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Background: The death rate (the number of deaths per 100 000 people per year) of colorectal cancer (CRC) has been dropping since 1980 due to increased screening, lifestyle-related risk factors, and improved treatment options; however, CRC is the third leading cause of cancer-related deaths in men and women in the United States. Therefore, successful therapy for CRC is an unmet clinical need. This study aimed to investigate the impacts of andrographolide (AGP) and melatonin (MLT) on CRC and the underlying mechanism. Methods: To investigate AGP and MLT anticancer effects, a series of metastatic colon cancer cell lines (T84, Colo 205, HT-29, and DLD-1) were selected. In addition, a metastatic patient-derived organoid model (PDOD) was used to monitor the anticancer effects of AGP and MLT. A series of bioassays including 3D organoid cell culture, MTT, colony formation, western blotting, immunofluorescence, and quantitative polymerase chain reaction (qPCR) were performed. Results: The dual therapy significantly promotes CRC cell death, as compared with the normal cells. It also limits CRC colony formation and disrupts the PDOD membrane integrity along with decreased Ki-67 expression. A significantly higher cleaved caspase-3 and the endoplasmic reticulum (ER) stress proteins, IRE-1 and ATF-6 expression, by 48 hours were found. This combinatorial treatment increased reactive oxygen species (ROS) levels. Apoptosis signaling molecules BAX, XBP-1, and CHOP were significantly increased as determined by qPCR. Conclusions: These findings indicated that AGP and MLT associated ER stress-mediated apoptotic metastatic colorectal cancer (mCRC) cell death through the IRE-1/XBP-1/CHOP signaling pathway. This novel combination could be a potential therapeutic strategy for mCRC cells.

Colorectal cancer (CRC) mortality has diminished for decades due to new and improved treatment profiles. However, CRC still ranks as the third most diagnosed cancer in the US. Therefore, a new therapeutic approach is needed to overcome colospheroids inhibition and drug resistance. It is well documented that andrographolide (AGP) and melatonin (MLT) have anti-carcinogenic properties. Our goal was to evaluate their synergistic effects on metastatic colon cancer cells (mCRC) and colospheroids. HT-29 and HCT-15 mCRC cells were simultaneously treated with serial dilutions of AGP and MLT for 24, 48 and 72 h. Cell viability was monitored using the MTT assay. The Chou-Talalay method for drug combination is based on the median effect equation, providing a theoretical basis for the combination index and the isobologram equation. This allows quantitative determination of drug interactions using the CompuSyn software, where CI 1 indicates synergistic, additive, and antagonistic effects respectively. Our results demonstrate that AGP and MLT in combination show synergism with CI values of 0.35293 and 0.34152 for HT-29 and HCT-15 respectively and a fractional inhibition of Fa = 0.50–0.90, as shown by the Fa-CI plot and isobologram. The synergism value was validated in colospheroids (HT-29-s and HCT-15-s) based on morphology, viability, and colony formation and in 5-FU drug resistant cell (HT-29R and HCT-116R) viability. The mechanism(s) of decreased cell viability are due to the induction of ER stress proteins and angiogenic inhibition. Our results provide rationale for using AGP in combination with MLT on mCRC.

// Aditi Banerjee 1 , Vivekjyoti Banerjee 1 , Steven Czinn 1 , Thomas Blanchard 1 1 Department of Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A Correspondence to: Aditi Banerjee, email: abanerjee@peds.umaryland.edu Keywords: andrographolide, chemotherapy, reactive oxygen species, endoplasmic reticulum stress, unfolded protein response Received: August 15, 2016 Accepted: January 30, 2017 Published: February 16, 2017 ABSTRACT Chemotherapy continues to play an essential role in the management of many cancers including colon cancer, the third leading cause of death due to cancer in the United States. Many naturally occurring plant compounds have been demonstrated to possess anti-cancer cell activity and have the potential to supplement existing chemotherapy strategies. The plant metabolite andrographolide induces cell death in cancer cells and apoptosis is dependent upon the induction of endoplasmic reticulum stress (ER stress) leading to the unfolded protein response (UPR). The goal of the present study was to determine the mechanism by which andrographolide induces ER stress and to further evaluate its role in promoting cell death pathways. The T84 and COLO 205 cancer cell lines were used to demonstrate that andrographolide induces increased ROS levels, corresponding anti-oxidant response molecules, and reduced mitochondrial membrane potential. No increases in ROS levels were detected in control colon fibroblast cells. Andrographolide-induced cell death, UPR signaling, and CHOP, Bax, and caspase 3 apoptosis elements were all inhibited in the presence of the ROS scavenger NAC. Additionally, andrographolide-induced suppression of cyclins B1 and D1 were also reversed in the presence of NAC. Finally, Akt phosphorylation and phospho-mTOR levels that are normally suppressed by andrographolide were also expressed at normal levels in the absence of ROS. These data demonstrate that andrographolide induces ER stress leading to apoptosis through the induction of ROS and that elevated ROS also play an important role in down-regulating cell cycle progression and cell survival pathways as well.

Proper diagnosis in the pediatric population is required to eradicate Helicobacter pylori (H. pylori) and prevent gastric cancer. Our aim was to assess the performance of non-invasive tests to diagnose H. pylori infection in pediatric patients. A retrospective analysis was performed on 141 pediatric patients requiring endoscopic evaluation and diagnostic tests for H. pylori infection to define the cause of abdominal symptoms. Non-invasive tests included the 13C-urea breath test (UBT), a monoclonal stool antigen test using enzyme-linked immuno-sorbent assay (mSAT), and a serum immunoglobulin G antibody test using antigens derived from Japanese individuals (S-Ab). This study investigated sensitivity, specificity, likelihood ratios for a positive and a negative test (LR+ and LR-), and accuracy of non-invasive tests, in comparison with invasive tests. Eighty two of 141 patients (58%) were recognized as H. pylori positive by invasive methods. When UBT, mSAT or S-Ab were analyzed, all were found to be effective over 94% accurate. Specificity ranged between 86.7% and 95.8%, and sensitivity ranged between 93.8% and 97.1%. When subjects were stratified into three distinct age groups, the best performance was achieved for 1-6 years old with mSAT at 100% for sensitivity, specificity, and accuracy. S-Ab yielded the best results for children 7-12 years old and the UBT test performed best for 13-18 years old. These results demonstrate the utility of UBT, mSAT, and S-Ab non-invasive tests in diagnosing H. pylori but suggest that certain tests may be optimal for children of distinct ages. Three non-invasive tests, UBT, mSAT and S-Ab showed sufficient sensitivity, specificity and accuracy for the initial diagnosis of H. pylori infection among pediatric patients. Non-invasive tests may contribute to achieving minimum invasive diagnosis with combining with a histological test and a culture test in children.

Helicobacter pylori (H. pylori) eradication rates achieved with a proton pump inhibitor (PPI), amoxicillin and clarithromycin have recently decreased to about 75% because of the increase in clarithromycin resistance in Japan. In the present study, H. pylori resistance rates against clarithromycin, amoxicillin and metronidazole were investigated in pediatric patients and eradication rates were evaluated when tailored antibiotics regimens based on antibiotic sensitivity data were used. We isolated H. pylori endoscopically from 77 pediatric patients suffering from abdominal symptoms. The susceptibility tests of H. pylori strains to clarithromycin, amoxicillin and metronidazole were examined and eradication therapy was tailored using the appropriate antibiotics. Seventy-seven patients were treated with a mean age of 12.16 ± 3.34 years (range, 4.92-19.75) consisting of 40 males and 37 females. The average resistance rates between 1998 and 2016 to clarithromycin, amoxicillin and metronidazole were 54.5% (42 of 77), 6.5% (5 of 77) and 5.2% (4 of 77) respectively. The prevalence of clarithromycin resistance increased significantly over time to reach 88.9% by 2013-2016. Successful eradication rates using tailored antibiotic treatment was 93.8% (61 of 65). Clarithromycin-based eradication therapy rate reached 92.6% against clarithromycin-sensitive strains. Metronidazole-based initial eradication therapy also had a high successful rate (97.0%) to clarithromycin-resistant strains. Although high rates of clarithromycin resistant H. pylori reaching about 90% were observed in Japanese children, tailored eradication therapy using the appropriate antibiotic agents were highly successful. H. pylori sensitivity testing and eradication therapy with appropriate antibacterial agents may contribute to accomplishment of high initial eradication rates and consequently reducing the incidence of developing gastric cancer.

It has been over 30 years since a link was established between H. pylori infection of the gastric mucosa and the development of chronic gastric diseases. Research in rodent models supported by data from human tissue demonstrated that the host immune response to H. pylori is limited by host regulatory T cells. Immunization has been shown to induce a potent Th1- and Th17-mediated immune response capable of eradicating or at least significantly reducing the bacterial load of H. pylori in the stomach in small animal models. These results have not translated well to humans. Clinical trials employing many of the strategies used in rodents for oral immunization including the use of a mucosal adjuvant such as Escherichia coli LT or delivery by attenuated enteric bacteria have failed to limit H. pylori infection and have highlighted the potential toxicity of exotoxin-based mucosal adjuvants. A recent study, however, utilizing a recombinant fusion protein of H. pylori urease and the subunit B of E. coli LT, was performed on over 4000 children. Efficacy of over 70% was demonstrated against naturally acquired infection compared to control volunteers one year post-immunization. Efficacy was reduced, but still above 50% at three years. This study provided new insight into the strategies for developing an improved vaccine for widespread use in countries with high infection rates and where gastric cancer (GC) remains one of the most common causes of death due to cancer.

Metastatic colorectal cancer (mCRC) is characterized by the expression of cellular oncogenes, the loss of tumor suppressor gene function. Therefore, identifying integrated signaling between onco-suppressor genes may facilitate the development of effective therapy for mCRC. To investigate these pathways we utilized cell lines and patient derived organoid models for analysis of gene/protein expression, gene silencing, overexpression, and immunohistochemical analyses. An inverse relationship in expression of oncogenic FoxM1 and tumor suppressor RASSF1A was observed in various stages of CRC. This inverse correlation was also observed in mCRC cells lines (T84, Colo 205) treated with Akt inhibitor. Inhibition of FoxM1 expression in mCRC cells as well as in our ex vivo model resulted in increased RASSF1A expression. Reduced levels of RASSF1A expression were found in normal cells (RWPE-1, HBEpc, MCF10A, EC) stimulated with exogenous VEGF165. Downregulation of FoxM1 also coincided with increased YAP phosphorylation, indicative of tumor suppression. Conversely, downregulation of RASSF1A coincided with FoxM1 overexpression. These studies have identified for the first time an integrated signaling pathway between FoxM1 and RASSF1A in mCRC progression, which may facilitate the development of novel therapeutic options for advanced colon cancer therapy.

It has been over 30 years since a link was established between H. pylori infection of the gastric mucosa and the development of chronic gastric diseases. Research in rodent models supported by data from human tissue demonstrated that the host immune response to H. pylori is limited by host regulatory T cells. Immunization has been shown to induce a potent Th1- and Th17-mediated immune response capable of eradicating or at least significantly reducing the bacterial load of H. pylori in the stomach in small animal models. These results have not translated well to humans. Clinical trials employing many of the strategies used in rodents for oral immunization including the use of a mucosal adjuvant such as Escherichia coli LT or delivery by attenuated enteric bacteria have failed to limit H. pylori infection and have highlighted the potential toxicity of exotoxin-based mucosal adjuvants. A recent study, however, utilizing a recombinant fusion protein of H. pylori urease and the subunit B of E. coli LT, was performed on over 4000 children. Efficacy of over 70% was demonstrated against naturally acquired infection compared to control volunteers one year post-immunization. Efficacy was reduced, but still above 50% at three years. This study provided new insight into the strategies for developing an improved vaccine for widespread use in countries with high infection rates and where gastric cancer (GC) remains one of the most common causes of death due to cancer.

There is increasing evidence that unconscious bias can affect realworld decision-making processes in publication just as in many other fields.1 In response, the editorial board of Pediatric Research is working to investigate and reduce the bias in the publication acceptance rates in order to preserve the integrity of the peer review process and publication. As news items have suggested that gender bias is a major problem in academia,2 we reviewed papers submitted between 1 November 2017 and 9 August 2018 to Pediatric Research. Encouragingly, we found that the acceptance rates of manuscripts were not significantly different between corresponding authors who were male or female. However, we incidentally uncovered a higher rejection rate in the manuscripts where the corresponding author had a name that could not be identified as either male or female and did not have a picture on their website so that we could identify their gender.3 It is important to point out that we do not know the reason for this, but its identification is the first step to further exploration, including assessing whether unconscious bias may play a role […]

The plant metabolite andrographolide induces cell cycle arrest and apoptosis in cancer cells. The mechanism(s) by which andrographolide induces apoptosis however, have not been elucidated. The present study was performed to determine the molecular events that promote apoptosis in andrographolide treated cells using T84, HCT116 and COLO 205 colon cancer cell lines. Andrographolide was determined to limit colony formation and Ki67 expression, alter nuclear morphology, increase cytoplasmic histone-associated-DNA-fragments, and increase cleaved caspase-3 levels. Andrographolide also induced significantly higher expression of endoplasmic reticulum (ER) stress proteins GRP-78 and IRE-1 by 48 h but not PERK or ATF6. Apoptosis signaling molecules BAX, spliced XBP-1 and CHOP were also significantly increased. Moreover, chemical inhibition of ER stress or IRE-1 depletion with siRNA in andrographolide treated cells significantly limited expression of IRE-1 and CHOP as determined by immunofluorescence staining, real time PCR, or immunobloting. This was accompanied by a decreased BAX/Bcl-2 ratio. Andrographolide significantly promotes cancer cell death compared to normal cells. These data demonstrate that andrographolide associated ER stress contributes to apoptosis through the activation of a pro-apoptotic GRP-78/IRE-1/XBP-1/CHOP signaling pathway.

Background Murine models of Helicobacter pylori infection are used to study host–pathogen interactions, but lack of severe gastritis in this model has limited its usefulness in studying pathogenesis. We compared the murine gastric epithelial cell line GSM06 to the human gastric epithelial AGS cell line to determine whether similar events occur when cultured with H. pylori. Materials and Methods The lysates of cells infected with H. pylori isolates or an isogenic cagA-deficient mutant were assessed for translocation and phosphorylation of CagA and for activation of stress pathway kinases by immunoblot. Results Phosphorylated CagA was detected in both cell lines within 60 minutes. Phospho-ERK 1/2 was present within several minutes and distinctly present in GSM06 cells at 60 minutes. Similar results were obtained for phospho-JNK, although the 54 kDa phosphoprotein signal was dominant in AGS, whereas the lower molecular weight band was dominant in GSM06 cells. Conclusion These results demonstrate that early events in H. pylori pathogenesis occur within mouse epithelial cells similar to human cells and therefore support the use of the mouse model for the study of acute CagA-associated host cell responses. These results also indicate that reduced disease in H. pylori-infected mice may be due to lack of the Cag PAI, or by differences in the mouse response downstream of the initial activation events.

The outbreak of COVID-19 caused by 2019–nCov/SARS-CoV-2 has become a pandemic with an urgent need for understanding the mechanisms and identifying a treatment. Viral infections including SARS-CoV are associated with increased levels of reactive oxygen species, disturbances of Ca++ caused by unfolded protein response (UPR) mediated by endoplasmic reticulum (ER) stress and is due to the exploitation of virus's own protein i.e., viroporins into the host cells. Several clinical trials are on-going including testing Remdesivir (anti-viral), Chloroquine and Hydroxychloroquine derivatives (anti-malarial drugs) etc. Unfortunately, each drug has specific limitations. Herein, we review the viral protein involvement to activate ER stress transducers (IRE-1, PERK, ATF-6) and their downstream signals; and evaluate combination therapies for COVID-19 mediated ER stress alterations. Melatonin is an immunoregulator, anti-pyretic, antioxidant, anti-inflammatory and ER stress modulator during viral infections. It enhances protective mechanisms for respiratory tract disorders. Andrographolide, isolated from Andrographis paniculata, has versatile biological activities including immunomodulation and determining SARS-CoV-2 binding site. Considering the properties of both compounds in terms of anti-inflammatory, antioxidant, anti-pyrogenic, anti-viral and ER stress modulation and computational approaches revealing andrographolide docks with the SARS-CoV2 binding site, we predict that this combination therapy may have potential utility against COVID-19., Graphical abstract Unlabelled Image

Background/Aims: Silencing of tumor suppressor genes (TSGs) and promotion of angiogenesis are associated with tumor development and metastasis. However, little is known if angiogenic molecules directly control TSGs and vice versa. Methods: A regulatory link between angiogenesis and down regulation of TSGs was evaluated using an anti-cancer agent, andrographolide (AGP) in cancer cells, mouse xenograft tissues and patient derived organoids through gene/protein expression, gene silencing, and immunohistochemical analyses. Results: AGP treatment demonstrated significant expression of RASSF1A and PTEN TSGs in colon cancer and other cancer cells, mouse tissues and organoids. Depletion of RASSF1A with siRNA limited cyclin D1 and BAX expression. SiRNA depletion of PTEN, upstream regulator of RASSF1A resulted in a 50% reduction in RASSF1A expression. Histopathological analysis of the AGP treated tumor sections showed significant reduction in vessel size, microvascular density and tumor mitotic index suggesting suppression of angiogenesis. This was corroborated by protein analysis demonstrating significant reductions in angiogenesis signaling pathway molecules VEGF165, FOXM1, and pAkt, but significant elevation of the endogenous angiogenesis inhibitor Tsp-2. Treatment of cells with exogenous VEGF prevented the suppression of angiogenesis signaling by AGP, resulting in sustained expression of pAkt, an upstream down-regulator of RASSF1A. RASSF1A expression remained low in VEGF treated cells despite the addition of AGP. Conclusion: Our results demonstrate for the first time that AGP induces RASSF1A expression in colon cancer cells and is dependent on angiogenesis signaling events. Therefore, our research may facilitate novel therapeutic options for advanced colon cancer therapy.

Helicobacter pylori colonizes mucosa, activates Toll-like and Nod-like receptors, and usually elicits a gastric T-helper 1/17 (Th1/Th17) type of immune response. Among several bacterial factors, the secreted peptidyl prolyl cis, trans-isomerase of H. pylori represents a key factor driving Th17 inflammation. A complex and fascinating balance between H. pylori and host factors takes part in the gastric niche and is responsible for the chronicity of the infection. Novel insights into the innate and adaptive responses against H. pylori, dealing with gastric epithelial cells, cytokines, and immune evasion have been elucidated over the past year and are discussed for the development of an effective vaccine.

Helicobacter pylori infects more than half the world's population and in developing nations the incidence can be over 90%. The morbidity and mortality associated with H. pylori-associated diseases including ulcers and gastric cancer therefore, disproportionately impact the developing world. Mice have been used extensively to demonstrate the feasibility of developing a vaccine for H. pylori infection, and for testing antigens, routes of immunization, dose, and adjuvants. These successes however, have not translated well in clinical trials. Although there are examples where immune responses have been activated, there are few instances of achieving a reduced bacterial load. In vivo and in vitro analyses in both mice and humans demonstrates that the host responds to H. pylori infection through the activation of immunoregulatory mechanisms designed to suppress the anti-H. pylori response. Improved vaccine efficacy therefore, will require the inclusion of factors that over-ride or re-program these immunoregulatory rersponse mechanisms.

Gastroesophageal reflux (GER) is defined as the involuntary retrograde passage of gastric contents into the esophagus with or without regurgitation or vomiting. It is a frequently experienced physiologic condition occurring several times a day, mostly postprandial and causes no symptoms. These infants are also called 'happy spitters'. GER disease (GERD) occurs when reflux of the gastric contents causes symptoms that affect the quality of life or pathologic complications, such as failure to thrive, feeding or sleeping problems, chronic respiratory disorders, esophagitis, hematemesis, apnea, and apparent life-threatening events. About 70-85 % of infants have regurgitation within the first 2 months of life, and this resolves without intervention in 95 % of infants by 1 year of age. The predominant mechanism causing GERD is transient lower esophageal sphincter (LES) relaxation, which is defined as an abrupt decrease in LES pressure to the level of intragastric pressure, unrelated to swallowing and of relatively longer duration than the relaxation triggered by a swallow. Regurgitation and vomiting are the most common symptoms of infant reflux. A thorough history and physical examination with attention to warning signals suggesting other causes is generally sufficient to establish a clinical diagnosis of uncomplicated infant GER. Choking, gagging, coughing with feedings or significant irritability can be warning signs for GERD or other diagnoses. If there is forceful vomiting, laboratory and radiographic investigation (upper gastrointestinal series) are warranted to exclude other causes of vomiting. Irritability coupled with back arching in infants is thought to be a non-verbal equivalent of heartburn in older children. Other causes of irritability, including cow's milk protein allergy, neurologic disorders, constipation and infection, should be ruled out. The presentation of cow's milk protein allergy overlaps with GERD, and both conditions may co-exist in 42-58 % of infants. In these infants, symptoms decrease significantly within 2-4 weeks after elimination of cow's milk protein from the diet. For non-complicated reflux, no intervention is required for most infants. Effective parental reassurance and education regarding regurgitation and lifestyle changes are usually sufficient to manage infant reflux. Sandifer syndrome, apnea and apparent life-threatening events are the extraesophageal manifestations of GERD in infants. Pharmacotherapeutic agents used to treat GERD encompass antisecretory agents, antacids, surface barrier agents and prokinetics. Currently, North American Society for Pediatric Gasroenterology, Hepatology and Nutrition (NASPGHAN) and European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) practice guidelines concluded that there is insufficient evidence to justify the routine use of prokinetic agents. Esomeprazole (Nexium) is now approved in the US for short-term treatment of GERD with erosive esophagitis in infants aged from 1 to 12 months. Although Nissen fundoplication is now well established as a treatment option in selected cases of GERD in children, its role in neonates and young infants is unclear and is only reserved for selective infants who did not respond to medical therapy and have life-threatening complications of GERD.

Establishment of Helicobacter pylori infection as an etiologic agent of peptic ulcer disease and other gastric pathologies marked a revolution in gastroenterology which spurred an enormous interest in gastric physiology and immunology research. The association was soon also demonstrated in children as well. Application of antimicrobial therapies have proven remarkably efficacious in eradicating H. pylori and curing pediatric patients of duodenal ulcers as well as gastritis, negating a lifetime of ineffective therapy and life-threatening disease. Countries with high H. pylori prevalence and where H. pylori associated gastric cancer remains a primary cause of death due to cancer however would benefit from childhood vaccination. Studies in rodents and humans utilizing oral vaccination with bacterial exotoxin adjuvants demonstrated potential for limiting H. pylori colonization in the stomach. Almost 25 y of vaccine research recently culminated in a phase III clinical trial of over 4,000 children aged 6-15 y old to test an oral vaccine consisting of the H. pylori urease B subunit genetically fused to the E. coli heat labile toxin. Vaccination was demonstrated to have an efficacy of over 70%. Vaccination may now serve as an effective strategy to significantly reduce H. pylori associated disease in children throughout the world.

Background: Helicobacter pylori (H. pylori) is a gram negative bacterium that can cause diseases such as peptic ulcers and gastric cancer. IL-17A, a proinflammatory cytokine that can induce the production of CXC chemokines for neutrophil recruitment, has recently been shown to be elevated in both H. pylori-infected patients and mice. Furthermore, studies in mouse models of vaccination have reported levels significantly increased over infected, unimmunized mice and blocking of IL-17A during the challenge phase in immunized mice reduces protective immunity. Because many aspects of immunity had redundant or compensatory mechanisms, we investigated whether mice could be protectively immunized when IL-17A function is absent during the entire immune response using IL-17A and IL-17A receptor knockout (KO) mice immunized against H. pylori. Materials and Methods: Gastric biopsies were harvested from naive, unimmunized/challenged, and immunized/challenged wild type (WT) and KO mice and analyzed for inflammation, neutrophil, and bacterial levels. Groups of IL-17A KO mice were also treated with anti-IFNγ or control antibodies. Results: Surprisingly, all groups of immunized KO mice reduced their bacterial loads comparably to WT mice. The gastric neutrophil counts did not vary significantly between IL-17A KO and WT mice, whereas IL-17RA KO mice had on average a four-fold decrease compared to WT. Additionally, we performed an immunization study with CXCR2 KO mice and observed significant gastric neutrophils and reduction in bacterial load. Conclusion: These data suggest that there are compensatory mechanisms for protection against H. pylori and for neutrophil recruitment in the absence of an IL-17A-CXC chemokine pathway.

Helicobacter pylori (H. pylori) can be maintained on both solid [1-4] and liquid media [5-7] but maintaining viable bacteria for prolonged periods can be expensive or impractical. Growth of plate cultures of H. pylori under aerobic conditions has been reported [8], and we routinely grow cultures in standard tissue culture incubators set at either 5% or 10% CO2 (unpublished results). Most laboratories however must rely on the use of closed container systems complete with gas generating envelope technology to create the appropriate microaerobic environment. These envelope systems can constitute significant costs when performing frequent or multiple cultures. Additionally, one of the manufacturers of these systems has recently discontinued their product, resulting in limited options in the market place. Continuous culture of H. pylori requires frequent subculture of viable bacteria to fresh plates. Many laboratory and pathogenic bacterial strains can be kept viable on sealed plates and stored at 4 °C to arrest growth. H. pylori survival at 4 °C, however, varies significantly with the media being used and it typically dies rapidly within 2 days [3,9-12]. Optimal methods for storing plate cultures at 4 °C have achieved viability for up to 9 days [13]. Typically, however, H. pylori must be passaged every 3–5 days to prevent the culture from dying. Although liquid cultures of H. pylori can be maintained in standard tissue culture incubators under static conditions, long-term maintenance requires frequent passage and ultimately consumes media supplemented with fetal bovine serum. While many of these issues can be circumvented simply by recovering H. pylori from frozen stocks when needed, viability varies depending upon the freezing media being employed [5,14,15]. Additionally, recovery times can take up to 7 days, result in light growth, and occasionally fail outright (unpublished observations). The purpose of this study was to assess a mechanism for maintaining viable H. pylori cultures in the laboratory without having to perform frequent passaging. We now report that H. pylori strains grown in agar stabs can be stored for at least 8 weeks in agar stab cultures kept at 37 °C.

The gastric pathogen Helicobacter pylori infects over half the world's population. The lifelong infection induces gastric inflammation but the host fails to generate protective immunity. To study the lack of protective H. pylori immunity, CD4(+)CD25(+) T(reg) cells were investigated for their ability to down-regulate H. pylori-specific CD4(+)CD25(-) cells in a murine model. CD25(-) lymphocytes from infected mice were hyporesponsive to antigenic stimulation in vitro even in the absence of CD25(+) T(reg) cells unless treated with high-dose IL-2. Transfer of CD45RB(hi) naïve CD25(-) cells from infected mice into rag1(-/-) mice challenged with H. pylori resulted in severe gastritis and reduced bacterial loads, whereas transfer of CD45RB(lo) memory CD25(-) cells from H. pylori-infected mice resulted in only mild gastritis and persistent infection. CD25(-) cells stimulated in the absence of CD25(+) cells in rag1(-/-) mice promoted bacterial clearance, but lost this ability when subsequently transferred to WT mice harboring CD25(+) cells. These results demonstrate that CD25(+) cells induce anergy in CD25(-) cells in response to H. pylori infection but are not required to maintain hyporesponsiveness. In addition, CD25(+) cells are able to suppress previously activated CD25(-) cells when responding to H. pylori challenge in vivo.

The North American Society for Pediatric Gastroenterology, Hepatology and Nutrition launched a provider and public education campaign in 2002 to raise awareness of gastroesophageal reflux disease (GERD). To determine the effectiveness of campaign messages, we conducted a knowledge, attitudes, and practice styles (KAPS) survey of pediatric providers. Understanding the spectrum of management styles of GERD in children is critical to achieve better health outcomes and reduce health care costs.The KAPS questionnaire was administered to 6000 randomly selected members of the American Academy of Pediatrics.A total of 1245 members responded; 82% worked in a primary care setting and 18% in subspecialty practices. Overall, 66% of the members order diagnostic testing in routine practice, 54% start testing for GERD in neonates, and 38% start testing after 1 month of age. The most common tests ordered were barium esophagram (45%) and esophageal pH monitoring (37%). GERD treatment with acid suppression before ordering diagnostic testing was a choice of 82% of the respondents. However, 19% believed acid suppression was best achieved by H2 blockers. If acid suppression was indicated, then only 36% followed guideline recommendations for therapy duration and 52% followed guideline recommendations for dosing. Antireflux surgery was recommended only as a last resort by 92%. Overall, 69% of providers believed the amount of GERD-related information available was not enough. Respondents who were not aware of available GERD practice guidelines ranged from 74% to 92%.Pediatric providers appear to frequently order diagnostic testing and treatment for GERD, yet knowledge about evidence-based GERD management among this random sample appeared limited. Moreover, a significant number of providers were not aware of different guideline publications.

Lymphoid tissue inducer (LTi) cells are activated by accessory cell IL-23, and promote lymphoid tissue genesis and antibacterial peptide production by the mucosal epithelium. We investigated the role of LTi cells in the gastric mucosa in the context of microbial infection. Mice deficient in IRAK-M, a negative regulator of TLR signaling, were investigated for increased LTi cell activity, and antibody mediated LTi cell depletion was used to analyze LTi cell dependent antimicrobial activity. H. pylori infected IRAK-M deficient mice developed increased gastric IL-17 and lymphoid follicles compared to wild type mice. LTi cells were present in naive and infected mice, with increased numbers in IRAK-M deficient mice by two weeks. Helicobacter and Candida infection of LTi cell depleted rag1(-/-) mice demonstrated LTi-dependent increases in calprotectin but not RegIII proteins. However, pathogen and commensal microbiota populations remained unchanged in the presence or absence of LTi cell function. These data demonstrate LTi cells are present in the stomach and promote lymphoid follicle formation in response to infection, but are limited by IRAK-M expression. Additionally, LTi cell mediated antimicrobial peptide production at the gastric epithelium is less efficacious at protecting against microbial pathogens than has been reported for other tissues.

Nitazoxanide is a Food and Drug Administration approved Thiazolide to treat parasitic infections such as giardiasis and cryptosporidiosis. Since nitazoxanide is available as a liquid suspension, this medication is uniquely suited for the treatment of children. However, there has been a paucity of published studies since its approval. We completed a systematic literature search using a variety of sources including PubMed to identify studies reporting the impact of nitazoxanide in treating parasitic infections in children ages 1 to 11 years. A similar search was conducted on other available treatment for parasitic infection. In total, 28 publications were found to meet the criteria above for nitazoxanide. A similar number of papers were identified to address other therapy commonly used regardless of product information on approved use. Despite the ease of administration and superiority of nitazoxanide to other Non FDA approved therapies for the treatment of Giardia or Cryptosporidium among children, this medication continues to be underutilized. These data indicated the need for additional clinical studies to raise awareness.

Background: We are not aware of any survey data that has assessed the general awareness by pediatricians of Giardia and/or Cryptosporidium infection, the most common causes of human intestinal parasitic infections in the United States. The aim of this survey was to evaluate pediatrician knowledge and perceptions on the treatment of parasitic infections in children 1-12 years of age. Methods: A 44-question online survey was conducted from July 1 to July 14, 2014 among 1000 pediatricians. Results: Almost all respondents identified viruses as the most likely cause of diarrhea (95.0%) in young patients and therefore reported treating with supportive therapy (82.4%). Approximately only 10% of respondents would suspect parasites if the diarrhea became persistent, with the duration of persistence generally considered as more than 1-2 weeks. If they considered parasites as the cause of diarrhea, they would most commonly test for both Cryptosporidium and Giardia, and initiate therapy after confirmation (70.9%). The drug of first choice for empiric therapy was most commonly metronidazole (78.4%) followed by paromomycin (10.4%) and nitazoxanide (6.4%). Notably, 72.0% of pediatricians considered the availability of nitazoxanide would change their potential treatment choice for diarrhea. Respondents preferred additional print materials (56.2%) followed by live events including web-based education (35.6%) to receive additional education concerning parasitic infection in children. Conclusion: This survey supports the need to enhance efforts to provide education among pediatricians on how best to manage and treat childhood diarrhea caused by parasites such as Giardia and Cryptosporidium.

Background: We undertook an online survey to gain a greater understanding of the knowledge, attitudes, and willingness to seek care among caregivers of small children. Methods: Caregivers were invited to participate via direct e-mail from Harris Poll, a company that specializes in on-line surveys. This was a prospective, web-based survey of a defined number of responders on a first come, first included basis. The survey focused on caregiver’s awareness of the potential causes of diarrheal illnesses and of available treatment options. Caregivers were required to be US residents, have a child living in the home who was 1 -12 years old and who suffered at least one episode of diarrhea in the last year. Results: The survey included 1,048 complete responses were accumulated. Responders were mostly female (59%) with one (51%) or two (39%) children in the home. The results of the survey showed a general lack of awareness of what constitutes clinical diarrhea and how best to care for a child who presents with persistent diarrhea. Conclusion: The results of this survey point to the need for broad public education on the importance of recognition, treatment, and prevention of childhood diarrhea caused by parasites such as Giardia and Cryptosporidium.

Intestinal immune responses are normally regulated to maintain a state of immune balance. Dendritic cells (DC) are antigen-presenting cells, which induce immune responses against microbes and other stimuli and are key players in the regulation of tolerance in the gut. These cells influence the differentiation of cytokine responses in T cells, and in the gut, in particular, such interactions may be critical to the course of inflammatory bowel disease (IBD). Using the CD45RBhi CD4+ T cell-reconstituted severe combined immunodeficient mouse model of colitis, we investigated the ability of isolated colon DC to stimulate immune responses in syngeneic and allogeneic spleen CD4+ T cells, as well as in colon T cells isolated from the same tissue as DC in IBD mice. We found that the frequency of DC in IBD mice colons and spleens was elevated in comparison with control mice, but colon and spleen DC exhibited different phenotypic and functional properties. Colon DC stimulated significantly higher levels of interferon-γ and interleukin-6 when cocultured with autologous colon T cells than in cocultures with syngeneic or allogeneic spleen T cells. These data suggest that in the IBD colon, DC-T cell interactions may create conditions with an abundance of proinflammatory cytokines, which favor the inflammatory state.

Background. Helicobacter pylori has been shown to induce pronounced gastric inflammation in the absence of interleukin-10 (IL-10) by 6 weeks post inoculation. The ability of IL-10−/– mice to eradicate H. pylori has not been demonstrated, possibly due to early sacrifice. Therefore, the long-term effect of enhanced gastritis on H. pylori colonization was determined in IL-10−/– mice. Methods. C57BL/6 and IL-10−/– mice were infected with H. pylori and assessed for the degree of gastritis, bacterial load, and in vitro T-cell recall response at 4 and 16 weeks of infection. Results. Infection of IL-10−/– mice resulted in significantly more severe gastritis than wild-type control mice and eradication of H. pylori by 4 weeks post inoculation. By 16 weeks, the level of gastritis in IL-10−/– was reduced to the levels observed in wild-type mice. Splenocytes from IL-10−/– mice were prone to produce significantly greater amounts of IFN-γ than wild-type mice when stimulated with bacterial antigens. Conclusions. These results indicate that the host is capable of spontaneously eradicating H. pylori from the gastric mucosa when inflammation is elevated beyond the chronic inflammation induced in wild-type mice, and that the gastritis dissipates following bacterial eradication. Additionally, these data provide support for a model of gastrointestinal immunity in which naturally occurring IL-10-producing regulatory T cells modulate the host response to gastrointestinal bacteria.

Helicobacter pylori infection causes gastritis and peptic ulcers and is associated with the development of gastric cancer. Approximately 50% of the world population is infected with H pylori , with the highest prevalence rates in developing countries. In the vast majority of individuals, infection is acquired during childhood with those of low socioeconomic means and having infected family members being at highest risk for early childhood acquisition. Definitive routes of transmission of the infection are unclear, with evidence suggesting oral-oral, gastric-oral, and fecal-oral routes. If untreated, H pylori infection is lifelong. Although clinical disease typically occurs decades after initial infection acquisition, children infected with H pylori may have gastritis, ulcers, mucosal-associated lymphoid type lymphoma, and, rarely, gastric atrophy with/without intestinal metaplasia (ie, both precursor lesions for gastric cancer). Controversy persists regarding testing for and treating H pylori , if found, in the large number of children who present with recurrent abdominal pain. Because young children (ie, younger than 5 years of age) who are treated and cured of their H pylori infection may be at risk for reinfection, the current recommendations do not recommend treatment unless an ulcer or gastric atrophy is present. However, despite the lack of clinical evidence, the trend is to more aggressively screen children for the presence of H pylori and to treat those children who are found to have the infection. H pylori infection can be eradicated by antimicrobial therapy plus a proton pump inhibitor, but no treatment regimen is 100% effective. Multiple drugs, frequent dosing, and length of treatment often contribute to poor patient compliance, and antibiotic eradication therapy is associated with increasing drug resistance.

Helicobacter pylori (H. pylori) is among the most common bacterial infections in humans. In 1982, H. pylori was discovered by Marshal and Warren, demonstrating an association between H. pylori and ulcer disease. H. pylori is a gram-negative, S-shaped rod that produces enzymes like urease, catalase and oxidase. The mechanism of acquisition and transmission of H. pylori is unclear, although the most likely mode of transmission is fecal-oral and oral-oral. The mode of transmission is supported by studies that demonstrate viable H. pylori organisms can be cultured from the stool or vomitus of infected patients. Risk factors such as minimal education and low socio-economic status during childhood affect the prevalence. Children infected with H. pylori develop histologic chronic active gastritis despite the fact that they are generally asymptomatic. A small percentage of these children will go on to develop peptic ulcer disease, and even gastric cancer. In contrast, the association of abdominal pain and H. pylori infection remains controversial. In the year 2000, the North American Society of Pediatric Gastroenterology guidelines on H. pylori reported that there is no evidence demonstrating a link between H. pylori-associated gastritis and abdominal pain, except in rare cases in which gastric or duodenal ulcer disease is present. Currently, treatment with a combination of two antimicrobial agents in conjunction with a proton pump inhibitor (PPI) continues to be recommended for the treatment of H. pylori associated peptic ulcer disease.

Background. Some Helicobacter pylori strains can survive within macrophage phagosomes for up to 24 hours. The factors that play a role in this survival remain ill-defined. Therefore, the contribution of catalase in mediating the survival of H. pylori following phagocytosis was investigated in vitro. Methods. An isogenic, catalase-deficient strain of H. pylori was generated and tested for sensitivity to hydrogen peroxide and susceptibility to macrophage-mediated killing. Results. The isogenic, catalase-deficient strain of H. pylori was effectively killed by hydrogen peroxide within 3 minutes compared to wild-type H. pylori which maintained 100% survival up to 21 minutes. The catalase-deficient mutant was also significantly more susceptible to macrophage-mediated killing than the parent strain, even when the ratio of bacteria to macrophage was increased. Conclusion. These results indicate that although some strains of H. pylori are capable of survival within the macrophage phagosome, survival is dependent on virulence factors such as catalase for evasion of innate host defense.

There is now considerable evidence that suggests that the H. pylori organism isa human pathogen. The strong association between H. pylori and gastroduodenal disease is well documented. A number of hypotheses have been suggested for the pathogenic mechanisms of H. pylori-induced gastroduodenal disease, including the presence of bacterial virulence factors, the production of inflammatory mediators, disregulation of acid secretion, and the host immune response. At the present time, treatment with a combination of a proton pump inhibitor and antimicrobial agents continues to be recommended for the treatment of H. pylori-associated peptic ulcer disease.

Pneumatosis intestinalis is an uncommon finding beyond the neonatal period, but it has been reported in immunocompromized pediatric patients. The association of pneumatosis intestinalis in children following renal transplantation has to the best of our knowledge been only reported once in children. We describe a 4-year-old female who developed intermittent emesis, weight loss, and intermittently loose bloody stools after cadaveric renal transplantation at age 3.5 years. An abdominal x-ray demonstrated extensive pneumatosis in the colon. The infectious work-up was negative. Histologically, she had increased eosinophils throughout the lamina propria in the rectum. A glucose breath test was suggestive of small bowel bacterial overgrowth. She was treated with 10 days of metronidazole with resolution of the diarrhea and occult blood in stools. One month after the treatment she had radiologic resolution of her pneumatosis. Based on this report, pneumatosis intestinalis should be considered in the differential diagnosis of children after organ transplant suffering from diarrhea, abdominal pain, or blood in the stool.

Infection with Helicobacter pylori results in antibody production and an inflammatory infiltrate of neutrophils, lymphocytes, plasma cells, macrophages, and eosinophils in the gastric mucosa. In spite of this immune response, H. pylori infection in most untreated people is lifelong. Mice can be protected from H. pylori infection by immunization, and based on the lack of protection in major histocompatibility complex class II-deficient immunized mice, the protection is presumed to require CD4+ T cells (9, 15, 26, 29, 33). The need for CD4+ T cells suggests that the cytokines they secrete play a role in immunity. Numerous studies of biopsies from H. pylori-infected patients have demonstrated proinflammatory cytokines and chemokines such as gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-2, IL-6, IL-12, IL-8, MIP-1α, and GROα in the gastric mucosa (6, 19, 31, 36, 45-47). The methods, i.e., reverse transcription-PCR (RT-PCR) and enzyme-linked immunosorbent assay, used in these studies to detect cytokines and chemokines do not allow discrimination of which cells are producing the mediators. However, immunohistochemistry of biopsies has revealed cytokine-containing cells in the epithelium and lamina propria. Increased IL-8 immunofluorescence was observed in the epithelia from patients with H. pylori-induced gastritis compared to normal gastric epithelium (7). Also, greater numbers of epithelial cells, intraepithelial lymphocytes, and lamina propria cells positive for IL-1β, IL-6, IL-8, and TNF-α were found in H. pylori-infected patients than in uninfected individuals (24). T cells isolated from biopsies of infected patients produced IL-2, IFN-γ, and TNF-α but rarely Th2 cytokines (IL-4 and IL-5), leading to the conclusion that T cells in the infected gastric mucosa have a Th1 phenotype (3, 39). In contrast, one study found an increase in CD8+ T cells and NK cells by fluorescence-activated cell sorter analysis of isolated gastric lymphocytes from infected patients and an increase of IL-4 and IL-6 in supernatants of cultured antral biopsies (1). Finally, H. pylori-specific T-cell clones generated from biopsies of infected patients expressed mainly IFN-γ (20 of 24 clones tested) or a combination of IFN-γ, IL-4, and IL-5 (4 of 24 clones) when stimulated with H. pylori antigens (8). TNF-α was secreted by all the H. pylori-reactive clones. Overall, the preponderance of proinflammatory cytokines and Th1 T cells has lead to the idea that H. pylori-induced gastritis is a Th1-mediated disease (16, 25). Much less is known about cytokines and chemokines in the gastric mucosa of H. pylori-infected or immunized mice. High levels of IFN-γ were constitutively produced by lymphocytes isolated from the gastric lamina propria of Helicobacter felis-infected or immunized-challenged (I/C) C57BL/6 mice (28). mRNA expression for IFN-γ, TNF-α, IL-1β, RANTES, MIP-2, IP-10, and MCP-1 was detected in C57BL/6 mice infected with H. felis (11). Immunized mice protected from H. pylori had relatively high levels of mRNA for IFN-γ but undetectable to low levels of IL-4 and IL-5 (14). In situ hybridization for IFN-γ and IL-4 mRNAs in C57BL/6 mice demonstrated a greater prevalence of IFN-γ-expressing cells in H. pylori-infected mice than in naive controls but no difference in IL-4-positive cells (38). Taken together these results suggest that IFN-γ is produced in the gastric mucosa of mice in response to Helicobacter sp. and may be related to gastritis and/or protection. To determine which cytokines are associated with protection from H. pylori infection, gastric samples from protected and unprotected I/C mice, unimmunized-not challenged (U/NC) mice, and unimmunized-challenged (U/C) mice were screened for a panel of cytokines and chemokines. Elevated levels of IL-12p40, IFN-γ, TNF-α, and inducible nitric oxide synthase (iNOS) mRNAs appeared to be associated with protection, and the biologic effects of IL-12p40, IFN-γ, and iNOS were studied in mice genetically deficient in these products.

Helicobacter pylori infection of the stomach is associated with the development of gastritis, peptic ulcers, and gastric adenocarcinomas, but the mechanisms are unknown. MUC1 is aberrantly overexpressed by more than 50% of stomach cancers, but its role in carcinogenesis remains to be defined. The current studies were undertaken to identify the genetic mechanisms regulating H. pylori-dependent MUC1 expression by gastric epithelial cells. Treatment of AGS cells with H. pylori increased MUC1 mRNA and protein levels, and augmented MUC1 gene promoter activity, compared with untreated cells. H. pylori increased binding of STAT3 and MUC1 itself to the MUC1 gene promoter within a region containing a STAT3 binding site, and decreased CpG methylation of the MUC1 promoter proximal to the STAT3 binding site, compared with untreated cells. These results suggest that H. pylori upregulates MUC1 expression in gastric cancer cells through STAT3 and CpG hypomethylation.

1. Gisela Chelimsky, MD* 2. Steven Czinn, MD† 1. 2. *Department of Pediatrics. 3. 4. †Chief, Division of Pediatric Gastroenterology, Department of Pediatrics, Rainbow Babies and Children’s Hospital and Case Western Reserve University, Cleveland, OH. Objectives After completing this article, readers should be able to: 1. Describe the different mechanisms involved in the pathogenesis of peptic ulcer disease. 2. Recognize the presenting symptoms of peptic ulcer disease and the differential diagnosis of those symptoms in children. 3. Clarify the role of endoscopic procedures in pediatric peptic ulcer disease. 4. Describe the role of Helicobacter pylori in the pathogenesis of peptic ulcer disease. 5. Review the medications and dosages commonly used in the treatment of peptic ulcer disease and H pylori infection in children. During the past decade, it has been estimated that peptic ulcer disease has affected more than 4 million people in the United States annually. For much of the last century, the pathogenesis of this disease was believed to be due to a number of predisposing factors, the most important of which was the hypersecretion of gastric acid. The development of pediatric flexible fiberoptic endoscopes that allowed the gastroenterologist to obtain gastric biopsies and identification of the microorganism Helicobacter pylori have changed our understanding of gastritis in children. Acid-peptic inflammation is believed to occur when there is an imbalance between cytotoxic and cytoprotective factors in the upper gastrointestinal tract. The toxic mechanisms include acid, pepsin, medications such as aspirin and nonsteroidal anti-inflammatory drugs, bile acids, and infection with H pylori . The defensive or cytoprotective mechanisms include the mucous layer, local bicarbonate secretion, and mucosal blood flow. Acid secretion from the parietal cells can be stimulated by three secretagogues: histamine via the paraendocrine pathway, acetylcholine via the neuroendocrine pathway, and gastrin via the endocrine pathway. Each employs a different mode of delivery to its target, the oxyntic cell. Acethylcholine is released at the vagal cholinergic terminals near the oxyntic cells (or parietal cell). Gastrin is released by the G cells in the antral and duodenal mucosa and is …

To determine whether systemic immunization against Helicobacter pylori could be achieved with an adjuvant approved for human use, the efficacy of vaccination with Helicobacter antigen in combination with aluminum hydroxide (AlOH) was evaluated in a murine model of Helicobacter infection. Immunization with antigen and AlOH induced interleukin-5-secreting, antigen-specific T cells, and immunization with antigen and complete Freund's adjuvant induced interferon-gamma-secreting, antigen-specific T cells, as determined by ELISPOT assay. Both immune responses conferred protection after challenge with either H. pylori or H. felis, as confirmed by the complete absence of any bacteria, as assessed by both histology and culture of gastric biopsy samples. Protection was antibody independent, as demonstrated with antibody-deficient muMT mice (immunoglobulin-gene knockout mice), and CD4(+) spleen T cells from immunized mice were sufficient to transfer protective immunity to otherwise immunodeficient rag1(-/-) recipients. These results suggest an alternative and potentially more expeditious strategy for development of a human-use H. pylori vaccine.