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Richard Freeman Research Institute, Alton Ochsner Medical Institutions, New Orleans, LA 70121
2To whom correspondence should be addressed. E-mail: amason{at}ochsner.org.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONHCV infection and type...Retroviral etiology of primary...LITERATURE CITED |
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KEY WORDS: • diabetes • hepatitis • infection • liver • cirrhosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONHCV infection and type...Retroviral etiology of primary...LITERATURE CITED |
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Today, the problem of establishing causal relationships is compounded further with the ease of cloning occult pathogens, mandating the development of new strategies to determine disease associations without wasting time and resources. For example, several novel viruses have been cloned from patients with post-transfusion hepatitis without any obvious link to chronic liver disease. The problem of proving causality is confounded further as Koch’s postulates are not readily applicable to chronic disorders unless the pathogen can be cultured and a small animal model convincingly develops the phenotypic manifestation of disease. Indeed, the discovery of hepatitis C virus (HCV)3 was notable for the very reason that the cloning was performed without the virus ever being isolated, visualized, grown in tissue culture system or passaged in a small animal model. In this review, we suggest that in vitro coculture models and clinical trials with antiviral therapy can provide sufficient evidence to help establish a causal association for viral induction of chronic idiopathic disorders. Specifically, we evaluate the epidemiologic, pathophysiology and immunogenetic data suggesting an association of HCV infection with type 2 diabetes and suggest the use of vitro coculture systems and clinical trials that have helped to implicate a recently isolated human retrovirus in the etiology of primary biliary cirrhosis.
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HCV infection and type 2 diabetes |
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TOPABSTRACTINTRODUCTIONHCV infection and type...Retroviral etiology of primary...LITERATURE CITED |
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–7)
, type 2 diabetes (6
–9)
and
thalassemia (10)
, as well as a national survey
(11)
. A 2- to 10-fold increase in diabetes has been
reported worldwide (Table 1
) in HCV-positive patients compared with liver disease control
subjects (1
–7)
, although a single study failed to support
the association (12)
. In both of the largest retrospective
studies conducted to date, which incorporate >1,000 patients, HCV
infection was found to be an independent risk factor associated with
type 2 diabetes by multivariate analysis (4
,6)
.
Collectively the studies outlined in Table 1
have been criticized for
the lack of matching age, sex, BMI, socioeconomic status, race and
stage of liver disease (12)
. However, the association of
HCV infection with diabetes has been observed in noncirrhotic
populations with thalassemia, for example, who have a fourfold
increased risk of diabetes with HCV infection (10)
, and in
a study of liver transplant recipients, in which HCV-positive
patients have a reported four- to eightfold increased prevalence of
diabetes as compared with patients with other viral or cholestatic
liver disease 1 y after liver transplantation (13)
.
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). In English type 2 diabetic subjects with abnormal serum
aminotransferases, HCV antibody was detected in 28% of patients of
African origin, 12% of Caucasians and 8% of Asians (8)
.
In a large U. S. clinic-based population, 4.2% of consecutive
patients with diabetes were found to be HCV antibody positive compared
with 1.6% in the control group undergoing thyroid scan
(6)
. These seroprevalence studies should also be
interpreted with caution because of the lack of data on cirrhosis,
ascertainment bias and the difficulty in selecting adequate control
groups. However, a cross-sectional study involving nearly 10,000
subjects from the Third National Health and Nutrition Examination
Survey addresses some of these concerns (11)
. Multivariate
analysis with matching of known risk factors showed that HCV infection
provided a more than threefold increased risk of developing diabetes in
individuals aged >40 y and twofold for those aged <40 y, whereas
hepatitis B virus infection had no discernible effect
(11)
. None of the patients with type 1 diabetes had
evidence of HCV infection, suggesting that the diagnosis of diabetes
per se had little impact on the subsequent development of HCV
infection. However, no data were available to assess the temporal
relationship for the onset of each disease or the role of cirrhosis on
the development of diabetes.
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. The same linkage in two independent databases
prompts the speculation that type 2 diabetes can occur as a result of
viral infection in genetically predisposed individuals with liver
disease. From a mechanistic standpoint, HCV-positive patients with
diabetes do not develop specific autoantibodies; therefore, autoimmune
destruction of the pancreas does not appear to be an important
mechanism (2
,6)
. There is evidence that HCV-positive
diabetic patients have both peripheral insulin resistance as well as
ß-cell dysfunction, as indicated by decreased C-peptide levels
and limited acute insulin responses (4
,15
,16)
.
Furthermore, postmortem studies reveal that HCV replicates in the
pancreas (17)
and clinical trials report improvement in
several measures of glucose metabolism after antiviral treatment
(16)
. However, it remains to be determined whether the
reversible component of diabetes is attributable to HCV directly
perturbing the synthetic function in ß-cells in patients with chronic
liver disease secondary to HCV infection. In summary, the epidemiologic association of HCV with type 2 diabetes seems convincing. Coculture studies with clonal ß-cells and HCV may answer the question of whether viral infection of the pancreas directly correlates with diminished acute insulin responses seen in patients with HCV infection. A small animal model may ultimately be required to investigate the intricate relationship between liver and endocrine disease to establish whether HCV infection causes diabetes beyond the risk attributable to liver disease alone. It is also possible that transgenic mouse studies using expressed HCV proteins or prospective longitudinal studies assessing endocrine responses to antiviral therapy will provide further insight into the pathogenesis of viral induction of diabetes. However, a multifaceted attack is clearly warranted to investigate a causal association of HCV infection and diabetes to stem the epidemics of both common disorders.
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Retroviral etiology of primary biliary cirrhosis |
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TOPABSTRACTINTRODUCTIONHCV infection and type...Retroviral etiology of primary...LITERATURE CITED |
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. The development of progressive granulomatous
destruction of bile ducts usually leads to cirrhosis, but it is
questionable how autoantibodies reactive to a ubiquitous intracellular
organelle can cause a disease limited to biliary epithelium. It is also
important to note that the role of autoimmunity plays in causing PBC is
controversial because autoantibody levels and immunosuppression have
little impact on the disease process (19)
. Of interest,
immunohistochemistry studies using AMAs demonstrate a protein
resembling the mitochondrial autoantigen, PDC-E2, on PBC patients’
bile ducts and perihilar lymph nodes, which may be responsible for
precipitating the formation of autoantibodies in the first place. This
aberrant expression of the AMA reactive proteins in diseased tissues is
unique to PBC patients and is considered a phenotypic manifestation of
PBC.
There are several lines of evidence to suggest an infectious etiology
for PBC (20)
. Reports have documented the clustering of
disease in nonrelated family members and specific geographic locations
as well as an increased incidence of PBC in migrant populations moving
to higher prevalence countries and disease recurrence in the hepatic
allograft after liver transplantation (20)
. To investigate
a potential infectious etiology of PBC, we have used a combination of
immunologic, cloning and electron micrograph techniques to identify a
putative agent. In initial studies, we found that the majority of PBC
patients had demonstrable antibody reactivity to the human
intracisternal A-type particle, a retrovirus isolated from
Sjogren’s syndrome patients, and we also observed virus-like
particles by electron microscopy in extracted biliary epithelial cells
(BECs) from PBC patients (21)
. Subsequently, we cloned a
novel exogenous retrovirus from a PBC biliary epithelium cDNA library
and used reverse transcriptase–polymerase chain reaction (RT-PCR) and
immunohistochemistry to demonstrate that the viral infection was
specifically detected in perihilar lymph nodes of patients with PBC
(22)
.
We have also established an in vitro model for PBC by coculturing
normal BECs with perihilar lymph nodes from patients with chronic liver
disease. The BECs cocultured with PBC lymph nodes were found to express
increased levels of PDC-E2 autoantigen by Western blot and
immunocytochemistry after 5 d (23)
. Induction of the
PBC phenotype occurred specifically with PBC lymph nodes and not with
other liver disease controls. The supernatants from the PBC cocultures
also induced PDC-E2 expression in fresh BECs, and this was abrogated by
gamma irradiation (24)
. Electron microscopy studies
revealed virus-like particles in the BECs and supernatants derived
from the PBC cocultures and reverse transcriptase activity in PBC
supernatants processed on sucrose gradients ranging from 1.14 to 1.17
g/mL, where enveloped retroviruses cosediment (24)
. Using
RT-PCR, we found that the novel viral sequences were detected in
the BECs and supernatants cocultivated with PBC samples
(22)
.
Further support for a retroviral etiology of PBC has been provided by
pilot studies with antiretroviral therapies. One year of therapy with
Lamivudine 150 mg/d in 10 PBC patients resulted in a temporary
improvement in liver biochemistry tests, a significant reversal of bile
duct loss and an improvement in histologic stage was observed in three
patients (25)
. In a second pilot study, 5 of the 10 PBC
patients have been treated for 6 mo on Combivir twice daily with
significant reductions in serum aminotransferases and alkaline
phosphatase levels, and 3 patients have completely normalized their
hepatic biochemistry teats. In the Lamivudine study, a significant log
fold reduction in serum AMA levels was observed at the end of treatment
(25)
. Immunohistochemistry studies were performed to
investigate whether the reduction in autoantibodies corresponded with a
diminished expression of the PDC-E2 autoantigen in biliary epithelium,
and four of the eight patients with identifiable bile ducts before and
after therapy had no evidence of the AMA reactive protein on their
biliary epithelium after treatment. Collectively, these data support
the model that a retrovirus mediates the aberrant expression of the
major autoantigen associated with PBC. Even though the results of these
antiviral studies are encouraging, it should be recognized that the
expression of AMA reactivity in PBC patients’ biliary epithelium can
be heterogeneous, the histologic stage may vary in different biopsies
from the same liver, and only a few patients have been treated to date.
Can we assume a causal association of the novel human retrovirus with the development of PBC based on the partial resolution of the disease in vivo with antiretroviral therapy and the generation of the disease phenotype in the in vitro model? The answer depends on the specificity of the so-called PBC phenotype and the proof that antiretroviral therapy really works for patients with PBC. However, the notion that an in vitro coculture model can substitute as a modification of Koch’s postulates merits further consideration. The detection of the virus in the perihepatic lymph nodes by RT-PCR and immunohistochemistry fulfills the first of Koch’s postulates, and the isolation of the transmissible agent in supernatants of BEC cocultured with PBC lymph nodes partially fulfills the second of Koch’s postulates to isolate the organism in pure culture. The demonstration that inoculation of the pure culture into a host causes the disease constitutes the third postulate, and the forth postulate is the subsequent recovery of the microbial agent from the host lesion. Accordingly, if we substitute the latter with serial passage of supernatants containing the novel virus onto the BECs in our in vitro model, then the development of the PBC phenotype and subsequent recovery of the virus partially fulfill the last two postulates.
In summary, we have identified an agent with the morphologic,
hydrodynamic, enzymatic and genomic features of an exogenous retrovirus
that is detected in PBC patients’ lymph nodes in vivo and is
associated with the development of the PBC phenotype in vitro. The
establishment of a causal association of infection and disease is
supported by the preliminary antiviral studies and the modified in
vitro postulates with the development of the PBC phenotype in BECs. The
specificity of PBC phenotype could be enhanced further using data from
a microarray study showing specific activation of wnt
pathways in PBC patients’ hepatic cDNA (26)
, with
evaluation of infected and uninfected BECs for the specific microarray
transciptional fingerprint. Also, the use of antiviral therapy or
neutralizing antibodies to prevent the development of the phenotype in
vitro would also strengthen the hypothesis considerably. Certainly more
flexible and feasible models for determining microbial causality are
eagerly awaited.
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FOOTNOTES |
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3 Abbreviations: AMA, antimitochondrial antibody;
BEC, biliary epithelial cell;HCV, hepatitis C virus; PBC, primary
biliary cirrhosis; PDC-E2, pyruvate dehydrogenase complex E2;
RT-PCR, reverse transcriptase–polymerase chain reaction.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONHCV infection and type...Retroviral etiology of primary...LITERATURE CITED |
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