Identification of novel serological biomarkers for inflammatory bowel disease using Escherichia coli proteome chip.

Specific antimicrobial antibodies present in the sera of patients with inflammatory bowel disease (IBD) have been proven to be valuable serological biomarkers for diagnosis/prognosis of the disease. Herein we describe the use of a whole Escherichia coli proteome microarray as a novel high throughput proteomics approach to screen and identify new serological biomarkers for IBD. Each protein array, which contains 4,256 E. coli K12 proteins, was screened using individual serum from healthy controls (n = 39) and clinically well characterized patients with IBD (66 Crohn disease (CD) and 29 ulcerative colitis (UC)). Proteins that could be recognized by serum antibodies were visualized and quantified using Cy3-labeled goat anti-human antibodies. Surprisingly significance analysis of microarrays identified a total of 417 E. coli proteins that were differentially recognized by serum antibodies between healthy controls and CD or UC. Among those, 169 proteins were identified as highly immunogenic in healthy controls, 186 proteins were identified as highly immunogenic in CD, and only 19 were identified as highly immunogenic in UC. Using a supervised learning algorithm (k-top scoring pairs), we identified two sets of serum antibodies that were novel biomarkers for specifically distinguishing CD from healthy controls (accuracy, 86 +/- 4%; p < 0.01) and CD from UC (accuracy, 80 +/- 2%; p < 0.01), respectively. The Set 1 antibodies recognized three pairs of E. coli proteins: Era versus YbaN, YhgN versus FocA, and GabT versus YcdG, and the Set 2 antibodies recognized YidX versus FrvX. The specificity and sensitivity of Set 1 antibodies were 81 +/- 5 and 89 +/- 3%, respectively, whereas those of Set 2 antibodies were 84 +/- 1 and 70 +/- 6%, respectively. Serum antibodies identified for distinguishing healthy controls versus UC were only marginal because their accuracy, specificity, and sensitivity were 66 +/- 5, 69 +/- 5, and 61 +/- 7%, respectively (p < 0.04). Taken together, we identified novel sets of serological biomarkers for diagnosis of CD versus healthy control and CD versus UC.

[Botulinic toxin type A in the treatment of children with neurogenic bladder]. / Toxina botulínica-A en el tratamiento de niños con vejiga neurogénica.

UNLABELLED: INTRODUCTION. Botulinum toxin A (BoNTA) is an alternative in children with neurogenic bladder refractory to anticholinergics. OBJECTIVE. To evaluate the initial effects of BoNTA in neurogenic bladders. PATIENTS AND METHODS 12 patients (9 myelomeningoceles, 1 tethered cord, 1 medullary astrocytoma, 1 Ewing's sarcoma) were studied. The pretreatment evaluation included: voiding diary, incontinence score, renovesical ultrasound, cystourethrography and urodynamic parameters. Patients received 300 UI of BoNTA via detrusor injections. They were re-evaluated at months 1, 3 and 6. After the third control, if improvement was confirmed, patients were injected again. Otherwise, surgery was indicated. RESULTS: After the initial injection, 50% of patients achieved complete continence and 20% remained with minimal leaks. The mean maximum bladder capacity increased from 264 +/- 117 ml to 324 +/- 170 ml (P = 0.322). The mean detrusor pressure decreased from 46 +/- 17 cm H2O to 42 +/- 14 cm H2O (P=0.596). The mean bladder compliance increased from 7.6 +/- 5.9 ml/cm H2O to 10.4 +/- 6.4 ml/cm H2O (P = 0.290). The reflex voiding contractions disappeared in 2 patients; 8 presented trabeculae. Ten patients were reinjected and two underwent surgery. CONCLUSION: 70% urinary continence was achieved after the initial injection. No significant urodynamic changes were observed. A longer follow-up with subsequent injections is needed.

NOD2 mutations and anti-Saccharomyces cerevisiae antibodies are risk factors for Crohn's disease in African Americans.

OBJECTIVES: NOD2 mutations and anti-Saccharomyces cerevisiae antibodies (ASCAs) are established risk factors of Crohn's disease (CD) in whites but have not been assessed in African-American (AA) adults with CD. METHODS: AAs with CD and controls were recruited by the Mid-Atlantic African-American IBD Study as part of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) IBD Genetics Consortium. Genotyping for the three common CD NOD2 mutations (Leu1007fsinsC, G908R/2722g>c, and R702W/2104c>t) and ASCA enzyme-linked immunosorbent assays were performed in 183 AA CD patients and in 143 controls. Logistic regression was used to calculate adjusted odds ratios (ORs) for the association between ASCA and disease phenotype. RESULTS: ASCA sensitivity and specificity values were 70.5 and 70.4%, respectively. On univariate analysis, ASCA was significantly associated with younger age at diagnosis, ileal involvement, and complicated (stricturing/penetrating) behavior. On multivariate analysis, ASCA titer (per 25 Units) was associated with ileal involvement (OR 1.18, 95% confidence interval (CI): 1.04-1.34), complicated behavior (OR 1.13, 95% CI: 1.01-1.28), and surgery (hazard ratio: 1.11, 95% CI: 1.02-1.21). Cigarette smoking and CD family history were also significantly associated with surgery. NOD2 carriers (all heterozygotes) were more common among CD cases than controls (8.2 vs. 2.1%; OR 4.17%, 95% CI: 1.18-14.69). The NOD2 mutation population attributable risk was 6.2%. CONCLUSIONS: In comparison with whites, ASCA in AAs has a similar sensitivity but a lower specificity for CD. ASCA is associated with ileal involvement, complicated behavior, and surgery in AAs with CD. NOD2 is a risk gene for AA CD, although mutation frequency and population attributable risk are much lower than in whites.

Environmental triggers of autoimmune thyroiditis.

Autoimmune thyroiditis is among the most prevalent of all the autoimmunities. Autoimmune thyroiditis is multifactorial with contributions from genetic and environmental factors. Much information has been published about the genetic predisposition to autoimmune thyroiditis both in experimental animals and humans. There is, in contrast, very little data on environmental agents that can serve as the trigger for autoimmunity in a genetically predisposed host. The best-established environmental factor is excess dietary iodine. Increased iodine consumption is strongly implicated as a trigger for thyroiditis, but only in genetically susceptible individuals. However, excess iodine is not the only environmental agent implicated as a trigger leading to autoimmune thyroiditis. There are a wide variety of other synthetic chemicals that affect the thyroid gland or have the ability to promote immune dysfunction in the host. These chemicals are released into the environment by design, such as in pesticides, or as a by-product of industry. Candidate pollutants include polyaromatic hydrocarbons, polybrominated biphenols, and polychlorinated biphenols, among others. Infections are also reputed to trigger autoimmunity and may act alone or in concert with environmental chemicals. We have utilized a unique animal model, the NOD.H2(h4) mouse to explore the influence of iodine and other environmental factors on autoimmune thyroiditis.

Autoimmune thyroiditis and ROS.

Autoimmune thyroiditis, also known as chronic lymphocytic or Hashimoto's thyroiditis, is characterized by infiltration of the thyroid gland by inflammatory cells and production of autoantibodies to thyroid-specific antigens thyroglobulin and thyroperoxidase. It is accompanied by hypothyroidism due to destruction and eventual fibrous replacement of the follicle cells. Autoimmune thyroiditis is clearly multifactorial in etiology with genetic and environmental factors contributions. Excess dietary Iodine can exacerbate thyroiditis in genetically susceptible hosts such as the NOD.H2(h4) mouse. In this mouse excess dietary iodine leads to an increased immunogenicity of the thyroglobulin molecule and enhanced expression of ICAM-1 on thyroidal follicle cells. We present evidence here that ICAM-1 expression is enhanced by the elevated generation of reactive oxygen species (ROS). The anti-oxidant diphenyleneiodium (DPI), an inhibitor of NADPH oxidase, reduced both the generation of ROS and of ICAM-1 expression in cultures of NOD.H2(h4) mouse thyrocytes. These results suggest that antioxidants may have therapeutic value in preventing autoimmune thyroiditis.

Intracellular adhesion molecule-1 up-regulation on thyrocytes by iodine of non-obese diabetic.H2(h4) mice is reactive oxygen species-dependent.

Intracellular adhesion molecule-1 (ICAM-1) expression on the thyroid follicular cells of non-obese diabetic (NOD).H2(h4) mice is enhanced by iodide treatment, which correlates with autoimmune thyroid disease in genetically susceptible NOD.H2(h4) mice. The current study examines the mechanism of iodine-enhanced up-regulation of ICAM-1 on the surface of thyroid cells. We hypothesized that the up-regulation of ICAM-1 is due to a transient increase in production of reactive oxygen species (ROS). ROS may initiate signalling of the ICAM-1 gene promoter, enhancing up-regulated ICAM-1 protein on the cell surface. Single-cell suspensions of thyroid follicular cells from thyroiditis-susceptible NOD.H2(h4) or non-susceptible BALB/c mice were treated in vitro with sodium iodide. Extracellular and intracellular ROS were assessed by luminol-derived chemiluminescence and flow cytometry assays respectively. Our results demonstrate that thyroid follicular cells of NOD.H2(h4) generate higher levels of ROS compared with cells from non-susceptible strains of mice. Expression of a subunit protein of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, p67(phox), was analysed by Western blot immunoassay. A constitutive expression of the p67(phox) subunit protein was observed in NOD.H2(h4) mice prior to iodine treatment. No such expression was found in BALB/c mice. Treatment of NOD.H2(h4) thyroid cells with diphenyleneiodium, an inhibitor of NADPH oxidase, reduced generation of ROS and of ICAM-1 protein expression. Thus, thyrocytes from NOD.H2(h4) mice produce enhanced levels of ROS that may be mediated by NADPH oxidase. Consequently, in NOD.H2(h4) mice the ROS-induced signal for ICAM-1 up-regulation may contribute to mononuclear cellular infiltration of the thyroid gland and the progression of autoimmune thyroid disease.

Triggering receptor expressed on myeloid cells-1 (TREM-1) expression on gastric epithelium: implication for a role of TREM-1 in Helicobacter pylori infection.

In Helicobacter pylori gastritis gastric epithelium plays a central role in the innate immunity to H. pylori. However, epithelial receptors interacting with H. pylori have been poorly characterized so far. Recently a new triggering receptor expressed on myeloid cells-1 (TREM-1) has been identified on human neutrophils and monocytes. On these cells TREM-1 triggers innate immunity by stimulating the secretion of interleukin (IL)-8 and tumour necrosis factor (TNF)-alpha and thus amplifies bacterial-induced inflammation. In this study expression and function of TREM-1 in gastric epithelium exposed to H. pylori has been investigated. TREM-1 mRNA and protein were expressed on gastric epithelial cell lines as demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence activated cell sorter analysis. Gastric epithelial TREM-1 expression was up-regulated directly by H. pylori and was independent of epithelial IL-8 induced by H. pylori. Immunohistochemistry and tissue RT-PCR demonstrated significantly stronger TREM-1 expression in H. pylori gastritis compared with the non-inflamed gastric mucosa supporting in vivo that epithelial TREM-1 is up-regulated during H. pylori infection. Stimulation of gastric epithelial TREM-1 receptor resulted in IL-8 up-regulation on mRNA and protein level, as shown by real-time PCR and immunoassay. This is the first study localizing TREM-1 on gastric epithelium. Functional data suggest that TREM-1 expressed on gastric epithelium amplifies inflammation of the underlying gastric mucosa by up-regulation of IL-8.

Apoptin-induced cell death is modulated by Bcl-2 family members and is Apaf-1 dependent.

Apoptin, a chicken anemia virus-derived protein, selectively induces apoptosis in transformed but not in normal cells, thus making it a promising candidate as a novel anticancer therapeutic. The mechanism of apoptin-induced apoptosis is largely unknown. Here, we report that contrary to previous assumptions, Bcl-2 and Bcl-xL inhibit apoptin-induced cell death in several tumor cell lines. In contrast, deficiency of Bax conferred resistance, whereas Bax expression sensitized cells to apoptin-induced death. Cell death induction by apoptin was associated with cytochrome c release from mitochondria as well as with caspase-3 and -7 activation. Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad spectrum caspase inhibitor, was highly protective against apoptin-induced cell death. Apoptosis induced by apoptin required Apaf-1, as immortalized Apaf-1-deficient fibroblasts as well as tumor cells devoid of Apaf-1 were strongly protected. Thus, our data indicate that apoptin-induced apoptosis is not only Bcl-2- and caspase dependent, but also engages an Apaf-1 apoptosome-mediated mitochondrial death pathway.

Antibodies to saccharomyces cerevisiae in Crohn's disease: higher titers are associated with a greater frequency of mutant NOD2/CARD15 alleles and with a higher probability of complicated disease.

BACKGROUND: Both antibodies to Saccharomyces cerevisiae (ASCA) and carriage of two mutated NOD2/CARD15 alleles are associated with ileal Crohn's disease (CD) and complications requiring bowel surgery. We assessed the ASCA titer as a marker of CD clinical behavior. METHODS: In a cross-sectional study, we phenotyped 117 unrelated CD patients. Titers (Units, U) of ASCA IgG and IgA were measured and patients were genotyped for three high-risk NOD2/CARD15 alleles. Multiple logistic regression and Cox regression analyses were used to assess the association of factors to CD phenotype and time to surgery. RESULTS: ASCA seropositivity was associated with younger age at diagnosis, ileal disease, and complicated (stricturing or penetrating) behavior. There was a dose-response between the number of mutant NOD2/CARD15 alleles and the prevalence and titers of ASCA. The ASCA titer and tobacco use were associated with ileal disease independently of NOD2/CARD15 status. The ASCA titer (odds ratio (OR): 2.7 per 25 U, 95% confidence interval (CI): 1.5-46.7) and ileal disease were associated with stricturing/penetrating behavior, independently of NOD2/CARD15 status. Patients with ileal CD and ASCA titers of 41 U and 60 U needed 10 and 5 years of disease, respectively, to accumulate a 50% risk of complications. CONCLUSIONS: ASCA+ patients had a greater frequency of mutant NOD2/CARD15 alleles. Nonetheless, higher ASCA titers were associated with higher probabilities of ileal CD and stricturing/penetrating behavior independently of NOD2/CARD15 status. Higher ASCA titers were associated with more rapid development of complications. This quantitative marker may prove useful in risk-stratifying patients to more aggressive antiinflammatory therapies.

Heritability of levels of autoantibodies to thyroid antigens using the method of plotting regression of offspring on midparent (ROMP).

Only a few methods can be applied in a simple manner to estimate the genetic control of autoimmunity in humans. Here we examined the heritability of autoantibodies to two thyroid antigens; thyroglobulin (Tg) and thyroperoxidase (TPO, formerly known as thyroid microsomal antigen), using methods of regression of offspring on mid-parental values (ROMP). With the data sets available, affected and unaffected siblings were compared by this rapid screening method using results determined by hemagglutination (HA). The presence of both types of autoantibodies showed positive heritability in patients with Graves' thyrotoxicosis (TT), but it was not observed in chronic lymphocytic or Hashimoto's thyroiditis (CLT) patients. Since these assays have been extensively used over the years by most diagnostic and research laboratories, they should provide some insight as to which quantifiable parameters may be usefully accumulated to help select groups of patients and their families for further genetic study. ROMP may also be useful to determine the sequential appearance of different types of antibody in predicting disease onset in other family members, and in distinguishing maternal and paternal effects on imprinting. The method may be extended to study epitope spreading and other measures of disease progression.

Checkpoint kinase 1 (CHK1) protein and mRNA expression is downregulated in aggressive variants of human lymphoid neoplasms.

CHK1: gene encodes for a serine/threonine kinase involved in the regulation of cell cycle progression and DNA damage checkpoints. To determine the role of CHK1 in the pathogenesis of lymphoid neoplasms and its relationship to other DNA damage response genes, we have analyzed the gene status, protein, and mRNA expression in a series of tumors and nonneoplastic lymphoid tissues. CHK1 protein and mRNA expression levels were very low in both reactive tissues and resting lymphoid cells, whereas tumor samples showed a variable pattern of expression related to their proliferative activity. However, seven aggressive tumors showed a dissociate pattern of extremely low or negative protein expression in spite of a high proliferative activity. Four of these tumors were diffuse large B-cell lymphomas (DLCLs) with concordant reduced levels of mRNA, whereas one blastoid mantle cell lymphoma (B-MCL) and two DLCLs had relatively normal levels of mRNA. No gene mutations, deletions, or hypermethylation of the promoter region were detected in any of these cases. In all these tumors ATM, CHK2, and p53 genes were wild type. These findings suggest that CHK1 inactivation in NHLs occurs by loss of protein expression in a subset of aggressive variants alternatively to ATM, CHK2, and p53 alterations.

The transcript release factor PTRF augments ribosomal gene transcription by facilitating reinitiation of RNA polymerase I.

Termination of murine rDNA transcription by RNA polymerase I (Pol I) requires pausing of Pol I by terminator-bound TTF-I (transcription termination factor for Pol I), followed by dissociation of the ternary complex by PTRF (Pol I and transcript release factor). To examine the functional correlation between transcription termination and initiation, we have compared transcription on terminator-containing and terminator-less rDNA templates. We demonstrate that terminated RNA molecules are more efficiently synthesized than run-off transcripts, indicating that termination facilitates reinitiation. Transcriptional enhancement is observed in multiple- but not single-round transcription assays measuring either promoter-dependent or promoter-independent Pol I transcription. Increased synthesis of terminated transcripts is observed in crude extracts but not in a PTRF-free reconstituted transcription system, indicating that PTRF-mediated release of pre-rRNA is responsible for transcriptional enhancement. Consistent with PTRF serving an important role in modulating the efficiency of rRNA synthesis, PTRF exhibits pronounced charge heterogeneity, is phosphorylated at multiple sites and fractionates into transcriptionally active and inactive forms. The results suggest that regulation of PTRF activity may be an as yet unrecognized means to control the efficiency of ribosomal RNA synthesis.

Evaluación del proceso de atención urológica de pacientes pediátricos con mielomeningocele / Evaluation of the urological care process of pediatric patients with myelomeningocele

Introducción: El mielomeningocele (MMC) es una de las malformaciones congénitas más severas compatible con la vida. El 90% de los pacientes presenta vejiga neurogénica que debe ser evaluada y tratada precozmente. Objetivos: Describir la evaluación y tratamiento nefrourológico recibido por pacientes con MMC hasta el momento de la primera consulta en el Hospital Garrahan (periodo pre-ingreso). Describir la evaluación realizada y el tratamiento urológico implementado a partir del ingreso al hospital Garrahan (periodo post-ingreso). Evaluar la prevalencia de Enfermedad Renal Crónica (ERC). Población y Métodos: Se realizó un estudio con diseño clínico analítico, retrospectivo, longitudinal sobre pacientes con MMC de 1 mes a 18 años derivados al Hospital Garrahan para atención ambulatoria en los años 2011 y 2012. Resultados: Se incluyeron115 pacientes. Al momento de la derivación al hospital ("pre-ingreso") 7% de los pacientes habían logrado completar evaluación nefrourológica, (ecografía vesicorenal, urodinamia, Cistouretrografía, Centellograma renal y Creatininemia). Tratamiento: 33% vaciaban vejiga por CIL o vesicostomía y 21% recibían Oxibutinina. A partir del ingreso al seguimiento en el Garrahan 83% lograron completar la evaluación, y en función del resultado de la misma se indicó CIL en 87% y Oxibutinina en el 66% de los pacientes. La prevalencia de ERC al ingreso fue de 43%; la mayoría en estadio I. Conclusiones: La mayoría de los pacientes con MMC fueron derivados al hospital de tercer nivel con evaluaciones urológicas incompletas y sin el tratamiento adecuado de la vejiga neurogénica. El inicio del seguimiento interdisciplinario en un hospital de alta complejidad facilitó la realización de las evaluaciones necesarias y la implementación del tratamiento adecuado (AU)

Introduction: Myelomeningocele (MMC) is one of the most severe congenital malformations compatible with life. Of all the patients, 90% presents with a neurogenic bladder requiring early evaluation and treatment. Objectives: To describe the uronephrological evaluation and treatment received by patients with MMC up to the first consultation at Garrahan Hospital (pre-follow-up period). To describe the urological evaluation and treatment implemented from referral to Garrahan Hospital (follow-up period). To evaluate the prevalence of chronic kidney disease (CKD). Population and Methods: A retrospective, longitudinal study with a clinical, analytical design was conducted in patients with MMC between 1 months and 18 years of age referred to Garrahan Hospital for outpatient care in 2011 and 2012. Results: 115 patients were included. At the time of referral to the hospital ("pre-follow-up") 7% of the patients had undergone complete uronephrological evaluation (kidney-bladder ultrasonography, urodynamic studies, cystourethrography, renal scintigraphy, and creatininemia levels). Treatment: 33% emptied their bladder by CIC or vesicostomy and 21% received oxybutynin. From follow-up initiation at Garrahan Hospital, 83% underwent complete evaluation, and based on the results CIC was indicated in 87% and oxybutynin in 66% of the patients. On admission, prevalence of CKD was 43%; with stage I in the majority of the patients. Conclusions: The majority of the patients with MMC were referred to a third-level hospital with incomplete urological studies and without adequate treatment of the neurogenic bladder. Initiation of interdisciplinary follow-up at a tertiary hospital allowed for the necessary studies and implementation of adequate treatment (AU)

The role of ceramide in receptor- and stress-induced apoptosis studied in acidic ceramidase-deficient Farber disease cells.

The activation of sphingomyelinases leading to the generation of ceramide has been implicated in various apoptotic pathways. However, the role of ceramide as an essential death mediator remains highly controversial. In the present study, we investigated the functional relevance of ceramide in a genetic model by using primary cells from a Farber disease patient. These cells accumulate ceramide as the result of an inherited deficiency of acidic ceramidase. We demonstrate that Farber disease lymphocytes and fibroblasts underwent apoptosis induced by various stress stimuli, including staurosporine, anticancer drugs and gamma-irradiation, equally as normal control cells. In addition, caspase activation by these proapoptotic agents occurred rather similarly in Farber disease and control fibroblasts. Interestingly, Farber disease lymphoid cells underwent apoptosis induced by the CD95 death receptor more rapidly than control cells. Our data therefore suggest that ceramide does not play an essential role as a second messenger in stress-induced apoptosis. However, in accordance with a role in lipid-rich microdomains, ceramide by altering membrane composition may function as an amplifier in CD95-mediated apoptosis.

Circulating heart-reactive antibodies in patients with myocarditis or cardiomyopathy.

Heart-reactive antibodies are commonly observed in patients with myocarditis or cardiomyopathy. Such antibodies may be important in the pathogenesis of these disorders, yet the specific antigens recognized have not been studied systematically. This report characterizes circulating heart autoantibodies from patients with myocarditis (n = 17) or idiopathic cardiomyopathy (n = 71) and from healthy volunteers (n = 15). Indirect immunofluorescence demonstrated that high titer (greater than or equal to 1:20) immunoglobulin G (IgG) antibody activity occurred in 59% of the myocarditis samples, 20% of the cardiomyopathy samples and none of the normal samples. All samples were tested by Western immunoblotting for IgG activity against a normal human heart extract. The number of antigens recognized by each sample was enumerated and the molecular weight of each antigen estimated; the prevalence of reactivity against antigens in selected molecular weight classes was determined. There was no difference in the mean number of heart antigens recognized by serum from each group. For most weight classes, prevalence either did not differ significantly among the various groups or subgroups or was greatest among samples from healthy volunteers. Prevalence of reactivity with 190 to 199 kilodalton (kd) antigens was greatest (p less than 0.05) among low titer serum samples from patients with myocarditis. High titer cardiomyopathy serum differed from normal serum by an increased (p less than 0.05) prevalence of antibodies to 40 to 49 and 100 to 109 kd antigens. These results suggest that western immunostaining may ultimately contribute substantively to identifying patients with myocarditis or cardiomyopathy.

Induction of major histocompatibility complex antigens within the myocardium of patients with active myocarditis: a nonhistologic marker of myocarditis.

The histologic diagnosis of active myocarditis is frequently difficult to establish. A nonhistologic marker of immune activation would be clinically useful in identifying cases of immune-mediated myocarditis. A viral etiology with subsequent autoimmunity to cardiac antigens has been implicated in human myocarditis. Because autoimmunity and viral disease are commonly associated with increased expression of major histocompatibility complex (MHC) antigens on targeted tissue, we examined endomyocardial biopsy samples from patients with active myocarditis for abnormal levels of MHC antigen expression. Thirteen patients with active myocarditis and eight control patients with other well-defined cardiac diagnoses (coronary disease, amyloidosis or neoplasm) were studied. A sensitive radioimmunoassay was developed that utilized monoclonal antibodies to human MHC class I and class II antigens in order to quantitate the expression of both of these antigens within each biopsy. Abnormal MHC class I and class II antigen expression was present in 11 of 13 myocarditis specimens and 1 of 8 control samples (specificity 88%, sensitivity 84.6%). Active myocarditis samples had approximately a 10-fold increase in MHC class I and class II expression. Immunoperoxidase staining localized abnormal MHC expression primarily within microvascular endothelium and along myocyte surfaces (11 of 13). This study is the first to demonstrate a marked increase in major histocompatibility complex antigen expression within the myocardium of patients with active myocarditis. The identification of abnormal histocompatibility antigen expression within an endomyocardial biopsy may prove a useful adjunct to the histologic diagnosis of myocarditis.

Heritability of levels of autoantibodies using the method of plotting regression of offspring on midparent (ROMP).

The genetic control of the levels of autoantibodies has rarely been examined. We examined the heritability of autoantibodies to glutamic acid decarboxylase (GAD65) in type 1 diabetes, and to thyroglobulin (Tg) in chronic lymphocytic thyroiditis and thyrotoxicosis, using regression of offspring on midparent (ROMP) methods. Levels of autoantibodies in patients and their parents were significantly correlated in thyrotoxicosis (R2 = 0.569, p = 0.001), consistent with the reported Gm association, but not in chronic lymphocytic thyroiditis or type 1 diabetes. Extension of the procedure to other autoantibody disorders could be informative.

The role of iodine in autoimmune thyroiditis.

Like most cancers, autoimmune diseases generally are due to the interaction of a number of genetic traits with an environmental trigger. Autoimmune thyroiditis, a model of organ-specific autoimmune disease, is associated with iodine as a precipitating environmental factor. T cells from patients with chronic thyroiditis proliferate in response to normal human thyroglobulin, but fail to react with non-iodinated thyroglobulin. Using a selected monoclonal antibody, we were able to identify a binding site on thyroglobulin containing iodinated thyronine. The greatest affinity was for tetraiodothyronine and binding depended upon the number as well as the positions of iodines. We have also studied an inbred strain of mice, NOD-H2h4, that developed thyroiditis spontaneously. The onset of disease was hastened in a dose-dependent manner by adding iodine to the drinking water. The occurrence of disease was greater in conventional than in specific pathogen-free mice and correlated with T-cell proliferation and IgG2b antibody to thyroglobulin.

Transient thyrotoxicosis in an infant delivered to a long-acting thyroid stimulator (LATS)- and LATS protector-negative, thyroid-stimulating antibody-positive woman with Hashimoto's thyroiditis.

A thyrotoxic infant was delivered to a woman with a long-standing history of Hashimoto's thyroiditis, but with no evidence of Graves' disease. Long-acting thyroid stimulator (LATS) and LATS protector were absent, but thyroid-stimulating antibody was transiently present in the infant and markedly and persistently elevated in the mother. It is concluded that the maternal level of thyroid-stimulating immunoglobulins determines the presence and duration of transient neonatal thyrotoxicosis, and that thyroid-stimulating antibody is distinct from LATS and LATS protector.

Evidence for genetic transmission of thyroid peroxidase autoantibody epitopic "fingerprints".

Autoimmune thyroid disease is characterized by the tendency to cluster in families and by IgG class autoantibodies to antigens such as thyroid peroxidase (TPO). The epitopes recognized by polyclonal serum autoantibodies can be quantitatively fingerprinted using four recombinant human TPO autoantibodies (expressed as Fab) that define A and B domain epitopes in an immunodominant region. To determine whether these fingerprints are genetically transmitted, we analyzed fingerprints of 63 members of 7 multiplex Old Order Amish families and 17 individuals from 4 Hashimoto thyroiditis families. Inhibition of serum autoantibody binding to [125I]TPO by the recombinant Fab was used to assess recognition of the TPO immunodominant region (4 Fab combined) and recognition of domain A or B (individual Fab). Complex segregation analysis was performed using a unified model (POINTER). For the 4 Fab combined inhibition phenotype, the no transmission model was rejected (chi2(4) = 20.67; P < 0.0032), and the most parsimonious model includes a major gene effect. More importantly, evidence for genetic transmission was obtained for the phenotype defined by the ratio of inhibition by subdomain Fab B1:B2. Thus, for this ratio (reflecting recognition of the B domain), the no transmission model was rejected chi2(4) = 63.59; P < 0.000008). Moreover, the polygenic hypothesis could be rejected, but not the major locus hypothesis, suggesting that major genes might be involved in familial transmission of this trait. In conclusion, our findings suggest that autoantibody recognition of the TPO immunodominant region and the TPO B domain is genetically transmitted. These data may open the way to the identification by candidate analysis or positional cloning of at least one gene responsible for the development of Hashimoto's thyroiditis.