Patients with high levels of circulating malignant B cells were identified, one with chronic lymphocytic leukemia (CLL) and one with a marginal PCI-32765 cell line zone B-cell lymphoma (MZL). Blood was collected from these patients with informed consent and under local ethics committee approval. Peripheral blood lymphocytes were isolated, as previously described,13 by density-gradient centrifugation over Lympholyte (VH Bio) for 25 minutes at 800×g. Harvested lymphocytes

were washed in PBS and resuspended in RPMI 1640 with 10% FCS. T cells were depleted using anti-CD3 Abs (OKT3; Janssen Cilag, High Wycombe, UK) and antimouse immunoglobulin G (IgG)-coated beads (Invitrogen). Flow cytometry demonstrated that >90% of the isolated peripheral lymphocyte population in these patients was positive for the B-cell marker, CD19. Cell lines and peripheral KU-60019 ic50 blood mononuclear cells were washed, resuspended, and labeled with different fluorochrome-labeled primary Abs against chemokine receptors at optimal dilutions at 4°C, followed by a washing step with PBS and 5% FCS. Samples were analyzed on a Dako Cyan Flow cytometer using Summit 4.3 Software

(DakoCytomation, Glostrup, Denmark). The following Abs were used for fluorescence-activated cell-sorting (FACS) analysis of chemokine receptors and B-cell subsets: CCR6 (CTC5/FAB 1802P); CCR7 (150503/FAB197A); CXCR3 (49801/FAB160A); CXCR4 (12G5/FAB170P); and CXCR5 (51505/FAB190P) and were purchased from R&D Systems (Abingdon, selleck inhibitor UK). CD19 (MOPC-21/555413) was purchased from BD Pharmingen (Swindon, UK), and CD27 (O323/302822) was purchased from BioLegend (Cambridge, UK). The following Abs were used for integrin expression: alpha L/CD11a (clone 345913); beta 2/CD18 (clone 212701); beta 1/CD29 (clone P5D2); and alpha 4/CD49d (clone 265329) and were all purchased from R&D Systems. B-cell interaction with human HSECs was studied in flow-based adhesion assays using confluent monolayers of HSECs grown in chamber slides (Ibidi, Munich, Germany) and stimulated with tumor necrosis factor alpha (TNF-α) and interferon-gamma

(IFN-γ) for 24 hours at 10 ng/mL. We have previously demonstrated that cytokine treatment of human HSECs with TNF-α and IFN-γ led to increased cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) and CLEVER-1, whereas VAP-1 expression was unaffected by these cytokines.3, 4, 13 In some experiments, the endothelial monolayers were incubated with CXCL12 (300 ng/mL; Peprotech EC Ltd., London, UK) 2 hours before assays. Chamber slides were connected to a flow system, as previously described.4 Purified populations of B cells (1 × 106 cells/mL), lymphoma cell lines Karpas 422 and CRL-2261 (0.5 × 106 cells/mL), or primary malignant B cells (1 × 106 cells/mL) were perfused in flow media (endothelial-basal media supplemented with 0.01% human serum; Invitrogen) through the chamber slides over the ECs at a shear stress of 0.05 Pa, which mimics physiological flow in the sinusoids.

Patients with high levels of circulating malignant B cells were identified, one with chronic lymphocytic leukemia (CLL) and one with a marginal selleck chemical zone B-cell lymphoma (MZL). Blood was collected from these patients with informed consent and under local ethics committee approval. Peripheral blood lymphocytes were isolated, as previously described,13 by density-gradient centrifugation over Lympholyte (VH Bio) for 25 minutes at 800×g. Harvested lymphocytes

were washed in PBS and resuspended in RPMI 1640 with 10% FCS. T cells were depleted using anti-CD3 Abs (OKT3; Janssen Cilag, High Wycombe, UK) and antimouse immunoglobulin G (IgG)-coated beads (Invitrogen). Flow cytometry demonstrated that >90% of the isolated peripheral lymphocyte population in these patients was positive for the B-cell marker, CD19. Cell lines and peripheral STI571 solubility dmso blood mononuclear cells were washed, resuspended, and labeled with different fluorochrome-labeled primary Abs against chemokine receptors at optimal dilutions at 4°C, followed by a washing step with PBS and 5% FCS. Samples were analyzed on a Dako Cyan Flow cytometer using Summit 4.3 Software

(DakoCytomation, Glostrup, Denmark). The following Abs were used for fluorescence-activated cell-sorting (FACS) analysis of chemokine receptors and B-cell subsets: CCR6 (CTC5/FAB 1802P); CCR7 (150503/FAB197A); CXCR3 (49801/FAB160A); CXCR4 (12G5/FAB170P); and CXCR5 (51505/FAB190P) and were purchased from R&D Systems (Abingdon, check details UK). CD19 (MOPC-21/555413) was purchased from BD Pharmingen (Swindon, UK), and CD27 (O323/302822) was purchased from BioLegend (Cambridge, UK). The following Abs were used for integrin expression: alpha L/CD11a (clone 345913); beta 2/CD18 (clone 212701); beta 1/CD29 (clone P5D2); and alpha 4/CD49d (clone 265329) and were all purchased from R&D Systems. B-cell interaction with human HSECs was studied in flow-based adhesion assays using confluent monolayers of HSECs grown in chamber slides (Ibidi, Munich, Germany) and stimulated with tumor necrosis factor alpha (TNF-α) and interferon-gamma

(IFN-γ) for 24 hours at 10 ng/mL. We have previously demonstrated that cytokine treatment of human HSECs with TNF-α and IFN-γ led to increased cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) and CLEVER-1, whereas VAP-1 expression was unaffected by these cytokines.3, 4, 13 In some experiments, the endothelial monolayers were incubated with CXCL12 (300 ng/mL; Peprotech EC Ltd., London, UK) 2 hours before assays. Chamber slides were connected to a flow system, as previously described.4 Purified populations of B cells (1 × 106 cells/mL), lymphoma cell lines Karpas 422 and CRL-2261 (0.5 × 106 cells/mL), or primary malignant B cells (1 × 106 cells/mL) were perfused in flow media (endothelial-basal media supplemented with 0.01% human serum; Invitrogen) through the chamber slides over the ECs at a shear stress of 0.05 Pa, which mimics physiological flow in the sinusoids.

Transgenic mice in which the urokinase VX-809 order gene is driven by the human albumin promoter/enhancer were developed and shown to have accelerated hepatocyte death and consequent chronic stimulation of hepatocyte

growth.11 Transplanted rat hepatocytes proliferated and repopulated injured livers in immunodeficient uPA mice, which were produced by mating uPA transgenic mice with scid mice.12 Human hepatocytes were then transplanted into uPA/scid mice; these cells proliferated and replaced the apoptotic mice liver cells (Fig. 1). Such human hepatocyte chimeric mice have been shown to be susceptible to both HBV16 and HCV17 infections. Repopulation levels by human hepatocytes have been estimated by measuring human albumin levels in mouse serum. Replication levels of both HBV13 and HCV17 were higher in mice in which the repopulation index was higher. A unique attempt to remove mouse residual liver cells with the herpes simplex virus type-1 thymidine kinase (HSVtk)/ganciclovir selleck compound (GCV) system failed to result in a higher repopulation rate as a result of damage to the transplanted human hepatocyte caused by bystander effects.18 Despite this, mice with livers that have been highly repopulated with human hepatocytes are susceptible to infection with both HBV and HCV, and as

such comprised the most effective small animal model for chronic hepatitis so far developed.19,20 An example of a highly repopulated mouse liver that we are using in experiments is shown in Figure 2. Highly repopulated mice have been shown to be a valuable model for the study of drug metabolism.21–29 Advances in technology for human hepatocyte transplantation have enabled serial passage of human hepatocytes in uPA/scid selleck kinase inhibitor mice and have been shown to retain infectivity for HBV.30 This mouse model

and other animal models for the study of hepatitis viruses have been summarized in reviews by Meuleman and Leroux-Roels,31 Dandri et al.,32,33 Barth et al.,34 and Kneteman and Toso.35 The present review will focus on key issues and updated information. Since the initial reports of successful transmission of HBV to human hepatocyte chimeric mice in 2001 and 2004,16,27 several researchers have reported transmission of HBV into similar mice.13,36,37 In these studies, passage experiments studies show that HBV replicating in mice retain infectivity.13,36 Further, the presence of viral proteins has been shown immunohistochemically in human hepatocytes transplanted into mouse livers, but these are not present in mouse hepatocytes.13,36,37 Formation of viral particles in infected mouse livers can be shown by electron microscopy.36,37 Genetically engineered viruses lacking HBe-antigen have also been shown to infect chimeric mice, proving that e antigen is dispensable for viral infection and replication.

Transgenic mice in which the urokinase Selleck Maraviroc gene is driven by the human albumin promoter/enhancer were developed and shown to have accelerated hepatocyte death and consequent chronic stimulation of hepatocyte

growth.11 Transplanted rat hepatocytes proliferated and repopulated injured livers in immunodeficient uPA mice, which were produced by mating uPA transgenic mice with scid mice.12 Human hepatocytes were then transplanted into uPA/scid mice; these cells proliferated and replaced the apoptotic mice liver cells (Fig. 1). Such human hepatocyte chimeric mice have been shown to be susceptible to both HBV16 and HCV17 infections. Repopulation levels by human hepatocytes have been estimated by measuring human albumin levels in mouse serum. Replication levels of both HBV13 and HCV17 were higher in mice in which the repopulation index was higher. A unique attempt to remove mouse residual liver cells with the herpes simplex virus type-1 thymidine kinase (HSVtk)/ganciclovir Everolimus manufacturer (GCV) system failed to result in a higher repopulation rate as a result of damage to the transplanted human hepatocyte caused by bystander effects.18 Despite this, mice with livers that have been highly repopulated with human hepatocytes are susceptible to infection with both HBV and HCV, and as

such comprised the most effective small animal model for chronic hepatitis so far developed.19,20 An example of a highly repopulated mouse liver that we are using in experiments is shown in Figure 2. Highly repopulated mice have been shown to be a valuable model for the study of drug metabolism.21–29 Advances in technology for human hepatocyte transplantation have enabled serial passage of human hepatocytes in uPA/scid this website mice and have been shown to retain infectivity for HBV.30 This mouse model

and other animal models for the study of hepatitis viruses have been summarized in reviews by Meuleman and Leroux-Roels,31 Dandri et al.,32,33 Barth et al.,34 and Kneteman and Toso.35 The present review will focus on key issues and updated information. Since the initial reports of successful transmission of HBV to human hepatocyte chimeric mice in 2001 and 2004,16,27 several researchers have reported transmission of HBV into similar mice.13,36,37 In these studies, passage experiments studies show that HBV replicating in mice retain infectivity.13,36 Further, the presence of viral proteins has been shown immunohistochemically in human hepatocytes transplanted into mouse livers, but these are not present in mouse hepatocytes.13,36,37 Formation of viral particles in infected mouse livers can be shown by electron microscopy.36,37 Genetically engineered viruses lacking HBe-antigen have also been shown to infect chimeric mice, proving that e antigen is dispensable for viral infection and replication.

5 hepatoma cells and primary hepatocytes by cell-culture-derived HCV (HCVcc). Using an Huh7.5 coculture system we demonstrated that mAb16-71 interferes with direct cell-to-cell transmission of HCV. Finally we evaluated the in vivo efficacy of mAb16-71 in “human liver urokinase-type plasminogen activator, severe combined immune deficiency (uPA-SCID) mice” (chimeric mice). A 2-week anti-SR-BI therapy that was initiated 1 day before viral inoculation completely protected all Selleckchem BTK inhibitor chimeric mice from infection with serum-derived HCV of different genotypes. Moreover, a 9-day postexposure therapy that was initiated 3 days after viral inoculation (when viremia was already observed in the animals)

suppressed the rapid viral spread

observed in untreated control animals. After cessation of anti-SR-BI-specific antibody therapy, a rise of the viral load was observed. Conclusion: Using in vitro cell culture and human liver-chimeric mouse models, we show that a human mAb targeting the HCV coreceptor SR-BI completely prevents infection and intrahepatic spread of multiple HCV genotypes. This strategy may be selleck screening library an efficacious way to prevent infection of allografts following liver transplantation in chronic HCV patients, and may even hold promise for the prevention of virus rebound during or following antiviral therapy. (HEPATOLOGY 2012) With approximately 3% of the world’s population infected with the hepatitis C virus (HCV), endstage liver disease caused by this infection is currently the most common indication for liver transplantation.1 However, the donor liver almost inevitably

becomes infected by circulating virus and disease progression is accelerated in immune-suppressed transplant patients.2 Less than 30% of liver transplant patients treated with pegylated interferon therapy with or without ribavirin will achieve a sustained virological response and this combination therapy is often not well tolerated.3-5 selleck chemicals Therefore, new strategies to prevent graft reinfection are urgently needed. In the coming years, new direct antiviral compounds will considerably improve therapy outcome in patients without severe liver disease,6-8 but the side effects and potential drug-drug interactions associated with triple therapy may severely complicate their use in liver transplant patients with endstage liver disease.9-12 Because of the extreme genetic diversity of HCV and its ability to spread by way of cell-cell contacts, successful immunotherapy with polyclonal or monoclonal HCV-specific antibodies may be difficult to achieve.13-17 In contrast, viral (co-)receptors are genetically conserved and may represent better therapeutic targets. HCV entry is a multistep process in which different putative attachment factors and viral receptors are involved (reviewed18-20).

(2002) found a

similar range in Δ15N values. In contrast, Kurle (2002) found that Δ15N values for various Sorafenib manufacturer blood components in captive northern fur seals (Callorhinus ursinus) ranged from 4.1‰ to 5.2‰. Focusing on blood serum, Zhao et al. (2006) also found relatively large Δ15Nserum-diet values for captive harbor seals (Phoca vitulina), ranging from 3.9‰ to 4.6‰. Recently, Newsome et al. (in review) found a mean Δ15Nvibrissae-diet value of 3.5‰ for a wild population of California sea otters (Enhydra lutris nereis). Whereas the nitrogen in an animal’s diet is mainly sourced from the proteins it consumes, the carbon for an animal’s tissues is supplied by dietary proteins, lipids, and carbohydrates, which may differ in their carbon isotope composition. In addition, carbon occurs

in tissues composed of materials other than protein, such as bioapatite and lipids, which have a greater isotopic range than that observed for nitrogen from protein-rich tissues. In terrestrial mammals, the δ13C value of bioapatite reflects that of bulk diet, whereas that of proteins and lipids is often biased toward the protein or lipid portion selleck products of the diet, respectively, as a result of dietary routing of these components. For most lipids, there is usually a balance between routing of dietary lipids to tissue and de novo synthesis of new lipids; bone cholesterol is the one lipid that strongly reflects bulk diet (Jim et al. 2003). For proteins, there is a similar balance between routing of amino acids—particularly indispensible amino acids that cannot be produced through de novo synthesis—and production of the R-groups of dispensable amino acids from bulk diet or carbohydrate and lipid carbon (Howland et al. 2003, Jim et al. 2006). For pinnipeds, cetaceans and otters, which consume protein-rich diets with variable amounts of fat, the δ13C value of body protein should closely track that of bulk diet, but perhaps with different tissue-to-diet fractionations depending on dietary lipid content. Herbivorous sirenians would receive bulk dietary carbon from carbohydrates along with a smaller

quantity of proteins from plants or protein-rich epizooans, which should, in turn, reflect plant-derived carbon. Measured tissue-to-diet isotope discriminations for bioapatite, lipids and proteins are significantly different. For bioapatite, tissue-to-diet isotope fractions in terrestrial mammals differ between carnivores selleck inhibitor (+9‰) and herbivores (+12‰–+14‰) (reviewed in Koch 2007). The Δ13Capatite-diet value has been measured in manatees (Trichechus manatus latirostrius) on controlled diets and is +14‰ (MacFadden et al. 2004). While Δ13Capatite-diet values have not been determined experimentally for other marine mammals, field studies suggest they are similar to values for land carnivores (Clementz and Koch 2001, Clementz et al. 2007). In contrast, bulk consumer lipid is 13C-depleted by 2‰–5‰ relative to bulk diet (DeNiro and Epstein 1978, Tieszen et al. 1983, Howland et al.

(2002) found a

similar range in Δ15N values. In contrast, Kurle (2002) found that Δ15N values for various Osimertinib datasheet blood components in captive northern fur seals (Callorhinus ursinus) ranged from 4.1‰ to 5.2‰. Focusing on blood serum, Zhao et al. (2006) also found relatively large Δ15Nserum-diet values for captive harbor seals (Phoca vitulina), ranging from 3.9‰ to 4.6‰. Recently, Newsome et al. (in review) found a mean Δ15Nvibrissae-diet value of 3.5‰ for a wild population of California sea otters (Enhydra lutris nereis). Whereas the nitrogen in an animal’s diet is mainly sourced from the proteins it consumes, the carbon for an animal’s tissues is supplied by dietary proteins, lipids, and carbohydrates, which may differ in their carbon isotope composition. In addition, carbon occurs

in tissues composed of materials other than protein, such as bioapatite and lipids, which have a greater isotopic range than that observed for nitrogen from protein-rich tissues. In terrestrial mammals, the δ13C value of bioapatite reflects that of bulk diet, whereas that of proteins and lipids is often biased toward the protein or lipid portion Midostaurin supplier of the diet, respectively, as a result of dietary routing of these components. For most lipids, there is usually a balance between routing of dietary lipids to tissue and de novo synthesis of new lipids; bone cholesterol is the one lipid that strongly reflects bulk diet (Jim et al. 2003). For proteins, there is a similar balance between routing of amino acids—particularly indispensible amino acids that cannot be produced through de novo synthesis—and production of the R-groups of dispensable amino acids from bulk diet or carbohydrate and lipid carbon (Howland et al. 2003, Jim et al. 2006). For pinnipeds, cetaceans and otters, which consume protein-rich diets with variable amounts of fat, the δ13C value of body protein should closely track that of bulk diet, but perhaps with different tissue-to-diet fractionations depending on dietary lipid content. Herbivorous sirenians would receive bulk dietary carbon from carbohydrates along with a smaller

quantity of proteins from plants or protein-rich epizooans, which should, in turn, reflect plant-derived carbon. Measured tissue-to-diet isotope discriminations for bioapatite, lipids and proteins are significantly different. For bioapatite, tissue-to-diet isotope fractions in terrestrial mammals differ between carnivores selleck screening library (+9‰) and herbivores (+12‰–+14‰) (reviewed in Koch 2007). The Δ13Capatite-diet value has been measured in manatees (Trichechus manatus latirostrius) on controlled diets and is +14‰ (MacFadden et al. 2004). While Δ13Capatite-diet values have not been determined experimentally for other marine mammals, field studies suggest they are similar to values for land carnivores (Clementz and Koch 2001, Clementz et al. 2007). In contrast, bulk consumer lipid is 13C-depleted by 2‰–5‰ relative to bulk diet (DeNiro and Epstein 1978, Tieszen et al. 1983, Howland et al.

The classifications and recommendations are based on three categories: the source

of evidence in levels I through III; the quality of evidence designated by high (A), moderate (B), or low quality (C); and the strength of recommendations classified as strong (1) or weak (2). The literature databases and search strategies are outlined below. The resulting literature database was available to all members of the writing group (i.e., the authors). They selected references within their field of expertise and experience and graded the references according to the GRADE system.[1] The selection of references for the guideline was based on a validation of the appropriateness of the study design for the stated purpose, a relevant number of patients under study, and confidence in the participating centers and authors. References on original data were preferred and those that were found unsatisfactory selleck kinase inhibitor in any of these respects were excluded from further evaluation. There may be limitations in this approach when recommendations

are needed on rare problems or problems on which scant original data are available. In such cases, it may be necessary to rely on less-qualified references with a low grading. As a result of the important changes in the treatment of complications of cirrhosis (renal failure, infections, and variceal bleeding [VB]), studies performed more than 30 years ago have generally not been considered for these guidelines. Hepatic encephalopathy (HE) is a frequent complication and one of the most debilitating MK-8669 clinical trial manifestations of liver disease, severely affecting the lives of patients and their caregivers. Furthermore, selleckchem cognitive impairment associated with cirrhosis results in utilization of more health care resources

in adults than other manifestations of liver disease.[2] Progress in the area has been hindered by the complex pathogenesis that is not yet fully elucidated. Apart from such biological factors, there remains the larger obstacle that there are no universally accepted standards for the definition, diagnosis, classification, or treatment of HE, mostly as a result of insufficient clinical studies and standardized definitions. Clinical management tends to be dependent on local standards and personal views. This is an unfavorable situation for patients and contrasts with the severity of the condition and the high level of standardization in other complications of cirrhosis. The lack of consistency in the nomenclature and general standards renders comparisons among studies and patient populations difficult, introduces bias, and hinders progress in clinical research for HE. The latest attempts to standardize the nomenclature were published in 2002 and suggestions for the design of HE trials in 2011.

” Systematic “protocol” evaluations at each visit, liver tissue examinations at 6 month intervals, uniform treatment schedules, predefined responses to disease behavior, regular surveillance schedules using mailed serum specimens,

this website serum and liver tissue banks, and commitment to indefinite patient follow-up were the manifestations of this “tenacity.” I remain convinced that rigid adherence to protocol and compulsive follow-up are essential components of successful clinical investigation (Table 1). Bill Summerskill died suddenly in March 1977, and I was abruptly launched solo into the realm of CALD. Life-saving therapy had now been established by three controlled clinical trials; the disease was rare; funding sources were limited or uninterested,

and my principal initial concern was that there was nothing more to study.21,39,40 The practical clinical problems that required answers became obvious quickly through routine patient care, and they generated a compelling urgency for further clinical studies. Remarkable work from Meyer zum Buschenfelde’s group in Mainz, Germany,41-44 and Roger Williams’ group in London, England,45-49 invigorated the concept of autoimmune hepatitis, and I suddenly realized anew that I was in an exciting place at an exciting time. The first objective was to describe the clinical phenotype of autoimmune hepatitis and to distinguish it from other diseases. This was done by describing its autoantibodies,50 selleck products histological manifestations,51,52 clinical presentations,53-55 and response to corticosteroid treatment.56-58 The Selleck Dabrafenib disease had to be distinguished from systemic lupus

erythematosus59 and chronic viral hepatitis60; it had to be released from the early restrictive requirement for 6 months of disease activity61; subtypes based on mutually exclusive serological markers had to be explored62-64; and it had to accommodate patients with nonclassical manifestations.65 This was the era of serological exploration, and collaborations with Mikio Nishioka,66-70 Francesco Bianchi,71,72 Michael Manns62-64 and their coworkers were essential to understand the nature and clinical significance of antinuclear reactivities, including antibodies to ribonucleoproteins, histones, single-stranded DNA and doubled-stranded DNA, antibodies to actin, antibodies to liver kidney microsome type 1 (anti-LKM1), antibodies to soluble liver antigen (anti-SLA), and antimitochondrial antibodies in autoimmune hepatitis. These efforts complemented studies performed elsewhere, and they supported concepts of an acute autoimmune hepatitis,54 two serologically distinct forms of the disease,73 variant syndromes characterized by antimitochondrial antibodies,74 bile duct changes,75-79 or concurrent viral infection,80-82 and a seronegative state frequently misclassified as cryptogenic chronic hepatitis.

” Systematic “protocol” evaluations at each visit, liver tissue examinations at 6 month intervals, uniform treatment schedules, predefined responses to disease behavior, regular surveillance schedules using mailed serum specimens,

BAY 57-1293 clinical trial serum and liver tissue banks, and commitment to indefinite patient follow-up were the manifestations of this “tenacity.” I remain convinced that rigid adherence to protocol and compulsive follow-up are essential components of successful clinical investigation (Table 1). Bill Summerskill died suddenly in March 1977, and I was abruptly launched solo into the realm of CALD. Life-saving therapy had now been established by three controlled clinical trials; the disease was rare; funding sources were limited or uninterested,

and my principal initial concern was that there was nothing more to study.21,39,40 The practical clinical problems that required answers became obvious quickly through routine patient care, and they generated a compelling urgency for further clinical studies. Remarkable work from Meyer zum Buschenfelde’s group in Mainz, Germany,41-44 and Roger Williams’ group in London, England,45-49 invigorated the concept of autoimmune hepatitis, and I suddenly realized anew that I was in an exciting place at an exciting time. The first objective was to describe the clinical phenotype of autoimmune hepatitis and to distinguish it from other diseases. This was done by describing its autoantibodies,50 find more histological manifestations,51,52 clinical presentations,53-55 and response to corticosteroid treatment.56-58 The CH5424802 order disease had to be distinguished from systemic lupus

erythematosus59 and chronic viral hepatitis60; it had to be released from the early restrictive requirement for 6 months of disease activity61; subtypes based on mutually exclusive serological markers had to be explored62-64; and it had to accommodate patients with nonclassical manifestations.65 This was the era of serological exploration, and collaborations with Mikio Nishioka,66-70 Francesco Bianchi,71,72 Michael Manns62-64 and their coworkers were essential to understand the nature and clinical significance of antinuclear reactivities, including antibodies to ribonucleoproteins, histones, single-stranded DNA and doubled-stranded DNA, antibodies to actin, antibodies to liver kidney microsome type 1 (anti-LKM1), antibodies to soluble liver antigen (anti-SLA), and antimitochondrial antibodies in autoimmune hepatitis. These efforts complemented studies performed elsewhere, and they supported concepts of an acute autoimmune hepatitis,54 two serologically distinct forms of the disease,73 variant syndromes characterized by antimitochondrial antibodies,74 bile duct changes,75-79 or concurrent viral infection,80-82 and a seronegative state frequently misclassified as cryptogenic chronic hepatitis.