Get your own Free Home PageAccording to James G. Fox, Professor and Director of Division of Comparative Medicine, Massachusetts Institute of Technology, they are currently actively pursuing the possibility of infection with Helicobacter hepaticus( discovered earlier in laboratory animals ) in several human patient populations who have idiopathic liver disease. Another group at University of Missouri School of Medicine( Michael Cooperstock, MD, MPH ) is also presently investigating such cases.
HELICOBACTER HEPATICUS, A RECENTLY RECOGNIZED BACTERIAL PATHOGEN, ASSOCIATED WITH CHRONIC HEPATITIS AND HEPATOCELLULAR NEOPLASIA IN LABORATORY MICE
Jerry M. Rice, Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick,Maryland, USA, Emerging Infectious Diseases, Volume 1 * Number 4, October-December 1995
Gastric carcinoma, one of the most prevalent human cancers worldwide, is among the neoplasms for which epidemiologic evidence of environmental causes is strongest. The exact nature of these environmental causes was obscure until mounting evidence recently linked chronic infection of the gastric antrum mucosa by Helicobacter pylori (a microaerobic, gram-negative, spiral bacterium) with elevated cancer risk (1). It is now recognized that gastric B-cell lymphoma of mucosa-associated lymphoid tissue is also closely linked to gastric H. pylori infection, and eradication of the infection with antibiotics can result in regression of the lymphoma (2,3). This startling finding has stimulated intense interest in the genus Helicobacter and related organisms; as a result, additional species of Helicobacter are now frequently isolated and characterized from many non-human hosts. Until 1994, however, only H. pylori was known to be associated with tumor development, in humans or in any other animal species.
In 1992, at the National Cancer Institute's Frederick Cancer Research and Development Center (FCRDC) in Frederick, Maryland, a high prevalence of liver disease was observed among certain strains of mice; these mice were untreated controls in long-term chemical carcinogenesis experiments. Affected strains, notably A/JCr, had been bred at FCRDC under pathogen-free conditions and were free of known serologically detectable murine viruses and parasites; moreover, they had no histologically demonstrable hepatic abnormalities, except for a very low incidence (1% to 2%) of hepatocellular tumors in mice 15 months of age or older. Over a very short period, the prevalence of a histologically distinctive form of hepatitis increased to virtually 100% in male mice at 1 year of age (Table 1). The earliest demonstrable lesions were small, undistinctive foci of hepatic necrosis seen in young mice aged 2 to 6 months. In older mice, aged 6 to 10 months, there was a highly distinctive pericholangitis, consisting of abundant mononuclear cell infiltrates around bile ducts within portal triads. The biliary epithelium within affected ducts was focally swollen, and the luminal surfaces of damaged epithelial cells were poorly defined in hematoxylin and eosin-stained sections (4,5). In livers with extensive lesions, bile ductular (oval cell) hyperplasia was also prominent. Moreover, mice with hepatitis usually had hepatocellular tumors, often multiple, that included both adenomas and carcinomas (4).
Hepatocellular tumors in mice are one of the most common endpoints in bioassays for chemical carcinogens. They were not, at that time, known to be associated with infectious agents. Accordingly, initial efforts to identify the cause of the hepatitis/hepatocellular tumor syndrome were directed toward possible sources of chemical exposure. The possibility of accidental exposure to experimental substances within the research animal facilities was ruled out when liver disease was identified in mice that had never left the breeding areas which are located in separate buildings. Extensive chemical analyses of food, bedding, water, and other possible sources of toxic substances had negative results.
Detailed pathologic examination by light microscopy of tissue sections from diseased livers was continued, and many special stains were used. One such stain, Steiner's silver impregnation procedure for spirochetes (6), revealed in hepatic tissue uniform bodies that were consistent in size and shape with bacteria. Homogenates of fresh liver tissue from diseased mice proved effective in transmitting hepatitis to A/J mice purchased from commercial sources outside FCRDC, when given by intraperitoneal injection (5). In addition, from these homogenates, a motile, spiral bacterium could be cultivated on blood agar plates incubated at 37ºC under anaerobic or microaerobic conditions.
This organism was subsequently characterized by ultrastructural morphologic examination, biochemical characteristics, and 16S rRNA gene sequence. Determined to be a new species related to H. pylori, it was given the name H. hepaticus (7). The bacterium is motile and gram negative, 0.2 to 0.3 µm in diameter, 1.5 to 5.0 µm long, and curved to spiral in shape, with one to several spirals; it has bipolar sheathed flagella (one at each end) but lacks the periplasmic fibers that envelope the bacterial cells in other mouse Helicobacter species. H. hepaticus has strong urease activity, is oxidase and catalase positive, produces H2S, reduces nitrate to nitrite, and grows microaerobically at 37º C but not at 25º C or 42º C. It is resistant to cephalothin and nalidixic acid but sensitive to metronidazole. Photographs illustrating its morphologic structure by light (4,5) and electron (4,5,7) microscopy have been published. The species-defining characteristic of the organism, the nucleotide sequence of its 16S rRNA gene, has been used to develop a diagnostic assay based on polymerase chain reaction (8).
Systematic examination of rodents of all species and strains produced at FCRDC, especially retired breeders, showed that the characteristic hepatitis and associated bacteria were present in mice of several strains (A/JCr, DBA/2NCr, C3H/HeNCr) and that within these strains, the male mice were more severely affected than the female. Mice with severe combined immunodeficiencies were especially vulnerable. The precise location of organisms demonstrable by Steiner stain within infected liver parenchyma was shown by transmission electron microscopy to be invariably extracellular and characteristically within bile canaliculi (4,5). No liver disease was seen in some strains (e.g., C57BL/6NCr) or in F1 hybrids between sensitive and resistant strains (e.g., B6C3F1). Rodent species other than mice (e.g., rats, Syrian hamsters, and guinea pigs) were not affected.
In infected mice with severe combined immunodeficiency, cecal inflammation was histologically demonstrable (5), and organisms were isolated from the mucosa of the large intestine (7), which may mean that the usual ecologic niche occupied by H. hepaticus is that of a commensal colonizer of the intestinal tract (8). Since mice are coprophagic, it appears highly likely that natural transmission of the organisms is the oral-fecal route. Why and how H. hepaticus invades the liver in mice of certain strains remain to be determined. Hepatitis is also characteristic of certain other enteric pathogenic bacteria, such as Campylobacter jejuni (9) that, unlike H. hepaticus, have not been associated with liver tumor development. The tissue damage that accompanies persistent infection by H. hepaticus, H. pylori, and certain other Helicobacter species may be due, at least in part, to a soluble, trypsin-sensitive cytotoxin of high molecular weight produced by these organisms (10). There is no precedent for any direct role of such a toxin in carcinogenesis. On the other hand, chronic infections by viruses, bacteria, or certain parasites are recognized risk factors for human cancers at various sites. The hypothesis that chemically reactive, potentially genotoxic, substances of low molecular weight (including nitric oxide and active oxygen species) generated by inflammatory cells at the site of chronic infection may initiate or enhance carcinogenesis has been examined (11). The hypothesis is under active investigation in the context of H. hepaticus-associated liver disease.
H. hepaticus is susceptible to a number of antibiotics; treatment of susceptible, naturally infected 8-to 10-week-old strain A/JCr mice with single or combined antimicrobial agents has been evaluated for efficacy in eradicating established infections (12). Amoxicillin, metronidazole, and tetracycline administered singly failed to eradicate bacteria from the gastrointestinal tract, but either amoxicillin or tetracycline, in combination with metronidazole and bismuth, was effective in eradicating H. hepaticus from the liver, cecum, and colon when given by oral gavage for a period of 2 weeks (12). The effect of antibiotic therapy on the carcinogenic process, or in older animals, remains to be established.
The importance of H. hepaticus to humans is not yet completely known. The organism clearly has the potential to confound bioassays for chemical carcinogens, but this potential has no direct effect on humans. Even though most Helicobacter species identified to date are characteristically associated with (and named after) specific mammalian host species in which they generally inhabit the gastrointestinal tract (with or without causing gastritis or other chronic inflammatory disease), the potential host range for some species is quite broad. H. pylori, originally isolated from humans, has recently been isolated also from the domestic cat; this raises the possibility that Helicobacter pylori may be a zoonotic pathogen that can be transmitted from companion animals to humans (13). Exploring the possibility of zoonotic transmission of H. pylori, H. hepaticus, or any other Helicobacter species would require isolation of the organism in question by culture methods. Serologic methods have not yet been refined to the level of species specificity. Humans infected with H. pylori mount a serum antibody response to the bacteria that is readily detected by enzyme-linked immunosorbent assays and is considered evidence of ongoing disease (1); mice infected with H. hepaticus similarly produce serum antibodies to that species that have been demonstrated by Western blotting (5). Antisera to H. pylori can be used to visualize H. hepaticus in mouse liver tissue sections stained by the avidin-biotin complex immunohistochemical procedure (5). The cross- reactivity between these two species precludes use of available serologic methods to establish whether H. hepaticus has infected humans.
Regardless of whether H. hepaticus is itself capable of infecting humans, it serves to demonstrate that liver tissue can be persistently infected by at least one member of the genus Helicobacter, and that liver cancer can be a long-term consequence of such infection. This discovery raises questions about the existence of a comparable relationship between liver cancer in humans and unrecognized bacterial infections. Reviews are under way of tissue blocks from pathology archives in search of organisms demonstrable by the Steiner stain in liver sections from human populations at high risk for liver cancer.
SERODIAGNOSIS OF HELICOBACTER HEPATICUS INFECTION IN MICE BY AN ENZYME LINKED IMMUNOSORBENT ASSAY
Robert S. Livingston, Lela K. Riley, Earl K. Steffen, Cynthia L. Besch-Williford, Reuel R. Hook, Jr., and Craig L. Franklin; Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211; Journal of Clinical Microbiology, May 1997, p. 1236-1238, Vol. 35, No. 5
Helicobacter hepaticus is a newly recognized bacterium associated
with chronic active hepatitis, hepatic carcinoma, and inflammatory
bowel disease in mice. Currently, fecal or tissue PCR, fecal culture,
or histologic examination of silver-stained liver sections is
used to diagnose H. hepaticus infection. In this report, we describe
an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis
of H. hepaticus infection in mice with a membrane digest preparation
of H. hepaticus as the antigen. Sera from mice positive for H.
hepaticus by PCR or histologic examination (n = 88), positive
for Helicobacter bilis by PCR (n = 13), positive for other helicobacters
(not identifiable to species level) by PCR (n = 25), or negative
for all Helicobacter species by PCR (n = 162) were used to evaluate
the ELISA. Results indicated that ELISA provided 93.2% sensitivity,
94% specificity, 87.2% positive predictive value, and 96.9% negative
predictive value. Cross-reactive antibodies were detected in some
mice infected with helicobacters not identifiable to species level.
To further define ELISA sensitivity and specificity, groups of
10 C57BL/6 mice were inoculated per os with H. hepaticus, Helicobacter
muridarum, or H. bilis. Sera were collected and examined by the
ELISA. H. hepaticus-infected mice seroconverted by 2 weeks and
maintained ELISA reactivity throughout the 18-week study, while
mice infected with H. muridarum and H. bilis were negative by
ELISA. These results indicate that this reported ELISA is highly
sensitive and specific for the serodiagnosis of H. hepaticus infection
in mice.
INFLAMMATORY BOWEL DISEASE: AN IMMUNITY-MEDIATED CONDITION TRIGGERED BY BACTERIAL INFECTION WITH HELICOBACTER HEPATICUS
Rachel J. Cahill,1 Charmaine J. Foltz,1 James G. Fox,1,2, Charles A. Dangler,1 Fiona Powrie(3) and David B. Schauer1,2; Division of Comparative Medicine(1) and Division of Toxicology(2), Massachusetts Institute of Technology, Cambridge, Massachusetts 03129, DNAX Research Institute of Molecular and Cellular Biology Inc.(3), Palo Alto, California 943043; Infection and Immunity, Aug. 1997, p. 3126-3131, Vol. 65, No. 8
Inflammatory bowel disease (IBD) is thought to result from either an abnormal immunological response to enteric flora or a normal immunological response to a specific pathogen. No study to date has combined both factors. The present studies were carried out with an immunologically manipulated mouse model of IBD. Mice homozygous for the severe combined immunodeficiency (scid) mutation develop IBD with adoptive transfer of CD4+ T cells expressing high levels of CD45RB (CD45RBhigh CD4+ T cells). These mice do not develop IBD in germfree conditions, implicating undefined intestinal flora in the pathogenesis of lesions. In controlled duplicate studies, the influence of a single murine pathogen, Helicobacter hepaticus, in combination with the abnormal immunological response on the development of IBD was assessed. The combination of H. hepaticus infection and CD45RBhigh CD4+ T-cell reconstitution resulted in severe disease expression similar to that observed in human IBD. This study demonstrates that IBD develops in mice as a consequence of an abnormal immune response in the presence of a single murine pathogen, H. hepaticus. The interaction of host immunity and a single pathogen in this murine system provides a novel model of human IBD, an immunity-mediated condition triggered by bacterial infection.
HELICOBACTER CANIS ISOLATED FROM A DOG LIVER WITH MULTIFOCAL NECROTIZING HEPATITIS
Fox JG; Drolet R; Higgins R; Messier S; Yan L; Coleman BE; Paster BJ; Dewhirst FE Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge 02139, USA. J Clin Microbiol, 1996 Oct, 34:10, 2479-82
On the basis of biochemical, phenotypic, and 16S rRNA analysis, a novel gram-negative bacterium, isolated from normal and diarrheic dogs as well as humans with gastroenteritis, has been recently named Helicobacter canis. A 2-month-old female crossbred puppy was submitted to necropsy with a history of weakness and vomiting for several hours prior to death. The liver had multiple and slightly irregular yellowish foci up to 1.5 cm in diameter. Histologically, the liver parenchyma contained randomly distributed, occasionally coalescing hepatocellular necrosis, often accompanied by large numbers of mononuclear cells and neutrophils. Sections of liver stained by the Warthin-Starry silver impregnation technique revealed spiral- to curve-shaped bacteria predominantly located in bile canaliculi and occasionally in bile ducts. Aerobic culture of liver was negative, whereas small colonies were noted on Campylobacter selective media after 5 days of microaerobic incubation. The bacteria were gram negative and oxidase positive but catalase, urease, and indoxyl acetate negative; nitrate was not reduced to nitrite, and the organism did not hydrolyze hippurate. The bacteria were also resistant to 1.5% bile. Electron microscopy revealed spiral-shaped bacteria with bipolar sheathed flagella. By 16S rRNA analysis, the organism was determined to be H. canis. This is the first observation of H. canis in active hepatitis in a dog and correlates with recent findings of Helicobacter hepaticus- and Helicobacter bilis-related hepatic disease in mice. Further studies are clearly warranted to ascertain whether H. canis-associated hepatitis is more widespread in canines as well as a cause of previously classified idiopathic liver disease in humans.
EVALUATION OF ANTIBIOTIC THERAPIES FOR ERADICATION OF H. HEPATICUS.
Foltz CJ; Fox JG; Yan L; Shames B Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge 02139, USA. Antimicrob Agents Chemother, 1995 Jun, 39:6, 1292-4
The newly recognized murine pathogen Helicobacter hepaticus is known to colonize the ceca and colons of several strains of mice from a variety of commercial suppliers. Additionally, the organism persistently infects mice, causes a chronic hepatitis, and is linked to hepatic tumors in the A/JCr inbred mouse strain. For this reason, eradication of the organism from infected mouse colonies is desirable. Treatment modalities for eradication of H. hepaticus from the gastrointestinal system consisted of oral administration of various antibiotic combinations previously evaluated for eradication of experimental H. felis gastric infection in mice. A/JCr mice (8 to 10 weeks old) naturally infected with H. hepaticus were divided into six treatment groups of 10 animals each. Animals received monotherapy of amoxicillin, metronidazole, or tetracycline or triple therapy of amoxicillin-metronidazole-bismuth (AMB) or tetracycline-metronidazole-bismuth (TMB). All medications were administered by oral gavage three times daily for 2 weeks. One month after the final treatment, mice were euthanatized and livers, ceca, and colons were cultured for H. hepaticus. All untreated control animals had H. hepaticus isolated from the cecum and/or colon. H. hepaticus was not recovered from the livers, ceca, or colons of the AMB or TMB treatment groups. All animals receiving the various antibiotic monotherapies had H. hepaticus isolated from the cecum and colon. We conclude that at the doses and the route evaluated, AMB and TMB triple therapies are effective for eradication of H. hepaticus in 8- to 10-week old A/JCr mice.
THE GOOD NEWS IS THAT ONCE DIAGNOSED, H. HEPATICUS APPEARS TO BE TREATABLE WITH THE SIMILAR 2 WEEK TREATMENT APPROVED BY FDA FOR H. PYLORI:
from the Journal of American Medical Association( AMA ), June 18, 1997:
The topics in gastrointestinal tract disease that continue to generate the most interest are Helicobacter pylori and the increasing evidence and acceptance that Helicobacter pylori gastritis is recognized as a precursor lesion to peptic ulcer, atrophic gastritis, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. A search for H pylori genetic markers of pathogenicity continues, with 1 group demonstrating that certain alleles of vacA, the gene encoding the vacuolating cytotoxin of H pylori, are found in patients with ulcer disease more often than other alleles.[9] Another group has suggested that long-term use of proton pump inhibitors in reflux esophagitis puts H pylori-infected patients at increased risk of atrophic gastritis,[10] although a recent Food and Drug Administration (FDA) advisory panel concluded there was inadequate evidence to support that assertion. A urea breath test was approved by the FDA and now offers a means of evaluating the success of antimicrobial therapy without endoscopy. The FDA has also approved 3 combination regimens for treatment of H pylori infection: omeprazole plus clarithromycin(cure rate about 70%), ranitidine bismuth citrate plus clarithromycin (cure rate about 80%), and bismuth subsalicylate plus metronidazole plus tetracycline combined with an antisecretory drug (cure rate about 80%), each given for 2 weeks. However, most experts believe the best therapy may be a combination of ranitidine bismuth citrate or a proton pump inhibitor plus clarithromycin plus either amoxicillin or metronidazole given for 7 to 10 days (cure rate about 90%[11]). The question of whom to treat remains in a state of flux. Everyone concedes that recurrence of peptic ulcer and its complications is dramatically decreased after successful treatment of H pylori, and all such infected patients should be treated.[12] Studies demonstrating regression of low-grade MALT lymphomas justify antimicrobials as initial therapy. Much less secure are recommendations to screen and treat patients with nonulcer dyspepsia or the population as a whole to prevent gastric adenocarcinoma.[13]
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