Complications and chemotherapy have little impact on postoperative quality of life after pancreaticoduodenectomy - a cohort study.

BACKGROUND: With the poor prognosis of pancreatic cancer and the high rate of postoperative complications after pancreaticoduodenectomy, it is important to evaluate how the operation affects patients' quality of life. METHODS: This single-centre study included all patients undergoing pancreaticoduodenectomy from 2006 to 2016. Quality of life was measured with two questionnaires preoperatively, and at 6 and 12 months postoperatively. Comparisons between groups were made using a linear mixed models analysis. RESULTS: Of 279 patients planned for pancreaticoduodenectomy, 245 underwent the operation. The postoperative response rates were all 80% or more. Differences were found in one domain between the early and late time periods and three domains between patients receiving and not receiving adjuvant chemotherapy. No significant differences were found between patients with and without severe postoperative complications. However, the demographic variables of age group, sex, preoperative diabetes and smoking all exerted a significant impact on postoperative quality of life. CONCLUSION: While little or no impact was shown for the factors of postoperative complications, time period and adjuvant chemotherapy, demographic data, such as age, sex, preoperative diabetes and smoking, had considerable impacts on postoperative quality of life after pancreaticoduodenectomy.

Gastroduodenal Changes Two Years After Eradication of Helicobacter pylori in a Population-Based Cohort.

BACKGROUND: The main cause of chronic gastritis is Helicobacter pylori (H. pylori) infection. Some individuals with H. pylori-related chronic gastritis develop atrophy of the gastric mucosa, a risk factor for gastric neoplasia. When H. pylori-associated gastritis is encountered, it is important to be aware of its natural history and reversibility of associated histopathological and hormonal changes. METHODS: A sample of 501 volunteers from the general population in the municipality of Linkoping, Sweden, was examined with esophago-gastro-duodenoscopy (EGD) with biopsy. Blood samples were collected in the fasting state and the subjects answered a questionnaire concerning lifestyle factors, medications and disease history. At a primary follow-up examination, after 8 years, 314 participants were re-examined and those infected with H. pylori received eradication. Two years after successful eradication therapy, 82 participants attended re-examination with EGD and blood sampling, as in the previous examinations. RESULTS: In this prospective cohort study of a sample of volunteers from the general population, all of the 82 participants had chronic gastritis with at least one positive H. pylori test before eradication therapy. During follow-up, non-steroid-inflammatory-drug (NSAID) use had decreased significantly (P = 0.007, McNemar). The H. pylori serology was still positive in 79/82 subjects (P = 0.007, McNemar). The basal gastrin and cholecystokinin (CCK) concentrations both had decreased (P < 0.001 for both, Wilcoxon), whereas the P-somatostatin had increased (P < 0.001, Wilcoxon). Symptoms included in the self-administered symptom questionnaire concerned the last 3 months showed no big difference at all. The inflammation had decreased in both antrum (before 2/38/42/0 and after 60/22/0/0, P < 0.0001) and corpus (before 3/54/22/3 and after 58/23/1/0, P < 0.0001). Changes in the inflammatory activity had decreased significantly in both the antrum (P < 0.001) and the corpus (P < 0.001). Intestinal metaplasia was without changes. Regarding the duodenal bulb, the inflammation decreased. CONCLUSIONS: Being aware of the natural history of chronic gastritis, even beyond eradication of H. pylori, is important because of the associations to gastric neoplasia and ulcer disease. The blood mirror of gastroduodenal parameters showed decreased values, except for somatostatin that increased and a symptomatology with no significant changes although, morphologically determined, both inflammation and atrophy had decreased.

Host Gene Polymorphisms in Relation to Helicobacter Pylori Infection and Associated Diseases in a Population Based Cohort.

BACKGROUND: This prospective population based cohort study explores possible associations between host gene polymorphisms, blood group and life style factors on the one hand, and Helicobacter pylori infection, peptic ulcer, and the grade of inflammation, atrophy and intestinal metaplasia of the gastric mucosa, on the other hand. METHODS: The study population (472 volunteers) has previously undergone screening with gastroduodenoscopy, biopsy and blood sampling. The host gene polymorphisms of IL1B-31C/T, IFNGR1-56T/C, the IL1RN VNTR in exon 2 and the HLA-DRB1 gene alleles were analyzed using PCR and pyrosequencing. RESULTS: H. pylori infection was negatively related to HLA DRB1*03 (odds ratio (OR) 95% CI: 0.388 - 0.989) and was more frequent in individuals with blood group O than A (OR 95% CI: 1.121 - 2.677). There was a lower risk of moderate to severe inflammation in the antrum among individuals with IL1B-31 TC compared to CC carriers (OR 95% CI: 0.094 - 0.733). The IL1RN*L2 genotype was associated with higher risk of IM in the antrum than the *LL genotype (OR 95% CI: 1.570 - 15.878). There was a negative relation between the HLA DRB1 alleles *04 (OR 95% CI: 0.234 - 0.831) and *08 (OR 95% CI: 0.013 - 0.915), and IM in the antrum. CONCLUSION: The IL1RN VNTR and the IL1ß-31 alleles seem to be associated with intestinal metaplasia of the corpus mucosa and the grade of inflammation of the antrum, respectively. However, no unambiguous correlations could be identified between the host polymorphisms and the occurrence of H. pylori infection, peptic ulcer, and the grade of inflammation, atrophy and IM of the gastric mucosa.

Association between cagA and vacA genotypes and pathogenesis in a Helicobacter pylori infected population from South-eastern Sweden.

BACKGROUND: Chronic gastritis, peptic ulcer disease, and gastric cancer have been shown to be related to infection with Helicobacter pylori (H. pylori). Two major virulence factors of H. pylori, CagA and VacA, have been associated with these sequelae of the infection. In this study, total DNA was isolated from gastric biopsy specimens to assess the cagA and vacA genotypes. RESULTS: Variations in H. pylori cagA EPIYA motifs and the mosaic structure of vacA s/m/i/d regions were analysed in 155 H. pylori-positive gastric biopsies from 71 individuals using PCR and sequencing. Analysis of a possible association between cagA and vacA genotypes and gastroduodenal pathogenesis was made by logistic regression analysis. We found that H. pylori strains with variation in the number of cagA EPIYA motif variants present in the same biopsy correlated with peptic ulcer, while occurrence of two or more EPIYA-C motifs was associated with atrophy in the gastric mucosa. No statistically significant relation between vacA genotypes and gastroduodenal pathogenesis was observed. CONCLUSIONS: The results of this study indicate that cagA genotypes may be important determinants in the development of gastroduodenal sequelae of H. pylori infection. In contrast to other studies, vacA genotypes were not related to disease progression or outcome. In order to fully understand the relations between cagA, vacA and gastroduodenal pathogenesis, the mechanisms by which CagA and VacA act and interact need to be further investigated.

Variation in number of cagA EPIYA-C phosphorylation motifs between cultured Helicobacter pylori and biopsy strain DNA.

The Helicobacter pylori cagA gene encodes a cytotoxin which is activated by phosphorylation after entering the host epithelial cell. Phosphorylation occurs on specific tyrosine residues within EPIYA motifs in the variable 3'-region. Four different cagA EPIYA motifs have been defined according to the surrounding amino acid sequence; EPIYA-A, -B, -C and -D. Commonly, EPIYA-A and -B are followed by one or more EPIYA-C or -D motif. Due to observed discrepancies in cagA genotypes in cultured H. pylori and the corresponding DNA extracts it has been suggested that genotyping assays preferentially should be performed directly on DNA isolated from biopsy specimens. Gastric biopsies randomly selected from a Swedish cohort were homogenised and used for both direct DNA isolation and for H. pylori specific culturing and subsequent DNA isolation. In 123 of 153 biopsy specimens, the cagA EPIYA genotypes were in agreement with the corresponding cultured H. pylori strains. A higher proportion of mixed cagA EPIYA genotypes were found in the remaining 30 biopsy specimens. Cloning and sequencing of selected cagA EPIYA amplicons revealed variations in number of cagA EPIYA-C motifs in the mixed amplicons. The study demonstrates that culturing of H. pylori introduces a bias in the number of EPIYA-C motif. Consistent with other H. pylori virulence genotyping studies, we suggest that cagA EPIYA analysis should be performed using total DNA isolated from biopsy specimens.

The desmoplastic stroma plays an essential role in the accumulation and modulation of infiltrated immune cells in pancreatic adenocarcinoma.

Tumor microenvironment is composed of tumor cells, fibroblasts, and infiltrating immune cells, which all work together and create an inflammatory environment favoring tumor progression. The present study aimed to investigate the role of the desmoplastic stroma in pancreatic ductal adenocarcinoma (PDAC) regarding expression of inflammatory factors and infiltration of immune cells and their impact on the clinical outcome. The PDAC tissues examined expressed significantly increased levels of immunomodulatory and chemotactic factors (IL-6, TGFß, IDO, COX-2, CCL2, and CCL20) and immune cell-specific markers corresponding to macrophages, myeloid, and plasmacytoid dendritic cells (DCs) as compared to controls. Furthermore, short-time survivors had the lowest levels of DC markers. Immunostainings indicated that the different immune cells and inflammatory factors are mainly localized to the desmoplastic stroma. Therapies modulating the inflammatory tumor microenvironment to promote the attraction of DCs and differentiation of monocytes into functional DCs might improve the survival of PDAC patients.

Interleukin 1α sustains the expression of inflammatory factors in human pancreatic cancer microenvironment by targeting cancer-associated fibroblasts.

The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) is dynamic, with an extensive interaction between the stroma and tumor cells. The aim of this study was to delineate the cross talk between PDAC and cancer-associated fibroblasts (CAFs), with a focus on the mechanism creating the chronic inflammatory tumor milieu. We assessed the effects of the cross talk between PDAC and CAF cell lines on the creation and sustenance of the inflammatory tumor microenvironment in pancreatic cancer. The coculture of PDAC and CAF cell lines enhanced the levels of inflammatory factors including IL-1α, IL-6, CXCL8, VEGF-A, CCL20, and COX-2. CAFs were superior to tumor cells regarding the production of most inflammatory factors, and tumor cell-associated IL-1α was established as the initiator of the enhanced production of inflammatory factors through the binding of IL-1α to IL-1 receptor 1 (IL-1R1) expressed predominantly by CAFs. Furthermore, we found a correlation between IL-1α and CXCL8 expression levels in PDAC tissues and correlation between IL-1α expression and the clinical outcome of the patients. This confirmed an important role for the IL-1 signaling cascade in the creation and sustenance of a tumor favorable microenvironment. Neutralization of the IL-1α signaling efficiently diminished the cross talk-induced production of inflammatory factors. These data suggest that the cross talk between PDAC cells and the main stroma cell type, i.e. CAFs, is one essential factor in the formation of the inflammatory tumor environment, and we propose that neutralization of the IL-1α signaling might be a potential therapy for this cancer.

Expression of multiple forms of 3'-end variant CCK2 receptor mRNAs in human pancreatic adenocarcinomas.

BACKGROUND: Two main types of receptors for gastrin and cholecystokinin (CCK) have been cloned and identified. CCK1 (CCK-A) receptors are expressed in the pancreas, the gallbladder, and parts of the brain, while CCK2 (CCK-B/gastrin) receptors (CCK2R) are expressed in gastric glands and in most of the brain. A splice variant of the CCK2R designated CCKRi4sv (CCK-C), which is constitutively expressed in human pancreatic cancer cells, has also been described. The purpose of the present investigation was to study CCK2R, CCK2i4svR, and gastrin mRNA expression in human pancreatic adenocarcinoma on the assumption that co-expression of CCK2R and gastrin or constitutive CCK2i4svR mRNA expression plays a pivotal role in the progression of pancreatic cancer. FINDINGS: PCR amplification using CCK2R specific primer-pairs, followed by ethidium-bromide stained agarose gel electrophoresis revealed the expression of wild-type CCK2R mRNA in 12 of 17 biopsy specimens. A CCK2R intron 4 specific nested PCR assay revealed that CCK2i4svR mRNA was expressed in only one of the biopsy specimen. The authenticity of PCR amplicons was confirmed by cloning of selected amplicons and DNA sequence analysis. Moreover, we found that hitherto undescribed multiple forms of 3'-end variant CCK2R mRNAs with various deletions in the retained intron 4 and exon 5, tentatively generating truncated proteins, were expressed in the pancreatic adenocarcinomas. CONCLUSION: Cloning and DNA sequencing of selected amplicons revealed that CCK2R and multiple CCK2i4svR-like mRNAs are expressed in human pancreatic adenocarcinoma. The originally described CCK2i4svR mRNA was only expressed in one of 17 tumours and appears to be rarely expressed in pancreatic adenocarcinoma. We report that CCK2R- and gastrin mRNA co-expression may play a role in a portion, but not in all of these tumours, and that aberrant splicing takes places in these tissues generating multiple forms of 3'-end variant CCK2R mRNAs.

Semi mature blood dendritic cells exist in patients with ductal pancreatic adenocarcinoma owing to inflammatory factors released from the tumor.

BACKGROUND: Much evidence exists regarding the fact that blood DCs, both myeloid DCs (MDCs) and plasmacytoid DCs (PDCs), are negatively affected in different types of cancer, with both reduced numbers and impaired functionality. Functional impairment of DCs in patients with pancreatic ductal adenocarcinoma (PDAC), may contribute to the poor clinical outcome. The aim of this study was to examine the effects PDAC had on blood DCs and elucidate the underlying mechanism responsible for the DC impairment. METHODOLOGY/PRINCIPAL FINDINGS: We examined the systemic influence PDAC exerted on blood DCs by ex vivo measuring numerous activation and maturation markers expressed on these cells. Furthermore, the effect patient plasma and the inflammatory factors CXCL8 and PGE(2) had on purified MDCs and PDCs from healthy donors was assessed and compared to the DCs existing in PDAC patients. We found a partial maturation of the blood MDCs and PDCs in PDAC patients with significantly enhanced expression of CD83, CD40, B7H3, PDL-1, CCR6, and CCR7 and decreased expression of ICOSL, and DCIR. These changes lead to impairment in their immunostimulatory function. Furthermore, chronic pancreatitis gave rise to DCs with similar semi-mature phenotype as seen in PDAC. Low expression of ICOSL was associated with poor prognosis. We found that the mechanism underlying this semi-maturation of DCs was inflammatory factors existing in the PDAC patients' plasma. Of note, PGE(2), which is elevated PDAC patient plasma, was one contributing factor to the changes seen in MDCs and PDCs phenotype. CONCLUSION/SIGNIFICANCE: Our findings point to a role for the systemic inflammation in transforming blood MDCs and PDCs into semi-mature cells in PDAC patients and we show a correlation between maturation status and clinical outcome. Thus, means to preserve a functional blood DC compartment in PDAC patients by diminishing the inflammation could facilitate their ability to control the disease and improve survival.

Pancreatic adenocarcinoma exerts systemic effects on the peripheral blood myeloid and plasmacytoid dendritic cells: an indicator of disease severity?

BACKGROUND: Dendritic cells (DCs) isolated from tumor bearing animals or from individuals with solid tumors display functional abnormalities and the DC impairment has emerged as one mechanism for tumor evasion from the control of the immune system. Ductal pancreatic adenocarcinoma (PDAC), the most common pancreatic cancer, is recognized as a very aggressive cancer type with a mortality that almost matches the rate of incidence. METHODS: We examined the systemic influence ductal pancreatic adenocarcinoma (PDAC) exerted on levels of peripheral blood DCs and inflammatory mediators in comparison to the effects exerted by other pancreatic tumors, chronic pancreatitis, and age-matched controls. RESULTS: All groups examined, including PDAC, had decreased levels of myeloid DCs (MDC) and plasmacytoid DCs (PDC) and enhanced apoptosis in these cells as compared to controls. We found elevated levels of PGE2 and CXCL8 in subjects with PDAC, and chronic pancreatitis. Levels of these inflammatory factors were in part restored in PDAC after tumor resection, whereas the levels of DCs were impaired in the majority of these patients approximately 12 weeks after tumor removal. Our results prove that solid pancreatic tumors, including PDAC, systemically affect blood DCs. The impairments do not seem to be tumor-specific, since similar results were obtained in subjects with chronic pancreatitis. Furthermore, we found that PDAC patients with a survival over 2 years had significant higher levels of blood DCs compared to patients with less than one year survival. CONCLUSIONS: Our findings points to the involvement of inflammation in the destruction of the blood MDCs and PDCs. Furthermore, the preservation of the blood DCs compartment in PDAC patients seems to benefit their ability to control the disease and survival.

Natural history of chronic gastritis in a population-based cohort.

OBJECTIVE: To describe and explore the natural history of Helicobacter pylori infection and chronic gastritis in terms of gastric mucosal atrophy and ulcer development over time in a population-based cohort. MATERIAL AND METHODS: A population-based cohort of 314 volunteers was re-screened (median follow-up interval of 8.4 years) with gastroduodenoscopy with biopsy, assessment of H. pylori status, analysis of pepsinogens, and monitoring of a nonsteroidal anti-inflammatory drug (NSAID) use and alcohol and smoking habits. RESULTS: The incidence of duodenal or prepyloric ulcer was 0.45 per 100 person years and was associated with weekly NSAID use (odds ratios, OR 27.8), weekly alcohol consumption (OR 19.4) and smoking (OR 31.0), but not with H. pylori status. De novo infection with H. pylori was not observed, and the infection had disappeared in 11 of 113 subjects. Among subjects with chronic gastritis, the incidence of atrophy of the corpus mucosa was 1.4 per 100 person years. Atrophy development was related to age (OR 1.23) and to the severity of chronic inflammation in the corpus mucosa at baseline (OR 8.98). Substituting atrophy for subnormal S-pepsinogen I/S-pepsingen II gave similar results. CONCLUSIONS: In this cohort, the minimum incidence of ulcer was 0.45 per 100 person years. Smoking, alcohol, and NSAIDs, but not H. pylori infection were significant risk factors. The incidence of atrophy of the corpus mucosa was 1.4 per 100 person years with a positive relation to age and to the degree of chronic inflammation at baseline. Atrophy was stationary in advanced stages.

Application of PCR amplicon sequencing using a single primer pair in PCR amplification to assess variations in Helicobacter pylori CagA EPIYA tyrosine phosphorylation motifs.

BACKGROUND: The presence of various EPIYA tyrosine phosphorylation motifs in the CagA protein of Helicobacter pylori has been suggested to contribute to pathogenesis in adults. In this study, a unique PCR assay and sequencing strategy was developed to establish the number and variation of cagA EPIYA motifs. FINDINGS: MDA-DNA derived from gastric biopsy specimens from eleven subjects with gastritis was used with M13- and T7-sequence-tagged primers for amplification of the cagA EPIYA motif region. Automated capillary electrophoresis using a high resolution kit and amplicon sequencing confirmed variations in the cagA EPIYA motif region. In nine cases, sequencing revealed the presence of AB, ABC, or ABCC (Western type) cagA EPIYA motif, respectively. In two cases, double cagA EPIYA motifs were detected (ABC/ABCC or ABC/AB), indicating the presence of two H. pylori strains in the same biopsy. CONCLUSION: Automated capillary electrophoresis and Amplicon sequencing using a single, M13- and T7-sequence-tagged primer pair in PCR amplification enabled a rapid molecular typing of cagA EPIYA motifs. Moreover, the techniques described allowed for a rapid detection of mixed H. pylori strains present in the same biopsy specimen.

Homocysteine levels in chronic gastritis and other conditions: relations to incident cardiovascular disease and dementia.

BACKGROUND: Homocysteine levels in circulation are determined by several factors and hyperhomocysteinemia is reportedly associated with cardiovascular diseases and dementia. The aim of this study is to determine the relation of chronic gastritis and other conditions to homocysteine levels and their relation to incident cardiovascular diseases and dementia. METHODS: An adult population-based cohort (N = 488) was screened for H. pylori infection, gastro-duodenitis (endoscopic biopsies), disease history, and lifestyle factors. Blood samples were analyzed for pepsinogen I and II (gastric function), vitamin B12, folate, homocysteine, and cystatin C (renal function). The methylenetetrahydrofolate reductase C677T polymorphism reportedly associated with hyperhomocysteinemia was analyzed by pyrosequencing. Incident cardiovascular diseases and dementia were monitored during a median follow-up interval of 10 years. RESULTS: At baseline, there was a positive relation of S-homocysteine to male gender, age, S-cystatin C, methylenetetrahydrofolate reductase 677TT genotype and atrophic gastritis. During follow-up, cardiovascular diseases occurred in 101/438 and dementia in 25/488 participants, respectively. Logistic regression analysis (adjusting for gender, age at baseline, follow-up interval, BMI, smoking, alcohol consumption, NSAID use, P-cholesterol, and P-triglycerides) showed an association of S-homocysteine higher than 14.5 mumol/l to cardiovascular diseases (OR 2.05 [95% c.i. 1.14-3.70]), but not to dementia overall. CONCLUSIONS: Gender, age, vitamin B12, folate, renal function, atrophic gastritis and the methylenetetrahydrofolate 677TT genotype were significant determinants of homocysteine levels, which were positively related to incident cardiovascular diseases.

Characterization of the gastric cardia in volunteers from the general population. Type of mucosa, Helicobacter pylori infection, inflammation, mucosal proliferative activity, p53 and p21 expression, and relations to gastritis.

The aim of this research was to characterize the mucosa of the gastric cardia in relation to infection with Helicobacter pylori and the occurrence of chronic gastritis in other parts of the stomach in a sample of the general population. In this study, 80 adult volunteers underwent esophagogastroscopy with biopsies from the gastric cardia, corpus, and antrum. Gastritis was classified according to the Sydney system. Chronic gastritis (cardia excepted) was diagnosed in 35 subjects, 30 with H. pylori infection. Epithelial proliferative activity (Ki-67), p53- and p21 expression were examined quantitatively with cell counting after immunohistochemical stainings. Esophagitis was diagnosed macroscopically. Fourty eight subjects had cardia-type and 32 corpus-type mucosa in the anatomical cardia. The prevalence of esophagitis (nine cases) did not differ between these groups. Carditis was more prevalent among subjects with cardia-type mucosa (73 vs. 28%, P < 0.0001). H. pylori was present in 48% of those with cardia-type and 25% of those with corpus-type mucosa (P = 0.06). Of the 44 subjects with carditis, 31 had H. pylori in this location. The group with H. pylori infection had significantly higher mucosal proliferative activity when compared to uninfected subjects. Among the subjects with H. pylori-associated carditis, more p53-positive epithelial cells were detected compared to both the non-infected group (P = 0.0004) and to subjects with non-H. pylori-associated carditis (P = 0.03). In subjects with cardia-type mucosa, and both carditis and gastritis, the degree of chronic inflammation was higher in the cardia compared to the corpus and antrum and the p53 expression was significantly higher in the cardia compared to the corpus, but similar to that in the antrum. The proliferative activity was significantly higher in the antrum compared to the cardia and corpus, respectively. In conclusion, H. pylori infection, carditis, and increased p53 expression are more common in subjects with cardia- than corpus-type mucosa in the gastric cardia. Carditis is mainly related to H. pylori infection. There are some differences regarding inflammation, proliferative activity, and p53 expression between the cardia and other regions of the stomach, yet the significance of these differences remains to be clarified.

A morphometric study of antral G-cell density in a sample of adult general population: comparison of three different methods and correlation with patient demography, helicobacter pylori infection, histomorphology and circulating gastrin levels.

Helicobacter pylori infection has been linked to hypergastrinemia and either decreased or normal G-cell content in the antral mucosa. To clarify this controversial issue, we quantitatively determined antral G-cell content on the same biopsy specimens with three different methods and examined whether these methods are intercorrelated and the relation of these methods to plasma gastrin concentrations, demography, the occurrence of H. pylori infection and chronic gastritis. Gastric antral mucosal biopsy sections from 273 adults (188 with and 85 without H pylori infection) from a general population sample were examined immunohistochemically for G-cells using cell counting, stereology (point counting) and computerized image analysis. Gastritis was scored according to the updated Sydney system. Basal plasma gastrin concentrations were measured by radioimmunoassay. The three methods for G-cell quantification were poorly correlated and the results showed no correlation with basal plasma gastrin concentrations. The antral G-cell density and scores for H. pylori colonization were positively related to age. Neither the scores for chronic inflammation, nor the scores for inflammatory activity, atrophy or intestinal metaplasia were consistently related to the antral G-cell content. In conclusion, the results of three techniques for G-cell quantification in the gastric antral mucosa were poorly intercorrelated and none of the methods correlated with plasma gastrin concentrations. Age and scores for H pylori colonization seem to be determinants of the G-cell density. That common morphometric techniques correlate poorly is of utmost importance to bear in mind when quantitative morphological studies are planned, compared or interpreted.

Concurrent genotyping of Helicobacter pylori virulence genes and human cytokine SNP sites using whole genome amplified DNA derived from minute amounts of gastric biopsy specimen DNA.

BACKGROUND: Bacterial and cellular genotyping is becoming increasingly important in the diagnosis of infectious diseases. However, difficulties in obtaining sufficient amount of bacterial and cellular DNA extracted from the same human biopsy specimens is often a limiting factor. In this study, total DNA (host and bacterial DNA) was isolated from minute amounts of gastric biopsy specimens and amplified by means of whole genome amplification using the multiple displacement amplification (MDA) technique. Subsequently, MDA-DNA was used for concurrent Helicobacter pylori and human host cellular DNA genotyping analysis using PCR-based methods. RESULTS: Total DNA was isolated from gastric biopsy specimens of 12 subjects with gastritis and 16 control subjects having a normal mucosa. The DNA was amplified using a multiple displacement amplification (MDA) kit. Next, concurrent genotyping was performed using H. pylori-specific virulence gene PCR amplification assays, pyrosequencing of bacterial 16S rDNA and PCR characterisation of various host genes. This includes Interleukin 1-beta (IL1B) and Interferon-gamma receptor (IFNGR1) SNP analysis, and Interleukin-1 receptor antagonist (IL1RN) variable tandem repeats (VNTR) in intron 2. Finally, regions of the vacA-gene were PCR amplified using M13-sequence tagged primers which allowed for direct DNA sequencing, omitting cloning of PCR amplicons. H. pylori specific multiplex PCR assays revealed the presence of H. pylori cagA and vacA genotypic variations in 11 of 12 gastritis biopsy specimens. Using pyrosequencing, 16S rDNA variable V3 region signatures of H. pylori were found in 11 of 12 individuals with gastritis, but in none of the control subjects. Similarly, IL1B and IFNGR1-SNP and IL1RN-VNTR patterns could be established in all individuals. Furthermore, sequencing of M13-sequence tagged vacA-PCR amplicons revealed the presence of highly diverse H. pylori vacA-s/i/m regions. CONCLUSION: The PCR-based molecular typing methods applied, using MDA-amplified DNA derived from small amounts of gastric biopsy specimens, enabled a rapid and concurrent molecular analysis of bacterial and host genes in the same biopsy specimen. The principles and technologies used in this study could also be applied to any situation in which human host and microbial genes of interest in microbial-host interactions would need to be sequenced.

Hoarseness and misdirected swallowing in patients with hiatal hernia.

The purpose of this study was to elucidate whether misdirected swallowing is an extra-laryngeal cause of hoarseness and investigate whether the prevalence of misdirected swallowing and hoarseness in patients with hiatal hernias differ from those with and without pathological gastroesophageal reflux (GER). One hundred and ninety eight patients with hiatal hernias diagnosed via esophageal manometry and pH-reflux test and 262 subjects in the general population who did not have a hiatal hernia at endoscopy, filled in a questionnaire about symptoms on hoarseness, misdirected swallowing, and heartburn. Hoarseness (35%), misdirected swallowing to the larynx (MSL; 35%), misdirected swallowing to the nose (MSN; 22%) and heartburn (85%) were significantly more common in patients with hiatal hernia than in controls (13, 5, 1, and 6%, respectively, P<0.001). MSL and MSN in the patient group were significantly interrelated (P<0.0001). Hoarseness and MSL were not significantly associated (P<0.076). Hoarseness and MSL were as common in the hernia group with normal GER, as in the group with pathological GER. There is a predisposition for hoarseness and MSL in patients with hiatal hernias, but the cause-and-effect relationship is unclear. Hoarseness does not seem to be caused by pathological GER.

Multiple strand displacement amplification of DNA isolated from human archival plasma/serum: identification of cytokine polymorphism by pyrosequencing analysis.

BACKGROUND: DNA isolation from formalin-fixed paraffin-embedded tissue appears to be problematic due to degradation caused by fixative. Our aim was to investigate if the isolated genomic DNA from archival plasma/serum, combined with multiple strand displacement amplification (MDA) can be used for genotyping. METHODS: Nine archival plasma/serum samples and freshly frozen gastric biopsies from the same nine H. pylori-infected subjects were used for DNA isolation. Subsequently, MDA-DNA derived from the plasma/serum samples and DNA isolated from the antrum biopsies were analyzed by PCR amplification and pyrosequencing for the presence of interleukin-1beta gene (IL-1B) single nucleotide polymorphism (SNP). In addition, Southern blot and pyrosequencing analysis of H. pylori-specific PCR amplicons were performed. RESULTS: IL-1B SNP profiles obtained from the plasma/serum MDA-DNA and antrum biopsy DNA were identical. A C/C genotype was observed in 7 of 9 samples, and 2 of 9 revealed a C/T genotype for IL-1B -511. Similarly, 7 of 9 had a T/T, and 2 of 9 had a C/T genotype for IL-1B -31; 4 of 9 had a C/C, 4 of 9 had a C/T, and 1 of 9 had a T/T genotype, respectively, for IL-1B +3954. Moreover, pyrosequencing analysis revealed the presence of H. pylori 26695 and J99-like 16S rDNA variable V3 region sequence motifs in the antrum biopsies but not in the plasma/serum samples. CONCLUSIONS: We conclude that MDA combined with pyrosequencing enables a rapid and accurate molecular typing of cytokine single nucleotide polymorphisms from archival plasma/serum samples.

Long-term morpho-functional development of Helicobacter pylori-induced gastritis in Mongolian gerbils.

OBJECTIVE: Epidemiological studies have shown that Helicobacter pylori infection with associated chronic gastritis is the main risk factor for development of gastric cancer. The aim of this study was to investigate the long-term development of H. pylori-induced gastritis in Mongolian gerbils in terms of morphology, gastrin secretion, epithelial proliferation and gene expression of pro-inflammatory cytokines. MATERIAL AND METHODS: A total of 133 gerbils were inoculated with H. pylori and 62 served as controls. The gerbils were killed at different time-points between 6 and 94 weeks after inoculation. Serum concentrations of anti-H. pylori IgG and gastrin were determined by enzyme-linked immunoabsorbent assay (ELISA) and radioimmunoassay (RIA), respectively. Epithelial proliferation was evaluated immunohistochemically after labeling with 5-bromo-2'-deoxy-uridine. Gene expression of beta-actin, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) were measured by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Histological parameters of gastritis were assessed semiquantitatively and expressed as a "gastritis score". RESULTS: Serum concentrations of anti-H. pylori IgG and gastrin increased over time. Epithelial proliferation in the antrum was increased 6 weeks after inoculation, followed by increased proliferation in the corpus 32 weeks after inoculation. Gene expression of IL-1beta and TNF-alpha were increased in H. pylori-infected gerbils. Beta-actin was not a reliable endogenous control for RT-PCR. With time, gastritis expanded from the antrum to the corpus and the gastritis score increased to reach a peak 32 weeks after inoculation. Pseudopyloric metaplasia (loss of specialized cells) was a characteristic feature in the corpus mucosa. Gastric ulcers, but neither dysplasia nor carcinoma, were observed during 94 weeks of infection. CONCLUSIONS: Long-term H. pylori infection in Mongolian gerbils led to progressive gastritis, glandular atrophy, hypergastrinemia, increased epithelial proliferation and elevated gene expression of pro-inflammatory cytokines.