Exploring the potential of phytochemicals as inhibitors of 3'-phosphoadenosine 5'-phosphosulfate synthase 1 targeting cancer therapy.

3'-phosphoadenosine 5'-phosphosulfate synthase 1 (PAPSS1) is an enzyme that critically synthesises the biologically active form of sulfate (PAPS) for all sulfation reactions. The discovery of PAPSS1 as a possible drug target for cancer therapy, specifically in non-small cell lung cancer, has prompted us to investigate potential small-molecule inhibitors of PAPSS1. Here, a structure-based virtual screening method was used to search for phytochemicals in the IMPPAT database to find potential inhibitors of PAPSS1. The primary hits were selected based on their physicochemical, ADMET, and drug-like properties. Then, the binding affinities were calculated and analyzed the interactions to identify safer and more effective hits. The research identified two phytochemicals, Guggulsterone and Corylin, that exhibited significant affinity and specific interaction with the ATP-binding pocket of PAPSS1. Structural observations made by molecular docking were further accompanied by molecular dynamics (MD) simulations and principal component analysis (PCA) to examine the conformational changes and stability of PAPSS1 with the elucidated compounds Guggulsterone and Corylin. MD simulation results suggested that the binding of Guggulsterone and Corylin stabilizes the PAPSS1 structure, leading to fewer conformational changes. This implies that these compounds may be useful in developing PAPSS1 inhibitors for the therapeutic development against non-small cell lung cancer (NSCLC). This study highlights the potential of phytochemicals as PAPSS1 inhibitors and the utility of computational approaches in drug discovery.Communicated by Ramaswamy H. Sarma.

Discovering potential inhibitors of Raf proto-oncogene serine/threonine kinase 1: a virtual screening approach towards anticancer drug development.

Raf proto-oncogene serine/threonine kinase 1 (RAF1 or c-Raf) is a serine/threonine protein kinase crucial in regulating cell growth, differentiation, and survival. Any disruption or overexpression of RAF1 can result in neoplastic transformation and other disorders such as cardiomyopathy, Noonan syndrome, leopard syndrome, etc. RAF1 has been identified as a potential therapeutic target in drug development against various complex diseases, including cancer, due to its remarkable role in disease progression. Here, we carried out a multitier virtual screening study involving different in-silico approaches to discover potential inhibitors of RAF1. After applying the Lipinski rule of five, we retrieved all phytocompounds from the IMPPAT database based on their physicochemical properties. We performed a molecular docking-based virtual screening and got top hits with the best binding affinity and ligand efficiency. Then we screened out the selected hits using the PAINS filter, ADMET properties, and other druglike features. Eventually, PASS evaluation identifies two phytocompounds, Moracin C and Tectochrysin, with appreciable anti-cancerous properties. Finally, all-atom molecular dynamics simulation (MDS) followed by interaction analysis was performed on the elucidated compounds in complex with RAF1 for 200 ns to investigate their time-evolution dynamics and interaction mechanism. Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) and Dynamical Cross-Correlation Matrix (DCCM) analyses then followed these results from the simulated trajectories. According to the results, the elucidated compounds stabilize the RAF1 structure and lead to fewer conformational alterations. The results of the current study indicated that Moracin C and Tectochrysin could serve as potential inhibitors of RAF1 after required validation.Communicated by Ramaswamy H. Sarma.

Antioxidative and ROS-dependent apoptotic effects of Cuscuta reflexa Roxb. stem against human lung cancer: network pharmacology and in vitro experimental validation.

Lung cancer remains a formidable global health challenge, necessitating the exploration of novel therapeutic approaches. This study investigates the potential of Cuscuta reflexa Roxb. stem extract as an anticancer agent against human lung cancer, focusing on its antioxidative and ROS-dependent apoptotic effects. Utilizing a combination of network pharmacology and in-vitro experimental validation, we delineate the multifaceted molecular mechanisms underlying the observed effects. The antioxidant potential of C. reflexa stem extract was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH•), 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS•+) and ferric reducing/antioxidant power (FRAP), hydroxyl free radical scavenging, reactive nitrogen oxide scavenging and super oxide anion radical scavenging assays. Furthermore, the antiproliferative and proapoptotic effect of C. reflexa stem extract was evaluated against A549 lung adenocarcinoma cell line using the consecrated sulforhodamine B (SBR) and Annexin V-PI assays. Additionally, the mitochondrial membrane potential (MMP) and the total reactive oxygen species (ROS) estimation assays were performed. As a result, network pharmacology analysis revealed a complex interaction network between the bioactive constituents of C. reflexa and key proteins implicated in lung cancer progression. The C. reflexa stem extract showed dose-dependent antioxidant activity against DPPH• (IC50 - 87.38 µg/mL), reactive nitrogen oxide (IC50 - 318.34 µg/mL), FRAP (IC50 - 359.96 µg/mL), hydroxy free radicals (IC50 - 526.12 µg/mL) than ABTS●+ (IC50 - 698.45 µg/mL) and super oxide anion (IC50 - 892.71 µg/mL) as well as cytotoxic activity against A549 cells (IC50 - 436.80 µg/mL). Observations of morphological features in treated cells have revealed hallmark of apoptosis properties. Furthermore, as a result of treatment with C. reflexa stem extract, ROS generation and mitochondrial depolarization were increased in A549 cells, suggesting that this treatment has significant apoptotic properties. . These findings highlight the potential utility of this natural extract as an innovative therapeutic strategy for lung cancer treatment. The integration of network pharmacology and experimental validation enhances our understanding of the underlying mechanisms and provide the way for further translational research.Communicated by Ramaswamy H. Sarma.

Genome-Wide Analysis of Kidney Renal Cell Carcinoma: Exploring Differentially Expressed Genes for Diagnostic and Therapeutic Targets.

Kidney renal cell carcinoma (KIRC) is the most common type of renal cancer. Kidney malignancies have been ranked in the top 10 most frequently occurring cancers. KIRC is a prevalent malignancy with a poor prognosis. The disease has risen for the last 40 years, and robust biomarkers for KIRC are needed for precision/personalized medicine. In this bioinformatics study, we utilized genomic data of KIRC patients from The Cancer Genome Atlas for biomarker discovery. A total of 314 samples were used in this study. We identified many differentially expressed genes (DEGs) categorized as upregulated or downregulated. A protein-protein interaction network for the DEGs was then generated and analyzed using the Search Tool for the Retrieval of Interacting Genes plugin of Cytoscape. A set of 10 hub genes was selected based on the Maximum Clique Centrality score defined by the CytoHubba plugin. The elucidated set of genes, that is, CALCA, CRH, TH, CHAT, SLC18A3, FSHB, MYH6, CAV3, KCNA4, and GBX2, were then categorized as potential candidates to be explored as KIRC biomarkers. The survival analysis plots for each gene suggested that alterations in CHAT, CAV3, CRH, MYH6, SLC18A3, and FSHB resulted in decreased survival of KIRC patients. In all, the results suggest that genomic alterations in selected genes can be explored to inform biomarker discovery and for therapeutic predictions in KIRC.

Identification of novel c-Kit inhibitors from natural sources using virtual screening and molecular dynamics simulations.

The Mast/Stem cell growth factor receptor Kit (c-Kit), a Proto-oncogene c-Kit, is a tyrosine-protein kinase involved in cell differentiation, proliferation, migration, and survival. Its role in developing certain cancers, particularly gastrointestinal stromal tumors (GISTs) and acute myeloid leukemia (AML), makes it an attractive therapeutic target. Several small molecule inhibitors targeting c-Kit have been developed and approved for clinical use. Recent studies have focused on identifying and optimizing natural compounds as c-Kit inhibitors employing virtual screening. Still, drug resistance, off-target side effects, and variability in patient response remain significant challenges. From this perspective, phytochemicals could be an important resource for discovering novel c-Kit inhibitors with less toxicity, improved efficacy, and high specificity. This study aimed to uncover possible c-Kit inhibitors by utilizing a structure-based virtual screening of active phytoconstituents from Indian medicinal plants. Through the screening stages, two promising candidates, Anilinonaphthalene and Licoflavonol, were chosen based on their drug-like features and ability to bind to c-Kit. These chosen candidates were subjected to all-atom molecular dynamics (MD) simulations to evaluate their stability and interaction with c-Kit. The selected compounds Anilinonaphthalene from Daucus carota and Licoflavonol from Glycyrrhiza glabra showed their potential to act as selective binding partners of c-Kit. Our results suggest that the identified phytoconstituents could serve as a starting point to develop novel c-Kit inhibitors for developing new and effective therapies against multiple cancers, including GISTs and AML. The use of virtual screening and MD simulations provides a rational approach to discovering potential drug candidates from natural sources.Communicated by Ramaswamy H. Sarma.

Integrating network pharmacology approaches for the investigation of multi-target pharmacological mechanism of 6-shogaol against cervical cancer.

Traditional treatment of cancer has been plagued by a number of obstacles, such as multiple drug resistance, toxicity and financial constraints. In contrast, phytochemicals that modulate a variety of molecular mechanisms are garnering increasing interest in complementary and alternative medicine. Therefore, an approach based on network pharmacology was used in the present study to explore possible regulatory mechanisms of 6-shogaol as a potential treatment for cervical cancer (CC). A number of public databases were screened to collect information on the target genes of 6-shogaol (SuperPred, Targetnet, Swiss target prediction and PharmMapper), while targets pertaining to CC were taken from disease databases (DisGeNet and Genecards) and gene expression omnibus (GEO) provided expression datasets. With STRING and Cytoscape, protein-protein interactions (PPI) were generated and topology analysis along with CytoNCA were used to identify the Hub genes. The Gene Ontology (GO) database Enrichr was used to annotate the target proteins, while, using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, signaling pathway enrichment analysis was conducted. Molecular docking and survival analysis for the Hub genes revealed four genes (HSP90AA1, HRAS, ESR1 and EGFR) with lowest binding energy and majority of the Hub genes (EGFR, SRC, CASP-3, HSP90AA1, MTOR, MAPK-1, MDM2 and ESR1) were linked with the overall survival of CC patients. In conclusion, the present study provides the scientific evidence which strongly supports the use of 6-shogoal as an inhibitor of cellular proliferation, growth, migration as well as inducer of apoptosis via targeting the hub genes involved in the growth of CC.Communicated by Ramaswamy H. Sarma.

Discovering Gummadiol and Isoarboreol as potential inhibitors of sphingosine kinase 1: virtual screening and MD simulation studies.

Sphingosine kinase 1 (SphK1) dysfunction is well-known to be linked to various severe diseases, including breast, lung, prostate, and hematological cancers. Due to its crucial function in the onset of cancer and its progression, it is considered a notable drug target for anticancer therapy. Small molecule inhibitors with high specificity and efficacy towards SphK1 are needed for their therapeutic use. In order to find possible SphK1 inhibitors, we conducted a stepwise structure-based virtual screening of plant-based molecules available from the IMPPAT library. A multi-step virtual screening, including physicochemical and ADMET evaluation, PAINS, molecular docking, PASS analysis followed by molecular dynamics (MD) simulation and principal component analysis, identifies two compounds, Gummadiol and Isoarboreol, against SphK1. All-atom MD simulations were performed for 100 ns which examined the structural changes and stability of the docked complexes in the aqueous environment. The time evolution data of structural deviations and compactness, PCA and free energy landscapes suggested that the binding of Gummadiol and Isoarboreol with SphK1 is considerably stable throughout the trajectory. The study highlighted the use of phytochemicals in anticancer therapeutics and presented Gummadiol and Isoarboreol as promising inhibitors of SphK1.Communicated by Ramaswamy H. Sarma.

Antibiofilm Potential and Exoenzyme Inhibition by Elattaria cardamomum Essential Oil in Candida spp. Strains.

Fungal infections caused by Candida species have attracted great interest due to their resistance to commercial antifungal agents. Essential oils from aromatic and medicinal plants have many bioactive compounds that are known for their important biological activities, mainly their antimicrobial effects. In the present study, we aimed to evaluate the antifungal ability of Elettaria cardamomum essential oil (EO) against different clinical Candida isolates. Then, we investigated the anti-phospholipase, anti-protease, and anti-biofilm activity of E. cardamomum EO against the selected isolates. Twenty-four Candida strains (clinical and reference) were tested for virulence factors such as biofilm formation, protease, and phospholipase activity. The minimum inhibitory (MIC) and fungicidal (MFC) concentrations of E. cardamomum were determined, and their effects were tested against all Candida strains. Our results revealed that E. cardamomum EO was rich in α-terpinyl acetate (56.5%), limonene (12.6%), and mentha-2.4(8)-diene (7.65%). The tested EO showed activity against all tested Candida strains in their planktonic form and against exoenzymes and biofilm production. Based on our findings, we promote the use of E. cardamomum EO as a treatment against clinical Candida isolates active on the virulence factors of this fungus.

Cytotoxic Activity, Cell Cycle Inhibition, and Apoptosis-Inducing Potential of Athyrium hohenackerianum (Lady Fern) with Its Phytochemical Profiling.

In the present study, we investigated the cytotoxic effects of Athyrium hohenackerianum ethanolic extract (AHEE) on the proliferation of breast, lung, and colon cancer cells. The AHEE was tested for its effect on the progression of the cell cycle, followed by induction of apoptosis determination by flow cytometry. Real-time qRT-PCR was also utilized to observe the initiation of apoptosis. In addition, GC-MS was performed in order to identify the active phytochemicals present in the AHEE. A cytotoxic activity with an IC50 value of 123.90 µg/mL against HCT-116 colon cancer cells was exhibited by AHEE. Following propidium iodide staining, annexin-V/PI, and clonogenic assays, AHEE treatment results in cell arrest in the S phase, causing an increase in the early and late phases of apoptosis and displaying antiproliferative potential, respectively. The morphological alterations were further monitored using acridine orange/ethidium bromide (AO/EB) staining. When compared with the control cells, features of apoptotic cell death, including nuclear fragmentation, in the AHEE-treated cells were noticed. The apoptosis was also confirmed by detecting the increased expression of p53 and caspase-3 along with the downregulation of Bcl-2. GC-MS analysis revealed that trans-linalool oxide, loliolide, phytol, 4,8,12,16-tetramethylheptadecan-4-olide, and gamma-sitosterol were the major phytochemical constituents. Based on these findings, it can be suggested that AHEE causes cellular death via apoptosis, which should be further explored for the identification of active compounds responsible for these observed effects. Therefore, AHEE can be used in the pharmaceutical development of anticancer agents for cancer therapeutics.

Bax/Bcl-2 Cascade Is Regulated by the EGFR Pathway: Therapeutic Targeting of Non-Small Cell Lung Cancer.

Non-small cell lung carcinoma (NSCLC) comprises 80%-85% of lung cancer cases. EGFR is involved in several cancer developments, including NSCLC. The EGFR pathway regulates the Bax/Bcl-2 cascade in NSCLC. Increasing understanding of the molecular mechanisms of fundamental tumor progression has guided the development of numerous antitumor drugs. The development and improvement of rationally planned inhibitors and agents targeting particular cellular and biological pathways in cancer have been signified as a most important paradigm shift in the strategy to treat and manage lung cancer. Newer approaches and novel chemotherapeutic agents are required to accompany present cancer therapies for improving efficiency. Using natural products as a drug with an effective delivery system may benefit therapeutics. Naturally originated compounds such as phytochemicals provide crucial sources for novel agents/drugs and resources for tumor therapy. Applying the small-molecule inhibitors (SMIs)/phytochemicals has led to potent preclinical discoveries in various human tumor preclinical models, including lung cancer. In this review, we summarize recent information on the molecular mechanisms of the Bax/Bcl-2 cascade and EGFR pathway in NSCLC and target them for therapeutic implications. We further described the therapeutic potential of Bax/Bcl-2/EGFR SMIs, mainly those with more potent and selectivity, including gefitinib, EGCG, ABT-737, thymoquinone, quercetin, and venetoclax. In addition, we explained the targeting EGFR pathway and ongoing in vitro and in vivo and clinical investigations in NSCLC. Exploration of such inhibitors facilitates the future treatment and management of NSCLC.

Rifaximin has minor effects on bacterial composition, inflammation, and bacterial translocation in cirrhosis: A randomized trial.

BACKGROUND AND AIM: Decompensated cirrhosis is characterized by disturbed hemodynamics, immune dysfunction, and high risk of infections. Translocation of viable bacteria and bacterial products from the gut to the blood is considered a key driver in this process. Intestinal decontamination with rifaximin may reduce bacterial translocation (BT) and decrease inflammation. A randomized, placebo-controlled trial investigated the effects of rifaximin on inflammation and BT in decompensated cirrhosis. METHODS: Fifty-four out-patients with cirrhosis and ascites were randomized, mean age 56 years (± 8.4), and model for end-stage liver disease score 12 (± 3.9). Patients received rifaximin 550-mg BD (n = 36) or placebo BD (n = 18). Blood and fecal (n = 15) sampling were conducted at baseline and after 4 weeks. Bacterial DNA in blood was determined by real-time qPCR 16S rRNA gene quantification. Bacterial composition in feces was analyzed by 16S rRNA gene sequencing. RESULTS: Circulating markers of inflammation, including tumor necrosis factor alpha, interleukins 6, 10, and 18, stromal cell-derived factor 1-α, transforming growth factor ß-1, and high sensitivity C-reactive protein, were unaltered by rifaximin treatment. Rifaximin altered abundance of bacterial taxa in blood marginally, only a decrease in Pseudomonadales was observed. In feces, rifaximin decreased bacterial richness, but effect on particular species was not observed. Subgroup analyses on patients with severely disturbed hemodynamics (n = 34) or activated lipopolysaccharide binding protein (n = 37) revealed no effect of rifaximin. CONCLUSION: Four weeks of treatment with rifaximin had no impact on the inflammatory state and only minor effects on BT and intestinal bacterial composition in stable, decompensated cirrhosis (NCT01769040).

Detection of Helicobacter rodentium-like DNA in the liver tissue of patients with chronic liver diseases by polymerase chain reaction-denaturing gradient gel electrophoresis and DNA sequence analysis.

Many Helicobacter spp. were isolated from the stomach, intestinal tract, and liver of different animals and humans. The association between Helicobacter spp. and hepatobiliary diseases, including hepatocellular carcinoma, was thoroughly examined, indicating a potential role of the bacteria in the progression toward cancer. In our work, we screened 97 liver biopsies from patients with chronic liver diseases for the presence of Helicobacter spp. DNA. With the use of genus-specific polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing, we found that the majority of Helicobacter spp. DNA detected was similar to Helicobacter rodentium DNA (71%). The DNA of other detected Helicobacter spp. was similar to Helicobacter pylori DNA. This is the first indication of H. rodentium-like DNA presence in human liver tissue. We also conclude that PCR-DGGE is a useful screening method for assigning species designation and heterogeneity.

Determination of lamivudine-resistant variants of hepatitis B virus by denaturing gradient gel electrophoresis: a novel approach to monitoring drug resistance.

BACKGROUND: A DGGE-based assay for hepatitis B virus (HBV) drug-resistance monitoring was designed and checked for feasibility. It detects mutations within the YMDD motif related to lamivudine resistance. MATERIAL/METHODS: The YMDD motif of HBV polymerase was amplified by the set of primers designed in this study. DGGE analysis of the amplicons was performed on 9% polyacrylamide gels containing a 20-40% gradient of urea plus formamide and electrophoresis was performed. DNA sequencing was performed using a standard protocol. RESULTS: Based on the DGGE pattern of previously sequenced HBV variants, a reference ladder consisting of bands was constructed within and near the YMDD motif of HBV with excellent resolution. The genotypes of all the fragments included in the ladder were confirmed by sequencing after DGGE analysis. The flexibility of DGGE was demonstrated by the ability to add more bands to the migration ladder when new variants were discovered during the analysis of patient specimens. Clinical samples from HBV-infected patients were also used to demonstrate the performance of this approach. CONCLUSIONS: This preliminary feasibility study of HBV drug-resistance monitoring by means of DGGE shows the potential advantage of this approach for low-cost screening for viral drug resistance in clinical settings. The presented example can be extended to detect other mutations related to drug resistance in the HBV genome as well as other viruses.

Detection of Helicobacter species in liver and stomach tissues of patients with chronic liver diseases using polymerase chain reaction-denaturing gradient gel electrophoresis and immunohistochemistry.

OBJECTIVE: Helicobacter DNA has been detected in the hepatobiliary tree of patients with chronic liver diseases (CLD). The presence of H. pylori in the stomach compared with in the liver of the same patients with CLD has not been studied, therefore to the aim of this study was to investigate the presence of Helicobacter DNA and antigens in the liver and stomach of Polish patients with chronic liver diseases using molecular and immunological methods. MATERIAL AND METHODS: Gastric mucosa and liver tissue samples and sera were collected from 97 Polish patients with CLD. Anti-H. pylori antibodies were detected by enzyme immunoassay (EIA), and H. pylori-like antigens detected by immunohistochemistry. Helicobacter DNA was detected in stomach and liver samples using a semi-nested Helicobacter genus-specific polymerase chain reaction (PCR) assay, and Helicobacter species identified by denaturing gradient gel electrophoresis (DGGE) and sequencing analysis of amplified PCR products. RESULTS: H. pylori was identified by DGGE and sequence analysis in 60/62 (97%) and 25/25 (100%) of the gastric and liver Helicobacter genus-positive samples, respectively, whereas DNA of H. heilmannii was detected in 2/62 (3%) of the Helicobacter genus-positive gastric samples. H. pylori cagA gene was detected in 23/62 (36%) and 3/25 (12%) gastric and liver tissue samples, respectively. H. pylori-like antigens were detected in 61/97 (63%) gastric mucosa and in 40/97 (41%) liver tissue samples. CONCLUSIONS: H. pylori-like organisms appeared to dominate the gastric mucosa and liver tissue of Polish patients with CLD. The prevalence of the cagA gene was higher in stomach compared with liver samples, which suggests a possible role of cagA negative H. pylori-like organisms in CLD. On the other hand, no significant correlation was found between the presence of H. pylori-like DNA and antigens in the liver and liver function tests.

Expression of matrix metalloprotease-2, -7 and -9 on human colon, liver and bile duct cell lines by enteric and gastric Helicobacter species.

Gastric and enteric Helicobacter species have been associated with malignant and inflammatory diseases of the stomach, liver, gall bladder and intestine. Matrix metalloproteinases (MMPs) participate in degradation of extracellular matrix, which allows bacteria to come in contact with and interact with the cells. Enhanced level of MMPs facilitates metastasis and cell invasion of tumor cells by removal of physical barriers, as well as modulation of biologic activities of the proteins residing in the extracellular matrix. The aim of this study was to evaluate the effect of gastric and enteric Helicobacter on induction of MMPs in hepatocytes and epithelial cells of gall bladder and colon. Human hepatocytes HepG2, gall bladder epithelial cells TFK-1, and colon epithelial cells HT29 were infected with strains of H. pylori cagA+, cagE+, H. pylori cagA-, cagE-, H. pullorum, H. cholecystus, H. bilis and H. hepaticus. Protein levels of MMPs were analyzed by enzyme-linked immunosorbent assay and immunohistochemistry. Reverse transcription-quantitative polymerase chain reaction was used to study mRNA levels. Increased expression of MMP-2 and MMP-9 was observed on HepG2, TFK-1 and HT29 infected with H. pylori cagA+, cagE+ and H. cholecystus strains. H. pylori cagA+, cagE+, H. cholecystus, H. pullorum, H. bilis and H. hepaticus strains increased expression of MMP-7 on HT29, compared to uninfected control cells. The effect of MMP upregulation on HepG2, TFK-1 and HT29 was bacterial dose dependent. H. pylori cagA-, cagE- strain did not increase expression of MMPs. Inducible MMPs on colon and bile duct epithelial cells as well as hepatocytes may play an important role in facilitating invasion and progression of cancer by Helicobacter species colonizing the hepatobiliary and gastrointestinal tract.

Helicobacter pylori and other Helicobacter species in gallbladder and liver of patients with chronic cholecystitis detected by immunological and molecular methods.

OBJECTIVE: Despite several recent reports on the detection of Helicobacter DNA in human bile, there are still uncertainties concerning the correlation of these findings with biliary tract and liver diseases. MATERIAL AND METHODS: Using molecular methods and immunohistochemistry (IHC), we investigated gallbladder and liver biopsy specimens from 22 adult Ukrainian patients with chronic cholecystitis for the presence of Helicobacter species. Patient sera were collected and tested for antibody reactivity to antigens of three Helicobacter spp. Detection of Helicobacter DNA was performed using a Helicobacter genus-specific 16S rDNA PCR. Amplified DNA was identified by PCR-denaturating gradient gel electrophoresis (DGGE) and DNA sequencing. Tissue sections of gallbladder and liver were examined by IHC with antibodies specific to H. pylori, the CagA and VacA cytotoxins of H. pylori, H. hepaticus and to Campylobacter jejuni. Patient sera were analysed by immunoblot for IgG antibodies to soluble surface proteins of H. pylori, H. hepaticus and H. bilis. RESULTS: Helicobacter DNA was found in 16/22 (73%) of the gallbladder samples and in 11/22 (50%) of the liver samples. IHC showed the presence of the H. pylori specific cytotoxins CagA and VacA inside the gallbladder epithelial cells without co-localization of H. pylori at the epithelial lining. Immunoblot analysis of the patient sera did not show any correlation between the presence of Helicobacter DNA and IgG antibody responses. CONCLUSIONS: The high prevalence of Helicobacter DNA and the positive findings by IHC in gallbladder and liver raise questions concerning an infectious role of Helicobacter in patients with chronic cholecystitis.

Detection of Helicobacter ganmani-like 16S rDNA in pediatric liver tissue.

BACKGROUND: To determine the presence of Helicobacter species in the liver biopsy specimens from children with various chronic liver diseases as data in adult literature suggests a possible role of these bacteria in their pathogenesis. MATERIALS AND METHODS: Paraffin sections of 61 liver biopsies of pediatric patients with miscellaneous diseases and autopsy liver tissue from 10 control subjects with no evidence of preexisting liver disease were examined for the presence of Helicobacter species by a genus-specific seminested polymerase chain reaction (PCR) assay. PCR-products of positive samples were further characterized by denaturing gradient gel electrophoresis (DGGE) and DNA-sequence analysis. Based on those results, a seminested PCR assay for H. ganmani was developed and applied to the samples. RESULTS: On analysis, 40/61 patient samples were positive in the genus-specific Helicobacter PCR and 4/10 from the control group. The nucleotide sequences of 16S rDNA fragments were 99-100% similar to mainly Helicobacter sp. 'liver' and H. ganmani. PCR-products similar to H. canis and H. bilis were also found. The 16S rDNAs of control specimens showed similarity to Helicobacter sp. 'liver'. In the H. ganmani-specific PCR analysis 19 patients, but none of the controls, were positive. CONCLUSIONS: Amplified Helicobacter 16S rDNAs were related to Helicobacter sp. 'liver' or H. ganmani in liver biopsy specimens of pediatric patients. The possible significance of Helicobacter species in pediatric liver diseases needs to be evaluated further in prospective studies.