Human parainfluenza virus type 3 infections in a haemato-oncology unit: social distancing measures needed in outpatient clinics.

BACKGROUND: Human parainfluenza virus type 3 (HPIV3) infections are associated with high mortality in immunocompromised settings, especially in bone marrow transplant recipients. Asymptomatic infection and lack of effective antiviral treatment makes HPIV3 prevention and treatment a real challenge. AIM: To retrospectively investigate the epidemiological characteristics, clinical characteristics and outcomes of 51 haematology patients with confirmed HPIV3 infections, detected between February and May 2019 in the haematology unit at King's College Hospital, London. METHODS: Between February and May 2019, HPIV3 RNA was detected in combined nose and throat swab samples collected from 51 symptomatic haematology patients, 41 of whom attended the haematology outpatient unit. Clinical data were reviewed retrospectively and a timeline of patients' appointments drawn up to investigate transmission. Sequencing analysis was performed on 14 stored samples. FINDINGS: Fifty-one patients were identified with HPIV3 infection. Mean age was 54 years (SD: 12; range: 19-72) and 60% (31/51) were male. There were 41 (80%) bone marrow transplant recipients, 24 had an allograft, and 17 an autograft. Thirty-day and 3-month mortality post HPIV3 was 6% and 14%, respectively. Lower respiratory tract infection and inpatient acquisition were associated with higher mortality (6/7 vs 1/7, P = 0.010; and 5/7 vs 2/7, P = 0.031). Onset of HPIV3 infection in patients within 6 days of attending the clinic was associated with the clusters identified in phylogenetic analysis (64% (9/14) vs 21% (8/37); odds ratio: 6.5 (confidence interval: 95% 1.7-25); P = 0.006). CONCLUSION: Timelines suggested community transmission, but also possible transmission patterns within the outpatients and subsequent nosocomial transmission within the same ward. Early recognition of HPIV3 infection and the use of polymerase chain reaction and sequence analysis is fundamental in identifying respiratory virus outbreaks and person-to-person transmission. Careful planning of outpatient clinic attendance is required to minimize contact and prevent respiratory virus transmission in immunosuppressed patients.

Towards developing standardized protocols for evaluation of cellular mucosal immune responses - Recommendations from a DAIDS/NIH workshop, June 15-16, 2009.

Although 80% of HIV infections occur through mucosal routes and vaccine strategies need to be designed for inducing protective immune responses at the site of the viral entry, it has proven to be very challenging to measure these responses. A 2-day workshop was convened by Division of AIDS, National Institutes of Health on June 15-16, 2009 to address the challenges encountered in the evaluation of mucosal T cell immune responses. The goal of the workshop was to obtain recommendations/consensus for developing standardized protocols for the assessment of mucosal immunity. This report summarizes the areas of consensus and recommendations that should assist in developing standardized methodologies for the evaluation of mucosal immune responses.

Childbearing and risk of cataract in young women: an epidemiological study in central India.

BACKGROUND/AIMS: Observations in central India, over a period of more than a decade, suggested that the frequency of sight restoring cataract surgery was substantially higher in women of childbearing age compared to men of the same age. Formal surveys in the subcontinent of India have confirmed a higher prevalence of cataract in women. The present study was conducted to explore possible effects of childbearing and associated adverse factors on cataract risk. METHODS: A case-control study design was used. Cases were mothers aged 35-45 with bilateral "senile" cataract. Controls were mothers of the same age but with clear lenses, attending the hospital services with other, mostly minor, complaints. RESULTS: A significant association was found between childbearing and risk of sight impairing cataract in mothers. Having more than three babies doubled the risk (adjusted odds ratio 2.0, p=0.012), and the risk increased by an estimated 20% for each additional birth. The birth effect was independent of age, socioeconomic status (occupation and income level), body mass index, and multiple episodes of severe dehydration, all regarded as putative risk factors for cataract. CONCLUSIONS: Having more than three babies may substantially increase the risk of sight impairing cataract in mothers of childbearing age in central India. The findings open new research challenges to identify cataract risk factors to which mothers may be exposed during pregnancy and childbirth, particularly under poor socioeconomic conditions.

Anthrax-toxin-mediated delivery of a 19 kDa antigen of Mycobacterium tuberculosis into the cytosol of mammalian cells.

PA63, the proteolytically activated 63 kDa fragment of protective antigen (PA, 83 kDa), mediates translocation of lethal factor (LF) and oedema factor into the cytosol. The N-terminal 254 amino acids of LF (LFn) are required for binding to PA63 and mediating translocation of active ligands fused to either the N- or C-terminus. Here we report translocation of a 19 kDa antigen of Mycobacterium tuberculosis into the cytosol of mammalian cells when fused to the C-terminus of LFn (LFn-19kDa). The fusion protein was non-toxic to J774A.1 macrophage cells in combination with PA and retained the ability to bind to PA63 when incubated with Chinese hamster ovary K1 cells. The data show the efficacy of anthrax toxin to mediate translocation of M. tuberculosis antigens into the cytosol of mammalian cells and may prove useful in delivering proteins and peptides carrying immunodominant mycobacterial antigens into the cytosol.

A dominant negative mutant of Bacillus anthracis protective antigen inhibits anthrax toxin action in vivo.

PA63, a proteolytically activated 63-kDa form of anthrax protective antigen (PA), forms heptameric oligomers and has the ability to bind and translocate the catalytic moieties, lethal factor (LF), and edema factor (EF) into the cytosol of mammalian cells. Acidic pH triggers oligomerization and membrane insertion by PA63. A disordered amphipathic loop in domain II of PA (2beta2-2beta3 loop) is involved in membrane insertion by PA63. Because conditions required for membrane insertion coincide with those for oligomerization of PA63 in mammalian cells, residues constituting the 2beta2-2beta3 loop were replaced with the residues of the amphipathic membrane-inserting loop of its homologue iota-b toxin secreted by Clostridium perfringens. It was hypothesized that such a molecule might assemble into hetero-heptameric structures with wild-type PA ultimately leading to the inhibition of cellular intoxication. The mutation blocked the ability of PA to mediate membrane insertion and translocation of LF into the cytosol but had no effect on proteolytic activation, oligomerization, or binding LF. Moreover, an equimolar mixture of purified mutant PA (PA-I) and wild-type PA showed complete inhibition of toxin activity both in vitro on J774A.1 cells and in vivo in Fischer 344 rats thereby exhibiting a dominant negative effect. In addition, PA-I inhibited the channel-forming ability of wild-type PA on the plasma membrane of CHO-K1 cells thereby indicating protein-protein interactions between the two proteins resulting in the formation of mixed oligomers with defective functional activity. Our findings provide a basis for understanding the mechanism of translocation and exploring the possibility of the use of this PA molecule as a therapeutic agent against anthrax toxin action in vivo.

Long-range magnetic order and the darwin lagrangian

We simulate a finite system of N confined electrons with inclusion of the Darwin magnetic interaction in two and three dimensions. The lowest-energy states are located using the steepest descent quenching adapted for velocity dependent potentials. Below a critical density the ground state is a static Wigner lattice. For supercritical density the ground state has a nonzero kinetic energy. The critical density decreases with N for exponential confinement but not for harmonic confinement. The lowest-energy state also depends on the confinement and dimension: an antiferromagnetic cluster forms for harmonic confinement in two dimensions.

Antimicrobial activity of MHC class I-restricted CD8+ T cells in human tuberculosis.

Studies of mouse models of tuberculosis (TB) infection have indicated a central role for MHC class I-restricted CD8+ T cells in protective immunity. To define antigens and epitopes of Mycobacterium tuberculosis (MTB) proteins that are presented by infected cells to CD8+ T cells, we screened 40 MTB proteins for HLA class I A*0201-binding motifs. Peptides that bound with high affinity to purified HLA molecules were subsequently analyzed for recognition by CD8+ cytotoxic T lymphocytes. We identified three epitopes recognized by CD8+ T cells from patients recovering from TB infection. Those three epitopes were derived from three different antigens: thymidylate synthase (ThyA(30-38)), RNA polymerase beta-subunit (RpoB(127-135)), and a putative phosphate transport system permease protein A-1 (PstA1(75-83)). In addition, CD8+ T cell lines specific for three peptides (ThyA(30-38), PstA1(75-83), and 85B(15-23)) were generated from peripheral blood mononuclear cells of normal HLA-A*0201 donors. These CD8+ T cell lines specifically recognized MTB-infected macrophages, as demonstrated by production of IFN-gamma and lysis of the infected target cells. Finally, CD8+ cytotoxic T lymphocytes reduced the viability of the intracellular MTB, providing evidence that CD8+ T cell recognition of MHC class I-restricted epitopes of these MTB antigens can contribute to effective immunity against the pathogen.

Recent developments in mycobacterial research.

Tuberculosis remains a major health problem in the world, which is compounded further by the alarmingly high rate of M. tuberculosis infections in AIDS patients. Thus, there is an urgent need to advance our understanding of the mycobacterium to develop new drugs. The extraordinary recent developments in mycobacterial genetic research, particularly in genomics will greatly facilitate this goal. The knowledge of the entire genome sequence of M. tuberculosis will help in designing new chemotherapeutic and immunotherapeutic interventions. This review highlights recent developments in genomics, mycobacterial genetics, novel vaccine strategies, and our understanding of tuberculous dormancy.

Determinants of T cell reactivity to the Mycobacterium leprae GroES homologue.

The 10-kDa protein Ag of Mycobacterium leprae, a human GroES hsp10 cognate, is a major T cell Ag in human leprosy infection. We investigated the mechanism for T cell responsiveness to this Ag according to the trimolecular interaction between T cell, peptide, and Ag-presenting element. This research was accomplished by mapping T cell epitopes in leprosy patients and correlating these responses with peptide-MHC binding affinities. We found that the majority of tuberculoid leprosy patients responded to peptides corresponding to residues 25-39 and 28-42. Truncation analysis of these peptides mapped the exact epitope to be within the overlapping region comprising residues 28-39. Responsiveness was correlated with the HLA-DRB5*0101 allele, which bound the peptides with moderate affinity. This allele is linked to HLA-DR2, which is associated with the resistant form of leprosy. Therefore, T cell responsiveness in tuberculoid leprosy may be mediated by the ability of HLA-DRB5*0101 to bind and present peptides of the immunodominant 10-kDa Ag.

Immune response to recombinant mycobacterial proteins in patients with tuberculosis infection and disease.

The capacity of four Mycobacterium tuberculosis recombinant antigens to elicit proliferation and cytokine production by human T cells was evaluated. Proliferative responses of peripheral blood mononuclear cells (PBMC) to all antigens were greater in healthy tuberculin reactors than in pulmonary tuberculosis patients, and proliferative responses of pleural fluid cells were greater than those of PBMC from patients with tuberculous pleuritis. The proliferative responses to the four recombinant antigens were similar in all patient groups, and there was no selective unresponsiveness to any antigen in pulmonary tuberculosis patients. The 38-kDa antigen induced less interferon-gamma than did the 10-, 30-, and 65-kDa antigens, and all four antigens induced similar amounts of interleukin-10. These results suggest that none of the four recombinant antigens are immunodominant, and that the 10-, 30-, and 65-kDa antigens are similar in their capacity to induce a potentially protective Th1-like response.

Structure of the heat shock protein chaperonin-10 of Mycobacterium leprae.

Members of the chaperonin-10 (cpn10) protein family, also called heat shock protein 10 and in Escherichia coli GroES, play an important role in ensuring the proper folding of many proteins. The crystal structure of the Mycobacterium leprae cpn10 (Ml-cpn10) oligomer has been elucidated at a resolution of 3.5 angstroms. The architecture of the Ml-cpn10 heptamer resembles a dome with an oculus in its roof. The inner surface of the dome is hydrophilic and highly charged. A flexible region, known to interact with cpn60, extends from the lower rim of the dome. With the structure of a cpn10 heptamer now revealed and the structure of the E. coli GroEL previously known, models of cpn10:cpn60 and GroEL:GroES complexes are proposed.

Vaccination with pure Mycobacterium leprae proteins inhibits M. leprae multiplication in mouse footpads.

In this study, we evaluated vaccination with a number of purified, as well as recombinant, Mycobacterium leprae proteins for protective efficacy in mice. BALB/c mice were immunized intradermally with various native somatic (purified) or recombinant M. leprae proteins and their synthetic polypeptides emulsified in Freund's incomplete adjuvant. The protective efficacy of these preparations was assessed by enumeration of bacilli in the footpads of mice challenged with viable M. leprae 1 to 2 months following immunization. Protection was afforded by the purified and recombinant 10-kDa M. leprae cytoplasmic heat shock protein, the recombinant cell wall-associated 65-kDa M. leprae heat shock protein, and to a lesser extent, the purified 28-kDa M. leprae cytoplasmic protein (superoxide dismutase). Vaccination with either the purified or recombinant 35-kDa M. leprae cell membrane protein, the synthetic 27-amino-acid N-terminal peptide of the 10-kDa protein, the recombinant 18-kDa M. leprae protein, or the purified 22-kDa cell membrane protein was ineffective. When the interval between immunization and challenge was increased to 6 months, the purified 10-kDa M. leprae protein and the recombinant 65-kDa M. leprae protein lost vaccine efficacy, while a sodium dodecyl sulfate-soluble protein fraction of the M. leprae cell wall (soluble proteins), as had been found previously, continued to protect, suggesting that multiple M. leprae protein epitopes are critical for solid vaccine protection.

Chemical definition, cloning, and expression of the major protein of the leprosy bacillus.

The decline in prevalence of leprosy is not necessarily matched by a fall in incidence, emphasizing the need for new antigens to measure disease transmission and reservoirs of infection. Mycobacterium leprae obtained from armadillo tissues was disrupted and subjected to differential centrifugation to arrive at preparations of cell wall, cytoplasmic membrane, and cytosol. By committing 0.3 g of M. leprae to the task, it was possible to isolate from the cytosol and fully define the major cytosolic protein. Amino-terminus sequencing and chemical and enzymatic cleavage, followed by more sequencing and fast atom bombardment-mass spectrometry of fragments, allowed description of the entire amino acid sequence of a protein of 10,675-Da molecular mass. The sequence derived by chemical means is identical to that deduced previously from DNA analysis of the gene of a 10-kDa protein, a GroES analog. The work represents the first complete chemical definition of an M. leprae protein. PCR amplification of the 10-kDa protein gene, when cloned into Escherichia coli with a pTRP expression vector, allowed production of the recombinant protein. Chemical analysis of the expressed protein demonstrated that it exactly reflected the native protein. The recombinant major cytosolic protein appears to be a promising reagent for skin testing, still probably the most appropriate and pragmatic means of measuring incidence of leprosy.

Comparison of fresh autogenous with formalin preserved allogeneic bone grafts in rabbits. An experimental study.

A complete circumferential gap was produced in the diaphysis of the ulna in rabbits and the radius was left intact. The gap on the right side was filled with formalin preserved allogeneic (FPA) bone graft, and fresh autogenous bone taken from the right ulna was put into the gap on the left side. The gap was left untouched in a control group. Up to 8 weeks, the rate of union was slower in the FPA grafts compared with the autogenous grafts. Maximum fluorescence was seen at 10 to 16 weeks and was the same in both groups. These results indicate that osteoinduction occurs with normal bony repair in 80% of both groups, although the onset of healing is delayed in the FPA grafts.

Immunoreactivity of a 10-kDa antigen of Mycobacterium tuberculosis.

Identification of Ag of Mycobacterium tuberculosis recognized by T cells is essential to understanding the pathogenesis of tuberculosis and mechanism(s) of resistance to infection. Previous studies evaluating the immunoreactivity of nitrocellulose transfers of M. tuberculosis Ag separated by SDS-PAGE indicated that a high proportion of M. tuberculosis-reactive T cell lines proliferate in response to a 10-kDa Ag. We therefore purified this Ag from M. tuberculosis culture filtrates and evaluated its immunoreactivity in patients with tuberculous infection. Proliferative responses of PBMC to the 10-kDa Ag were similar to those induced by whole M. tuberculosis and greater than those elicited by other proteins isolated from culture filtrate. Furthermore, in patients with tuberculous pleuritis, proliferative responses to the 10-kDa Ag were higher in pleural fluid mononuclear cells than in PBMC, indicating that T cell reactivity to this Ag is enhanced at the site of disease. The first 15 amino acids of the 10-kDa Ag were identical to those defined previously for Bacillus Calmette-Guérin-a (BCG-a), and a T cell clone recognized the 10-kDa Ag and a peptide of BCG-a, indicating that the 10-kDa Ag corresponds to BCG-a. This Ag elicited IFN-gamma production by pleural fluid mononuclear cells and by PBMC from healthy tuberculin reactors, suggesting that the 10-kDa Ag can enhance macrophage activation and resistance to mycobacterial infection. Our findings indicate that the 10-kDa Ag of M. tuberculosis is highly immunoreactive and should be evaluated for its capacity to elicit protective immunity.

A major T cell antigen of Mycobacterium leprae is a 10-kD heat-shock cognate protein.

Several mycobacterial antigens, identified by monoclonal antibodies and patient sera, have been found to be homologous to stress or heat-shock proteins (hsp) defined in Escherichia coli and yeast. A major antigen recognized by most Mycobacterium leprae-reactive human T cell lines and cell wall-reactive T cell clones is a 10-kD protein that has now been cloned and sequenced. The predicted amino acid sequence of this protein is 44% homologous to the hsp 10 (GroES) of E. coli. The purified native and recombinant 10-kD protein was found to be a stronger stimulator of peripheral blood T cell proliferation than other native and recombinant M. leprae proteins tested. The degree of reactivity paralleled the response to intact M. leprae throughout the spectrum of leprosy. Limiting-dilution analysis of peripheral blood lymphocytes from a patient contact and a tuberculoid patient indicated that approximately one third of M. leprae-reactive T cell precursors responded to the 10-kD antigen. T cell lines derived from lepromin skin tests were strongly responsive to the 10-kD protein. T cell clones reactive to both the purified native and recombinant 10-kD antigens recognized M. leprae-specific epitopes as well as epitopes crossreactive with the cognate antigen of M. tuberculosis. Further, the purified hsp 10 elicited strong delayed-type hypersensitivity reactions in guinea pigs sensitized to M. leprae. The strong T cell responses against the M. leprae 10-kD protein suggest a role for this heat-shock cognate protein in the protective/resistant responses to infection.

Mortality and cataract: findings from a population-based longitudinal study.

The study was carried out in a rural population in central India. A random sample of 11 village communities provided 1020 persons aged 40-64 years, who were examined in 1982 and again reassessed in 1986. Statistical analysis, based on the Mantel-Haenszel method for stratified data, showed increased mortality in persons who had central lens opacities, compared with those who had trivial or no central lens opacities. The significant age-adjusted death ratio was just over 2 (2.2), as were the age/sex-adjusted and age/vision-adjusted estimates, which indicate doubling of mortality in the cataract cohort. Multiple regression analysis using the Cox proportional-hazards model gave very similar results. Statistical tests for homogeneity of death ratios across the various age/sex/vision strata were carried out, and the observed association between cataract and mortality was found to be consistent, both in males and in females, in the youngest and oldest age groups, and among those with adequate vision of 6/18 or better as well as among persons with serious visual impairment. There were no known diabetics in the study sample, which came from what could reasonably be regarded as a non-diabetic population.