Thyroid-stimulating hormone and thyroid-stimulating hormone receptor structure-function relationships

This review focuses on recent advances in the structure-function relationships of thyroid-stimulating hormone (TSH) and its receptor. TSH is a member of the glycoprotein hormone family constituting a subset of the cystine-knot growth factor superfamily. TSH is produced by the pituitary thyrotrophs and released to the circulation in a pulsatile manner. It stimulates thyroid functions using specific membrane TSH receptor (TSHR) that belongs to the superfamily of G protein-coupled receptors (GPCRs). New insights into the structure-function relationships of TSH permitted better understanding of the role of specific protein and carbohydrate domains in the synthesis, bioactivity, and clearance of this hormone. Recent progress in studies on TSHR as well as studies on the other GPCRs provided new clues regarding the molecular mechanisms of receptor activation. Such advances are a result of extensive site-directed mutagenesis, peptide and antibody approaches, detailed sequence analyses, and molecular modeling as well as studies on naturally occurring gain- and loss-of-function mutations. This review integrates expanding information on TSH and TSHR structure-function relationships and summarizes current concepts on ligand-dependent and -independent TSHR activation. Special emphasis has been placed on TSH domains involved in receptor recognition, constitutive activity of TSHR, new insights into the evolution of TSH bioactivity, and the development of high-affinity TSH analogs. Such structural, physiological, pathophysiological, evolutionary, and therapeutic implications of TSH-TSHR structure-function studies are frequently discussed in relation to concomitant progress made in studies on gonadotropins and their receptors.     

Telepathology for routine light microscopic and frozen section diagnosis

Telepathology (TP) uses telecommunication linkages to electronically capture, store, retrieve, and transmit images to distant sites. We assessed the feasibility of a dynamic real-time TP system for light microscopic (LM) diagnosis of anatomic pathology specimens, including frozen sections. Six pathologists, in 2 separate periods, read a set of 160 retrospectively retrieved slides (80 of which were frozen sections) by TP and LM. Reading times were recorded. Diagnoses were compared with the reference diagnosis (established by a group of 5 independent pathologists) and graded on a scale of 0 to 2 (2, correct; 1, incorrect but no clinical impact; 0, incorrect with clinical impact). Overall, LM was more accurate than TP compared with the reference diagnosis (score, 1.68 vs 1.54). There was no difference in accuracy between frozen section and paraffin-embedded tissue. Intraobserver agreement ranged from 82.5% to 88.2%. The average reading time was 6.0 minutes for TP and 1.4 minutes for LM. During the study, reading time decreased for TP but not for LM. These results show that despite marginally lower accuracy and longer reading times, TP isfeasible for routine light microscopic diagnosis, including frozen sections.     

High genetic diversity of HIV-1 strains in Chad,West Central Africa

The genetic diversity of HIV-1 strains in Chad was documented with a total of 107 samples from patients attending the general hospital in N'Djamena, the capital city of Chad. The genetic subtypes were identified in the V3-V5 env and p24 gag regions by sequence and phylogenetic tree analyses. Of the 107 strains, 78 had the same subtype/CRF designation between env and gag. Four subtypes and three CRFs were found to cocirculate: subtype A, 20.5%; subtype D, 18.7%; CRF02_AG, 13.1%; CRF11_cpx, 13.1%; subtype G, 3.7%; CRF01_AE, 2.8%; and subtype F1, 0.9%. The remaining 29 strains (27%) had discordant subtypes or CRF designations between env and gag; in 15 of these 29 strains, a CRF was involved in the recombination event, and 10 were subtype G in gag and subtype A in env, forming a separate subcluster within subtypes G and A. Subtype D strains represent almost 20% of the HIV-1 strains circulating in Chad and form a separate subcluster in gag and env. Nearly full-length genome sequencing for two such strains (99TCD-MN011 and 99TCD-MN012) revealed that they represent nonrecombinant subtype D variants. Compared with neighboring countries, the genetic subtype distribution of HIV-1 strains in Chad is unique for several reasons: lower prevalence of CRF02, high prevalence of CRF11 and subtype D, and absence of CRF06. These data clearly show that subtype distribution is very heterogeneous in Africa, probably the result of different founder effects.     

Recovery of the endogenous beta cell function in the NOD model of autoimmune diabetes

In light of accumulating evidence that the endocrine pancreas has regenerative properties and that hematopoietic chimerism can abrogate destruction of beta cells in autoimmune diabetes, we addressed the question of whether recovery of physiologically adequate endogenous insulin regulation could be achieved in the nonobese diabetic (NOD) mice rendered allogeneic chimerae. Allogeneic bone marrow (BM) was transplanted into NOD mice at the preclinical and overtly clinical stages of the disease using lethal and nonlethal doses of radiation for recipient conditioning. Islets of Langerhans, syngeneic to the BM donors, were transplanted under kidney capsules of the overtly diabetic animals to sustain euglycemia for the time span required for recovery of the endogenous pancreas. Nephrectomies of the graft-bearing organs were performed 14 weeks later to confirm the restoration of endogenous insulin regulation. Reparative processes in the pancreata were assessed histologically and immunohistochemically. The level of chimerism in NOD recipients was evaluated by flow cytometric analysis. We have shown that as low as 1% of initial allogeneic chimerism can reverse the diabetogenic processes in islets of Langerhans in prediabetic NOD mice, and that restoration of endogenous beta cell function to physiologically sufficient levels is achievable even if the allogeneic BM transplantation is performed after the clinical onset of diabetes. If the same pattern of islet regeneration were shown in humans, induction of an autoimmunity-free status by establishment of a low level of chimerism, or other alternative means, might become a new therapy for type 1 diabetes.     

Cortical auditory processing and communication in children with autism: electrophysiological/behavioral relations.

The purpose of the present study was to investigate the relations between late auditory evoked potentials (AEPs) recorded at temporal sites (the N1c wave or Tb) and verbal and non-verbal abilities in children with autism. The study was performed in 26 mentally retarded children with autism (AUT) aged 4-8 years (mean age +/- S.E.M. = 71 +/- 2 months; mean verbal and non-verbal developmental quotient +/- S.E.M. = 36 +/- 4 and 48 +/- 3). The stimuli used were 750 Hz tone bursts of 200 ms duration delivered binaurally at different intensity levels (50, 60, 70, 80 dB SPL) with 3-5 s interstimulus intervals. Temporal AEPs were first compared to those of a group of 16 normal children (NOR) in the same age range (mean age +/- S.E.M. = 69 +/- 3 months). We then focused on the AUT group and considered relations between temporal AEPs and the severity of disorders of verbal and non-verbal communication assessed using a behavior rating scale. AEPs recorded on left and right temporal sites were of smaller amplitude in the AUT group than in the NOR group. Increasing intensity-related amplitude was observed on both sides in NOR and only on the right side in AUT. The lack of intensity effect on the left side resulted in a particular pattern of asymmetry at the highest level of intensity (80 dB SPL) with greater N1c amplitude on the right than on the left side (the reverse was found in the NOR group). Electro-clinical correlations indicated that the greater the amplitude of the right temporal N1c responses, the higher the verbal and non-verbal communication abilities. This suggests a developmental reorganization of left-right hemisphere functions in autism, with preferential activation of the right hemisphere for functions usually allocated to the left hemisphere, particularly those involving the secondary auditory areas situated on the lateral surface of the superior temporal gyrus where the N1c/Tb wave is generated.     

Differences in airway remodeling between subjects with severe and moderate asthma

BACKGROUND: Airway remodeling in asthma comprises a range of structural changes. Several studies have suggested an association between these changes and disease severity. The relationship between the extent of remodeling and lung function is not well defined. OBJECTIVE: We sought to contrast the structural changes in the airways of well-defined groups of subjects with severe and moderate asthma and to correlate the extent of remodeling with disease severity. METHODS: Endobronchial biopsy specimens were obtained from 15 subjects with severe and 13 subjects with moderate asthma. Epithelial integrity, cell-layer areas, subepithelial fibrosis, and the distance between epithelial and airway smooth muscle (ASM) layers were measured by means of image analysis. Collagen was identified by using Van Giesen stain, and ASM was defined by using smooth muscle alpha-actin immunostaining. Specific immunostains were performed for the evaluation of RANTES, IL-8, and eotaxin expression as markers of ASM phenotype. RESULTS: ASM area was greater in subjects with severe (0.24+/- 0.03 mm(2)) than in subjects with moderate (0.05+/- 0.01 mm(2)) asthma (P<.001). The distance between the epithelial and ASM layers was less in the severe group (0.12+/- 0.01 mm) than in the moderate group (0.24+/- 0.02, P<.001). A trend toward greater subepithelial fibrosis in subjects with severe asthma did not reach statistical significance. IL-8 and eotaxin expression, but not RANTES expression, were increased in the ASM of subjects with severe asthma compared with in subjects with moderate asthma. CONCLUSION: Smooth muscle alteration is the key structural change that distinguishes severe from moderate asthma, and phenotypic change in ASM might contribute to the difficulty in obtaining adequate control in some subjects with severe asthma.     

Discriminatory power and reproducibility of novel DNA typing methods for Mycobacterium tuberculosis complex strains

In recent years various novel DNA typing methods have been developed which are faster and easier to perform than the current internationally standardized IS6110 restriction fragment length polymorphism typing method. However, there has been no overview of the utility of these novel typing methods, and it is largely unknown how they compare to previously published methods. In this study, the discriminative power and reproducibility of nine recently described PCR-based typing methods for Mycobacterium tuberculosis were investigated using the strain collection of the interlaboratory study of Kremer et al. This strain collection contains 90 M. tuberculosis complex and 10 non-M. tuberculosis complex mycobacterial strains, as well as 31 duplicated DNA samples to assess reproducibility. The highest reproducibility was found with variable numbers of tandem repeat typing using mycobacterial interspersed repetitive units (MIRU VNTR) and fast ligation-mediated PCR (FLiP), followed by second-generation spoligotyping, ligation-mediated PCR (LM-PCR), VNTR typing using five repeat loci identified at the Queens University of Belfast (QUB VNTR), and the Amadio speciation PCR. Poor reproducibility was associated with fluorescent amplified fragment length polymorphism typing, which was performed in three different laboratories. The methods were ordered from highest discrimination to lowest by the Hunter-Gaston discriminative index as follows: QUB VNTR typing, MIRU VNTR typing, FLiP, LM-PCR, and spoligotyping. We conclude that both VNTR typing methods and FLiP typing are rapid, highly reliable, and discriminative epidemiological typing methods for M. tuberculosis and that VNTR typing is the epidemiological typing method of choice for the near future.     

Evaluation of a microsphere-based flow cytometric assay for diagnosis of celiac disease

The multiplexed particle-based flow cytometric technology proposes a new approach for the diagnosis of autoimmune diseases combining the advantages of conventional methods with the ability to quantitatively determine multiple autoantibodies in the same sample, simultaneously and rapidly. Recently, a commercial kit (FIDIS Celiac, Biomedical Diagnostics, Mane la Vallé, France) was introduced for the simultaneous detection of IgA anti-tissue transglutaminase (anti-tTG), IgG, and IgA anti-gliadin antibodies (AGA). This study was undertaken to evaluate and compare the FIDIS Celiac kit with standardized commercial ELISAs (QUANTA Lite, INOVA Diagnostics Inc., San Diego, CA). A disease group consisted of 21 samples from untreated patients with biopsy confirmed celiac disease (CD), and two control groups of historical sera (207 from regular blood donors and 181 from chronically infected hepatitis patients) were studied. All control sera were negative for IgA anti-endomysial antibodies (EmA) and had an IgA concentration above the lower normal limit. Concerning the reproducibility, intra- and inter-assay coefficients of variation (CVs) ranging between 2% and 12%, and between 3% and 21%, respectively, were observed. Regarding the diagnostic quality, each assay was compared to the disease diagnosis using the McNemar test and the kappa (K) parameter, while ROC analysis was applied. Generally, the performance of FIDIS assay was proved almost equally adequate to that of ELISA in the detection of IgA anti-tTG antibodies, IgA and IgG AGA. However, the performance of FIDIS assay was found surmounting that of ELISA among hepatitis patients, possibly due to the avoidance of debris and unbound cross contaminants and, hence, the "noise" of such materials in samples under analysis. Taking our results together with the simplicity and the high throughput of FIDIS assay, its overall performance in the diagnosis of CD seems better than that of ELISA.