Systematic review: recurrent autoimmune liver diseases after liver transplantation.

BACKGROUND: Autoimmune liver diseases (AILD) constitute the third most common indication for liver transplantation (LT) worldwide. Outcomes post LT are generally good but recurrent disease is frequently observed. AIMS: To describe the frequency and risk factors associated with recurrent AILD post-LT and provide recommendations to reduce the incidence of recurrence based on levels of evidence. METHODS: A systematic review was performed for full-text papers published in English-language journals, using the keywords 'autoimmune hepatitis (AIH)', 'primary biliary cholangitis and/or cirrhosis (PBC)', 'primary sclerosing cholangitis (PSC)', 'liver transplantation' and 'recurrent disease'. Management strategies to reduce recurrence after LT were classified according to grade and level of evidence. RESULTS: Survival rates post-LT are approximately 90% and 70% at 1 and 5 years and recurrent disease occurs in a range of 10-50% of patients with AILD. Recurrent AIH is associated with elevated liver enzymes and IgG before LT, lymphoplasmacytic infiltrates in the explants and lack of steroids after LT (Grade B). Tacrolimus use is associated with increased risk; use of ciclosporin and preventive ursodeoxycholic acid with reduced risk of PBC recurrence (all Grade B). Intact colon, active ulcerative colitis and early cholestasis are associated with recurrent PSC (Grade B). CONCLUSIONS: Recommendations based on grade A level of evidence are lacking. The need for further study and management includes active immunosuppression before liver transplantation and steroid use after liver transplantation in autoimmune hepatitis; selective immunosuppression with ciclosporin and preventive ursodeoxycholic acid treatment for primary biliary cholangitis; and improved control of inflammatory bowel disease or even colectomy in primary sclerosing cholangitis.

Frequent proviral integration of the human betaretrovirus in biliary epithelium of patients with autoimmune and idiopathic liver disease.

BACKGROUND: A human betaretrovirus (HBRV) has been linked with primary biliary cirrhosis (PBC) following the detection of viral particles in biliary epithelium by electron microscopy and cloning of the betaretrovirus genome from biliary epithelium and peri-hepatic lymph nodes. Evidence for viral infection was found in the majority of PBC patients' peri-hepatic lymph node samples. However, less than a third of the liver samples had detectable HBRV, whereas others were unable to detect betaretrovirus infection or noted the presence of virus in the liver of patients with other diagnoses. AIMS: To address the hypothesis that the betaretrovirus may be below the limits of detection in the liver, biliary epithelial cells (BEC) were investigated for the evidence of infection. METHODS: Ligation-mediated PCR and next generation sequencing were used to detect proviral integrations in liver, lymph nodes and BEC isolated from liver transplant recipients. Hybridisation-based assays were used to detect betaretroviral RNA in BEC. RESULTS: Unique HBRV integrations and betaretrovirus RNA were detected in the majority of biliary epithelia derived from patients with PBC, autoimmune hepatitis and cryptogenic liver disease but rarely in other liver transplant recipients with primary sclerosing cholangitis and other hepatic disorders. HBRV integrations were commonly found in PBC patients' lymph nodes but rarely in whole liver samples. CONCLUSIONS: Human betaretrovirus infection is frequently observed at the site of disease in patients with primary biliary cirrhosis and also in biliary epithelium of patients with autoimmune hepatitis and cryptogenic liver disease.

Vector-induced NT-3 expression in rats promotes collateral growth of injured corticospinal tract axons far rostral to a spinal cord injury.

Rewiring the injured corticospinal tract (CST) by promoting connections between CST axons and spared neurons is a strategy being explored experimentally to achieve improved recovery of motor function after spinal cord injury (SCI). Reliable interventions to promote and direct growth of collaterals from injured CST axons are in high demand to promote functionally relevant detour pathways. A promising tool is neurotrophin-3 (NT-3), which has shown growth-stimulating and chemo-attractive effects for spared CST axons caudal to a CST lesion. Yet, efforts to promote growth of injured CST axons rostral to a SCI with NT-3 have been less successful to date. Evidence indicates that immune activation in the local growth environment, either intrinsic or induced by the endotoxin lipopolysaccharide (LPS), can play a decisive role in the CST's responsiveness to NT-3. Here, we test the potential of NT-3 as a tool to enhance and direct collateral growth from the injured CST rostral to a SCI (1) using long-term expression of NT-3 by adeno-associated viral vectors, (2) with and without stimulating the immune system with LPS. Our results indicate that inducing a growth response from injured CST axons into a region of vector-mediated NT-3 expression is possible in the environment of the spinal cord rostral to a SCI, but seems dependent on the distance between the responding axon and the source of NT-3. Our findings also suggest that injured CST axons do not increase their growth response to NT-3 after immune activation with LPS in this environment. In conclusion, this is to our knowledge the first demonstration that NT-3 can be effective at promoting growth of injured CST collaterals far rostral to a SCI. Making NT-3 available in close proximity to CST target axons may be the key to success when using NT-3 to rewire the injured CST in future investigations.

Association of primary biliary cirrhosis with variants in the CLEC16A, SOCS1, SPIB and SIAE immunomodulatory genes.

We fine mapped two primary biliary cirrhosis (PBC) risk loci, CLEC16A (C-type lectin domain family 16 member A)-suppressor of cytokine signaling 1 (SOCS1) and Spi-B protein (SPIB) and sequenced a locus, sialic acid acetylesterase (SIAE), proposed to harbor autoimmunity-associated mutations. In all, 1450 PBC cases and 2957 healthy controls were genotyped for 84 single-nucleotide polymorphisms (SNPs) across the CLEC16A-SOCS1 and SPIB loci. All 10 exons of the SIAE gene were resequenced in 381 cases and point substitutions of unknown significance assayed for activity and secretion. Fine mapping identified 26 SNPs across the CLEC16A-SOCS1 and 11 SNPs across the SPIB locus with significant association to PBC, the strongest signals at the CLEC16A-SOCS1 locus emanating from a SOCS1 intergenic SNP (rs243325; P=9.91 × 10(-9)) and at the SPIB locus from a SPIB intronic SNP (rs34944112; P=3.65 × 10(-9)). Among the associated SNPs at the CLEC16A-SOCS1 locus, two within the CLEC16A gene as well as one SOCS1 SNP (rs243325) remained significant after conditional logistic regression and contributed independently to risk. Sequencing of the SIAE gene and functional assays of newly identified variants revealed six patients with functional non-synonymous SIAE mutations (Fisher's P=9 × 10(-4) vs controls) We demonstrate independent effects on risk of PBC for CLEC16A, SOCS1 and SPIB variants, while identifying functionally defective SIAE variants as potential factors in risk for PBC.

Linking human beta retrovirus infection with primary biliary cirrhosis.

Several environmental agents have been linked with primary biliary cirrhosis (PBC) that include bacteria, xenobiotics and viruses. A human beta retrovirus (HBRV) related to mouse mammary tumor virus has been cloned and characterized from patients with PBC. This agent can be detected in the majority of patients' perihepatic lymph nodes by immunochemistry and RT-PCR. The HBRV has recently been isolated in culture and integration sites have been identified in the genome of patients to provide convincing evidence of beta retrovirus infection in patients. Three lines of evidence support a role for the virus in PBC. First, the beta retrovirus is linked with aberrant expression of mitochondrial protein(s) on the biliary epithelium cell (BEC) surface, a disease specific phenotype. Second, the related agent, mouse mammary tumor virus has been linked with autoimmune biliary disease in the NOD.c3c4 mouse model for PBC. In this mouse model, the virus is localized to diseased biliary epithelium that also display aberrant expression of the mitochondrial autoantigens. In translational studies, both patients with PBC and NOD.c3c4 mice demonstrate significant improvement in biliary disease with combination antiviral therapy. An overview of the biological relevance of the beta retrovirus infection in PBC will be discussed in this review.

Cyclosporine A protects against primary biliary cirrhosis recurrence after liver transplantation.

Primary biliary cirrhosis (PBC) reoccurs in a proportion of patients following liver transplantation (LT). The aims of our study were to evaluate the risk factors associated with PBC recurrence and determine whether recurrent disease constitutes a negative predictor for survival. One hundred and eight patients receiving LT for end-stage PBC were studied. Recurrent disease was diagnosed in 28 patients (26%). Probability of recurrent PBC at 5 years was 13% and 29% at 10 years with an overall incidence of 3.97 cases per 100 patient years. By univariate Cox analysis use of tacrolimus (HR 6.28, 95% CI, 2.44-16.11, p < 0.001) and mycophenolate mofetil (HR 5.21, 95% CI, 1.89-14.33, p = 0.001) were associated with higher risk of recurrence; whereas use of cyclosporine A (CsA) and azathioprine were associated with reduced risk of recurrence (HR 0.13, 95% CI 0.05-0.35, p < 0.001 and HR 0.27, 95% CI 0.11-0.64, p = 0.003, respectively). In the multivariate Cox analysis, only CsA was independently associated with protection against recurrence (HR 0.17, 95% CI 0.06-0.71, p = 0.02). Five-year probability of survival was 83% and 96%, in patients without and with recurrence (log-rank test, p = 0.3). Although PBC transplant recipients receiving CsA have a lower risk of disease recurrence, the development of recurrent PBC did not impact on long-term patient survival.

Clinical trial: randomized controlled study of zidovudine and lamivudine for patients with primary biliary cirrhosis stabilized on ursodiol.

BACKGROUND: A human betaretrovirus has been characterized in patients with primary biliary cirrhosis (PBC). Uncontrolled studies using combination anti-retroviral therapy have reported significant biochemical and histological improvement. AIM: To conduct a double blind, randomized controlled trial as a proof of principal to link infection with PBC. METHODS: Fifty-nine patients with an alkaline phosphatase level>1.5 upper limits of normal stabilized on ursodeoxycholic acid therapy were randomized to either 300 mg zidovudine and 150 mg lamivudine B.I.D. or placebo for 6 months. RESULTS: None of the patients normalized alkaline phosphatase and no significant differences were observed in normalizing serum aminotransferase levels. Significant differences were observed in the antiviral versus placebo arms with improvements in serial alkaline phosphatase (p<0.04), ALT (p<0.03) and AST (p<0.04) as well as clinical score (p<0.02). After 6 months, 25% of patients in the placebo arm and 4% in the antiviral arm had evidence of virus in serum. CONCLUSIONS: The study endpoints for normalizing hepatic biochemistry were too stringent to show efficacy for zidovudine and lamivudine therapy despite the demonstrable impact on clinical and biochemical improvement. Accordingly, more potent anti-viral regimens will be required to confirm the efficacy of antiviral therapy in PBC patients with human betaretrovirus infection.

Reverse transcriptase activity in patients with primary biliary cirrhosis and other autoimmune liver disorders.

BACKGROUND: Patients with biliary disease make retroviral antibodies and the Human Betaretrovirus has been characterized in patients with primary biliary cirrhosis. AIM: To screen patients with autoimmune liver disease for evidence of retroviral infection. METHODS: Real-time reverse transcriptase polymerase chain reaction was used to detect Human Betaretrovirus, and a reverse transcriptase assay to measure reverse transcriptase activity in plasma. RESULTS: Using reverse transcriptase polymerase chain reaction, 24% of primary biliary cirrhosis samples were positive for Human Betaretrovirus when compared to 13% with autoimmune hepatitis, 5% of other liver diseases and 3% of the non-liver disease control subjects. Reverse transcriptase activity was found in 73% of patients with autoimmune hepatitis, 42% with primary biliary cirrhosis, 22% of liver patients without viral or autoimmune disease and 7% of subjects without liver disease. In patients with autoimmune liver disease, detection of reverse transcriptase activity was related to higher ALT levels, whereas others stabilized on immunosuppressive therapy either preliver or postliver transplantation were less likely to be reverse transcriptase-positive. CONCLUSIONS: Most patients with autoimmune hepatitis have detectable reverse transcriptase activity. Investigations will be required to assess whether this represents the expression of endogenous retroviruses and retrotransposable elements in inflamed tissue, or signifies the presence of exogenous retroviral infection.

Steroid-free liver transplantation using rabbit antithymocyte globulin induction: results of a prospective randomized trial.

Steroids have been 1 of the primary modes of immunosuppression since the inception of transplantation and have been credited with both the prevention and treatment of rejection. Steroids also have been held responsible for increased infections, posttransplantation diabetes, and recurrent hepatitis after orthotopic liver transplantation (OLT). The purpose of this ongoing prospective randomized trial is to eliminate steroid use in OLT through induction with rabbit antithymocyte globulin (RATG). This is the first report of a prospective randomized trial in OLT achieving complete absence of steroids. Seventy-one adult patients were prospectively randomized to administration of RATG or steroids. Thirty-six patients were randomized to the administration of RATG induction at a dose of 1.5 mg/kg intravenously (IV) beginning during the anhepatic phase. No steroids were administered. Patients were administered a second 1.5-mg/kg dose of RATG post-OLT day 1. Thirty-five patients were randomized to the administration of methylprednisolone, which had been our standard immunosuppressive protocol. These patients were administered methylprednisolone, 1,000 mg IV, initiated during the anhepatic phase and followed by steroid taper. Maintenance immunosuppression consisted of tacrolimus and mycophenolate, with or without prednisone. Three patients died in each group, for an overall survival rate of 91% in each group. One patient in each group required re-OLT, for a graft survival rate of 89% in each group. Seven patients administered RATG had biopsy-proven rejection (20.5%), all of whom were successfully treated by increasing tacrolimus doses. Eleven patients administered steroid had biopsy-proven rejection (32%), 7 (64%) of whom required additional steroids for treatment, whereas 4 patients (36%) were successfully treated by increasing tacrolimus doses. The incidence of rejection was not statistically significant; however, there was a significant difference in the incidence of steroid-requiring rejection (P =.01). The incidence of recurrent hepatitis C was 50% in RATG patients and 71% in steroid patients (P = not significant). The incidence and severity of infectious complications were slightly lower in RATG patients, accounted for by a greater incidence of cytomegalovirus (CMV) infection in the steroid patients. RATG induction enables complete avoidance of steroid use in OLT with a trend toward a lower rejection rate, decreased incidence of post-OLT diabetes and recurrent hepatitis C, and decreased CMV infection. This prospective randomized trial gives encouraging support that steroids can be safely eliminated in OLT.

Transplantation of livers from hbc Ab positive donors into HBc Ab negative recipients: a strategy and preliminary results.

Here we describe a strategy for using livers from hepatitis B core antibody (anti-HBc) positive donors in anti-HBc negative recipients and report our preliminary results. Adult anti-HBc negative recipients were immunized against hepatitis B virus (HBV) prior to transplantation. Liver biopsies from anti-HBc positive, HBs Ag negative donors were performed at the time of procurement to rule out acute hepatitis or chronic liver disease. Donor serum and liver samples were collected for HBV DNA analysis by PCR. Recipients were given HBIG (10000 units, i.v.) during the anhepatic phase of transplantation. Patients were treated with lamivudine (150 mg) beginning on postoperative day (POD) 1. If HBV DNA was not detected in either donor liver or serum by PCR, recipient antiviral therapy was stopped. If donor liver and serum were positive for HBV DNA by PCR, the recipient was maintained on combination lamivudine and HBIG therapy. If HBV DNA was detected in donor liver but not in donor serum, the patient was managed on lamivudine therapy alone. Between February 1999 and June 2000, six anti-HBc negative recipients received liver transplants from anti-HBc positive donors. PCR analysis of serum from the six donors was negative for HBV DNA in each, while donor liver PCR analysis was positive in five of six for HBV DNA. Accordingly, all patients were given HBIG in the anhepatic phase of transplantation and five of six were maintained on daily lamivudine therapy. Follow-up periods have ranged from 2 to 18 months. There has been no emergence of de novo hepatitis B. Serial serum HBs Ag and HBV DNA assays have all proven negative. Moreover, while on lamivudine therapy, 2 patients now have undetectable HBV DNA in hepatic allograft biopsies by PCR analysis. Our strategy for using livers from anti-HBc donors has yielded promising initial results. De novo hepatitis B has not occurred and our data suggest residual hepatitis B virus may be eradicated in recipients maintained on lamivudine therapy.

GP73, a novel Golgi-localized protein upregulated by viral infection.

We report the isolation and characterization of GP73, a novel 73kDa human Golgi protein. The GP73 cDNA was cloned by differential screening of a cDNA library derived from the liver of a patient with adult giant-cell hepatitis (GCH), a rare form of hepatitis with presumed viral etiology. In vitro transcription-translation studies indicate that GP73 is an integral membrane protein, and immunolocalization experiments using epitope-tagged GP73 demonstrate that the protein is localized to the Golgi apparatus. Northern blot analysis of RNA from multiple human tissues reveals a single GP73 mRNA transcript with a size of approximately 3.0kb. Immunohistochemical studies using rabbit polyclonal antisera directed against recombinant GP73 demonstrate that the protein is preferentially expressed by epithelial cells in many human tissues. In normal livers, GP73 is consistently present in biliary epithelial cells, whereas hepatocytes show little or no signal. In contrast, livers of patients with GCH display strong GP73 immunoreactivity in multinucleated hepatocytes. GP73 mRNA and protein are expressed in highly differentiated HepG2 hepatoma cells after infection with adenovirus in vitro. We conclude that GP73 represents a novel, epithelial cell-specific integral membrane Golgi protein that can be upregulated in response to viral infection.

Retroviruses in autoimmune liver disease: genetic or environmental agents?

Retroviruses have been implicated in the pathogenesis of several human autoimmune conditions including Sjögren's syndrome, primary biliary cirrhosis, immune mediated diabetes, and multiple sclerosis. The human intracisternal A type particle derived from Sjögren's syndrome patients' salivary glands was the first retrovirus to be isolated from a human autoimmune disorder but the agent has yet to be cloned. In primary biliary cirrhosis patients, virus like particles have been observed by electron microscopy in biliary epithelium, endogenous retroviral sequences have been cloned from liver samples, and antibody reactivity to the human intracisternal A type particle has been observed in the majority of patients tested. However, there is no evidence to link the endogenous retroviral sequences in primary biliary cirrhosis patients to the retroviral antibody reactivity or virus like particles. In other patients with liver disease, reactivity to the human intracisternal A type particle was observed in a small but significant proportion of patients with hepatitis C virus infection. If the intracisternal A type particle is an endogenous retrovirus, it is interesting to speculate that hepatitis C virus infection may modulate the endogenous retroviral expression, as chronic hepatitis C has been linked with the development of Sjögren's syndrome. Furthermore, many patients with chronic hepatitis C virus infection have reactivity to an autoantigen of unknown significance known as GOR that has protein sequence homology with both hepatitis C virus nucleocapsid protein as well as HTLV-1 gag. This may be an another example of an endogenous retroviral protein acting as an autoantigen in liver disease patients. At this time, there is little evidence to suggest that endogenous retroviruses are infectious agents that cause autoimmune disease but they may be implicated as either genetic elements or antigens. Further studies will be required to characterize the role that both exogenous and endogenous retroviruses play in the pathogenesis of autoimmune liver diseases.

Association of diabetes mellitus and chronic hepatitis C virus infection.

While patients with liver disease are known to have a higher prevalence of glucose intolerance, preliminary studies suggest that hepatitis C virus (HCV) infection may be an additional risk factor for the development of diabetes mellitus. To further study the correlation of HCV infection and diabetes, we performed a retrospective analysis of 1,117 patients with chronic viral hepatitis and analyzed whether age, sex, race, hepatitis B virus (HBV) infection, HCV infection, and cirrhosis were independently associated with diabetes. In addition, a case-control study was conducted to determine the seroprevalence of HCV infection in a cohort of 594 diabetics and 377 clinic patients assessed for thyroid disease. In the former study after the exclusion of patients with conditions predisposing to hyperglycemia, diabetes was observed in 21% of HCV-infected patients compared with 12% of HBV-infected subjects (P =.0004). Multivariate analysis revealed that HCV infection (P =.02) and age (P =.01) were independent predictors of diabetes. In the diabetes cohort, 4.2% of patients were found to be infected with HCV compared with 1.6% of control patients (P =.02). HCV genotype 2a was observed in 29% of HCV-RNA-positive diabetic patients versus 3% of local HCV-infected controls (P <.005). In conclusion, the data suggest a relatively strong association between HCV infection and diabetes, because diabetics have an increased frequency of HCV infection, particularly with genotype 2a. Furthermore, it is possible that HCV infection may serve as an additional risk factor for the development of diabetes, beyond that attributable to chronic liver disease alone.

A case of syncytial giant cell hepatitis with features of a paramyxoviral infection.

Adult syncytial giant cell hepatitis (GCH) is an uncommon and often fulminant form of hepatitis that may be caused by infection with a novel paramyxo-like virus. We present the case of a 69-yr-old man who presented with acute, community-acquired hepatitis and chronic lymphocytic leukemia. A liver biopsy showed the typical findings of panlobular syncytial giant cell hepatitis. Electron microscopic examination demonstrated abundant nucleocapsid-like protein material in the cytoplasm and nuclei of affected hepatocytes. These structures were similar to, but distinct from, those of known paramyxoviridae, suggesting infection with a novel, related virus. In situ hybridization studies with a probe directed against the measles fusion protein gene gave a positive signal with a hepatocyte distribution. No signal was obtained with the measles nucleocapsid protein probe, suggesting that the disease agent was genetically distinct from, but related to, the measles virus. Subsequent liver biopsies were characterized by the gradual disappearance of the giant cell changes and by the concomitant development of cirrhosis. This is a case of adult GCH that resolved spontaneously and led to cirrhosis, thus implicating GCH as a potential cause of "cryptogenic" liver disease. Our findings provide further support for the existence of a distinct, as yet unidentified viral species as a cause of this disease.

Detection of retroviral antibodies in primary biliary cirrhosis and other idiopathic biliary disorders.

BACKGROUND: Retroviruses have been implicated in the aetiology of various autoimmune diseases. We used immunoblots as a surrogate test to find out whether retroviruses play a part in the development of primary biliary cirrhosis. METHODS: We did western blot tests for HIV-1 and the human intracisternal A-type particle (HIAP), on serum samples from 77 patients with primary biliary cirrhosis, 126 patients with chronic liver disease, 48 patients with systemic lupus erythematosus, and 25 healthy volunteers. FINDINGS: HIV-1 p24 gag seroreactivity was found in 27 (35%) of 77 patients with primary biliary cirrhosis, 14 (29%) of 48 patients with systemic lupus erythematosus, 14 (50%) of 28 patients with chronic viral hepatitis, and nine (39%) of 23 patients with either primary sclerosing cholangitis or biliary atresia, compared with only one (4%) of 24 patients with alcohol-related liver disease or alpha1-antitrypsin-deficiency liver disease, and only one (4%) of 25 healthy volunteers (p=0.003). Western blot reactivity to more than two HIAP proteins was found in 37 (51%) of patients with primary biliary cirrhosis, in 28 (58%) of patients with systemic lupus erythematosus, in 15 (20%) of patients with chronic viral hepatitis, and in four (17%) of those with other biliary diseases. None of the 23 patients with either alcohol-related liver disease or alpha1-antitrypsin deficiency, and only one of the healthy controls showed the same reactivity to HIAP proteins (p<0.0001). Our results showed a strong association between HIAP seroreactivity and the detection of autoantibodies to double-stranded DNA. HIAP seroreactivity was also strongly associated with the detection of mitochondrial, nuclear, and extractable nuclear antigens. INTERPRETATION: The HIV-1 and HIAP antibody reactivity found in patients with primary biliary cirrhosis and other biliary disorders may be attributable either to an autoimmune response to antigenically related cellular proteins or to an immune response to uncharacterised viral proteins that share antigenic determinants with these retroviruses.

Molecular basis for persistent hepatitis B virus infection in the liver after clearance of serum hepatitis B surface antigen.

Hepatitis B virus (HBV) DNA has been detected by polymerase chain reaction (PCR) in the liver of patients with resolved chronic HBV infection and sustained clearance of hepatitis B surface antigen (HBsAg) from serum. However, it is unknown whether the virus is transcriptionally active at this time or if the covalently closed circular (CCC) replicative intermediate of HBV DNA can still be detected. Therefore, hepatic nucleic acid extracts from seven patients who had cleared serum HBsAg were assessed by (PCR) for either reverse-transcribed HBV RNA, or an intact direct repeat region of the HBV genome indicative of the CCC replicative intermediate of HBV DNA. HBV transcripts were detected in four of seven patients in the study group, whereas an intact direct repeat region of the HBV genome was detected in three. Evidence for viral transcription and replication was more frequently detected in patients who had recently cleared serum HBsAg, but HBV RNA was also detected in one patient 5 years after HBsAg clearance, and an intact direct repeat region of HBV DNA was detected in another subject at nearly 4 years after resolution of disease. Therefore, hepatic HBV transcription may be associated with replicative intermediates of persistent HBV DNA in patients who have cleared HBsAg from serum, suggesting that, on occasion, HBV may not be in a latent state but undergoing low-level replication.