Alterations of cholinergic receptors and the vesicular acetylcholine transporter after lateral fluid percussion injury in newborn piglets.

AIMS: Traumatic brain injury (TBI) is one of the leading causes of death and disability in children. Adult animal models of TBI showed cholinergic alterations. However, there is no comparable data on immature animals. Therefore, this study investigates cholinergic markers in a large animal model of juvenile TBI. METHODS: Twenty-seven female newborn piglets were subjected to lateral fluid percussion (FP) injury and compared with 12 untreated animals. After 6 h, animals were sacrificed and the brains removed. The hemispheres ipsilateral to FP-TBI from seven piglets and corresponding hemispheres from six control animals were used for autoradiography. Receptor density was determined with [(3)H]epibatidine (nicotinic acetylcholine receptors) or [(3)H]QNB (muscarinic acetylcholine receptors). The density of the vesicular acetylcholine transporter (vAChT) was assessed with (-)-[(3)H]vesamicol. Cerebral blood flow was measured by coloured microsphere method. RESULTS: Cerebral blood flow and brain oxygen delivery were transiently reduced early after FP-TBI (P < 0.05). TBI caused reductions of muscarinic acetylcholine receptor density (fmol/mg) in the basal forebrain (sham: 10797 +/- 1339, TBI: 8791 +/- 1031), while nicotinic acetylcholine receptor remained stable. Significant increases in vAChT density (fmol/mg) were observed in the basal forebrain (sham: 2347 +/- 171, TBI: 2884 +/- 544), putamen (sham: 2276 +/- 181, TBI: 2961 +/- 386), cortex (sham: 1928 +/- 262, TBI: 2377 +/- 294), thalamic areas (sham: 2133 +/- 272, TBI: 2659 +/- 413), hippocampus (sham: 2712 +/- 145, TBI: 3391 +/- 501) and hypothalamus (sham: 2659 +/- 139, TBI: 3084 +/- 304). CONCLUSIONS: Cholinergic markers are altered after mild-to-moderate TBI in the immature brain. Whereas the ACh receptors are stable in almost any brain region after TBI, vAChT expression increases after trauma at the employed severity of this specific trauma model.

Yersinia enterocolitica infection does not confer an increased risk of thyroid antibodies: evidence from a Danish twin study.

Understanding of the aetiological basis of thyroid autoimmunity may be gained by studying the early stages of the disease process. We aimed to (1) investigate the relationship between thyroid antibody status and Yersinia enterocolitica (YE) infection in euthyroid subjects and (2) explore the relative importance of genetic and environmental risk factors in the acquisition of YE infection. The association between thyroid antibody status and YE infection was explored using a case-control design. Furthermore, thyroid antibody-positive twins were compared with their thyroid antibody-negative co-twin. In 468 twins, IgA and IgG antibodies to virulence-associated outer-membrane proteins (YOPs) of YE were measured. Of these, 147 were thyroid antibody-positive (cases). A total of 147 age- and gender-matched twins were chosen as controls. The prevalence of YOP antibodies was lower among thyroid antibody-positive individuals than among controls. Yersinia infection was not associated with a positive thyroid antibody status: the odds ratio (with 95% CI) for YOP IgA-ab was 0.66 (0.42-1.05), P = 0.078 and for YOP IgG-ab it was 0.95 (0.60-1.50), P = 0.816. Within discordant twin pairs, the thyroid antibody-positive twin did not have an increased risk of Yersinia infection compared to the thyroid antibody-negative co-twin [odds ratio: YOP IgA-Ab: 0.94 (0.49-1.83), P = 0.866, and YOP IgG-Ab: 1.35 (0.72-2.53), P = 0.345]; 41% (95% CI 10-67% of the liability of being YOP antibody-positive was due to genetic effects. In conclusion, Yersinia infection does not confer an increased risk of thyroid antibodies. The genetic contribution in the acquisition of Yersinia infection is modest.

[Reduced radioiodine uptake at increased iodine intake and 131I-induced release of "cold" iodine stored in the thyroid]. / Verminderter Radioiod-Uptake durch erhöhte Iodzufuhr und 131I-induzierte Freisetzung von thyreoidal gespeichertem "kalten" Iod.

AIM: The extent of urinary iodine excretion (UIE) provides information about iodine supply and release. In the present study we investigated correlations between UIE and radioiodine uptake (RIU) as well as effects of radioiodine therapy on UIE in patients with autonomous goitre. PATIENTS, METHODS: In 197 consecutive patients with thyroid autonomy, UIE was measured twice during radioiodine test (RITe) and correlated with RIU. In 98 of these patients, thyroglobulin and thyroid volume (V) were determined prior to therapy. Individual changes in urinary iodine excretion (DeltaUIE) and TG (DeltaTG) could be investigated four weeks (4W) and six months (6M) after radioiodine therapy. Additionally, DeltaV was determined 6M after therapy. DeltaUIE, DeltaTG and DeltaV were correlated with target dose and target volume. RESULTS: Patients with higher iodine excretion exhibited significantly lower thyroidal radioiodine uptake values. Twofold increased UIE prior to therapy decreased radioiodine uptake by 25%. Compared with pretherapeutic values, UIE and TG were significantly increased four weeks after radioiodine therapy (p < 0.001). Median values of both parameters were found to be doubled. The product of target dose and target volume was not only correlated with a decrease of thyroid volume 6M after therapy, but also with an increase of UIE and TG in the early phase after therapy. CONCLUSIONS: It was confirmed that UIE during RITe is a measure for iodine intake and can be used to investigate the competition between stable iodine and radioiodine. The increase of UIE and TG four weeks after therapeutic administration of radioiodine can be explained by disintegrated thyroid follicles. The therapy-induced iodine release may be one important cause for the development of hyperthyroidism in some patients during the first weeks after radioiodine therapy. It may contribute to the known decrease of radioiodine uptake after preapplications of 131I in various thyroid diseases.

Expression of pendrin in benign and malignant human thyroid tissues.

The Pendred syndrome gene (PDS) encodes a transmembrane protein, pendrin, which is expressed in follicular thyroid cells and participates in the apical iodide transport. Pendrin expression has been studied in various thyroid neoplasms by means of immunohistochemistry (IHC), Western blot and RT-quantitative real-time PCR. The expression was related to the functional activity of the thyroid tissue. Follicular cells of normal, nodular goitre and Graves' disease tissues express pendrin at the apical pole of the thyrocytes. In follicular adenomas, pendrin was detected in cell membranes and cytoplasm simultaneously in 10 out of 15 cases. Pendrin protein was detected in 73.3 and 76.7% of the follicular (FTC) and papillary (PTC) thyroid carcinomas, respectively, where pendrin was solely localised inside the cytoplasm. An extensive intracellular immunostaining of pendrin was observed in six out of 11 (54.5%) of positive FTCs and 19 out of 23 (82%) of PTCs. Focal reactivity was detected in one follicular- and three papillary carcinomas, whereas pendrin protein was absent in three of 15 FTC and four of 30 PTC; mRNA of pendrin was detected in 92.4% of thyroid tumours. The relative mRNA expression of pendrin was lower in cancers than in normal thyroid tissues (P<0.001). The pendrin protein level was found to parallel its mRNA expression, which was not, however, related to the tumour size and tumour stage. In conclusion, pendrin is expressed in the majority of differentiated thyroid tumours with high individual variability but its targeting to the apical cell membrane is affected.

Increased prevalence of antibodies to enteropathogenic Yersinia enterocolitica virulence proteins in relatives of patients with autoimmune thyroid disease.

Infections have been implicated in the pathogenesis of a number of autoimmune diseases, and Yersinia enterocolitica (YE) might play a role in the development of autoimmune thyroid disease (AITD). Clinical evidence in support of this hypothesis has been inconclusive. We reasoned that looking earlier in the natural course of AITD might enhance chances of finding evidence for YE infection. Consequently, we determined seroreactivity against YE in subjects at risk of developing AITD, i.e. in 803 female relatives of AITD patients in self-proclaimed good health. As a comparison group we used 100 healthy women who participated in a program for reference values. IgG and IgA antibodies to virulence-associated outer membrane proteins (YOPs) of YE were measured by a specific assay. Serum thyroid peroxidase antibodies (TPO-Ab) as indicators of AITD were considered to be positive at levels of> 100 kU/l. The prevalence of YOP IgG-Ab was higher in AITD relatives than in controls (40.1% vs. 24%, P = 0.002), and the same was true for YOP IgA-Ab (22% vs. 13%, P < 0.05). Of the 803 AITD relatives, 44 had an increased or decreased plasma TSH, and 759 were euthyroid as evident from a normal TSH; the prevalence of YOP-Ab did not differ between these three subgroups. TPO-Ab were present in 10% of controls and in 27% of the AITD relatives (P < 0.001). The prevalence of TPO-Ab in the euthyroid AITD relatives was not different between YOP IgG-Ab positive and negative subjects (23.3% vs. 24.7%, NS), nor between YOP IgA-Ab positive and negative subjects (21.2% vs. 24.9%, NS). In conclusion, healthy female relatives of AITD patients have an increased prevalence of YOP antibodies, which, however, is not related to the higher prevalence of TPO antibodies in these subjects. The findings suggest a higher rate of persistent YE infection in AITD relatives. Susceptibility genes for AITD may also confer a risk for YE infection.

mAChRs in the grasshopper brain mediate excitation by activation of the AC/PKA and the PLC second-messenger pathways.

The species-specific sound production of acoustically communicating grasshoppers can be stimulated by pressure injection of both nicotinic and muscarinic agonists into the central body complex and a small neuropil situated posterior and dorsal to it. To determine the role of muscarinic acetylcholine receptors (mAChRs) in the control of acoustic communication behavior and to identify the second-messenger pathways affected by mAChR-activation, muscarinic agonists and membrane-permeable drugs known to interfere with specific mechanisms of intracellular signaling pathways were pressure injected to identical sites in male grasshopper brains. Repeated injections of small volumes of muscarine elicited stridulation of increasing duration associated with decreased latencies. This suggested an accumulation of excitation over time that is consistent with the suggested role of mAChRs in controlling courtship behavior: to provide increasing arousal leading to higher intensity of stridulation and finally initiating a mating attempt. At sites in the brain where muscarine stimulation was effective, stridulation could be evoked by forskolin, an activator of adenylate cyclase (AC); 8-Br-cAMP-activating protein kinase A (PKA); and 3-isobuty-1-methylxanthine, leading to the accumulation of endogenously generated cAMP through inhibition of phosphodiesterases. This suggested that mAChRs mediate excitation by stimulating the AC/cAMP/PKA pathway. In addition, muscarine-stimulated stridulation was inhibited by 2'-5'-dideoxyadenonsine and SQ 22536, two inhibitors of AC; H-89 and Rp-cAMPS, two inhibitors of PKA; and by U-73122 and neomycin, two agents that inhibit phospholipase C (PLC) by independent mechanisms. Because the inhibition of AC, PKA, or PLC by various individually applied substances entirely suppressed muscarine-evoked stridulation in a number of experiments, activation of both pathways, AC/cAMP/PKA and PLC/IP(3)/diacylglycerine, appeared to be necessary to mediate the excitatory effects of mAChRs. With these studies on an intact "behaving" grasshopper preparation, we present physiological relevance for mAChR-evoked excitation mediated by sequential activation of the AC- and PLC-initiated signaling pathways that has been reported in earlier in vitro studies.

A role for muscarinic excitation: control of specific singing behavior by activation of the adenylate cyclase pathway in the brain of grasshoppers.

Muscarinic acetylcholine receptors exert slow and prolonged synaptic effects in both vertebrate and invertebrate nervous systems. Through activation of G proteins, they typically decrease intracellular cAMP levels by inhibition of adenylate cyclase or stimulate phospholipase C and the turnover of inositol phosphates. In insects, muscarinic receptors have been credited with two main functions: inhibition of transmitter release from sensory neuron terminals and regulation of the excitability of motoneurons and interneurons. Our pharmacological studies with intact and behaving grasshoppers revealed a functional role for muscarinic acetylcholine receptors as being the basis for specific arousal in defined areas of the brain, underlying the selection and control of acoustic communication behavior. Periodic injections of acetylcholine into distinct areas of the brain elicited songs of progressively increasing duration. Coinjections of the muscarinic receptor antagonist scopolamine and periodic stimulations with muscarine identified muscarinic receptor activation as being the basis for the underlying accumulation of excitation. In contrast to reports from other studies on functional circuits, muscarinic excitation was apparently mediated by activation of the adenylate cyclase pathway. Stimulation of adenylate cyclase with forskolin and of protein kinase A with 8-Br-cAMP mimicked the stimulatory effects of muscarine whereas inhibition of adenylate cyclase with SQ22536 and of protein kinase A with H-89 and Rp-cAMPs suppressed muscarine-stimulated singing behavior. Activation of adenylate cyclase by muscarinic receptors has previously been reported from studies on membrane preparations and heterologous expression systems, but a physiological significance of this pathway remained to be demonstrated in an in vivo preparation.

Pharmacological brain stimulation releases elaborate stridulatory behaviour in gomphocerine grasshoppers--conclusions for the organization of the central nervous control.

Grasshoppers produce a variety of sounds generated by complex movements of the hindlegs. Stridulation, performed in the context of partner finding, mating and rivalry, can be released by pressure injection of cholinergic agonists into the protocerebrum. Particularly stimulation with muscarinic agonists induced long-lasting stridulation that resembled the natural behaviour to an astonishing degree, not only with respect to their temporal structure and right/left coordination, but also to changes in the song sequences according to the progress of courtship stridulation, even including accessory movements of other parts of the body. According to the complexity of their stridulatory behaviour ten gomphocerine species were chosen for this comparative study. The results indicate that the protocerebrum fulfils two important tasks in the control of stridulation: (1) it integrates sensory input relevant to stridulation that represents a certain behavioural situation and internal state of arousal, and (2) it selectively activates and deactivates the thoracic networks that generate the appropriate movement and sound patterns. With the knowledge of the natural behaviour and the accessibility to pharmacological and electrophysiological studies, the cephalic control system for stridulation in grasshoppers appears to be a suitable model for how the brain selects and controls appropriate behaviours for a given situation.

Comparison of glucose concentration in interstitial fluid, and capillary and venous blood during rapid changes in blood glucose levels.

The relationship between glucose concentrations in interstitial fluid (ISF) and blood has generated great interest due to its importance in minimally invasive and noninvasive techniques for measuring blood glucose. The relationship between glucose levels in dermal ISF, and capillary and venous blood was studied with the dermal ISF samples obtained using the suction blister technique. The study was conducted with intensely managed diabetics whose blood glucose levels were manipulated so as to induce rapid changes in blood glucose levels. Glucose levels in the three compartments exhibited high correlations both when individual subjects were considered separately and when data from all subjects were combined. No significant time lag during glucose excursions was observed among the ISF, and capillary and venous glucose levels.

Design and evolution of the data management systems in the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial.

This paper describes the design and evolution of the data management systems developed in support of the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. These systems span platforms from stand-alone computers to distributed systems on local area networks to mainframes. Allowing all of these systems to share appropriate information electronically introduces integration, synchronization, testing, and support challenges. For each platform, applications were developed to handle data entry, editing, trial management, reporting, telecommunications, and data sharing. Approaches to issues such as level of data access, integration with other, existing applications, and handling the expansion of the protocol are discussed.

Enhanced intestinal expression of heat shock protein 70 in patients with inflammatory bowel diseases.

Heat shock protein (Hsp) 70 is stress-inducible and exhibits both protective and antigenic properties. This study investigated the mucosal expression of the constitutive (Hsp70c) and inducible form (Hsp70i) as well as antibodies against human Hsp70 in inflammatory bowel disease and controls. Biopsies were assessed by immunoblot and immunofluorescence, resection specimens by immunohistochemistry, and mucosal antibody content by isoelectric focusing. Compared to controls, expression of Hsp70 was enhanced in ulcerative colitis (P<0.05), less so in Crohn's disease and infectious colitis. Strong epithelial staining was found for Hsp70c and Hsp70i in both diseases. Mucosal and submucosal mononuclear cells showed enhanced Hsp70c expression in Crohn's disease and to a lesser degree in ulcerative colitis. Antibodies of isotypes A or M were detected in nearly all patients and controls. The different pattern of Hsp70 expression in Crohn's disease compared to ulcerative colitis points to a distinct protective and immunological function, whereas a role in autoimmunity seems unlikely.

Substrate inhibition and affinities of the glyoxysomal beta-oxidation of sunflower cotyledons

During the glyoxysomal beta-oxidation of long-chain acyl-CoAs, short-chain intermediates accumulate transiently (Kleiter and Gerhardt 1998, Planta 206: 125-130). The studies reported here address the underlying factors. The studies concentrated upon the aspects of (i) chain length specificity and (ii) metabolic regulation of the glyoxysomal beta-oxidation of sunflower (Helianthus annuus L.) cotyledons. (i) Concentration-rate curves of the beta-oxidation of acyl-CoAs of various chain lengths showed that the beta-oxidation activity towards long-chain acyl-CoAs was higher than that towards short-chain acyl-CoAs at substrate concentrations <20 &mgr;M. At substrate concentrations >20 &mgr;M, long-chain acyl-CoAs were beta-oxidized more slowly than short-chain acyl-CoAs because the beta-oxidation of long-chain acyl-CoAs is subject to substrate inhibition which had already started at 5-10 &mgr;M substrate concentration and results from an inhibition of the multifunctional protein (MFP) of the beta-oxidation reaction sequence. However, low concentrations of free long-chain acyl-CoAs are rather likely to exist within the glyoxysomes due to the acyl-CoA-binding capacity of proteins. Consequently, the beta-oxidation rate towards a parent long-chain acyl-CoA will prevail over that towards the short-chain intermediates. (ii) Low concentrations (

Neurochemical control of cricket stridulation revealed by pharmacological microinjections into the brain.

Neuroactive substances were administered into the frontal protocerebrum of tethered male Gryllus bimaculatus by pressure injections from microcapillaries. All three types of species-specific song pattern (calling song, rivalry song and courtship song) could be elicited by injection of acetylcholine and cholinergic agonists. Injection of nicotine led to short bouts of calling song that occurred after a short latency. In contrast, muscarine elicited long-lasting stridulation that took longer to develop. The pharmacologically induced song patterns showed transitions from rivalry song to calling song and from calling song to courtship song, which also occur during natural behaviour. Stridulation induced by a cholinergic agonist could be immediately blocked by microinjection of (&ggr;)-aminobutyric acid (GABA) into the same neuropile sites. Administration of picrotoxin in resting crickets led to enhanced motor activity that incorporated the three different song patterns. We propose that, in the brain of the cricket, acetylcholine and GABA are putative transmitters involved in the control of stridulation. Histological analysis located the stimulation sites to an area between the pedunculus and the (&agr;)-lobe of the mushroom body in which the command neurons for calling song have dendritic arborizations.

Natural killer cell activity in patients with Graves' disease and Hashimoto's thyroiditis.

Natural killer (NK) cell activity of peripheral blood lymphocytes (PBL) against k562 human tumor cell targets was studied in patients with Graves' disease and Hashimoto's thyroiditis. NK activity was measured in a standard 4-hour 51chromium (Cr) release assay. Cytotoxicity was expressed as lytic units (LU)/10(6) PBL. Significantly decreased NK cell activity was demonstrated in both groups of patients, with mean (+/- SE) lytic units of 10.3 (+/- 9.1) and 13.3 (+/- 10.3) for patients with Graves' disease and Hashimoto's thyroiditis, respectively, compared with 36.0 (+/- 26.3) for age- and sex-matched normal subjects. When patients with Graves' disease were analyzed according to their thyroid status; NK activity was significantly depressed in (1) hyperthyroid patients before treatment; (2) hyperthyroid patients receiving antithyroid therapy; and (3) euthyroid patients receiving antithyroid therapy, compared with normal subjects. Graves' disease patients who were hypothyroid after radioactive iodine therapy or thyroidectomy had normal NK activity. No significant differences between hyperthyroid and euthyroid patients or between hypothyroid patients and normal subjects were demonstrated. NK activity in patients with Graves' disease did not correlate with serum levels of thyroxine, the presence or severity of ophthalmopathy, or titers of serum thyroid antibodies. In patients with Hashimoto's thyroiditis there was no correlation between NK activity and goiter size, titers of antithyroid antibodies, or thyroid status. These findings suggest that depression of NK activity in both disorders is secondary to abnormalities of thyroid hormone secretion, although an effect of the underlying autoimmune reactions has not been excluded.

In old age the majority of thyroid peroxidase autoantibodies are directed to a single TPO domain irrespective of thyroid function and iodine intake.

OBJECTIVE: We have examined (1) which epitopes on thyroid peroxidase (TPO) are recognized by TPO autoantibodies (TPO-Aab) in old age and to what extent? (2) Does the TPO-Aab pattern differ in euthyroid and hypothyroid elderly subjects or does it depend on their iodine intake? DESIGN: TPO-Aab positive sera obtained from a screening study of nursing-home residents living in areas of varying iodine intake were tested by competition studies with monoclonal antibodies (mAbs) recognizing different epitopes on TPO. SUBJECTS: The nursing-home residents with TPO-Aab positivity were from (A) an iodine abundant area (Eastern Hungary, median iodine excretion -MIE-: 0.462 mumol/mmol creatinine, N = 13); (B) an area of obligatory iodinated salt prophylaxis since the 1950s (Slovakia, MIE: 0.090 mumol/mmol creatinine, N = 11); (C) a moderately iodine-deficient area (Northern Hungary, MIE: 0.065 mumol/mmol creatinine, N = 13). MEASUREMENTS: Thirteen murine TPO antibodies generated against several epitopes of the four (A, B, C, D) antigenic domains on the TPO were co-incubated with the TPO-Aab positive sera on TPO coated microtitre plates. The amount of mAb bound was estimated after further incubation with goat anti-mouse antibodies, conjugated with horseradish peroxidase and tetramethylbenzidine as chromogen. The TPO-Aab positive sera were characterized by the pattern of percentage of inhibition of mAb binding caused by the TPO-Aabs. RESULTS: TPO-Aabs inhibited only the binding of mAbs raised against the antigenic domains A (mAb9, mAb2, mAb60) and B (mAb64, mAb59, mAb18, mAb15). The extent of inhibition depended upon the TPO-Aab titre but in all cases the binding of mAb9 was inhibited to the highest degree. The percentage inhibition of mAb9 was (a) 34 +/- 17% (M +/- SD) caused by sera (N = 8) with TPO-Aab titre 1/100-1/200 (higher than that of all mAbs recognizing domain B, P < 0.01-P < 0.001), (b) 76 +/- 18% caused by sera (N = 14) with TPO-Aab titre 1/1000 (higher than that of all other mAbs -P < 0.01-P < 0.001, except mAb64), (c) 99 +/- 4% caused by sera (N = 15) with TPO-Aab titre 1/4000-1/16,000 (higher than that of all other mAbs, P < 0.01-P < 0.001). Thus, only mAb9 was inhibited completely by high titres of TPO-Aabs. The qualitative and quantitative distribution pattern of mAb inhibition was similar in the subgroups of elderly hypothyroid and euthyroid subjects with comparable TPO-Aab levels, as well as in the subgroups with varying iodine intake. CONCLUSIONS: (1) In old age, there is a polyclonal TPO autoantibody response but the majority of the autoantibodies are directed to the TPO region mapped by or close to mAb9 (domain A); (2) the autoantibody response does not differ in elderly subjects with or without the clinical manifestations of autoimmune thyroid disease and does not depend on the iodine supply of the elderly subjects.

Antigen receptor variable region repertoires expressed by T cells infiltrating thyroid, retroorbital, and pretibial tissue in Graves' disease.

To date, it has remained unclear whether T cells infiltrating thyroid, retroorbital, and pretibial tissue of patients with Graves' ophthalmopathy and pretibial dermopathy represent a primary immune response that is directed against certain antigenic determinants shared among these involved tissues. To characterize these T cells at the molecular level, we compared the T cell antigen receptor (TcR) variable (V) region gene usage in thyroid, retroorbital, pretibial tissue, and peripheral blood mononuclear cells of two patients with Graves' disease, ophthalmopathy, and pretibial dermopathy. Ribonucleic acid was extracted, reverse transcribed, and amplified using the PCR and 22 V alpha and 23 V beta gene-specific oligonucleotide primers. The resulting TcR V alpha and V beta transcripts were verified by Southern hybridization analysis using TcR C region-specific, digoxigenin-labeled oligonucleotide probes. In addition, complementarity determining regions 3 and junctional regions of TcR V beta genes were sequenced. Marked similarities of intrathyroidal, retroorbital, and pretibial TcR V alpha and V beta gene repertoires were noted with respect to the degree of TcR V gene restriction and the patterns of individual V genes expressed. Sequence analysis of junctional domains of V beta families revealed oligoclonality of intrahyroidal, retroorbital, and pretibial T cells. In addition, certain conserved junctional motifs were shared by T cells derived the thyroid gland and the extrathyroidal sites. Our results suggest that in the two patients with Graves' disease and extrathyroidal manifestations studied, similar antigenic determinants may have contributed to the recruitment and oligoclonal expansion of T cells both within the thyroid gland and at the involved extrathyroidal sites.

Transgenic mice models in autoimmunity--discussion.

Transgenic mice are an important in vivo model for studying the function of single genes. Specific induction and tissue specific expression of the inserted genes are the great advantages of this system. However, there a risks in constructing transgenic mice and the interpretation of the experimental data. To prevent artefacts and to optimize the transgenic model, the experimental systems have to fulfill the following presets: 1. Experiments has to be done with a stable transgenic line and not with the first heterogeneous transgeniced generation, because they differ in their site of gene insertion and in the cellular response. 2. The number of experiments have to be statistically sufficient. Often only a few number of experiments are done, due to the bad reproduction of transgenic mice. 3. Health of transgenic mice has to be proven, since the transgenic animals are under laboratory conditions and not under professional breeding conditions. 4. As a control there should be a transgenic mouse with a comparable pseudogene insertion beside the normal littermate control, to exclude artefacts by the simple gene insertion. If all of these presets are fulfilled, a transgenic mice model will be one of the best experimental systems.

Bacterial virulence antigens and the pathogenesis of autoimmune thyroid diseases (AITD).

There is now substantial evidence that autoimmune thyroid diseases (AITD) coincide with a subclinical persisting infection with Yersinia enterocolitica (YE) which manifests through humoral and cellular immune reactions against YE at the onset of AITD. The humoral and cellular crossreactivities of YE with thyroid autoantigens are exclusively directed against conformational epitopes of YE membrane associated antigens and of YE plasmid encoded virulence proteins (YOPs). Especially, the outer membrane domain of the TSH-Receptor (THSR) appears to have conformational homologies with YE antigens. Immunological- and molecular findings, however, do not allow definite conclusions about a potential role of YE-infection in AITD, although the evidence is suggestive. Recent investigations on the effect of YE-superantigen (Sag) on T-cells from patients with AITD as well as AITD like manifestations in YE immunized mice and rats may yield more conclusive information about the role of YE infection in the pathogenesis of AITD.

Molecular analysis of antigen receptor variable region repertoires in T lymphocytes infiltrating the intrathyroidal and extrathyroidal manifestations in patients with Graves' disease.

To determine whether T cells infiltrating thyroid, orbital and pretibial tissue of patients with Graves' ophthalmopathy (GO) and pretibial dermopathy (PTD) represent a primary immune response that is directed against certain antigenic determinants shared between these involved tissues, we characterized these T cells at the molecular level. T cell antigen receptor (TcR) variable (V) region gene usage in thyroid, orbital, pretibial tissue and peripheral blood mononuclear cells of patients with GD, GO and PTD was assessed using RT-PCR and 22 V alpha and 23 V beta gene-specific oligonucleotide primers, followed by Southern hybridization analysis using TcR C-region-specific, digoxigenin-labelled oligonucleotide probes. In some instances, CDR3- and junctional regions of TcR V beta genes were sequenced. Marked restriction and similarities of V alpha and V beta gene usage were detected in samples derived from patients with active GO and PTD of recent onset. Moreover, sequence analysis of junctional domains of V beta families revealed oligoclonality of some intrathyroidal, orbital and pretibial T cell populations as well as the presence of conserved junctional motifs shared by T cells derived the thyroid gland and the extrathyroidal sites. These data suggest that similar antigenic determinants may be responsible for the recruitment and oligoclonal expansion of T cells both within the thyroid gland and at the involved extrathyroidal sites in Graves' disease.

Molecular cloning, sequencing and sequence analysis of the fox-2 gene of Neurospora crassa encoding the multifunctional beta-oxidation protein.

We present the molecular cloning and sequencing of genomic and cDNA clones of the fox-2 gene of Neurospora crassa, encoding the multifunctional beta-oxidation protein (MFP). The coding region of the fox-2 gene is interrupted by three introns, one of which appears to be inefficiently spliced out. The encoded protein comprises 894 amino acid residues and exhibits 45% and 47% sequence identity with the MFPs of Candida tropicalis and Saccharomyces cerevisiae, respectively. Sequence analysis identifies three regions of the fungal MFPs that are highly conserved. These regions are separated by two segments that resemble linkers between domains of other MFPs, suggesting a three-domain structure. The first and second conserved regions of each MFP are homologous to each other and to members of the short-chain alcohol dehydrogenase family. We discuss these homologies in view of recent findings that fungal MFPs contain enoyl-CoA hydratase 2 and D-3-hydroxyacyl-CoA dehydrogenase activities, converting trans-2-enoyl-CoA via D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA. In contrast to its counterparts in yeasts, the Neurospora MFP does not have a C-terminal sequence resembling the SKL motif involved in protein targeting to microbodies.