Genotypic analysis of Mycobacterium tuberculosis in Bangladesh and prevalence of the Beijing strain

Genotypic analysis was performed on 48 Mycobacterium tuberculosis complex strains collected from a hospital in Dhaka city. Deletion analysis showed that the isolates were all M. tuberculosis; 13 of them were found to be of the "ancestral" type, while 35 were of the "modern" type, indicating that both endemic (ancestral type) and epidemic (modern type) strains cause tuberculosis in Bangladesh. Genotyping based on the spoligotype and variable-number tandem repeats (VNTR) of mycobacterial interspersed repetitive units (MIRU) was also done. A total of 34 strains (71%) were grouped by spoligotyping into nine different clusters; the largest comprised 15 isolates of the Beijing genotype, whereas the remaining eight clusters consisted of two to five isolates. MIRU-VNTR typing detected 32 different patterns among 44 tested strains, and the 15 Beijing strains were further discriminated by MIRU-VNTR typing (7 distinct patterns for the 15 isolates). These results indicate that MIRU-VNTR typing, along with spoligotyping and deletion analysis, can be used effectively for molecular epidemiological studies to determine ongoing transmission clusters; to our knowledge, this is the first report about the type of strains prevailing in Bangladesh.     

Clonal Diversity and Turnover of Streptococcus mitis bv. 1 on Shedding and Nonshedding Oral Surfaces of Human Infants during the First Year of Life

Streptococcus mitis bv. 1 is a pioneer colonizer of the human oral cavity. Studies of its population dynamics within parents and their infants and within neonates have shown extensive diversity within and between subjects. We examined the genetic diversity and clonal turnover of S. mitis bv. 1 isolated from the cheeks, tongue, and primary incisors of four infants from birth to 1 year of age. In addition, we compared the clonotypes of S. mitis bv. 1 isolated from their mothers' saliva collected in parallel to determine whether the mother was the origin of the clones colonizing her infant. Of 859 isolates obtained from the infants, 568 were unique clones. Each of the surfaces examined, whether shedding or nonshedding, displayed the same degree of diversity. Among the four infants it was rare to detect the same clone colonizing more than one surface at a given visit. There was little evidence for persistence of clones, but when clones were isolated on multiple visits they were not always found on the same surface. A similar degree of clonal diversity of S. mitis bv. 1 was observed in the mothers' saliva as in their infants' mouths. Clones common to both infant and mothers' saliva were found infrequently suggesting that this is not the origin of the infants' clones. It is unclear whether mucosal immunity exerts the environmental pressure driving the genetic diversity and clonal turnover of S. mitis bv. 1, which may be mechanisms employed by this bacterium to evade immune elimination.     

The onset of polymorphonuclear leucocyte membrane-stimulated metabolic activity.

The time course of polymorphonuclear leucocyte oxidative metabolism following membrane stimulation by four different agents was examined using the techniques of luminol- and lucigenin-dependent chemiluminescence. After addition of opsonized zymosan, phorbol myristate acetate or digitonin to polymorphonuclear leucocytes there was a lag period of between 35 and 55 sec before the onset of chemiluminiscence. In contrast, after addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP), the lag period before chemiluminescence was less than 19 sec. Luminol-dependent chemiluminescence was reduced by superoxide dismutase and almost abolished by sodium azide. The inhibitory effect of the latter was less marked when using FMLP. Lucigenin-dependent chemiluminescence was inhibited by superoxide dismutase and enhanced by sodium azide. Cytochalasin B reduced zymosan and digitonin stimulated chemiluminescence but increased FMLP stimulated chemiluminescence. The results of the onset of polymorphonuclear leucocyte metabolic activity using other techniques.     

Purification and characterization of urease isolated from the pathogenic fungus Coccidioides immitis.

Coccidioides immitis, the causative agent of San Joaquin Valley fever (coccidioidomycosis), produces a urease which has been suggested to contribute to the virulence of this fungal pathogen. Urease catalyzes the hydrolysis of urea and has been proposed to at least partly account for alkalinity of the microenvironment in which C. immitis grows due to the release of ammonia and ammonium ions. The C. immitis urease was purified to homogeneity (1048-fold) from the mycelial cytosol by chromatographic fractionation. The sequence of 12 N-terminal amino-acid residues of the purified, native polypeptide was identical to that predicted by the translated urease gene sequence which has been reported. The isolated enzyme exhibited a specific activity in the presence of urea of 1750 micromol min(-1) mg(-1) protein, has a native molecular mass of 450 kDa, revealed a Km for urea of 4.1 mM, had a pH optimum of 8.0 and is heat stable. Hydroxyurea, acetohydroxamic acid (AHA) and boric acid each inhibited activity of the purified enzyme. Urease activity was enhanced by the presence of 5-10 mM concentrations of Mg2+ or Mn2+, but inhibited by Li+, Ni2+, Cu2+ or Zn2+. The reversible urease inhibitor, AHA, blocked enzyme activity in the crude mycelial cytosolic fraction when added at a concentration of 10 mM. On the other hand, 10 mM AHA added to 4-day-old mycelial cultures only partially decreased the amount of ammonium detected in the culture medium. It is evident, therefore, that C. immitis urease activity does not account for the total amount of ammonia secreted during in vitro growth of the pathogen. Other metabolic sources of ammonia, which may also contribute to the virulence of C. immitis, are under investigation.     

The response to lipopolysaccharide of mouse spleen lymphocytes fractionated on the basis of surface immunoglobulin and complement receptor using fluorescence-activated cell sorting and rosetting techniques.

Mouse spleen lymphocytes were stained with rabbit antisera specific for either μ chain or δ chain, followed by fluorescein-conjugated goat anti-rabbit immunoglobulin. The cells were analysed and fractionated using a fluorescence-activated cell sorter. Fifty-five per cent of the lymphocytes stained with a polyspecific anti-Ig reagent or with a combination of anti-μ and anti-δ reagents, while about 40% of the lymphocytes were stained when either the anti-μ reagent or the anti-δ reagent was used alone. Three per cent of the lymphocytes stained with the anti-μ reagent, but not with the anti-δ reagent, and eight per cent stained only with the anti-δ reagent. Unfractionated spleen cells and populations depleted of μ- or δ-bearing cells were cultured in the presence of lipopolysaccharide. All three populations responded by incorporating [³H]-thymidine and secreting IgM and IgG.Spleen cells were fractionated by a rosetting technique into complement receptor-positive and negative populations. Both populations were able to respond to lipopolysaccharide and to synthesize Ig of both the IgM and IgG classes. Unfractionated cells and complement receptor-negative populations were stained for surface μ or δ chain and analysed on the fluorescence-activated cell sorter. The distribution of staining intensity suggested that the complement receptor-bearing population was enriched in cells which stain weakly for μ and cells which stain with a low to intermediate intensity for δ chain.It is concluded that the precursors of IgM- and IgG-secreting cells are not limited to any one of the three populations of cells defined on the basis of surface immunoglobulin or to either of the populations defined on the basis of the complement receptor.     

Early cellular responses to intradermal injection of Kveim suspension in normal subjects and those with sarcoidosis.

In a detailed controlled study of the cellular response to Kveim suspension in vivo we used immunohistological and histochemical methods to examine cryostat sections of immature Kveim biopsy specimens in subjects with sarcoidosis and normal controls. Changes seen at 48 hours, at which time papular reactions have sometimes been reported, are described. Eight cases of sarcoidosis previously confirmed by a positive Kveim test were studied, in five of whom the test remained positive; plus two subjects with sarcoidosis studied prospectively; and four healthy controls. There were two main features of the 48 hour response: collagen disruption with associated histiocytes, which showed increased acid phosphatase activity; and perivascular infiltrates of lymphocytes and small groups of dendritic cells. The T4:T8 ratios in the infiltrates were similar to those found in the peripheral blood of the subjects, and few lymphocytes showed evidence of activation. T lymphocytes were also seen free in the dermis and migrating to the epidermis. Small juxtacapillary clumps of dendritic cells, identified by NA1/34 (= OKT6; Langerhans' cells) and RFD1 (interdigitating cell) monoclonal antibodies, were found. The Langerhans' cells in the epidermis were, however, normal in number and distribution. These features, which were found in all groups, are not consistent with pre-existing hypersensitivity to Kveim suspension in sarcoidosis. Subsequent differences between sarcoid and normal subjects in the development of granulomas in the Kveim response may therefore relate to the different handling of the foreign material by the cells affected, rather than to differences in the early non-specific recruitment of the cells to the test site.     

The screening of Duchenne muscular dystrophy patients for submicroscopic deletions.

We have probed the DNA of 156 Duchenne muscular dystrophy (DMD) patients, representing 140 kindreds, with cloned DNA sequences derived from Xp21 and known to show deletions in some DMD patients. Sixteen cases showed a deletion, as defined by lack of hybridisation to one or more of the four probes used. However, two of these cases were brothers, so 15 independent deletions (10.7%) are represented. The deletion map is compatible with the suggested order for the sites of the probes used in the study, that is, telomere----pERT87.15----pERT87.8----pERT87.1----pX J1.1----754----centromere. Further mapping of these deletions and characterisation of the deletion breakpoints should facilitate more accurate molecular localisation of the gene or genes which, when mutated, are responsible for causing DMD.     

Lymphocyte reactivity to influenza virus in man.

Dermal delayed hypersensitivity and in vitro lymphocyte reactivity, both to purified influenza antigens, have been observed in man. A correlation between these two indices of delayed hypersensitivity was found in subjects without known recent exposure to the virus, but neither correlated with levels of circulating haemagglutination-inhibiting (HAI) antibody. Results suggest that in man immunological sensitization of lymphocytes to influenza virus antigen is long-lived.     

Specific and nonspecific immune factors in dental plaque fluid and saliva from young and old populations.

Separate samples of supragingival dental plaque overtly free of blood were centrifuged to obtain the free fluid phase (plaque fluid). Bound protein was eluted from the plaque bacteria and matrix by washing the plaque with a low-pH buffer. The plaque fluid, low pH eluate, and whole saliva were assayed for immunoglobulins A, G, and M, the third component of complement, lysozyme, lactoferrin, and lactoperoxidase. Concentrations of total protein and albumin were also determined. Antibody reactive with specific plaque bacteria was detected by indirect immunofluorescent microscopy. Specific and nonspecific immune proteins were present in plaque fluid from adult subjects at significantly greater concentrations than in their saliva, which suggests that these proteins are concentrated in dental plaque. The results indicate that both saliva and gingival exudate contribute to the immunological proteins found in the free fluid phase of dental plaque. The observation that immunoglobulin A antibody reactive with plaque bacteria could be detected in plaque fluid suggests that a wide variety of immunological reactions may occur in the dental plaque. These potential interactions between host, plaque bacteria, and their products could serve to influence the plaque flora and its ability to induce disease.     

Congenital diaphragmatic hernia with probable autosomal recessive inheritance in an extended consanguineous Pakistani pedigree.

We report four cases of congenital diaphragmatic hernia occurring in two generations of a consanguineous Pakistani family. The affected subjects resembled no recognised genetic syndrome. This family provides further evidence for possible autosomal recessive inheritance of congenital diaphragmatic hernia in some cases.