Heritability analysis of IgG4 antibodies in autoimmune thyroid disease.

A study of IgG4 autoantibody levels in juvenile thyroid disease patients showed evidence of heritability using the ROMP screening method. These levels increased with time despite the fact that total IgG antibody decreased with time. Evidence of heritability was demonstrated only in patients with high titers of autoantibodies to both thyroglobulin (Tg) and thyroperoxidase (TPO) unlike family members who may show high titers of one or the other and be asymptomatic at the time of sampling. Since high and low IgG4 levels give different heritability plots, these findings may represent a more severe fibrotic form of thyroiditis with a distinct genetic background. Hence a simple predictive approach is offered by this screening tool for the disease in patients and family members which may be helpful in the future to identify IgG4-related thyroiditis early in the course of disease without the requirement for biopsy.

Exclusion of SIX6 hemizygosity in a child with anophthalmia, panhypopituitarism and renal failure.

We report a patient who presented with anophthalmia, panhypopituitarism, early onset of end stage renal failure, and craniofacial abnormalities. MRI at age 3 revealed that the pituitary was absent and renal biopsy demonstrated nephronophthisis as the cause of the renal failure. A similar syndrome has been associated with interstitial deletions of chromosome 14q22 and in one case hemizygosity for SIX6 was demonstrated. The patient reported here had a normal karyotype and Southern blot did not reveal loss of one copy of SIX6. We discuss other possible candidate genes that could be implicated in this syndrome.

Acute activation of peripheral lymphocytes during treatment of diabetic ketoacidosis.

Activated peripheral T-lymphocytes are increased in both pre-insulin-dependent diabetes mellitus (IDDM) patients and in recently diagnosed IDDM patients, as well as in various forms of acute stress. We studied the in vivo T-lymphocyte activation in six patients in severe diabetic ketoacidosis (DKA) prior to treatment, after 24 h of treatment and > or =5 days after admission. Five of the six patients showed an increased percentage of activated T-lymphocytes based on the expression of HLA-DR at 24 h of treatment when compared to the admission percentage of activation (P<.05). There was no correlation to the admission serum glucose, osmolality, or electrolytes. Serum pH showed a trend toward an inverse correlation, but was not statistically significant. We speculate that T-lymphocyte activation plays a role in the progression of the acute complications of subclinical brain edema and interstitial pulmonary edema of DKA. This process could also be another factor in the progression of the chronic complications of IDDM in addition to the well-established effects of hyperglycemia and hypertension.

The corepressor mSin3a interacts with the proline-rich domain of p53 and protects p53 from proteasome-mediated degradation.

While the transactivation function of the tumor suppressor p53 is well understood, less is known about the transrepression functions of this protein. We have previously shown that p53 interacts with the corepressor protein mSin3a (hereafter designated Sin3) in vivo and that this interaction is critical for the ability of p53 to repress gene expression. In the present study, we demonstrate that expression of Sin3 results in posttranslational stabilization of both exogenous and endogenous p53, due to an inhibition of proteasome-mediated degradation of this protein. Stabilization of p53 by Sin3 requires the Sin3-binding domain, determined here to map to the proline-rich region of p53, from amino acids 61 to 75. The correlation between Sin3 binding and stabilization supports the hypothesis that this domain of p53 may normally be subject to a destabilizing influence. The finding that a synthetic mutant of p53 lacking the Sin3-binding domain has an increased half-life in cells, compared to wild-type p53, supports this premise. Interestingly, unlike retinoblastoma tumor suppressor protein, MDMX, and p14(ARF), Sin3 stabilizes p53 in an MDM2-independent manner. The ability of Sin3 to stabilize p53 is consistent with the model whereby these two proteins must exist on a promoter for extended periods, in order for repression to be an effective mechanism of gene regulation. This model is consistent with our data indicating that, unlike the p300-p53 complex, the p53-Sin3 complex is immunologically detectable for prolonged periods following exposure of cells to agents of DNA damage.

Analysis of p53-regulated gene expression patterns using oligonucleotide arrays.

Oligonucleotide microarrays were employed to quantitate mRNA levels from a large number of genes regulated by the p53 transcription factor. Responses to DNA damage and to zinc-inducible p53 were compared for their transcription patterns in cell culture. A cluster analysis of these data demonstrates that genes induced by gamma radiation, UV radiation, and the zinc-induced p53 form distinct sets and subsets with a few genes in common to all these treatments. Cell type- or cell line-specific p53 responses were detected. When p53 proteins were induced with zinc, the kinetics of induction or repression of mRNAs from p53-responsive genes fell into eight distinct classes, five different kinetics of induction, and three different kinetics of repression. In addition, low levels of p53 in a cell induced or repressed only a subset of genes observed at higher p53 levels. The results of this study demonstrate that the nature of the p53 response in diverse mRNA species depends on the levels of p53 protein in a cell, the type of inducing agent or event, and the cell type employed. Of 6000 genes examined for p53 regulatory responses, 107 induced and 54 repressed genes fell into categories of apoptosis and growth arrest, cytoskeletal functions, growth factors and their inhibitors, extracellular matrix, and adhesion genes.

Transcriptional repression by wild-type p53 utilizes histone deacetylases, mediated by interaction with mSin3a.

There is growing evidence that the p53 tumor suppressor protein not only can function to activate gene transcription but also to repress the expression of specific genes. Although recent studies have implicated the transcriptional repression function of p53 in the pathway of apoptosis, the molecular basis of this activity remains poorly understood. This study takes a first step toward elucidating this mechanism. We report that trichostatin A (TSA), an inhibitor of histone deacetylases (HDACs), abrogates the ability of p53 to repress the transcription of two genes that it negatively regulates, Map4 and stathmin. Consistent with this finding, we report that p53 physically associates in vivo with HDACs. This interaction is not direct but, rather, is mediated by the corepressor mSin3a. Both wild-type p53 and mSin3a, but not mutant p53, can be found bound to the Map4 promoter at times when this promoter preferentially associates with deacetylated histones in vivo. Significantly, inhibition of p53-mediated transcriptional repression with TSA markedly inhibits apoptosis induction by p53. These data offer the first mechanistic insights for p53-mediated transcriptional repression and underscore the importance of this activity for apoptosis induction by this protein.

Acetoacetate and beta-hydroxybutyrate differentially regulate endothelin-1 and vascular endothelial growth factor in mouse brain microvascular endothelial cells.

Insulin-dependent diabetes mellitus (IDDM), is characterized by a lack of insulin production from beta cells in the pancreas. One of the metabolic consequences of this insulin deficit is an increased hepatic synthesis of ketone bodies, resulting in a serious medical complication, diabetic ketoacidosis (DKA). DKA, in turn, has been associated with the development of cerebral edema. The severity of this complication ranges from death to a subclinical presentation, but seems to be invariably present to some degree. The etiology of the cerebral edema is unknown, but changes in osmolality, pH, and insulin effects on the blood-brain barrier have all been suggested as possible culprits. Blood-brain barrier impermeability is maintained by the endothelial cells (EC) lining the blood vessels. Thus, it would seem likely that alterations in EC function would be necessary for the development of cerebral edema. However, no studies have examined the effects of ketone bodies on brain endothelial cells. The two major ketone bodies in DKA are acetoacetate (AcAc) and beta-hydroxybutyrate (BOHB). In the present study we examined the effect of these ketone bodies on a major intracellular signalling pathway. The changes in intracellular calcium concentration, and the production of two vasoactive peptides, endothelin-1 (ET-1) and vascular permeability factor (VPF/VEGF) in mouse brain microvascular endothelial cells (MBMEC). The present studies demonstrate the BOHB can increase vascular permeability factor. In contrast, AcAc increases the production of the potent vasoconstrictor, endothelin-1. This data would suggest that brain ECs are potential targets of the metabolic alterations in DKA.

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.

Computer analysis of magnetic resonance imaging of the brain in children and adolescents after treatment of diabetic ketoacidosis.

Cerebral vascular accidents are one of the causes of morbidity and mortality in children with diabetic ketoacidosis. We investigated the possible occurrence of asymptomatic cerebrovascular infarcts and the course of subclinical brain edema in six patients. Neurologic examinations and computer analysis of magnetic resonance imaging were performed immediately after, and again at 14 days after, correction of DKA. None of the patients had clinical evidence of a neurologic deficit. Neither radiologic evaluation nor computer analysis of MRI identified changes indicating asymptomatic ischemic events. However, a computer analysis of the MRI identified a significant increase of the total ventricle area between Day one and Day 14. Our study does not establish whether this change is a return to the baseline prior to DKA or a new baseline, representing an early manifestation of diabetic encephalopathy.

Kenny-Caffey syndrome and microorchidism.

We report on two adolescent boys with Kenny-Caffey syndrome and microorchidism. The first patient had elevated levels of serum follicle-stimulating hormone, but normal levels of luteinizing hormone and testosterone. There was no evidence of a microdeletion of the Y chromosome. The second patient had Leydig cell hyperplasia with normal seminiferous tubules and spermatogenesis, and normal pituitary histologic findings at autopsy. The presence of microorchidism in these patients confirms the previous observations and suggests subfertility, but does not fully clarify the pathogenesis.

HLA-DR alleles are associated with IDDM, but not with impaired neutrophil chemotaxis in IDDM.

Insulin-dependent diabetes mellitus (IDDM) is a risk factor for periodontitis. Depressed neutrophil chemotaxis has been demonstrated in IDDM and in early-onset periodontitis (EOP). HLA-DR antigens are associated with both IDDM and periodontitis. This investigation sought to determine an association of HLA-DR3, -DR4, and -DR53 with impaired neutrophil chemotaxis in an IDDM sample. The neutrophil chemotaxis index of 41 diabetics and 27 controls was determined by a modified Boyden chamber method, and certain class II HLA genotypes were determined by polymerase chain-reaction amplification of genomic DNA by means of sequence-specific primers (PCR-SSP). The mean chemotaxis index of the diabetics was significantly less than that of the controls (p < or = 0.02). HLA-DR3 (p < or = 0.002), -DR4 (p < 0.003), and -DR53 (p < or = 0.001) were associated with IDDM. Neutrophil chemotaxis and glucose metabolism were not significantly correlated. None of the HLA-DR alleles was associated with impaired neutrophil chemotaxis. Therefore, the neutrophil chemotaxis defect of IDDM appears to be independent of these HLA-DR-associated genes.

Interstitial pulmonary edema in children and adolescents with diabetic ketoacidosis.

The acute complications of diabetic ketoacidosis in children and adolescents are well recognized but not completely understood. Clinical studies have focused primarily on brain edema. We have investigated the prevalence and course of interstitial pulmonary edema in patients with severe diabetic ketoacidosis all of whom had uneventful clinical courses. High resolution computed tomography scans of the lungs were analyzed by determining the Hounsfield attenuation level and then converting to physical density values. All seven patients had evidence of interstitial pulmonary edema on the first scan, which was performed within 1 h of hydration and prior to receiving insulin; six of the seven patients had increased pulmonary density 6-8 h into treatment, and all had complete resolution of the interstitial changes at discharge. Our study suggests that subclinical interstitial pulmonary edema may be a frequent occurrence in children and adolescents with severe diabetic ketoacidosis and may very well be present prior to treatment. The study also supports the philosophy of cautious rehydration and the close monitoring of children and adolescents with diabetic ketoacidosis until a more complete understanding of this pathophysiologic event is achieved.

Characterization of monoclonal thyroid-stimulating and thyrotropin binding-inhibiting autoantibodies from a Hashimoto's patient whose children had intrauterine and neonatal thyroid disease.

A multiplicity of TSH receptor autoantibodies (TSHRAbs) have been characterized after subcloning heterohybridomas produced from the lymphocytes of a patient who has Hashimoto's thyroiditis and had three children with intrauterine or neonatal hyperthyroidism. Twelve clones produced stimulating TSHRAbs that increased cAMP levels and iodide uptake in rat FRTL-5 thyroid cells and increased cAMP levels in Chinese hamster ovary (CHO) cells transfected with the human TSHR; like 95% of Graves' stimulating TSHRAbs, all 12 have their functional epitope on the N-terminus of the TSHR extracellular domain, requiring residues 90-165 for activity. All 12 bind to human thyroid membranes in the absence, but not the presence, of TSH, but are only weak inhibitors of TSH binding in assays measuring TSH binding-inhibiting Igs (TBIIs). In contrast, 8 different clones produced TSHRAbs that did not increase cAMP levels, but, instead, exhibited significant TBII activity. Four inhibited the ability of TSH or a stimulating TSHRAb to increase cAMP levels and had their functional epitope on the C-terminal portion of the TSHR external domain, residues 261-370, mimicking the properties of blocking TSHRAbs that cause hypothyroidism in patients with idiopathic myxedema. The 4 other TBIIs inhibited the ability of TSH, but not that of a stimulating TSHRAb, to increase cAMP levels, like TBIIs in Graves' patients. The functional epitope for 3 of these Graves'-like TBIIs was residues 90-165; the functional epitope for the fourth was residues 24-89. The fourth also increased arachidonic acid release and inositol phosphate levels in FRTL-5 thyroid cells and exhibited conversion activity, i.e. the ability to increase cAMP levels in the presence of an anti-human IgG. Thus, this TBII exhibited signal transduction activity, unlike the other 3 Graves'-like TBIIs. The patient, therefore, has stimulating TSHRAbs and 3 different types of TBIIs, each with different functional properties and different epitopes on the TSHR.

Hyperlipidemia, insulin-dependent diabetes mellitus, and rapidly progressive diabetic retinopathy and nephropathy in Prader-Willi syndrome with del(15)(q11.2q13).

We report on a white man with Prader-Willi syndrome (PWS) and del(15)(q11.2q13), confirmed by fluorescence in situ hybridization (FISH), who had hyperlipidemia, insulin-dependent diabetes, and the early onset and rapid progression of diabetic retinopathy and nephropathy within 4 years after diagnosis of diabetes. The spectrum of glucose intolerance in patients with PWS is discussed, as well as those references which suggest that the prevalence of hyperlipoproteinemia in this condition may be greater than previously recognized. We suggest the need for clarification of both the prevalence and types of hyperlipoproteinemia, as well as the pathophysiology of glucose intolerance and correlation with molecular cytogenetic findings. We also encourage careful monitoring for diabetic complications to further clarify the prevalence and possible accelerated course of microvascular lesions.

Interleukin-10 promotes anti-collagen antibody production in type I diabetic peripheral B lymphocytes.

Previous studies have shown that type I diabetes (IDDM) increases the risk of developing periodontitis by 2-3-fold. IDDM patients exhibit destruction of the pancreatic beta cells, most probably caused by an autoimmune reaction. Evidence is accumulating to support the role of the autoimmune response in periodontal pathogenesis. A cytokine, interleukin (IL)-10, has been reported to selectively promote the expansion of a B lymphocyte lineage (CD5/LY1/B1) which has the propensity for secreting high levels of autoantibody. Therefore, the purpose of this project was to evaluate IL-10 production, percentage of CD5 B cells and the frequency of anti-collagen secreting cells in peripheral blood mononuclear cells of age, gender and race matched IDDM patients and controls. IL-10 production was evaluated by an ELISA using the supernatant of adherent peripheral blood cells cultured for 24 h in the presence of Porphyromonas gingivalis lipopolysaccharide (LPS). In 8 of 31 patients, IL-10 levels were significantly increased in IDDM compared to controls and a higher percentage of CD5 B cells was also observed by flow cytometry. In addition, these patients exhibited a higher frequency of anti-collagen secreting cells as elucidated by an ELISPOT. Moreover, treatment with a neutralizing anti-IL-10 antibody diminished the anti-collagen antibody response by 70%. These findings support the concept that a subset of IDDM patients possess an extremely robust IL-10 response following exposure to Gram-negative LPS, which could predispose them to the development of periodontitis through a heightened autoimmune mechanism.

Kindling-induced epileptiform potentials in piriform cortex slices originate in the underlying endopiriform nucleus.

1. Previous studies in vivo and in vitro have shown that kindling from several locations in the limbic system induces the onset of epileptiform activity in the piriform (olfactory) cortex in the rat. In the present study we tested the hypothesis that kindled epileptiform events in piriform cortex are initiated in the underlying endopiriform nucleus. The experiments were performed in slices taken from rats that were previously kindled by conventional means. 2. Both stimulus-evoked and spontaneous interictal-like epileptiform events were observed in most slices from the anterior piriform cortex, but in few slices from the posterior piriform cortex. These events resembled those described in unanesthetized and urethan-anesthetized rats in previous studies. 3. Findings in support of the hypothesis were as follows. Epileptiform events in the endopiriform nucleus preceded those in the piriform cortex. Epileptiform events could occur in endopiriform nucleus alone, but were only observed in the piriform cortex following occurrence in the endopiriform nucleus. A buildup in population activity preceded the onset of all-or-none epileptiform events in the endopiriform nucleus. Epileptiform events could be triggered by local application of glutamate in the endopiriform nucleus and adjacent claustrum, but not from the piriform cortex. Finally, local application of Co2+ in the endopiriform nucleus, but not in the piriform cortex or elsewhere in the slices, blocked the occurrence of epileptiform events. 4. Additional experiments were performed to further characterize the generation process. 6,7-Dinitroquinoxaline-2,3-dione (DNQX) blocked epileptiform events and the preceding accelerating buildup in multiunit activity at a concentration below that required to block the monosynaptic excitatory postsynaptic potential (EPSP). This suggests that EPSPs mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors underlie epileptiform events in slices of piriform cortex, and that multisynaptic interactions within the endopiriform nucleus are required for generation of these epileptiform EPSPs. By contrast, block of N-methyl-D-aspartate (NMDA) receptors decreased the amplitude of epileptiform EPSPs but did not block their occurrence, indicating that NMDA receptors contribute to generation but are not required. When membrane potential was depolarized to increase driving force, fast inhibitory postsynaptic potentials were found to consistently accompany the buildup process and epileptiform EPSPs. This indicates that if initiation of epileptiform activity in the endopiriform nucleus results from a compromise in feedback inhibition, this compromise is partial rather than complete. 5. Epileptiform EPSPs in slices of piriform cortex from kindled rats displayed similarities in properties, locus of origin, and mechanism of generation to those previously studied in slices from normal rats in which epileptiform activity was induced by a brief period of bursting activity. These similarities suggest that study of bursting-induced epileptiform EPSPs may provide insight into certain aspects of kindling-induced epileptogenesis.

Phenotypic evolution of classic 21-hydroxylase deficiency.

We describe a female patient who was diagnosed and treated at birth for a classic form of salt-losing congenital adrenal hyperplasia. At 17 years of age, against medical advice, she discontinued both mineralocorticoid and glucocorticoid replacement with no resulting clinical symptoms other than the occurrence of amenorrhoea. Steroid metabolites revealed significant abnormalities of the renin-angiotensin-aldosterone axis, as well as of pituitary-adrenal function. Analysis of our patient's DNA showed only one deleterious CYP21 mutation, an intron 2 base pair change activating a cryptic splice site. We speculate that expression of this patient's CYP21 genes may be altered by the effects of ageing or by changes in the steroid milieu.

Thyroid peroxidase autoantibody epitopic 'fingerprints' in juvenile Hashimoto's thyroiditis: evidence for conservation over time and in families.

In Hashimoto's thyroiditis, the humoral component is manifest by autoantibodies to thyroid peroxidase (TPO). Epitopic 'fingerprinting' of polyclonal serum TPO autoantibodies has been facilitated by the molecular cloning and expression as Fab of a repertoire of human TPO autoantibody genes. To investigate whether TPO autoantibody fingerprints are (i) stable over long periods of time (approximately 15 years), and (ii) inherited, we studied a cohort of nine patients with juvenile Hashimoto's thyroiditis and 21 first degree relatives of four of these patients. Fingerprints were determined by competition between four selected FAB and serum autoantibodies for binding to 125I-TPO. Regardless of titre, the TPO epitopic profile was stable in 10/12 individuals whose TPO autoantibody levels were sufficient for analysis on two or three occasions over 12-15 years. Although the TPO epitopic fingerprint profiles in two families raised the possibility of inheritance, overall the data from all four families did not reveal an obvious pattern of genetic control. In no family was the TPO epitopic fingerprint associated with HLA A, B or DR. In conclusion, TPO autoantibody epitopic fingerprints are frequently conserved over many years. Studies on additional families are necessary to establish whether or not the epitopic profiles of TPO autoantibodies are inherited.