Role of thyroid hormones and their receptors in peripheral nerve regeneration.

After peripheral nerve injury in adult mammals, reestablishment of functional connections depends on several parameters including neurotrophic factors, the extracellular matrix, and hormones. However, little is known about the contribution of hormones to peripheral nerve regeneration. Thyroid hormones, which are required for the development and maturation of the central nervous system, are also important for the development of peripheral nerves. The action of triiodothyronine (T3) on responsive cells is mediated through nuclear thyroid hormone receptors (TRs) which modulate the expression of specific genes in target cells. Thus, to study the effect of T3, it is first necessary to know whether the target tissues possess TRs. The fact that sciatic nerve cells possess functional TRs suggests that these cells can respond to T3 and, as a consequence, that thyroid hormone may be involved in peripheral nerve regeneration. The silicone nerve guide model provides an excellent system to study the action of local administration of T3. Evidence from such studies demonstrate that animals treated locally with T3 at the level of transection have more complete regeneration of sciatic nerve and better functional recovery. Among the possible regulatory mechanisms by which T3 enhances peripheral nerve regeneration is rapid action on both axotomized neurons and Schwann cells which, in turn, produce a lasting and stimulatory effect on peripheral nerve regeneration. It is probable that T3 up- or down-regulates gene expression of one or more growth factors, extracellular matrix, or cell adhesion molecules, all of which stimulate peripheral nerve regeneration. This could explain the greater effect of T3 on nerve regeneration compared with the effect of any one growth factor or adhesion molecule.

Mass spectrometry as a rapid and powerful alternative to antibodies for detecting LPXTG wall-associated proteins of Staphylococcus aureus

Functional characterization of transformed or natively present bacterial virulence proteins can be achieved employing various model systems. A prerequisite is to verify the correct expression of the transformed protein or the presence of the native protein in the microbe. Traditionally, antibodies are raised against the protein or a peptide thereof, followed by Western blot analysis or by fluorescence-activated cell sorting. Alternatively, the protein-coding gene can be fused with a downstream reporter gene, the expression of which reports the simultaneous expression of the upstream recombinant protein. Although being powerful, these methods are time consuming, especially when multiple proteins must be assessed. Here we describe a novel way to validate the expression of Gram-positive surface proteins covalently attached to the peptidoglycan. Eighteen out of the 21 known LPXTG-motif carrying cell wall-associated proteins of Staphylococcus aureus were cloned in Lactoccocus lactis either alone, in combinations or as truncated forms, and their correct expression was assessed by liquid chromatography coupled to mass spectrometry (LC-MS). The method is rapid, sensitive and precise. It can identify multiple proteins in transformed constructs without the time and cost needed for raising and testing multiple sets of antibodies.

Cerebrospinal fluid antimicroglial antibodies in Alzheimer disease: a putative marker of an ongoing inflammatory process.

Immunocompetent microglia play an important role in the pathogenesis of Alzheimer's disease (AD). Antimicroglial antibodies in the cerebrospinal fluid (CSF) in clinically diagnosed AD patients have been previously recorded. Here, we report the results of the analysis of the CSF from 38 autopsy cases: 7 with definite AD; 14 with mild and 10 with moderate Alzheimer's type pathology; and 7 controls. Antimicroglial antibodies were identified in 70% of patients with definite AD, in 80% of patients with moderate and in 28% of patients with mild Alzheimer's type pathology. CSF antimicroglial antibodies were not observed in any of the control cases. The results show that CSF antimicroglial antibodies are present in the majority of patients with definite AD and also in cases with moderate Alzheimer's type changes. They may also indicate dysregulation of microglial function. Together with previous observations, these findings indicate that compromised immune defense mechanisms play an important role in the pathogenesis of AD.

Feedback on hypothalamic TRH transcription is dependent on thyroid hormone receptor N terminus.

The beta thyroid hormone receptor (TRbeta), but not TRalpha1, plays a specific role in mediating T(3)-dependent repression of hypothalamic TRH transcription. To investigate the structural basis of isoform specificity, we compared the transcriptional regulation and DNA binding obtained with chimeric and N-terminally deleted TRs. Using in vivo transfection assays to follow hypothalamic TRH transcription in the mouse brain, we found that TRbeta1 and chimeras with the TRbeta1 N terminus did not affect either transcriptional activation or repression from the rat TRH promoter, whereas N-terminally deleted TRbeta1 impaired T(3)-dependent repression. TRalpha1 or chimeras with the TRalpha1 N terminus reduced T(3)-independent transcriptional activation and blocked T(3)-dependent repression of transcription. Full deletion of the TRalpha1 N terminus restored ligand-independent activation of transcription. No TR isoform specificity was seen after transcription from a positive thyroid hormone response element. Gel mobility assays showed that all TRs tested bound specifically to the main negative thyroid hormone response element in the TRH promoter (site 4). Addition of neither steroid receptor coactivator 1 nor nuclear extracts from the hypothalamic paraventricular nuclei revealed any TR isoform specificity in binding to site 4. Thus N-terminal sequences specify TR T(3)-dependent repression of TRH transcription but not DNA recognition, emphasizing as yet unknown neuron-specific contributions to protein-promoter interactions in vivo.

Polypeptides reactive with antibodies eluted from the surface of Babesia bovis-infected erythrocytes

A technique was sought that would enable identification of surface-exposed parasite antigens on Babesia bovis-infected erythrocytes (BbIE) that are not detectable by surface-specific immunoprecipitations. Antibodies which bind to the surface of BbIE were recovered from intact cells using a low pH wash procedure. The eluted antibodies were then used in conventional immunoprecipitation assays to identify parasite-synthesized polypeptides carrying epitopes that are exposed on the surface or are cross reactive with shuch epitopes. The results of these experiments support our previous data, obtained using a surface-specific immunoprecipitation technique, in the identification of a repertoire of parasite-derived antigens on the surface of infected erythrocytes (Allred et al., 1991). In addition, two polypeptides of Mr 68,000 and 185,000 were identified wich react strongly with the eluted antibodies but wich are not detected by surface-immunoprecipitation. These data illustrate the potential of this approach for identification of parasite polypeptides wich carry epitopes exposed on, or cross-reactive with exposed epitopes of the infected erythrocyte surface.

Antibodies in falciparum malaria: what matters most, quantity or quality?

In view of the recent demonstration that antibodies that are protective agains Plasmodium falciparum malaria may act in collaboration with blood monocytes, we have investigated the isotype content of sera from individuals with defined clinical states of resistance or susceptibility to malaria. Profound differences in the distribution of each Ig subclass and particulary in the ratio of cytophilic versus noncytophilic antibodies were found. In protected subjects, two cytophilic isotypes, IgG1 and IgG3 were found to predominate. In non-protected subjects, i.e. children and primary attack adults, three different situations were encountered: a) an imbalance in which IgG2, a non-cytophilic class, predominated (mostly seen in primary attacks); b) imbalance in which mostly IgM antibodies predominated (a frequent event in children) or c) less frequently, an overall low level of antimalarial antibodies. Of 33 non immune subjects studied all, except one, had one of the above defects. The function of total Ig presenting such an isotype imbalance was studied in vitro in Antibody-Dependent -Cellular-Inhibition assays. Not only did IgG from protected subjects cooperate efficiently with blood monocytes, whilst IgG from non-protected groups did not, but moreover the latter inhibit the in vitro effect of the former: in competition assays whole IgG from primary attack cases with increased IgG2 content, competed with IgG from immune adults, thus suggesting that non-protected subjects had antibodies to epitopes critical for protection, but that these antibodies are non functional.

Prevalence and levels of IgG and IgM antibodies against Plasmodium falciparum and P. vivax in blood donors from Rondônia, Brazilian Amazon

Antibodies of IgG and IgM isotopes reacting with Plasmodium falciparum and P. vivax thick-smear antigens were searched for by the indirect fluorescent antibody test (IFAT) in a random sample of 230 blood donors at the transfusion centre of Porto Velho (HEMERON), Rondônia State, western Brazilian Amazon. A high prevalence of IgG seropositivity (32% against P. falciparum, 24% against P. vivax and 37% against either P. falciparum or P. vivax antigens) was detected among them, despite the fact that candidates reporting recent (<12 months) malaria attacks were not elegible. Only a small proportion of them had also detectable IgM antibodies to these antigens. These data suggest an intense, relatively recent exposure to malaria in such an urban population sample. However, parasitaemia (as detected by microscopical examination of Giemsa-stained thick smears) was patent in only one prospective donor. The antibody profile of blood donors was compared with that of healthy subjects of all age groups, living in a close endemic area (Candeias village, 21 km east of Porto Velho). The villagers were classified into two groups according to their history of a recent (<12 months) or a remote (>12 months) past malaria attack due to either P. falciparum or P. vivax. Extensive overlapping was observed when the distribution of antibody titres of healthy subjects from Candeias village with a recent and remote malaria history was compared. In conclusion, subjects with a recent or a remote malaria history could not be distinguished by sorological criteria alone.

Prevalence of antibodies to Hepatitis C virus in populations at low and high risk for sexually transmited diseases in Rio de Janeiro

In order to investigate the sexual transmission of the Hepatitis C Virus (HCV), the prevalence of specific antibodies in populations at high and low risk for sexually transmitted diseases (STDs) was evaluated. The population at low risk for STDs was composed of persons who voluntarity donated blood at the Hospital Universitário Clementino Fraga Filho (HUCFF) between July and November, 1990 (n = 2494). The population at high risk for STDs was drawn from an ongoing study on the natural history of Human Immunodeficiency Virus (HIV) infection (n = 210, 187 with sexual risk factors for HIV infection). All samples were screened using a first generation ELISA. Repeat reactive samples were then tested in a second generation RIBA. For all ELISA positive samples, two sex and age-matched ELISA negative controls were selected. Data pertaining to the presence of antibodies to the Hepatitis B core antigen (anti-HBC antibodies) and to Treponema pallidum were abstracted from the medical records. The prevalence of RIBA 2 confirmed HCV infection among the blood donors was 2.08%, which is well above the reported prevalence in similar populations from developed western countries. Among the HIV infected homosexuals, the encountered prevalence was 7.96% (p < 0.0005). For the whole group with sexually acquired HIV infection, the prevalence was 8.02% (p < 0.000005). Anti-HBc antibodies were more frequently present in anti-HCV RIBA-2 confirmed positive blood donors than in controls (p < 0.001). 33.3% of the HCV-positive blood donors and 11.04% controls were found to be anti-HBc positive (p < 0.0005). As for the FTA-ABs, 17.6% HCV-positive donors and 4,9% controls were positive (p < 0.01). 5.9% samples from blood donors were both anti-HBc and FTA-ABS positive, whereas none of the controls reacted in both tests (p < 0.05). The association between HCV, Hepatitis B infection and syphilis in individuals at low risk for parenterally transmitted diseases suggests that sexual transmission contributes to the maintenance of the endemicity of HCV in the local population.

Standardization of serological tests for detecting anti-Trypanosoma cruzi antibodies in dogs

This paper reports on the standardization of four serological reactions currently used in human serodiagnosis for the detection of anti-Trypanosoma cruzi antibodies in naturally and experimentally infected dogs. Indirect immunofluorescence test (IFAT) and hemagglutination test (IHAT) were standardized, and complement fixation test (CFT) and direct agglutination test (DAT) were used for diagnostic confirmation. Four hundred and eighty one mongrel dogs that were studied by xenodiagnosis were used: (1) parasitemic dogs of two localities of endemic area (EA) of Santiago del Estero province in Argentina (n = 134); (2) non-parasitemic dogs of the same area (n = 285); (3) dogs experimentally infected with T. cruzi in the patent period (n = 6); (4) non-infected dogs (n = 56) which were born in the city of Buenos Aires (BA), one non-EA for Chagas' disease. For IFAT, parasitemic dogs EA showed 95% of reactive sera. Non parasitemic dogs EA showed 77% of non reactive sera. None sera from BA were reactive for dilutions higher than four. For IHAT, 84% of sera of parasitemic dogs EA showed serological reactivity and among non parasitemic dogs BA, 61% were non reactive, while the remainder showed at most titres of 1/16. The cut-off titres for IFAT and IHAT were 1/16 and 1/32 respectively, and for CFT and DAT 1/1 and 1/128 respectively. Sensitivity for IFAT, IHAT, CF and DAT were 95%, 84%, 97% and 95% respectively.