T3 Regulates a Human Macrophage-Derived TSH-ß Splice Variant: Implications for Human Bone Biology.

TSH and thyroid hormones (T3 and T4) are intimately involved in bone biology. We have previously reported the presence of a murine TSH-ß splice variant (TSH-ßv) expressed specifically in bone marrow-derived macrophages and that exerted an osteoprotective effect by inducing osteoblastogenesis. To extend this observation and its relevance to human bone biology, we set out to identify and characterize a TSH-ß variant in human macrophages. Real-time PCR analyses using human TSH-ß-specific primers identified a 364-bp product in macrophages, bone marrow, and peripheral blood mononuclear cells that was sequence verified and was homologous to a human TSH-ßv previously reported. We then examined TSH-ßv regulation using the THP-1 human monocyte cell line matured into macrophages. After 4 days, 46.1% of the THP-1 cells expressed the macrophage markers CD-14 and macrophage colony-stimulating factor and exhibited typical morphological characteristics of macrophages. Real-time PCR analyses of these cells treated in a dose-dependent manner with T3 showed a 14-fold induction of human TSH-ßv mRNA and variant protein. Furthermore, these human TSH-ßv-positive cells, induced by T3 exposure, had categorized into both M1 and M2 macrophage phenotypes as evidenced by the expression of macrophage colony-stimulating factor for M1 and CCL-22 for M2. These data indicate that in hyperthyroidism, bone marrow resident macrophages have the potential to exert enhanced osteoprotective effects by oversecreting human TSH-ßv, which may exert its local osteoprotective role via osteoblast and osteoclast TSH receptors.

Graves' Disease Mechanisms: The Role of Stimulating, Blocking, and Cleavage Region TSH Receptor Antibodies.

The immunologic processes involved in Graves' disease (GD) have one unique characteristic--the autoantibodies to the TSH receptor (TSHR)--which have both linear and conformational epitopes. Three types of TSHR antibodies (stimulating, blocking, and cleavage) with different functional capabilities have been described in GD patients, which induce different signaling effects varying from thyroid cell proliferation to thyroid cell death. The establishment of animal models of GD by TSHR antibody transfer or by immunization with TSHR antigen has confirmed its pathogenic role and, therefore, GD is the result of a breakdown in TSHR tolerance. Here we review some of the characteristics of TSHR antibodies with a special emphasis on new developments in our understanding of what were previously called "neutral" antibodies and which we now characterize as autoantibodies to the "cleavage" region of the TSHR ectodomain.

Thyroid and bone: macrophage-derived TSH-ß splice variant increases murine osteoblastogenesis.

It is now firmly established that TSH may influence the physiology and patho-physiology of bone by activating osteoblasts and inhibiting osteoclast activity resulting in relative osteoprotection. Whether this influence is directly exerted by pituitary-derived TSH in vivo is less certain, because we have previously reported that the suppression of pituitary TSH does not remove such protection. Here, we have characterized the functional relevance of a novel form of the TSH-ß subunit, designated TSH-ßv, known to be produced by murine bone marrow cells. We found that fresh bone marrow-derived macrophages (MØs) preferentially produced TSH-ßv and, when cocultured with CHO cells engineered to overexpress the full-length TSH receptor, were able to generate the production of intracellular cAMP; a phenomenon not seen in control CHO cells, such results confirmed the bioactivity of the TSH variant. Furthermore, cocultures of MØs and osteoblasts were shown to enhance osteoblastogenesis, and this phenomenon was markedly reduced by antibody to TSH-ß, suggesting direct interaction between MØs and osteoblasts as observed under the electron microscope. These data suggest a new paradigm of local modulation of bone biology by a MØ-derived TSH-like molecule and raise the question of the relative contribution of local vs pituitary-derived TSH in osteoprotection.

Antibodies against neural, nuclear, cytoskeletal, and streptococcal epitopes in children and adults with Tourette's syndrome, Sydenham's chorea, and autoimmune disorders.

BACKGROUND: Some cases of Tourette's syndrome (TS) are hypothesized to be caused by autoantibodies that develop in response to a preceding group A beta hemolytic streptococcal infection. METHODS: To test this hypothesis, we looked for the presence ot total and IgG antibodies against neural, nuclear, cytoskeletal and streptococcal epitopes using indirect immunofluorescent assays and Western blot techniques in three patient groups: TS (n = 81), SC (n = 27), and a group of autoimmune disorders (n = 52) and in normal controls (n = 67). Subjects were ranked after titrations of autoantibodies from 0 to 227 according to their level of immunoreactivity. RESULTS: TS patients had a significantly higher mean rank for total antineural and antinuclear antibodies, as well as antistreptolysin O titers. However, among children and adolescents, only the total antinuclear antibodies were increased in TS patients compared to age matched controls. Compared to SC patients, TS patients had a significantly lower mean rank for total and IgG class antineural antibodies, significantly lower IgG class anticytoskeletal antibodies, and a significantly higher rank for total antinuclear antibodies. Compared to a mixed group of autoimmune disorders, the TS patients had a significantly lower mean rank for total and IgG class antineural antibodies, total and IgG class antinuclear antibodies, IgG class anticytoskeletal antibodies, and a significantly higher rank for antistreptococcal antibodies. CONCLUSIONS: TS patients had significantly higher levels of total antineural and antinuclear antibodies than did controls. Their relation to IgG class antineural and antinuclear antibodies, markers for prior streptococcal infection, and other clinical characteristics, especially chronological age, was equivocal.

The question of PANDAS in adults.

BACKGROUND: Pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS) are a well-defined cause of obsessive-compulsive disorder in children. However, they have not been described or fully investigated in adults newly diagnosed with obsessive-compulsive disorder. METHODS: We describe an adult with onset of obsessive-compulsive disorder at 25 years of age after a severe antibiotic-responsive pharyngitis. He was evaluated with multiple psychiatric rating scales for obsessive-compulsive disorder and Tourette's syndrome, as well as with serologic assays and radiologic studies. RESULTS: In all respects except age our patient fulfilled established criteria for PANDAS. Assays for antibodies to group A beta-hematolytic streptococci, serum D8,17 lymphocytes, antistriatal (neuronal) antibodies, and anticytoskeletal antibodies all supported the hypothesis that a poststreptococcal process was active. Magnetic resonance imaging was abnormal and is described. CONCLUSIONS: The findings suggest that this patient's illness is similar to PANDAS in presentation and that poststreptococcal disease may result in adult-onset obsessive-compulsive disorder.

Endoscopic papillary dilation by balloon and isosorbide dinitrate drip infusion for removing bile duct stone.

BACKGROUND: Endoscopic papillary dilation (EPD) by balloon in the management of bile duct stones has recently been claimed to be effective for removing bile duct stones. METHODS: Without endoscopic sphincterotomy, we attempted to remove large or multiple bile duct stones through EPD combined with drip infusion of isosorbide dinitrate in 35 patients. Isosorbide dinitrate, at a rate of 5 mg/h, was administered intravenously, and a balloon dilator with a 10-mm diameter was inflated within 3 min across the papilla. Stones were then smashed using a mechanical lithotriptor, and the fragments were extracted with a basket or the balloon. RESULTS: Extraction of stones was successful in 33 (94%) of 35 patients by the combined therapy. Two of them (6%) developed mild pancreatitis. CONCLUSION: EPD combined with medical sphincter dilation was effective for large and multiple bile duct stones.

Antibodies to Ro/La, Cenp-B, and snRNPs antigens in autoimmune hepatitis of North America versus Asia: patterns of immunofluorescence, ELISA reactivities, and HLA association.

To assess whether demography is one of the important factors determining antibody response to nuclear antigens [ANA: SSA-Ro (52K and 60K), SSB-La, snRNPs (A, 70K, B'/B), and Cenp-B], we investigated 95 and 47 sera of autoimmune hepatitis (AIH) from North America and Asia, respectively, by immunofluorescent (IF) and recombinant ELISA. Correlations among nuclear IF patterns, ELISA, and disease indices were analyzed. The frequency and titer of individual antibodies differed significantly between the groups. Patients with speckled patterns were younger in both regions and had higher aspartate aminotransferase levels only in North America. HLA-A1, B8, DQ2, and DR4 or DR3 or both in North America, and A2, B61, DQ7, and DR4 in Asia were predominant. In Asia, B61 correlated with anti-70K, and DQ7 correlated with antibodies to 52K, Cenp-B, and B'/B. In North America, A1, B8, DR3 haplotype, and DQ2 correlated with antibodies to A and 70K. Anti-B'/B and DR4 in North America, and A2 in Asia, were associated with concurrent immunologic disorder. Individual ANA clusters correlated with individual HLA in the demography, and different HLA alleles might determine disease expression as well as different ANA being produced in AIH.

Endoscopic lithotomy of common bile duct stones with sublingual nitroglycerin and guidewire.

OBJECTIVE: In 21 patients, our objective was the endoscopic removal of common bile duct stones by sphincter dilation with the application of sublingual nitroglycerin. METHODS: Nitroglycerin 0.3-0.6 mg was needed for proper dilation of the orifice and for successful cannulation of the Dormia basket into the bile duct. Cannulation of the Dormia basket was simplified by placing the guidewire in the common bile duct beforehand. Because of possible stone impaction, a mechanical lithotriptor was applied smoothly in two patients. RESULTS: Complete stone removal was successful in 18 of the 21 (86%) patients. One patient who developed a mild form of acute pancreatitis recovered in a few days by conservative management with drip infusion of protease inhibitor. Blood pressure dropped transiently in a patient receiving nitroglycerin, but the general condition of the patient was stable. CONCLUSIONS: This procedure was found to be safe, easy, and effective in extracting common bile duct stones.

Antibodies to single-stranded and double-stranded DNA in antinuclear antibody-positive type 1-autoimmune hepatitis.

To determine the significance of antibodies to single-stranded (anti-ssDNA) and double-stranded DNA (anti-dsDNA) in antinuclear antibody (ANA)-positive type 1 autoimmune hepatitis, sera from 53 patients were tested by enzyme immunosorbent assay (ELISA) and indirect immunofluorescence using the Crithidia luciliae substrate. Anti-dsDNA were detected in 18 patients (34%) by ELISA and 12 patients (23%) by the Crithidia-based assay. Twenty patients with anti-dsDNA by either assay (38%) had higher serum levels of immunoglobulin G (3971 +/- 270 mg/dL vs. 3201 +/- 247 mg/dL, P = .05) than seronegative patients. They also had human leukocyte antigen (HLA) DR4 more commonly than other patients (83% vs. 41%, P = .006) and normal subjects (83% vs. 30%, P = .00007). In contrast to patients seropositive by the Crithidia-based assay, those seropositive by ELISA failed corticosteroid therapy more commonly (24% vs. 3%, P = .04). Anti-ssDNA were found in 45 patients (85%) and they did not distinguish patients with different clinical features or outcomes. We conclude that anti-dsDNA are common in ANA-positive type 1 autoimmune hepatitis. HLA DR4 is associated with their production, and seropositivity by ELISA characterizes patients who have a poorer immediate response to corticosteroid treatment. Anti-ssDNA are common but they do not have important clinical implications.

Viral hepatitis in Japan: update.

Viral hepatitis is defined as a hepatitis virus infection in which hepatic inflammation and necrosis lead to a characteristic feature. It is caused by at least five viral agents with specific epidemiological attributes and distinctive immunoserologic findings. The well-characterized forms are hepatitis A, B, C, D and E viruses. Recent status and the current progress of viral hepatitis in Japan are discussed in the present article.

Frequency and significance of antibodies to P450IID6 protein in Japanese patients with chronic hepatitis C.

BACKGROUND/AIMS: The aims of the current study were to assess the frequency and the significance of antibodies to cytochrome P450IID6 protein (anti-P450IID6) in various diseases among Japanese patients. METHODS: Sera from 541 patients were tested by indirect immunofluorescence, and the specificity of anti-P450IID6 was ascertained by either enzyme immunoassay (ELISA) or Western blot using recombinant antigen or rat liver microsomes. RESULTS: Anti-P450IID6 was found in only 6 of 235 patients (2.6%) with chronic active hepatitis (CAH) positive for hepatitis C virus (HCV) antibody and quantitative HCV-RNA with genotypes II and IV. The predominant epitopes on immunoblots were 66 and 50KD, a 10KD band being the newly underfined microsomal antigen. Even in the patients negative for autoantibodies to nuclear antigens (ANA) by routine indirect immunofluorescence test, various ANA were detected by the newly developed recombinant ELISA. These patients were younger, with lower gamma-globulin and IgG levels than patients with autoimmune hepatitis. Three of five patients with anti-P450IID6 responded well to interferon therapy and one received prednisone when interferon was ineffective. Interestingly, only this patient was diagnosed as definite autoimmune hepatitis according to the criteria proposed by the International Autoimmune Hepatitis Group (IAHG). The other five patients who did not satisfy the IAHG criteria might be considered as CAH-C with autoimmune features. No autoimmune hepatitis patients positive for anti-P450IID6 were identified in the current study, indicating that the variant is very rare in Japan. CONCLUSIONS: Anti-P450IID6 in CAH-C patients in Japan is not as rare as expected. Anti-P450IID6 among Japanese patients has uncertain significance and precludes further characterization of CAH-C with autoimmune features, which might require interferon therapy.

Immune response to cyclin B1 in hepatocellular carcinoma.

Proteins expressed by plasmids encoding human cyclins and cyclin-dependent kinase 2 (CDK2) were used as antigens in immunoblotting. Fifteen of 100 patients with hepatocellular carcinoma (HCC) were found to have autoantibodies reactive with cyclin B1 and with a 40-kd degradation product of cyclin B1-glutathione-S-transferase (GST) fusion protein. Only one serum was found to react with cyclin A and another single serum with CDK2 but no antibodies were detected to cyclin D1 and E. The basis for autoimmune responses to cyclin B1 in HCC are unknown at the present time but the possibilities might include aberrations in cyclin B1 regulation leading to altered product or its expression which resulted in stimulation of immune reactions.

Antibodies to P450IID6, SLA, PDH-E2 and BCKD-E2 in Japanese patients with chronic hepatitis.

Auto-antibodies specific to various antigens in chronic hepatitis (CH) have been detected but their specificities and implications were uncertain. The aims of the present study were to investigate the frequency and the significance of seropositivity of antibodies to P450IID6 or liver/kidney microsome 1 (LKM1), soluble liver antigen (SLA), pyruvate dehydrogenase (PDH) and branched-chain keto acid dehydrogenase (BCKD) in 188 Japanese patients with different forms of CH by western blot or enzyme immunoassay (EIA). Anti-LKM1 was also measured by indirect immunofluorescent test. Anti-P450IID6 was found in 6/188 (3.2%) CH patients including 5/104 (4.8%) with hepatitis C virus (C) infection and 1/12 (8.3%) CH-C patients with antibodies to nuclear and smooth muscle antigens and hypergammaglobulinaemia (> 2.5 g/dL). This patient was the only one diagnosed with autoimmune hepatitis (AIH). All CH patients with hepatitis B (B), hepatitis non-B non-C (NBNC) and AIH were seronegative for anti-LKM1. Antibodies to soluble liver antigen were found in two of 188 (1%) patients, one with AIH and one with CH-B. Anti-BCKD-E2 but not anti-PDH-E2 was found in four patients (2.5%), one with AIH, two with CH-C, and one with NBNC. There was no obvious difference in age, sex ratio and laboratory findings in patients with or without anti-SLA and anti-BCKD-E2. Antibodies to P450IID6, SLA, PDH-E2 and BCKD-E2 are uncommon in adult CH-C, CH-B, CH-NBNC and AIH patients in Japan. Some of these patients positive for auto-antibodies appear to have autoimmune features and might require a careful follow up. The heterogeneity of these antibodies in CH preclude further justification for subtyping of AIH by the presence of the distinct auto-antibodies.

Giant cell pneumonia: light microscopy, immunohistochemical, and ultrastructural study of an autopsy case.

An autopsy case of measles giant cell pneumonia with intranuclear inclusion bodies is reported. This case of giant cell pneumonia was studied by light microscopy and immunohistochemistry using monoclonal and polyclonal antibody to measles and by electron microscopy (EM). Light microscopic examination showed multinucleated epithelial giant cells with intranuclear and intracytoplasmic inclusions. The giant cells contained prominent, sharply marginated, eosinophilic intranuclear inclusions typical of classic measles pneumonia. Presence of measles antigen was confirmed using both monoclonal and polyclonal antibodies by peroxidase antiperoxidase method. Monoclonal antibody stained positively for intracytoplasmic and intranuclear inclusions. Electron microscopic examination of lung tissue showed intranuclear inclusions of filamentous or worm like nucleocapsid materials in multinucleated epithelial giant cells. The results suggest that this is a case of measles giant cell pneumonia and the intranuclear inclusion bodies are measles viral particles.

Increased expression of cytokines in liver and serum in patients with extrahepatic diseases.

To determine whether the liver plays an immunological role in certain extrahepatic disorders, we investigated the expression of interleukin (IL)-1 beta, IL-6, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha in 11 patients who had recovered from cholecystolithiasis, 12 patients with gastric cancer, 20 patients with chronic hepatitis, and 6 healthy controls. Cytokine mRNAs in the liver were detected by semiquantitative reverse transcribed-polymerase chain reaction. Serum cytokines and soluble IL-2 receptor (sIL-2R) were investigated by enzyme-linked immunosorbent assays. Increases in TNF-alpha, IL-6, IL-1 beta, and IFN-gamma mRNAs were found in the livers of patients with extrahepatic diseases. TNF-alpha and IL-6 peptides were increased in the sera of patients with gastric cancer. TNF-alpha in the sera and TNF-alpha mRNA in the liver were correlated in gastric cancer patients. Surprisingly, sIL-2R in the serum of gastric cancer patients was significantly higher than the level in healthy controls. Our findings suggest that the liver produces cytokines in reaction to extrahepatic lesions. Further, the increase in sIL-2R in gastric cancer patients indicates that malignancy may affect the immune network in vivo.

Scanning of polypoid gallbladder lesions by ultrasonic microprobes using transpapillary catheterization.

BACKGROUND AND STUDY AIMS: Polypoid lesions of the gallbladder can be diagnosed by transabdominal ultrasonography and endoscopic ultrasonography. However, the image resolution is limited, and the histopathology of these lesions is difficult to determine. We report here on our preliminary results in four patients using a high-frequency (20 MHz) ultrasonic microprobe introduced into the gallbladder via the transpapillary approach. PATIENTS AND METHODS: Insertion of the microprobe into the gallbladder through the papilla of Vater was attempted in four patients with gallbladder diseases without preceding endoscopic sphincterotomy. RESULTS: The lesions in the gallbladder were clearly visualized in three of the four patients. We tried to correlate the characteristic ultrasound findings with the histopathology of the lesions, which were found to be early cancer, adenoma, and adenomyomatosis on histopathology of the specimens after cholecystectomy. No complications occurred. CONCLUSIONS: High-resolution endoscopic transpapillary ultrasonography of the gallbladder is feasible and safe, and may contribute to the diagnosis of gallbladder diseases, particularly of small polypoid lesions.

Papillary dilation vs sphincterotomy in endoscopic removal of bile duct stones. A randomized trial with manometric function.

To circumvent the long-term effects of papillary ablation for extracting common bile duct stones (< 12 mm in diameter) in endoscopic sphincterotomy (EST), endoscopic papillary dilation (EPD) was attempted in 20 patients. To evaluate papillary function before and after the procedures, manometry of the sphincter of Oddi was carried out in 13 with EPD and 10 of 20 patients with EST. Extraction of all stones was successful (100%) in both groups at an equal rate. Repeated numbers of procedures were common in both groups. However, the mean duration of the procedure was high in EPD compared to EST (63 min vs 42 min, P < NS). Adjunctive therapies like mechanical lithotripsy (ML), nasobiliary drainage, and choledochoscopy were included in EPD, while EST required a basket catheter and ML. There was no significant difference on manometry before and after the procedures (P = NS), although papillary function was found to have decreased after the EPD. In contrast, all patients in the EST group lost papillary function after the procedure. Thirty-day morbidity and mortality rate were absent in both groups. Immediate and 2.5-year follow up complications were uncommon in both groups. As a simple method, EPD may be an effective and safe alternative to EST in the management of patients with bile duct stones who require maintenance of papillary function.

High prevalence of antibodies to recombinant CENP-B in primary biliary cirrhosis: nuclear immunofluorescence patterns and ELISA reactivities.

The purpose of the present study is to evaluate the centromeric pattern on human laryngeal tumour (HEp-2) cells by indirect immunofluorescent (IIF) test and to compare their reactivities with a newly developed recombinant centromere protein B enzyme linked immunosorbent assay (CENP-B ELISA) test using sera of antinuclear antibody (ANA)-reactive primary biliary cirrhosis (PBC) patients. Antimitochondrial antibody (AMA) subtypes (PDC-E2, BCOADC-E2, OGDC, protein X, and PDC-E1 alpha) by Western blot were also investigated to see whether they have any effect on the expression of CENP-B reactivities. A centromeric pattern (anticentromere antibody [ACA]) was detected in 11 of 25 (44%) PBC patients whereas CENP-B reactivity was found in 15 (60%) of them. There were some differences in IIF patterns and CENP-B reactivities. One PBC serum with indistinguishable ANA pattern reacted with CENP-B. Eight of 15 (53%) CENP-B reactive patients had other autoimmune-like disorders. Of 181 healthy sera, none was reactive for ACA either by IIF or by ELISA test. There was a correlation between ACA IIF and CENP-B ELISA titres (r = 0.824, P < 0.001). However, no correlation was observed between either CENP-B or AMA reactivities and/or between either autoantibodies or laboratory and histologic indices of PBC. These findings suggest that recombinant CENP-B ELISA appears to be more sensitive in identifying ACA than IIF, underlying its potential value as a screening test for the diagnosis of PBC complicated with other autoimmune-like disorders. The presence of multiple autoantibodies in PBC sera may reflect heterogeneous antigens recognition, and requires further study to identify target antigens at cellular and molecular levels.