Why Turner patients with 45, X monosomy should not be excluded from fertility preservation services.

In this case report, we highlight the practical dilemma, i.e. to perform ovarian tissue cryopreservation surgery in a 45, X Turner Syndrome patient or not, by reporting on the presence of follicles in a 13-year-old female diagnosed with 45, X monosomy and an unmeasurable anti-müllerian hormone serum level. We compare our results with previous research, highlight the challenges we faced in this case and provide recommendations for daily practice. Hereby, we demonstrate that excluding certain subgroups of Turner Syndrome patients (e.g. monosomy patients, and/or girls with an anti-müllerian hormone level below 2.0 ng/l) may be premature, especially based on the current state of published research data. This practical example of a challenging dilemma in the counselling of Turner Syndrome patients for fertility preservation is of interest for clinicians involved in fertility counselling and Turner Syndrome care.

International consensus: ovarian tissue cryopreservation in young Turner syndrome patients: outcomes of an ethical Delphi study including 55 experts from 16 different countries.

STUDY QUESTION: What is the standpoint of an international expert panel on ovarian tissue cryopreservation (OTC) in young females with Turner syndrome (TS)? SUMMARY ANSWER: The expert panel states that OTC should be offered to young females with TS, but under strict conditions only. WHAT IS KNOWN ALREADY: OTC is already an option for preserving the fertility of young females at risk of iatrogenic primary ovarian insufficiency (POI). Offering OTC to females with a genetic cause of POI could be the next step. One of the most common genetic disorders related to POI is TS. Due to an early depletion of the ovarian reserve, most females with TS are confronted with infertility before reaching adulthood. However, before offering OTC as an experimental fertility preservation option to young females with TS, medical and ethical concerns need to be addressed. STUDY DESIGN, SIZE, DURATION: A three-round ethical Delphi study was conducted to systematically discuss whether the expected benefits exceed the expected negative consequences of OTC in young females with TS. The aim was to reach group consensus and form an international standpoint based on selected key statements. The study took place between February and December 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: Anonymous panel selection was based on expertise in TS, fertility preservation or medical ethics. A mixed panel of 12 gynaecologists, 13 (paediatric) endocrinologists, 10 medical ethicists and 20 patient representatives from 16 different countries gave consent to participate in this international Delphi study. In the first two rounds, experts were asked to rate and rank 38 statements regarding OTC in females with TS. Participants were offered the possibility to adjust their opinions after repetitive feedback. The selection of key statements was based on strict inclusion criteria. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 46 participants completed the first Delphi round (response rate 84%). Based on strict selection criteria, six key statements were selected, and 13 statements were discarded. The remaining 19 statements and two additional statements submitted by the expert panel were re-evaluated in the second round by 41 participants (response rate 75%). The analysis of the second survey resulted in the inclusion of two additional key statements. After the approval of these eight key statements, the majority of the expert panel (96%) believed that OTC should be offered to young females with TS, but in a safe and controlled research setting first, with proper counselling and informed consent procedures, before offering this procedure in routine care. The remaining participants (4%) did not object but did not respond despite several reminders. LIMITATIONS, REASONS FOR CAUTION: The anonymous nature of this study may have led to lack of accountability. The selection of experts was based on their willingness to participate. The fact that not all panellists took part in all rounds may have resulted in selection bias. WIDER IMPLICATIONS OF THE FINDINGS: This international standpoint is the first step in the global acceptance of OTC in females with TS. Future collaborative research with a focus on efficacy and safety and long-term follow-up is urgently needed. Furthermore, we recommend an international register for fertility preservation procedures in females with TS. STUDY FUNDING/COMPETING INTEREST(S): Unconditional funding (A16-1395) was received from Merck B.V., The Netherlands. The authors declare that they have no conflict of interest.

To Freeze or Not to Freeze? An Update on Fertility Preservation In Females with Turner Syndrome.

Introduction Infertility is a major concern for females with Turner syndrome (TS), regardless of their age. While fertility preservation is now routinely offered to girls and young women with cancer, there are currently no recommendations on fertility preservation in girls and young women with TS who generally face an even higher risk for infertility. Despite the lack of international guidelines, preservation procedures have been performed experimentally in females with TS. Methods A systematic literature search based on the PRISMA-P methodology for systematic reviews was performed in order to collect all published data on fertility preservation options in females with TS between January 1980 and April 2018. A total number of 67 records were included in this review. The records were screened for information regarding cryopreservation of mature oocytes and ovarian tissue in females with TS. Two ongoing trials on fertility preservation in young females with TS were also included. Results Cryopreservation of oocytes or ovarian tissue has been performed experimentally in >150 girls and adolescents with TS over the last 16 years. The efficacy of fertility preservation options in females with TS is still unknown due to the lack of follow-up data. Conclusion The efficacy of fertility preservation procedures in females with TS is still unknown. Future studies with focus on efficacy, safety and long-term follow-up are desperately needed.

A preliminary study on a new model system to evaluate tumour-detection and tumour-purging protocols in ovarian cortex tissue intended for fertility preservation.

STUDY QUESTION: Is it possible to create a model system that mimics ovarian metastatic disease in order to devise new strategies to detect cancer cells and prevent cancer cell transmission via ovarian tissue autotransplantation in cancer survivors? SUMMARY ANSWER: Injection of bovine or human ovarian cortex fragments with cells from different cancer types led to the formation of proliferating tumour masses and newly formed small metastatic lesions. WHAT IS KNOWN ALREADY: Autotransplantation of ovarian tissue comes with the major concern of cancer cells possibly being present in the tissue. A model system to develop strategies aimed at enhancing the safety of ovarian tissue autotransplantation is currently lacking. STUDY DESIGN, SIZE, DURATION: The ability of injected human leukaemia, lymphoma, Ewing's sarcoma or breast cancer cells to proliferate and form tumour-like structures in bovine and human ovarian cortex tissue in vitro was assessed. The injected cells were from human cancer cell lines. After 4 days of culture, some tissue fragments were harvested for standard histological staining and immunohistochemical staining of tumour cell specific antigens and the Ki67 proliferation marker, while the remaining fragments were incubated for an additional 6 days (bovine tissue) or 3 days (human tissue) before analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Experiments were performed with ovarian tissue from women after prophylactic salpingo-oophorectomy. Bovine ovarian tissue was obtained at an abattoir. Glucose uptake during in vitro culture was monitored to quantify the viability of tissue. Tumour formation was assessed at Day 4 and Day 10 in bovine ovarian tissue and at Day 4 and Day 7 in human ovarian tissue, using histology and immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: We found that bovine and human ovarian cortex tissue could be cultured for up to 10 and 7 days, respectively, without any loss of viability. Our preliminary results show that all cell lines tested were capable of forming proliferating tumours in ovarian cortex tissue in vitro. Lymphoma and breast cancer cells produced small metastases near the original lesions. LIMITATIONS, REASONS FOR CAUTION: The tumour model presented was based on the growth of human cancer cell lines in ovarian cortex tissue. It is unknown whether these cells behave differently from malignant cells derived from primary tumours. In addition, the human ovarian tissue was derived from women over 39 years of age, which is obviously considerably older than patients opting for ovarian tissue cryopreservation. WIDER IMPLICATIONS OF THE FINDINGS: Our model system will facilitate the development of procedures to detect cancer cells in, or purge cancer cells from, human ovarian tissue. STUDY FUNDING/COMPETING INTERESTS: Unconditional funding was received from the Radboud Institute for Health Sciences, KiKa Foundation and Merck Serono. There are no conflicts of interest to declare.

Efficacy of ovarian tissue cryopreservation in a major European center.

PURPOSE: To evaluate the effect of cryopreservation and thawing of ovarian tissue from oncological patients opting for fertility preservation on ovarian tissue viability. METHODS: In this prospective cohort study, the ovarian tissue viability before and after cryopreservation and thawing was measured for 25 newly diagnosed oncological patients who had their ovarian tissue cryopreserved. Outcome measures were follicle integrity (histology), follicle viability (Calcein viability assay), steroid hormone production (estradiol and progesterone production in vitro) and overall tissue viability (glucose uptake in vitro). This study was conducted at a Cryobank for storage of ovarian tissue in a university hospital. RESULTS: Cryopreserved/thawed ovarian tissue showed a decreased glucose uptake when compared to tissue that had not been cryopreserved. In addition, a diminished E2 and P4 production was observed after cryopreservation and thawing, despite the fact that numbers of viable follicles as determined by the Calcein viability assay were comparable. Histological examination revealed a higher percentage of degenerated follicles after cryopreservation and thawing. CONCLUSIONS: Ovarian tissue cryopreservation and thawing impairs the viability of ovarian tissue in oncological patients opting for fertility preservation.

Chytrid infections exhibit historical spread and contemporary seasonality in a declining stream-breeding frog.

Species with extensive geographical ranges pose special challenges to assessing drivers of wildlife disease, necessitating collaborative and large-scale analyses. The imperilled foothill yellow-legged frog (Rana boylii) inhabits a wide geographical range and variable conditions in rivers of California and Oregon (USA), and is considered threatened by the pathogen Batrachochytrium dendrobatidis (Bd). To assess drivers of Bd infections over time and space, we compiled over 2000 datapoints from R. boylii museum specimens (collected 1897-2005) and field samples (2005-2021) spanning 9° of latitude. We observed a south-to-north spread of Bd detections beginning in the 1940s and increase in prevalence from the 1940s to 1970s, coinciding with extirpation from southern latitudes. We detected eight high-prevalence geographical clusters through time that span the species' geographical range. Field-sampled male R. boylii exhibited the highest prevalence, and juveniles sampled in autumn exhibited the highest loads. Bd infection risk was highest in lower elevation rain-dominated watersheds, and with cool temperatures and low stream-flow conditions at the end of the dry season. Through a holistic assessment of relationships between infection risk, geographical context and time, we identify the locations and time periods where Bd mitigation and monitoring will be critical for conservation of this imperilled species.

Management of precancerous conditions and lesions in the stomach (MAPS): guideline from the European Society of Gastrointestinal Endoscopy (ESGE), European Helicobacter Study Group (EHSG), European Society of Pathology (ESP), and the Sociedade Portuguesa de Endoscopia Digestiva (SPED).

Atrophic gastritis, intestinal metaplasia, and epithelial dysplasia of the stomach are common and are associated with an increased risk for gastric cancer. In the absence of guidelines, there is wide disparity in the management of patients with these premalignant conditions. The European Society of Gastrointestinal Endoscopy (ESGE), the European Helicobacter Study Group (EHSG), the European Society of Pathology (ESP) and the Sociedade Portuguesa de Endoscopia Digestiva (SPED) have therefore combined efforts to develop evidence-based guidelines on the management of patients with precancerous conditions and lesions of the stomach (termed MAPS). A multidisciplinary group of 63 experts from 24 countries developed these recommendations by means of repeat online voting and a meeting in June 2011 in Porto, Portugal. The recommendations emphasize the increased cancer risk in patients with gastric atrophy and metaplasia, and the need for adequate staging in the case of high grade dysplasia, and they focus on treatment and surveillance indications and methods.

Glucose/lactate metabolism of cryopreserved intact bovine ovaries as a novel quantitative marker to assess tissue cryodamage.

For some patients, the autotransplantation of a cryopreserved-thawed intact ovary might be the best option to preserve their reproductive potential after fertility-threatening treatment. The best procedure to successfully cryopreserve a human ovary without inflicting a devastating level of cryodamage is to date unknown. To optimize this procedure, this study developed an assay to monitor the extent of cryodamage inflicted on bovine ovarian tissue by different cryopreservation protocols. The assay measures glucose and lactate metabolism of ovarian tissue fragments in vitro and determines the extent of cryodamage in cryopreserved ovaries. This study tested the cryoprotective effect of two different routes of administration of the cryoprotectant dimethylsulphoxide (DMSO). The cryoprotective effect was assessed in different tissue layers of the ovary, namely the cortex, the subcortex and the medulla. Submersion of intact ovaries in DMSO prior to freezing-thawing resulted in the complete protection of the glucose/lactate metabolism of the cortex, but not of the inner ovarian mass. Perfusion without simultaneous submersion, resulted in partial protection of cortex, subcortex and medulla, while the combination of submersion and perfusion conveyed the highest level of protection for all three ovarian tissue layers.

In vitro 17ß-oestradiol release as a marker for follicular survival in cryopreserved intact bovine ovaries.

Transplantation of cryopreserved intact ovaries from cancer patients is a technically challenging option for restoring fertility after sterilizing cancer therapy. In this paper we describe an assay based on 17ß-oestradiol (oestradiol) production, to monitor the functional damage sustained by the ovarian tissue during the freeze/thawing procedure. To this end, fresh bovine ovarian cortical biopsies were cultured in vitro for 7 days. As a control, the oestradiol release of biopsies that had sustained maximal cryodamage was analyzed. In addition the oestradiol release by cortical biopsies from two ME2SO perfused and cryopreserved intact ovaries was analyzed. Oestradiol production could be measured in culture supernatants, while oestradiol release of maximal cryo-damaged biopsies was at background levels. In vitro oestradiol release by cortical biopsies can be used as a functional marker for cryo-damage and indicates that our assay is suitable to optimize the cryopreservation procedure of intact ovaries.

Helicobacter pylori CagA interacts with E-cadherin and deregulates the beta-catenin signal that promotes intestinal transdifferentiation in gastric epithelial cells.

Infection with Helicobacter pylori cagA-positive strains is associated with gastric adenocarcinoma. Intestinal metaplasia is a precancerous lesion of the stomach characterized by transdifferentiation of the gastric mucosa to an intestinal phenotype. The H. pylori cagA gene product, CagA, is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation by Src family kinases. Tyrosine-phosphorylated CagA specifically binds to and activates SHP-2 phosphatase, thereby inducing cell-morphological transformation. We report here that CagA physically interacts with E-cadherin independently of CagA tyrosine phosphorylation. The CagA/E-cadherin interaction impairs the complex formation between E-cadherin and beta-catenin, causing cytoplasmic and nuclear accumulation of beta-catenin. CagA-deregulated beta-catenin then transactivates beta-catenin-dependent genes such as cdx1, which encodes intestinal specific CDX1 transcription factor. In addition to beta-catenin signal, CagA also transactivates p21(WAF1/Cip1), again, in a phosphorylation-independent manner. Consequently, CagA induces aberrant expression of an intestinal-differentiation marker, goblet-cell mucin MUC2, in gastric epithelial cells that have been arrested in G1 by p21(WAF1/Cip1). These results indicate that perturbation of the E-cadherin/beta-catenin complex by H. pylori CagA plays an important role in the development of intestinal metaplasia, a premalignant transdifferentiation of gastric epithelial cells from which intestinal-type gastric adenocarcinoma arises.

Helicobacter pylori infection stimulates plasminogen activator inhibitor 1 production by gastric epithelial cells.

Chronic infection with the gastric pathogen Helicobacter pylori significantly increases the risk of developing atrophic gastritis, peptic ulcer disease, and gastric adenocarcinoma. H. pylori strains that possess the cag pathogenicity island, which translocates CagA into the host cells, augment these risks. The aim of this study was to determine the molecular mechanisms through which H. pylori upregulates the expression of plasminogen activator inhibitor 1 (PAI-1), a member of the urokinase activator system that is involved in tumor metastasis and angiogenesis. Levels of PAI-1 mRNA and protein were examined in tissues from H. pylori-infected patients and in vitro using AGS gastric epithelial cells. In vitro, cells were infected with toxigenic cag-positive or nontoxigenic cag-negative strains of H. pylori or isogenic mutants. The amount of PAI-1 secretion was measured by enzyme-linked immunosorbent assay, and mRNA levels were determined using real-time PCR. The regulation of PAI-1 was examined using the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor and small interfering RNA. Analysis of human biopsy samples revealed an increase in both PAI-1 mRNA and protein levels in patients with H. pylori gastritis compared to those of uninfected controls. Infection of AGS cells with H. pylori significantly increased PAI-1 mRNA expression and the secretion of PAI-1 protein. Moreover, PAI-1 mRNA and protein production was more pronounced when AGS cells were infected by H. pylori strains carrying a functional cag secretion system than when cells were infected by strains lacking this system. PAI-1 secretion was also reduced when cells were infected with either cagE-negative or cagA-negative mutants. The ectopic overexpression of CagA significantly increased the levels of PAI-1 mRNA and protein, whereas blockade of the ERK1/2 pathway inhibited H. pylori-mediated PAI-1 upregulation. These findings suggest that the upregulation of PAI-1 in H. pylori-infected gastric epithelial cells may contribute to the carcinogenic process.

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

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

An LGG-derived protein promotes IgA production through upregulation of APRIL expression in intestinal epithelial cells.

p40, a Lactobacillus rhamnosus GG (LGG)-derived protein, transactivates epidermal growth factor receptor (EGFR) in intestinal epithelial cells, leading to amelioration of intestinal injury and inflammation. To elucidate mechanisms by which p40 regulates mucosal immunity to prevent inflammation, this study aimed to determine the effects and mechanisms of p40 on regulation of a proliferation-inducing ligand (APRIL) expression in intestinal epithelial cells for promoting immunoglobulin A (IgA) production. p40 upregulated April gene expression and protein production in mouse small intestine epithelial (MSIE) cells, which were inhibited by blocking EGFR expression and kinase activity. Enteroids from Egfrfl/fl, but not Egfrfl/fl-Vil-Cre mice with EGFR specifically deleted in intestinal epithelial cells, exhibited increased April gene expression by p40 treatment. p40-conditioned media from MSIE cells increased B-cell class switching to IgA+ cells and IgA production, which was suppressed by APRIL receptor-neutralizing antibodies. Treatment of B cells with p40 did not show any effects on IgA production. p40 treatment increased April gene expression and protein production in small intestinal epithelial cells, fecal IgA levels, IgA+B220+, IgA+CD19+, and IgA+ plasma cells in lamina propria of Egfrfl/fl, but not of Egfrfl/fl-Vil-Cre, mice. Thus p40 upregulates EGFR-dependent APRIL production in intestinal epithelial cells, which may contribute to promoting IgA production.

Neonatal colonization of mice with LGG promotes intestinal development and decreases susceptibility to colitis in adulthood.

Development of the intestinal microbiota during early life serves as a key regulatory stage in establishing the host-microbial relationship. This symbiotic relationship contributes to developing host immunity and maintaining health throughout the life span. This study was to develop an approach to colonize conventionally raised mice with a model probiotic bacterium, Lactobacillus rhamnosus GG (LGG), and to determine the effects of LGG colonization on intestinal development and prevention of colitis in adulthood. LGG colonization in conventionally raised was established by administering LGG to pregnant mice starting at gestational day 18 and pups at postnatal days 1- 5. LGG colonization promoted bodyweight gain and increased diversity and richness of the colonic mucosa-associated microbiota before weaning. Intestinal epithelial cell proliferation, differentiation, tight junction formation, and mucosal IgA production were all significantly enhanced in LGG-colonized mice. Adult mice colonized with LGG showed increased IgA production and decreased susceptibility to intestinal injury and inflammation induced in the dextran sodium sulfate model of colitis. Thus, neonatal colonization of mice with LGG enhances intestinal functional maturation and IgA production and confers lifelong health consequences on protection from intestinal injury and inflammation. This strategy might be applied for benefiting health in the host.

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

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

Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach.

To determine whether infection with a Helicobacter pylori strain possessing cagA is associated with an increased risk of development of adenocarcinoma of the stomach, we used a nested case-control study based on a cohort of 5443 Japanese-American men in Oahu, Hawaii, who had a physical examination and a phlebotomy during 1967 to 1970. We matched 103 H. pylori-infected men who developed gastric cancer during a 21-year surveillence period with 103 H. pylori-infected men who did not develop gastric cancer and tested stored serum specimens from patients and controls for the presence of serum IgG to the cagA product of H. pylori using an ELISA. The serum IgG assay using a recombinant CagA fragment had a sensitivity of 94.4% and a specificity of 92.5% when used in a clinically defined population; serological results were stable for more than 7 years. For men with antibodies to CagA, the odds ratio of developing gastric cancer was 1.9 (95% confidence interval, 0.9-4.0); for intestinal type cancer of the distal stomach, the odds ratio was 2.3 (95% confidence interval, 1.0-5.2). Age < 72 years and advanced tumor stage at diagnosis were significantly associated with CagA seropositivity. We conclude that infection with a cagA-positive H. pylori strain in comparison with a cagA-negative strain somewhat increases the risk for development of gastric cancer, especially intestinal type affecting the distal stomach.

Helicobacter pylori strain-specific genotypes and modulation of the gastric epithelial cell cycle.

Helicobacter pylori cag+ strains enhance gastric epithelial cell proliferation and attenuate apoptosis in vivo, which may partially explain the increased risk of gastric cancer associated with these strains. The goals of this study were to identify specific H. pylori genes that regulate epithelial cell cycle events and determine whether these effects were dependent upon p53-mediated pathways. AGS gastric epithelial cells were cultured alone or in the presence of 21 clinical H. pylori isolates, H. pylori reference strain 60190, or its isogenic cagA-, picB-, vacA-, or picB-/vacA- derivatives. Coculture of H. pylori with AGS cells significantly decreased cell viability, an effect most prominent with cag+ strains (P < 0.001 versus cag-strains). cag+ strains significantly increased progression of AGS cells from G1 into G2-M at 6 h and enhanced apoptosis by 72 h. Compared with the parental 60190 strain, the picB- mutant attenuated cell cycle progression at 6 h (P < or = 0.05), and decreased apoptosis with enhanced AGS cell viability at 24 h (P < or = 0.04). The vacA- mutant decreased apoptosis and enhanced viability at later (48-72 h) time points (P < or = 0.05). Compared with the wild-type strain, the picB-/vacA- double mutant markedly attenuated apoptosis and increased cell viability at all time points (P < or = 0.05). Furthermore, cocolonization with H. pylori had no significant effect on expression of p53, p21, and MDM2. The diminished AGS cell viability, progression to G2-M, and apoptosis associated with cag+ H. pylori strains were dependent upon expression of vacA and genes within the cag pathogenicity island. These results may explain heterogeneity in levels of gastric epithelial cell proliferation and apoptosis found within H. pyloricolonized mucosa.

Pathogenic Helicobacter pylori strains translocate DNA and activate TLR9 via the cancer-associated cag type IV secretion system.

Helicobacter pylori (H. pylori) is the strongest identified risk factor for gastric cancer, the third most common cause of cancer-related death worldwide. An H. pylori constituent that augments cancer risk is the strain-specific cag pathogenicity island, which encodes a type IV secretion system (T4SS) that translocates a pro-inflammatory and oncogenic protein, CagA, into epithelial cells. However, the majority of persons colonized with CagA+ H. pylori strains do not develop cancer, suggesting that other microbial effectors also have a role in carcinogenesis. Toll-like receptor 9 (TLR9) is an endosome bound, innate immune receptor that detects and responds to hypo-methylated CpG DNA motifs that are most commonly found in microbial genomes. High-expression tlr9 polymorphisms have been linked to the development of premalignant lesions in the stomach. We now demonstrate that levels of H. pylori-mediated TLR9 activation and expression are directly related to gastric cancer risk in human populations. Mechanistically, we show for the first time that the H. pylori cancer-associated cag T4SS is required for TLR9 activation and that H. pylori DNA is actively translocated by the cag T4SS to engage this host receptor. Activation of TLR9 occurs through a contact-dependent mechanism between pathogen and host, and involves transfer of microbial DNA that is both protected as well as exposed during transport. These results indicate that TLR9 activation via the cag island may modify the risk for malignancy within the context of H. pylori infection and provide an important framework for future studies investigating the microbial-epithelial interface in gastric carcinogenesis.

Retinal pigment epithelium-immune system interactions: cytokine production and cytokine-induced changes.

Vision is dependent on proper function of several intraocular structures. Immune responses to eliminate invading pathogens from the eye may threat vision by causing damage to these structures. Therefore, immunological defence of the eye should be carefully balanced between efficacy and maintenance of functional integrity. The eye is equipped with several regulatory mechanisms to prevent certain immune and inflammatory responses and is, therefore, regarded as an immune privileged site. The retinal pigment epithelium (RPE) contributes to the immune privileged status of the eye as part of the blood-eye barrier and by the secretion of immunosuppressive factors inside the eye. RPE cells, however, may also play an important role in the development of immune and inflammatory responses in the posterior part of the eye. During the last decade it has become clear that RPE cells are highly sensitive to a variety of inflammatory cytokines. Under inflammatory conditions, RPE cells produce a myriad of cytokines that may activate the resident ocular cells or attract and activate leukocytes. Cytokine stimulation of RPE cells causes profound effects, including nitric oxide secretion, cell surface expression of MHC class II and adhesion molecules and abrogation of barrier function. This article provides a comprehensive review of the literature concerning RPE cells and cytokines.

Expression of macrophage-derived chemokine (MDC) mRNA in macrophages is enhanced by interleukin-1beta, tumor necrosis factor alpha, and lipopolysaccharide.

A cDNA encoding the C-C chemokine MDC was isolated from a human macrophage cDNA library by differential hybridization using monocyte- and macrophage-specific cDNA probes. During monocyte to macrophage differentiation in vitro, MDC expression is first detected after 1 day of culturing and reaches maximum levels after 6 days when macrophages have fully matured, as judged from the expression of known macrophage marker genes. Exposure of macrophages to lipopolysaccharide (LPS) results in a dose-dependent increase in MDC mRNA levels, with maximum induction occurring after 6-8 h, whereas expression levels of macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-2, interleukin-1beta (IL-1beta), and tumor necrosis factor alpha (TNF-alpha) respond much faster to LPS. Furthermore, MDC expression in macrophages is enhanced by the inflammatory mediators TNF-alpha and IL-1beta. Similar to other TNF-alpha/IL-1beta-inducible genes, costimulation of macrophages with both cytokines leads to higher MDC expression levels than stimulation with a single cytokine. By contrast, both resting and activated monocytes do not express MDC mRNA.