0.5%) of phase variation, resulting in the presence of several LPS variants in one bacterial cell population. One type of phase variation implied the loss of alpha1,3-linked fucose, resulting in variants that expressed nonsubstituted polylactosamines (also called the i antigen), i.e., Le(x) minus fucose; LPS: (lactosamine)n-core-lipid A. The switch of Le(x) to i antigen was reversible. A second group of variants arose by loss of polymeric main chain which resulted in expression of monomeric Le(y); LPS: (Le(y))-core-lipid A. A third group of variants arose by acquisition of alpha1,2-linked fucose which hence expressed Le(x) plus Le(y); LPS: (Le(y))(Le(x))n-core-lipid A. The second and third group of variants switched back to the parental phenotype [(Le(x))-core-lipid A] in lower frequencies. Part of the variation can be ascribed to altered expression levels of glycosyltransferase levels as assessed by assaying the activities of galactosyl-, fucosyl-, and N-acetylglucosaminyltransferases. Clearly phase variation increases the heterogeneity of H. pylori, and this process may be involved in generating the very closely related yet genetically slightly different strains that have been isolated from one patient.

Improving the success of culturing Helicobacter pylori from gastric biopsies.

Factors influencing the successful isolation of Helicobacter pylori from human gastric biopsies were studied. Within 24 h, each of the gastric biopsies was inoculated onto chocolate blood agar media and incubated for up to 2 weeks. Among 63 (70%) culture positive cases in 90 patients, 58 (64%) cases were culture positive for both specimens, while five (6%) cases were culture positive in only one biopsy. Of the 63 positive cultures, 51 H. pylori strains (81%) grew on both media with and without antibiotics. Eight strains (13%) grew only on medium without antibiotics, while four isolates (6%) were obtained only from medium with antibiotics. These results support the previous histological observation of patchy colonization of H. pylori in the stomach. The success rate for culture of H. pylori from gastric biopsies increased when two biopsies were taken and inoculated on chocolate blood agar media with and without antibiotics.

The lipopolysaccharide of Helicobacter mustelae type strain ATCC 43772 expresses the monofucosyl A type 1 histo-blood group epitope.

The lipopolysaccharide of Helicobacter mustelae type strain ATCC 43772 was obtained by phenol-water extraction of bacterial cells. Structural investigations were made on the lipid A free saccharide moiety released from the lipopolysaccharide by mild acetic acid hydrolysis. Nuclear magnetic resonance, gas liquid chromatography-mass spectrometry and fast atom bombardment-mass spectrometry were employed in the characterization of products from chemical manipulations. A monoclonal antibody specific for blood group A reacted strongly with lipopolysaccharide of H. mustelae. Chemical and serological data showed that the outer core region of the lipopolysaccharide from H. mustelae ATCC 43772 expresses the monofucosyl A type 1, alpha-D-GalNAc-(1-->3)-[alpha-L-Fuc-(1-->2]-beta-D-Gal-(1-->3)- beta-D-GlcNAc, blood group determinant, a mimic of animal cell surface glycolipids and glycoproteins.