Clinical associations and potential novel antigenic targets of autoantibodies directed against rods and rings in chronic hepatitis C infection.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is frequently associated with extrahepatic autoimmune disorders while interferon (IFN) and ribavirin treatment may exacerbate these conditions. Autoantibodies from HCV patients identify a novel indirect immunofluorescence (IIF) pattern on HEp-2 cells characterized by cytoplasmic rods and rings (RR). Our objectives were to determine the prevalence and clinical associations of RR autoantibodies in HCV patients, and identify related novel autoantibody targets. METHODS: Sera from 315 patients with HCV (301 treatment naive, 14 treated with interferon and/or ribavirin) were analyzed for the presence of RR antibodies by IIF on commercially available HEp-2 cell substrates. Antibodies to inosine monophosphate dehydrogenase 2 (IMPDH2) and cytidine triphosphate synthase 1 (CTPS1) were detected by addressable laser bead assay and other potential targets were identified by immunoscreening a protein microarray. Clinical and demographic data including HCV genotype, mode of infection, prior antiviral therapy, and histological findings were compared between RR antibody positive (RR+) and negative (RR-) patients. RESULTS: The median age of the HCV cohort was 51 years, 61% were male, and 76% were infected with HCV genotype 1 (G1). Four percent (n=14) had been treated with IFN-based therapy (IFN monotherapy, n=3; IFN/ribavirin, n=11); all had a sustained virologic response. In total, 15 patients (5% of the cohort) were RR+. RR+ and RR- patients had similar demographic and clinical characteristics including age, sex, mode of HCV infection, prevalence of the G1 HCV genotype, and moderate to severe fibrosis. Nevertheless, RR+ patients were significantly more likely than RR- cases to have been treated with IFN-based therapy (33% vs. 3%; adjusted odds ratio 20.5 [95% confidence interval 5.1-83.2]; P<0.0005). Only 1/10 RR positive sera had detectable antibodies to IMPHD2 and none had antibodies to CTPS1. Potentially important autoantibody targets identified on protein arrays included Myc-associated zinc finger protein (MAZI) and ankyrin repeat motif. CONCLUSION: The majority of HCV patients with RR autoantibodies previously received IFN/ribavirin antiviral therapy. Further studies are necessary to determine the genesis of intracellular RR and elucidate the clinically relevant autoantigens as well as the clinical and prognostic significance of their cognate autoantibodies.

Baseline Cannabinoid Use Is Associated with Increased Sedation Requirements for Outpatient Endoscopy.

Background and Aims: Given the underlying properties of cannabinoids, we aimed to assess associations between cannabinoid use and sedation requirements for esophagogastroduodenoscopy (EGD) and colonoscopy. Methods: A prospective cohort study was conducted at three endoscopy units. Adult outpatients undergoing EGD or colonoscopy with endoscopist-directed conscious sedation (EDCS) were given questionnaires on cannabinoid use and relevant parameters. Outcomes included intraprocedural midazolam, fentanyl, and diphenhydramine use, procedural tolerability, and adverse events. Multivariable logistic regression was performed to yield adjusted odds ratios (AORs) of outcomes. Results: A total of 419 patients were included. Baseline cannabinoid use was associated with high midazolam use, defined as ≥5 mg, during EGD (AOR 2.89, 95% confidence interval, CI: 1.19-7.50), but not during colonoscopy (AOR 0.89, 95% CI 0.41-1.91). Baseline cannabinoid use was associated with the administration of any diphenhydramine during EGD (AOR 3.04, 95% CI: 1.29-7.30) with a similar nonsignificant trend for colonoscopy (AOR 2.36, 95% CI: 0.81-7.04). Baseline cannabinoid use was associated with increased odds of requiring high total sedation, defined as any of midazolam ≥5 mg, fentanyl ≥100 mcg, or any diphenhydramine during EGD (AOR 3.72, 95% CI: 1.35-11.68). Cannabinoid use was not independently associated with fentanyl use, intraprocedural awareness, discomfort, or adverse events. Conclusions: Baseline cannabinoid use was associated with higher sedation use during endoscopy with EDCS, particularly with midazolam and diphenhydramine. Given increasingly widespread cannabinoid use, endoscopists should be equipped with optimal sedation strategies for this population. As part of the informed consent process, cannabis users should be counseled that they may require higher sedation doses to achieve the same effect.

Real-world tertiary referral centre experience stopping nucleos(t)ide analogue therapy in patients with chronic hepatitis B.

BACKGROUND: Identifying strategies for stopping nucleos(t)ide analogues (NUC) in patients with chronic hepatitis B (CHB) is a major goal in CHB management. Our study describes our tertiary-centre experience stopping nucleos(t)ide analogues (NUC) in CHB. METHODS: We conducted a retrospective cohort study of all individuals with CHB seen at the Calgary Liver Unit between January 2009 and May 2020 who stopped NUC. We collected baseline demographics and HBV lab parameters before and after stopping NUC with results stratified by off-treatment durability. Clinical flare was defined as alanine aminotransferase (ALT) over twice the upper limit of normal and virological flare as HBV DNA >2000 IU/mL. RESULTS: Forty-seven (3.5%) of the 1337 individuals with CHB stopped NUC therapy. During follow-up, six patients (12.8%) restarted NUCs because of a flare. All flares occurred within six months of discontinuation. Median time to restart treatment was 90 days (Q1 65, Q3 133). Upon restarting, all showed suppression of HBV DNA and ALT normalization. Factors associated with restarting NUC therapy included hepatitis B e antigen (HBeAg) positive status at first appointment and longer NUC consolidation therapy. Age, sex, ethnicity, liver stiffness measurement, choice of NUC, and quantitative hepatitis B surface antigen (qHBsAg) level at stopping were not associated with sustained response off-treatment. Six patients had functional cure with HBsAg loss. CONCLUSIONS: Stopping long-term NUC is feasible in HBeAg negative CHB. Hepatic flares can occur despite low levels of qHBsAg. Finite NUC therapy can be considered in eligible patients who are adherent to close monitoring and follow-up, particularly in the first six months after stopping NUC therapy.

Real-world clinical and virological outcomes in a retrospective multiethnic cohort study of 341 untreated and tenofovir disoproxil fumarate-treated chronic hepatitis B pregnant patients in North America.

BACKGROUND: There are limited long-term data on outcomes of chronic hepatitis B (CHB) in untreated and tenofovir disoproxil fumarate (TDF)-treated women during pregnancy. AIMS: To assess clinical outcomes in a multiethnic cohort of patients during pregnancy and post-partum in a low HBV endemic region. METHODS: Retrospective real-world study of women with CHB (treated or untreated with TDF) from 2011 to 2019; data including ALT, HBV DNA, HBeAg and liver stiffness measurement were collected during pregnancy and post-partum. RESULTS: In 341 women (446 pregnancies) followed for a median of 33 months (IQR: 26.7-39.5) post-partum, 19% (65/341) received TDF (11 initiated pre-pregnancy, 53 for mother-to-child transmission (MTCT) prevention). During follow-up, 72/341 had subsequent pregnancy, including 18/53 on TDF for MTCT risk, of whom 7/18 were re-treated. In all TDF-treated women, HBV DNA declined but rebounded after TDF withdrawal (median baseline, near birth and early follow-up levels were 7.2, 3.0 and 5.5 log IU/mL respectively [P < 0.01]). In HBeAg+ patients (65/341) ALT flares were more common (P = 0.03), especially for those who stopped TDF post-partum, requiring re-treatment in 21% (11/53). In comparison, 54% (116/215) of untreated women had a post-partum ALT flare; one with fulminant hepatitis underwent transplant 13 months post-partum. HBsAg clearance occurred in 2.6% (9/341, 3/9 HBeAg+, 2/9 TDF treated) at median 30 months (IQR: 23-40) and 37% (24/65) of HBeAg+ patients had HBeAg loss at median 17 months (IQR: 12-26) post-partum. CONCLUSIONS: Post-partum ALT flares were common, especially after TDF withdrawal. Overall, 37% achieved HBeAg clearance and 2.9% had HBsAg loss during long-term follow-up.

The Prevalence of Anti-Hexokinase-1 and Anti-Kelch-Like 12 Peptide Antibodies in Patients With Primary Biliary Cholangitis Is Similar in Europe and North America: A Large International, Multi-Center Study.

Primary biliary cholangitis (PBC), formerly known as primary biliary cirrhosis, is present worldwide. Autoantibodies, in particular anti-mitochondrial antibodies (AMA) detected by indirect immunofluorescence assays or newer solid phase immunoassays can detect most, but not all individuals with PBC. Detection of antibodies to the anti-nuclear antigens sp100 and gp210 can identify additional PBC patients, but some seronegative patients remain, often resulting in delayed diagnosis and treatment. Antibodies to kelch-like 12 (KLHL12) and hexokinase 1 (HK-1) were recently identified as new biomarkers for PBC and notably identify patients who are negative for conventional autoantibodies. To become globally adopted, it is important to validate these new biomarkers in different geographic areas. In the present study we evaluated the prevalence of anti-KLHL12 (measured by a KLHL12-derived peptide referred to as KL-p) and anti-HK-1 antibodies by ELISA at five sites within Europe and North America and demonstrated the presence of these antibodies in patients with PBC in all geographies.

A clinical approach to autoantibody testing in systemic autoimmune rheumatic disorders.

Musculoskeletal (MSK) disorders constitute one of the most common clinical presentations to clinical care givers. Within this category of illnesses, systemic autoimmune rheumatic diseases (SARD) such as systemic lupus erythematosus (SLE), systemic sclerosis (SSc), Sjögren's syndrome (SjS) and rheumatoid arthritis (RA) are included in the differential diagnosis. A hallmark of SARD is the production of autoantibodies, which are routinely requested as a guide to diagnosis and clinical decision making. The field of serological tests, including the detection of autoantibodies, is complex and often leads to confusion and misunderstanding. When used appropriately, autoantibodies can be a valuable adjunct to the diagnosis, and occasionally therapy and prognosis, of SARD. The role of autoantibody testing and a 'practical' approach to using these tests is the focus of this paper.

Spectrum of centrosome autoantibodies in childhood varicella and post-varicella acute cerebellar ataxia.

BACKGROUND: Sera from children with post-varicella infections have autoantibodies that react with centrosomes in brain and tissue culture cells. We investigated the sera of children with infections and post-varicella ataxia and related conditions for reactivity to five recombinant centrosome proteins: gammagamma-enolase, pericentrin, ninein, PCM-1, and Mob1. METHODS: Sera from 12 patients with acute post-varicella ataxia, 1 with post-Epstein Barr virus (EBV) ataxia, 5 with uncomplicated varicella infections, and other conditions were tested for reactivity to cryopreserved cerebellum tissue and recombinant centrosome proteins. The distribution of pericentrin in the cerebellum was studied by indirect immunofluorescence (IIF) using rabbit antibodies to the recombinant protein. Antibodies to phospholipids (APL) were detected by ELISA. RESULTS: Eleven of 12 children with post-varicella ataxia, 4/5 children with uncomplicated varicella infections, 1/1 with post-EBV ataxia, 2/2 with ADEM, 1/2 with neuroblastoma and ataxia, and 2/2 with cerebellitis had antibodies directed against 1 or more recombinant centrosome antigens. Antibodies to pericentrin were seen in 5/12 children with post-varicella ataxia but not in any of the other sera tested. IIF demonstrated that pericentrin is located in axons and centrosomes of cerebellar cells. APL were detected in 75% of the sera from children with post-varicella ataxia and 50% of children with varicella without ataxia and in none of the controls. CONCLUSION: This is the first study to show the antigen specificity of anti-centrosome antibodies in children with varicella. Our data suggest that children with post-varicella ataxia have unique autoantibody reactivity to pericentrin.

PR3-ANCA: a promising biomarker in primary sclerosing cholangitis (PSC).

BACKGROUND AND AIMS: The only recognized biomarker for primary sclerosing cholangitis (PSC) is atypical anti-neutrophil cytoplasmic antibodies (aANCA), which, in addition to having low sensitivity and specificity, is an indirect immunofluorescence (IIF) test lacking the advantages of high throughput and objectivity. Recent reports have shown that antibodies to proteinase-3 (PR3-ANCA) might add diagnostic value in inflammatory bowel disease (IBD), specifically in ulcerative colitis (UC). As PSC is associated with IBD, the objective of this study was to evaluate the frequency and clinical significance of PR3-ANCA in a large cohort of patients. METHODS: A total of 244 PSC and 254 control [autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), hepatitis C viral infection (HCV), hepatitis B viral infection (HBV), and healthy controls] sera and their clinical correlations were retrospectively analyzed for PR3-ANCA determined by ELISA and a new chemiluminescence immunoassay (CIA). Testing was also performed for aANCA by IIF. RESULTS: When measured by CIA, PR3-ANCA was detected in 38.5% (94/244) of PSC patients compared to 10.6% (27/254) controls (p<0.0001). By ELISA, PR3-ANCA was detected in 23.4% (57/244) of PSC patients compared to 2.7% (6/254) controls (p<0.0001). PR3-ANCA in PSC patients was not associated with the presence or type of underlying IBD, and, in fact, it was more frequent in Crohn's disease (CD) patients with PSC than previously reported in CD alone. PR3-ANCA in PSC measured by CIA correlated with higher liver enzymes. CONCLUSION: PR3-ANCA is detected in a significant proportion of PSC patients compared to other liver diseases including PBC and AIH. PR3-ANCA is associated with higher liver enzyme levels in PSC, and is not solely related to underlying IBD.

Minimal hepatic encephalopathy.

Minimal hepatic encephalopathy (MHE) is the earliest form of hepatic encephalopathy and can affect up to 80% of cirrhotic patients. By definition, it has no obvious clinical manifestation and is characterized by neurocognitive impairment in attention, vigilance and integrative function. Although often not considered to be clinically relevant and, therefore, not diagnosed or treated, MHE has been shown to affect daily functioning, quality of life, driving and overall mortality. The diagnosis of MHE has traditionally been achieved through neuropsychological examination, psychometric tests or the newer critical flicker frequency test. A new smartphone application (EncephalApp Stroop Test) may serve to function as a screening tool for patients requiring further testing. In addition to physician reporting and driving restrictions, medical treatment for MHE includes non-absorbable disaccharides (eg, lactulose), probiotics or rifaximin. Liver transplantation may not result in reversal of the cognitive deficits associated with MHE.

Epidemiology of gallbladder disease: cholelithiasis and cancer.

Diseases of the gallbladder are common and costly. The best epidemiological screening method to accurately determine point prevalence of gallstone disease is ultrasonography. Many risk factors for cholesterol gallstone formation are not modifiable such as ethnic background, increasing age, female gender and family history or genetics. Conversely, the modifiable risks for cholesterol gallstones are obesity, rapid weight loss and a sedentary lifestyle. The rising epidemic of obesity and the metabolic syndrome predicts an escalation of cholesterol gallstone frequency. Risk factors for biliary sludge include pregnancy, drugs like ceftiaxone, octreotide and thiazide diuretics, and total parenteral nutrition or fasting. Diseases like cirrhosis, chronic hemolysis and ileal Crohn's disease are risk factors for black pigment stones. Gallstone disease in childhood, once considered rare, has become increasingly recognized with similar risk factors as those in adults, particularly obesity. Gallbladder cancer is uncommon in developed countries. In the U.S., it accounts for only ~ 5,000 cases per year. Elsewhere, high incidence rates occur in North and South American Indians. Other than ethnicity and female gender, additional risk factors for gallbladder cancer include cholelithiasis, advancing age, chronic inflammatory conditions affecting the gallbladder, congenital biliary abnormalities, and diagnostic confusion over gallbladder polyps.

Epidemiology of gallstones.

Gallstones are common with prevalences as high as 60% to 70% in American Indians and 10% to 15% in white adults of developed countries. Ethnic differences abound with a reduced frequency in black Americans and those from East Asia, while being rare in sub-Saharan Africa. Certain risk factors for gallstones are immutable: female gender, increasing age, and ethnicity/family (genetic traits). Others are modifiable: obesity, the metabolic syndrome, rapid weight loss, certain diseases (cirrhosis and Crohn disease), gallbladder stasis (from spinal cord injury or drugs, such as somatostatin), and lifestyle.

Identification of GRASP-1 as a novel 97 kDa autoantigen localized to endosomes.

We have identified an autoantigen that is recognized by antibodies from an 18-year-old female with a history of recurrent infections who later in her clinical course developed Raynaud's phenomenon and telangiectasias. By indirect immunofluorescence (IIF), the index serum produced a unique cytoplasmic discrete speckled (CDS) staining pattern that partially colocalized with early endosome antigen 1 (EEA1) but not Golgi complex or other cytoplasmic organelles in HEp-2 cells. When HEp-2 cells were treated with 0.1 N HCl, the cytoplasmic speckled staining of the index serum was markedly decreased, suggesting that the reactive antigen was soluble. Western blot analysis showed a reactive approximately 97 kDa protein in a saline soluble protein preparation from HeLa cells. Mass spectrometric analysis of the excised 97 kDa band that was immunoprecipitated from HeLa cell extracts identified GRASP-1 as a possible target. The index serum and anti-GRASP-1 antibodies colocalized to structures in the cytoplasm of HEp-2 cells. Synthetic peptides representing the full-length GRASP-1 protein were used to identify reactive epitopes. Like many other cytoplasmic autoantigens, GRASP-1 has numerous coiled-coil domains throughout the protein with the exception of short segments at the amino and carboxyl terminus.

Autoantibodies to protein transport and messenger RNA processing pathways: endosomes, lysosomes, Golgi complex, proteasomes, assemblyosomes, exosomes, and GW bodies.

Over 50 years ago the lupus erythematosus (LE) cell phenomenon was described and this was quickly followed by the introduction of the LE cell test and indirect immunofluorescence (IIF) to detect antinuclear antibodies (ANA) in clinical laboratories. Recently, attention has turned to the identification of the autoantigens that bind to cytoplasmic organelles such as the Golgi complex, endosomes and other "cytoplasmic somes". Three endosome autoantigens include early endosome antigen 1 (EEA1, 160 kDa), cytoplasmic linker protein-170 (CLIP-170, 170 kDa), and lysobisphosphatidic acid (LBPA). Antibodies to EEA1 were seen in a variety of conditions but approximately 40% of the patients had a neurological disease. Despite the prominence of lysosomes in cells and tissues, reports of autoantibodies are limited to the lysosomal antigen h-LAMP-2 and the cytoplasmic antineutrophil antibodies (cANCA). Autoantigens in the Golgi complex include giantin/macrogolgin, golgin-245, golgin 160, golgin-97, golgin 95/gm130, and golgin-67. More recently, there has been an interest in autoantibodies that bind components of the "SMN complex" or the "assemblyosome". Arginine/glycine (RG)-rich domains in components of the SMN complex interact with Sm, like-Sm (LSm), fibrillarin, RNA helicase A (Gu), and coilin proteins, all of which are antigen targets in a variety of diseases. More recently, components of a novel cytoplasmic structure named GW bodies (GWBs) have been identified as targets of human autoantibodies. Components of GWBs include GW182, a unique mRNA-binding protein, like Sm proteins (LSms), and decapping (hDcp1) and exonuclease (Xrn) enzymes. Current evidence suggests that GWBs are involved in the cytoplasmic processing of mRNAs. Autoantibodies to the "cytoplasmic somes" are relatively uncommon and serological tests to detect most of them are not widely available.