Peter Timms

Chlamydial Persistence beyond the Biphasic Paradigm

The chlamydiae are an evolutionarily distinct group of eubacteria sharing an obligate intracellular lifestyle and a unique developmental cycle that has been well characterized under favorable cell culture conditions. This cycle begins when infectious, metabolically inert elementary bodies (EB)

High-dose vitamin D(3) during intensive-phase antimicrobial treatment of pulmonary tuberculosis: a double-blind randomised controlled trial.

BACKGROUND Vitamin D was used to treat tuberculosis in the pre-antibiotic era, and its metabolites induce antimycobacterial immunity in vitro. Clinical trials investigating the effect of adjunctive vitamin D on sputum culture conversion are absent. METHODS We undertook a multicentre randomised controlled trial of adjunctive vitamin D in adults with sputum smear-positive pulmonary tuberculosis in London, UK. 146 patients were allocated to receive 2·5 mg vitamin D(3) or placebo at baseline and 14, 28, and 42 days after starting standard tuberculosis treatment. The primary endpoint was time from initiation of antimicrobial treatment to sputum culture conversion. Patients were genotyped for TaqI and FokI polymorphisms of the vitamin D receptor, and interaction analyses were done to assess the influence of the vitamin D receptor genotype on response to vitamin D(3). This trial is registered with ClinicalTrials.gov number NCT00419068. FINDINGS 126 patients were included in the primary efficacy analysis (62 assigned to intervention, 64 assigned to placebo). Median time to sputum culture conversion was 36·0 days in the intervention group and 43·5 days in the placebo group (adjusted hazard ratio 1·39, 95% CI 0·90-2·16; p=0.14). TaqI genotype modified the effect of vitamin D supplementation on time to sputum culture conversion (p(interaction)=0·03), with enhanced response seen only in patients with the tt genotype (8·09, 95% CI 1·36-48·01; p=0·02). FokI genotype did not modify the effect of vitamin D supplementation (p(interaction)=0·85). Mean serum 25-hydroxyvitamin D concentration at 56 days was 101·4 nmol/L in the intervention group and 22·8 nmol/L in the placebo group (95% CI for difference 68·6-88·2; p<0·0001). INTERPRETATION Administration of four doses of 2·5 mg vitamin D(3) increased serum 25-hydroxyvitamin D concentrations in patients receiving intensive-phase treatment for pulmonary tuberculosis. Vitamin D did not significantly affect time to sputum culture conversion in the whole study population, but it did significantly hasten sputum culture conversion in participants with the tt genotype of the TaqI vitamin D receptor polymorphism. FUNDING British Lung Foundation.Summary Background Vitamin D was used to treat tuberculosis in the pre-antibiotic era, and its metabolites induce antimycobacterial immunity in vitro. Clinical trials investigating the effect of adjunctive vitamin D on sputum culture conversion are absent. Methods We undertook a multicentre randomised controlled trial of adjunctive vitamin D in adults with sputum smear-positive pulmonary tuberculosis in London, UK. 146 patients were allocated to receive 2·5 mg vitamin D 3 or placebo at baseline and 14, 28, and 42 days after starting standard tuberculosis treatment. The primary endpoint was time from initiation of antimicrobial treatment to sputum culture conversion. Patients were genotyped for Taq I and Fok I polymorphisms of the vitamin D receptor, and interaction analyses were done to assess the influence of the vitamin D receptor genotype on response to vitamin D 3 . This trial is registered with ClinicalTrials.gov number NCT00419068. Findings 126 patients were included in the primary efficacy analysis (62 assigned to intervention, 64 assigned to placebo). Median time to sputum culture conversion was 36·0 days in the intervention group and 43·5 days in the placebo group (adjusted hazard ratio 1·39, 95% CI 0·90–2·16; p=0.14). Taq I genotype modified the effect of vitamin D supplementation on time to sputum culture conversion (p interaction =0·03), with enhanced response seen only in patients with the tt genotype (8·09, 95% CI 1·36–48·01; p=0·02). Fok I genotype did not modify the effect of vitamin D supplementation (p interaction =0·85). Mean serum 25-hydroxyvitamin D concentration at 56 days was 101·4 nmol/L in the intervention group and 22·8 nmol/L in the placebo group (95% CI for difference 68·6–88·2; p Interpretation Administration of four doses of 2·5 mg vitamin D 3 increased serum 25-hydroxyvitamin D concentrations in patients receiving intensive-phase treatment for pulmonary tuberculosis. Vitamin D did not significantly affect time to sputum culture conversion in the whole study population, but it did significantly hasten sputum culture conversion in participants with the tt genotype of the TaqI vitamin D receptor polymorphism. Funding British Lung Foundation.

Vitamin D accelerates resolution of inflammatory responses during tuberculosis treatment

Calcidiol, the major circulating metabolite of vitamin D, supports induction of pleiotropic antimicrobial responses in vitro. Vitamin D supplementation elevates circulating calcidiol concentrations, and thus has a potential role in the prevention and treatment of infection. The immunomodulatory effects of administering vitamin D to humans with an infectious disease have not previously been reported. To characterize these effects, we conducted a detailed longitudinal study of circulating and antigen-stimulated immune responses in ninety-five patients receiving antimicrobial therapy for pulmonary tuberculosis who were randomized to receive adjunctive high-dose vitamin D or placebo in a clinical trial, and who fulfilled criteria for per-protocol analysis. Vitamin D supplementation accelerated sputum smear conversion and enhanced treatment-induced resolution of lymphopaenia, monocytosis, hypercytokinaemia, and hyperchemokinaemia. Administration of vitamin D also suppressed antigen-stimulated proinflammatory cytokine responses, but attenuated the suppressive effect of antimicrobial therapy on antigen-stimulated secretion of IL-4, CC chemokine ligand 5, and IFN-α. We demonstrate a previously unappreciated role for vitamin D supplementation in accelerating resolution of inflammatory responses during tuberculosis treatment. Our findings suggest a potential role for adjunctive vitamin D supplementation in the treatment of pulmonary infections to accelerate resolution of inflammatory responses associated with increased risk of mortality.

Epizootiology of Chlamydia infections in two free-range koala populations.

The prevalence of Chlamydia pecorum and Chlamydia pneumoniae infections in two free-range koala populations was assessed using genus-specific PCR combined with species-specific DNA probe hybridisation. Population A had a very high overall level of chlamydial infection (85%) with significantly more of these infections being due to C. pecorum (73%) compared to C. pneumoniae (24%). The second population had a much lower prevalence of infection (10%) with equal levels of both species. An important finding of this study was that. while five of 24 C. pecorum-infected koalas had clinical signs of the disease (both ocular and urogenital sites), none out of seven C. pneumoniae-infected koalas had signs of clinical disease. This suggests that C. pecorum may be the more pathogenic of the two chlamydial species infecting this host. The level of infection (assessed by intensity of the specific hybridisation signal) also differed between chlamydial species, with C. pecorum infections ranging from low to high grade whereas C. pneumoniae infections were always low grade. When the age of infected koalas was examined, 58% of young, sexually immature koalas were found to have C. pecorum infections, increasing to 100% of koalas in the older age groups. This suggests that, in this population at least, young koalas are readily infected with C. pecorum from their mothers. While the infection levels with C. pneumoniae were too low to be statistically significant, again, sexually immature koalas were found to be infected. The recent separation of chlamydial infections in koalas into two species is beginning to indicate different epizootiologies for koala C. pecorum compared to koala C. pneumoniae.

Molecular Evidence to Support the Expansion of the Hostrange of Chlamydophila pneumoniae to Include Reptiles as Well as Humans, Horses, Koalas and Amphibians

The Chlamydiales are a family of unique intracellular pathogens that cause significant disease in humans, birds and a wide range of animal hosts. Of the currently recognized species, Chlamydophila (previously Chlamydia) pneumoniae, unlike the other chlamydial species, has been previously considered to be solely a pathogen of humans, causing significant respiratory disease and has also been strongly connected with cardiovascular disease. Here we report the finding that strains of C. pneumoniae are widespread in the environment, being detected by molecular methods in a range of reptiles (snakes, iguanas, chameleons) and amphibians (frogs, turtles). Of particular interest was the finding that genotyping of the chlamydial major outer membrane protein gene in these newly identified C. pneumoniae strains showed that many were genetically very similar, if not identical to the human respiratory strains. Whether these reptilian and amphibian strains of C. pneumoniae are still capable of infecting humans, or crossed the host barrier some time ago, remains to be determined but may provide further insights into the relationship of this common respiratory infection with its human host.

1α,25-dihydroxyvitamin D3 inhibits matrix metalloproteinases induced by Mycobacterium tuberculosis infection

Matrix metalloproteinases (MMP) can degrade all components of pulmonary extracellular matrix. Mycobacterium tuberculosis induces production of a number of these enzymes by human macrophages, and these are implicated in the pathogenesis of pulmonary cavitation in tuberculosis. The active metabolite of vitamin D, 1α,25‐dihydroxyvitamin D3 [1α,25(OH)2D3], has previously been reported to inhibit secretion of MMP‐9 in human monocytes (MN), but its influence on the secretion and gene expression of MMP and tissue inhibitors of MMP (TIMP) in M. tuberculosis‐infected cells has not previously been investigated. We therefore determined the effects of 1α,25(OH)2D3 on expression, secretion and activity of a number of MMP and TIMP in M. tuberculosis‐infected human leucocytes; we also investigated the effect of 1α,25(OH)2D3 on the secretion of interleukin‐10 (IL‐10) and prostaglandin E2 (PGE2), both transcriptional regulators of MMP expression. We found that M. tuberculosis induced expression of MMP‐1, MMP‐7 and MMP‐10 in MN and MMP‐1 and MMP‐10 in peripheral blood mononuclear cells (PBMC). 1α,25(OH)2D3 significantly attenuated M. tuberculosis‐induced increases in expression of MMP‐7 and MMP‐10, and suppressed secretion of MMP‐7 by M. tuberculosis‐infected PBMC. MMP‐9 gene expression, secretion and activity were significantly inhibited by 1α,25(OH)2D3 irrespective of infection. In contrast, the effects of 1α,25(OH)2D3 on the expression of TIMP‐1, TIMP‐2 and TIMP‐3 and secretion of TIMP‐1 and TIMP‐2 were small and variable. 1α,25(OH)2D3 also induced secretion of IL‐10 and PGE2 from M. tuberculosis‐infected PBMC. These findings represent a novel immunomodulatory role for 1α,25(OH)2D3 in M. tuberculosis infection.

Recent advances in understanding the biology, epidemiology and control of chlamydial infections in koalas

The koala (Phascolarctos cinereus) is recognised as a threatened wildlife species in various parts of Australia. A major contributing factor to the decline and long-term viability of affected populations is disease caused by the obligate intracellular bacteria, Chlamydia. Two chlamydial species infect the koala, Chlamydia pecorum and Chlamydia pneumoniae, and have been reported in nearly all mainland koala populations. Chlamydial infections of koalas are associated with ocular infections leading to blindness and genital tract infections linked to infertility, among other serious clinical manifestations. Diagnosis can be based on clinical presentation alone, however, it is complicated by the observation that many koala chlamydial infections occur with no overt signs of clinical disease. Instead, accurate diagnosis requires detailed clinical assessment and confirmatory testing by a range of PCR-based assays. Antibiotic treatment for koala chlamydial infection is possible, however, results on its success are mixed. A more practical solution for the protection of diseased populations is the application of a koala Chlamydia vaccine, with recent trials indicating promising results. Interestingly, molecular epidemiology studies of koala C. pecorum infections and recent comparative genomic analyses of koala C. pneumoniae have revealed potential differences in their origin that will have wider ramifications for our understanding of human chlamydial infections and host adaptation of the chlamydiae. This review summarises changes to the taxonomy of koala chlamydial infections and recent advances in our understanding of the epidemiology, diagnosis, treatment, control and evolution of Chlamydia infections in this iconic wildlife species.

Chlamydia trachomatis infection: incidence, health costs and prospects for vaccine development

Chlamydia trachomatis infection is now the most common sexually transmitted disease worldwide. World Health Organisation figures estimated that 89 million new cases of genital Chlamydia infections occurred in 1995, highlighting the worldwide prevalence of infections and the economic burden on healthcare delivery. A number of methods have been developed for detection of chlamydial infection, which vary in sensitivity and specificity. No single method has yet gained general acceptance and in many countries Chlamydia infections are not reported, suggesting that the above figures may be an underestimate of the problem. As yet there is no consensus as to what constitutes a protective immune response against genital Chlamydia infection. Studies in animal models have shown that cell-mediated immunity, both Th1-driven macrophage activation and cytotoxic T cell responses, as well as antibody can mediate protection at different stages of the chlamydial life cycle. A successful vaccine would probably need to elicit both a systemic cell-mediated immune response to limit/resolve established infections and a mucosal IgA response to reduce bacterial shedding and the resulting spread of infection to partners of infected individuals. The immune response to Chlamydia, either through natural infection or following immunisation, also has the potential to enhance inflammation and to act as a driving force for constant mutation in the variable regions of the major outer membrane protein. As a result a constant prevalence of infection is maintained even in an immune population through the emergence of new allelic variants. Immune responses against antigens such as the 60 kDa heat shock protein can exacerbate inflammation through molecular mimicry and must not be elicited as a result of vaccination. Thus there are many challenges for the development of a successful vaccine which must elicit immunity against multiple serovars while at the same time minimising damaging pro-inflammatory immune responses.

IMMUNOPATHOGENESIS OF CHLAMYDIA TRACHOMATIS INFECTIONS IN WOMEN

OBJECTIVE To develop a model of pathogenesis by which Chlamydia trachomatis progresses from acute to chronic infection, and finally serious disease (salpingitis, tubal occlusion). DESIGN Review of current literature located through web-based Medline searches using key words: Chlamydia trachomatis, immunology, cytokines, heat shock protein, infertility. RESULT(S) Cell-mediated immune mechanisms appear to be critical in determining whether acute infection is resolved or progresses into chronicity with pathological outcome. What determines the particular immune pathway depends on a range of determinants-HLA subtype and human genetics, cytokine profile, infectious load, route of infection, and endocrinology. A clearer picture of the natural history of chlamydial pathology may assist in providing better predictors of those women who may go on to develop significant sequelae after infection. CONCLUSION(S) Predicting those who may develop serious disease, including infertility, may contribute to improved management of such persons during earlier stages of infection and assist in prevention.

Chlamydia muridarum Infection-Induced Destruction of Male Germ Cells and Sertoli Cells Is Partially Prevented by Chlamydia Major Outer Membrane Protein-Specific Immune CD4 cells

ABSTRACT Chlamydia trachomatis infections are increasingly prevalent worldwide. Male chlamydial infections are associated with urethritis, epididymitis, and orchitis; however, the role of Chlamydia in prostatitis and male factor infertility remains controversial. Using a model of Chlamydia muridarum infection in male C57BL/6 mice, we investigated the effects of chlamydial infection on spermatogenesis and determined the potential of immune T cells to prevent infection-induced outcomes. Antigen-specific CD4 T cells significantly reduced the infectious burden in the penile urethra, epididymis, and vas deferens. Infection disrupted seminiferous tubules, causing loss of germ cells at 4 and 8 wk after infection, with the most severely affected tubules containing only Sertoli cells. Increased mitotic proliferation, DNA repair, and apoptosis in spermatogonial cells and damaged germ cells were evident in atrophic tubules. Activated caspase 3 (casp3) staining revealed increased (6-fold) numbers of Sertoli cells with abnormal morphology that were casp3 positive in tubules of infected mice, indicating increased levels of apoptosis. Sperm count and motility were both decreased in infected mice, and there was a significant decrease in morphologically normal spermatozoa. Assessment of the spermatogonial stem cell population revealed a decrease in promyelocytic leukemia zinc finger (PLZF)-positive cells in the seminiferous tubules. Interestingly, adoptive transfer of antigen-specific CD4 cells, particularly T-helper 2-like cells, prior to infection prevented these effects in spermatogenesis and Sertoli cells. These data suggest that chlamydial infection adversely affects spermatogenesis and male fertility, and that vaccination can potentially prevent the spread of infection and these adverse outcomes.