Significance of PBMC response of rheumatic fever patients and control individuals to immunodominant streptococcal M5 protein epitopes

β-hemolytic streptococcal infection in developing countries still causes thousands of cases of Rheumatic Fever (RF). Molecular mimicry between streptococcal M protein (strep M) and heart components has been proposed as the triggering factor leading to autoimmunity in individuals with genetic susceptibility, which is linked to different HLA-DR alleles in different populations. In our hands, RF was significantly associated to HLA-DR7/53. Previous work in our lab has shown that heart-infiltrating T cells that simultaneously recognize strep M and heart proteins. Further, such T cells predominantly recognized the 81-103 strep M5 epitope. In this work, we analysed the proliferative response of peripheral blood mononuclear cells of 99 RF patients and 40 normal controls. Eighty-nine of the RF patients were HLA-typed. As among heart-infiltrating T cells, the 81-103 strep M5 protein epitope is the most frequently recognized epitope among RF PBMC (35.4%), against a 7.5% frequency of proliferation among normal controls (p=0.0018, chi square). However, the 81-103 epitope was as frequently recognized by HLA-DR7,53 positive as by negative individuals (45.2% vs 54.8%, respectively). Taken together, the results suggest that the 81-103 strep M5 epitope may be the immunodominant epitope, “promiscuously” recognized by T cells in a genetically diverse population. The demonstration that molecular mimicry is targeted to a discrete immunodominant “promiscuous” epitope in strep M5 may allow the development of a safe anti-streptococcal synthetic vaccine devoid of such epitopes.     

Postnatal development of hypoglossal motoneurons that innervate the hyoglossus and styloglossus muscles in rat

Postnatal development of hyoglossus and styloglossus motoneurons was studied in this investigation of the hypoglossal nucleus. Our findings show separate and distinct locations for hyoglossus and styloglossus motoneurons within the retrusor (dorsal) subdivision of the hypoglossal nucleus for all age groups. Hyoglossus and styloglossus motoneuron cross-sectional area reached their adult size at different times (by weeks 2 and 3, respectively). Cell roundness, as measured by form factor (measure of cell perimeter relative to its area), decreased with advancing postnatal age for both populations of motoneurons. Differences in the direction of the dendritic projection between hyoglossus and styloglossus motoneurons were found. Hyoglossus and styloglossus motoneuron development was compared to genioglossus motoneuron postnatal development.     

Serological heterogeneity of the IgM components of mixed (monoclonal IgM–polyclonal IgG) cryoglobulins

Mixed IgG–IgM cryoglobulins were isolated from the sera of seven patients with macroglobulinaemia or cryoglobulinaemia. The IgM components of all seven cryoproteins were monoclonal, containing κ light chains only, whereas the IgG components were polyclonal, containing both κ and λ light chains. Despite their apparent immunological homogeneity, the IgM components showed a wide range of antiglobulin activity. The data indicate that serological specificity may vary from one mixed cryoglobulin to another and that the monoclonal IgM components of different mixed cryoglobulins represent a heterologous group of antiglobulins.     

Avirulence of rough mutants of Shigella flexneri: requirement of O antigen for correct unipolar localization of IcsA in the bacterial outer membrane.

Mutations in the lipopolysaccharide (LPS) of Shigella spp. result in attenuation of the bacteria in both in vitro and in vivo models of virulence, although the precise block in pathogenesis is not known. We isolated defined mutations in two genes, galU and rfe, which directly affect synthesis of the LPS of S. flexneri 2a, in order to determine more precisely the step in virulence at which LPS mutants are blocked. The galU and rfe mutants invaded HeLa cells but failed to generate the membrane protrusions (fireworks) characteristic of intracellular motility displayed by wild-type shigellae. Furthermore, the galU mutant was unable to form plaques on a confluent monolayer of eucaryotic cells and the rfe mutant generated only tiny plaques. These observations indicated that the mutants were blocked in their ability to spread from cell to cell. Western immunoblot analysis of expression of IcsA, the protein essential for intracellular motility and intercellular spread, demonstrated that both mutants synthesized IcsA, although they secreted less of the protein to the extracellular medium than did the wild-type parent. More strikingly, the LPS mutants showed aberrant surface localization of IcsA. Unlike the unipolar localization of IcsA seen in the wild-type parent, the galU mutant expressed the protein in a circumferential fashion. The rfe mutant had an intermediate phenotype in that it displayed some localization of IcsA at one pole while also showing diffuse localization around the bacterium. Given the known structures of the LPS of wild-type S. flexneri 2a, the rfe mutant, and the galU mutant, we hypothesize that the core and O-antigen components of LPS are critical elements in the correct unipolar localization of IcsA. These observations indicate a more precise role for LPS in Shigella pathogenesis.     

Cloning and expression of the major 47-kilodalton surface immunogen of Treponema pallidum in Escherichia coli.

Monoclonal antibodies directed against the 47-kilodalton (kDa) major outer membrane surface immunogen of virulent Treponema pallidum subsp. pallidum were used to select Escherichia coli recombinant clones expressing the 47-kDa immunogen. The phenotype of the clones was dependent on the presence of recombinant plasmid in the host cell. Southern hybridization revealed that the cloned T. pallidum subsp. pallidum DNA sequence was an accurate representation of the T. pallidum subsp. pallidum genomic DNA arrangement. Purified immunoglobulin G from rabbits experimentally infected with T. pallidum subsp. pallidum and human secondary syphilitic sera specifically reacted with the clones, while normal human serum or immunoglobulin G from normal rabbit serum did not. Results of Southern hybridization indicated that a homologous 47-kDa immunogen gene was absent in at least four species of nonpathogenic treponemes tested, as well as from total rabbit genomic DNA. Rabbit anti-T. phagedenis biotype Reiter (treponemal nonpathogen) antiserum and a monoclonal antibody directed against a common treponemal determinant were unreactive with the clones. Western blotting and radioimmunoprecipitation experiments with specific monoclonal antibodies revealed that the recombinant (E. coli) and native (T. pallidum subsp. pallidum) forms of the antigen had identical electrophoretic mobilities. The availability of recombinant 47-kDa immunogen provides a new opportunity for biochemical analysis of the protein, structure-function studies, examination of its role in microbial pathogenesis, and assessment of its diagnostic and vaccinogenic potentials.     

Prenatal diagnosis and characterization of an unbalanced whole arm translocation resulting in monosomy for 18p

Monosomy for the short arm of chromosome 18 is one of the most frequent autosomal deletions observed. While most cases result from terminal deletion of 18p, 16% of cases reported were as a result of an unbalanced whole arm translocation resulting in monosomy 18p. The origin and structure of these derivative chromosomes were reported in only a few cases. We report the prenatal diagnosis and characterization of a new case of monosomy 18p as a result of an unbalanced whole arm translocation. Amniocentesis was performed at 15 weeks of gestation on a 34-year-old woman initially referred for advanced maternal age. Holoprosencephaly was identified by ultrasound at the time of amniocentesis. Karyotype analysis showed an unbalanced whole arm translocation between the long arm of one chromosome 18 and the long arm of one chromosome 22, 45,XX,der(18;22)(q10;q10), in all metaphases. In effect, the fetus had monosomy for 18p. Parental karyotypes were normal, suggesting a de novo origin for the der(18;22). Fluorescence in situ hybridization (FISH) analysis was performed with alpha-satellite probes D18Z1 and D14Z1/D22Z1 to identify the origin of the centromere on the der(18;22). Signal was observed with both probes, indicating that the centromere was composed of alpha-satellite DNA from both constituent chromosomes. Genotyping of the fetus and her parents with chromosome 18p STS marker D18S391 showed only the paternal 187 bp allele was present in the fetus, indicating that it was the maternal chromosome 18 involved in the der(18;22). This case and previous reports show that de novo unbalanced whole arm translocations are more likely to retain alpha-satellite sequences from the two chromosomes involved.     

New classification for mental disorders with management guidelines for use in primary care: ICD-10 PHC chapter five.

A primary care version of the International classification of diseases (10th revision) chapter five for mental and behavioural disorders (ICD-10 PHC chapter five) has been developed. This provisional version focuses on 24 conditions which are frequently seen in primary care and which can be managed effectively by general practitioners. The classification is accompanied by a flipcard for each of the conditions. The cards have diagnostic guidelines on one side and management guidelines on the other. The latter provide information which should be given to the patient, advice on the content of counselling, the available treatment methods, and indications for specialist referral. This classification system is also supported by diagnostic decision making aids, medication cards, and patient leaflets to facilitate the recognition and management of patients with mental disorders in primary care settings. The draft version of ICD-10 PHC chapter five will be finalized after field trials which will test the applicability and usefulness of the system in different primary care settings in various countries.     

The relationship between depression and aerobic capacity in hemodialysis patients

Depression and a reduction in the performance of pleasant activities have been reported in patients with end-stage renal disease receiving hemodialysis therapy. The purpose of this study was to examine the relationship between depression and the patients' capacity to engage in physical activity. The patients' VO2max, or maximal oxygen consumption, was found to be positively associated with three standardized self-report measures of depression in a sample of 15 patients receiving hemodialysis. The clinical and theoretical implications of this finding are considered.     

Temperature-sensitive membrane association of pp60src in tsNY68-infected cells correlates with increased tyrosine phosphorylation of membrane-associated proteins

Incubation of membrane vesicles from normal and Rous sarcoma virus-transformed chick embryo fibroblasts (CEF) with [gamma-32P]ATP resulted in the phosphorylation of a large number of proteins. The major differences observed between the membrane vesicles of untransformed and transformed cells were: (1) a 5- to 10-fold increase in the proportion of labeled phosphotyrosine in transformed vesicles and (2) the phosphorylation of pp60src in vesicles from transformed cells. Of the many proteins labeled in vitro, only pp60src was immunoprecipitated by TBR serum. Phosphorylation of the immunoprecipitated pp60src occurred on tyrosine in the 26-kDa carboxy-terminal Staphylococcus aureus V8 protease fragment. pp60src was not phosphorylated in vitro in membrane vesicles prepared from tsNY68-infected cells grown at the nonpermissive temperature. The proportion of labeled phosphotyrosine in membrane proteins from tsNY68-infected cells grown at the nonpermissive temperature was only slightly increased relative to that observed in membranes prepared from normal cells. Subcellular fractionation indicated that while pp60src was membrane associated in tsNY68-infected cells grown at the permissive temperature, pp60src was chiefly soluble in tsNY68-infected cells grown at the nonpermissive temperature. Temperature-sensitive membrane association of pp60src in tsNY68-infected cells was also observed by indirect immunofluorescence microscopy. When membranes were prepared from tsNY68-infected cells that had been downshifted from the nonpermissive to the permissive temperature, the reappearance of in vitro phosphorylated pp60src and the increase in the proportion of labeled phosphotyrosine in membrane vesicles correlated with the kinetics of src immune complex kinase reactivation and membrane association of pp60src.     

Dynamic properties of vestibular reflexes in the decerebrate cat

Summary Vestibulocollic (VCR) and vestibulo-ocular (VOR) reflexes were studied during angular rotation in the horizontal plane in precollicular decerebrate cats. Angular position was modulated by sinusoids or sums of sinusoids with frequencies ranging from 0.05 to 5 Hz.Reflex motor output was measured by recording electromyographic (EMG) activity of the lateral rectus and dorsal neck muscles and discharge of abducens motoneurons. Measured with respect to input angular acceleration VCR motor output displayed a second order lag at low frequencies, bringing mean EMG phase (–136 °) and gain slope (–35 dB/ decade) close to those of an angular position signal at 0.2 Hz. At higher frequencies the lag was counteracted by a second order lead bringing mean phase (–52 °) and gain slope (–5.6 dB/decade) back close to those of an angular acceleration signal at 3 Hz. By contrast, mean phase (–113 ° to –105 °) and gain slope (–21 to –28 dB/decade) of the VOR motor output remained close to those of an angular velocity signal across the entire frequency range.The data suggest that neural pathways producing the VCR receive selective input from irregular type horizontal semicircular canal afferents which provide one lag and one lead in the overall transfer function while the other lag and lead are produced by central pathways.Transaction of the medial longitudinal fasciculus (MLF), which eliminates all of the most direct (three neuron) arcs of the horizontal VCR, did not cause any detectable change in the horizontal VCR at either low or high frequencies. Reductions in overall gain occurred in some cases but these could be attributed to damage to axons outside the MLF. Less direct pathways, probably including vestibulo-reticulospinal pathways, are thus able to produce both the low-frequency, phase-lagging and high-frequency, phase-leading components of the horizontal VCR.Supported in part by NIH grants EY 02249, EY 00100, and NS 02619Recipient of NIH Fellowship NS 06030