Sequence variation and immunogenicity of the Mycoplasma genitalium MgpB and MgpC adherence proteins during persistent infection of men with non-gonococcal urethritis.

Mycoplasma genitalium is a sexually transmitted bacterial pathogen that infects men and women. Antigenic variation of MgpB and MgpC, the immunodominant adherence proteins of M. genitalium, is thought to contribute to immune evasion and chronic infection. We investigated the evolution of mgpB and mgpC sequences in men with non-gonococcal urethritis persistently infected with M. genitalium, including two men with anti-M. genitalium antibodies at enrollment and two that developed antibodies during follow-up. Each of the four patients was persistently infected with a different strain type and each patient produced antibodies targeting MgpB and MgpC. Amino acid sequence evolution in the variable regions of MgpB and MgpC occurred in all four patients with changes observed in single and multiple variable regions over time. Using the available crystal structure of MgpC of the G37 type strain we found that predicted conformational B cell epitopes localize predominantly to the variable region of MgpC, amino acids that changed during patient infection lie in these epitopes, and variant amino acids are in close proximity to the conserved sialic acid binding pocket. These findings support the hypothesis that sequence variation functions to avoid specific antibodies thereby contributing to persistence in the genital tract.

Neisseria meningitidis Urethritis Outbreak Isolates Express a Novel Factor H Binding Protein Variant That Is a Potential Target of Group B-Directed Meningococcal (MenB) Vaccines.

Factor H binding protein (FHbp) is an important Neisseria meningitidis virulence factor that binds a negative regulator of the alternative complement pathway, human factor H (FH). Binding of FH increases meningococcal resistance to complement-mediated killing. FHbp also is reported to prevent interaction of the antimicrobial peptide (AMP) LL-37 with the meningococcal surface and meningococcal killing. FHbp is a target of two licensed group B-directed meningococcal (MenB) vaccines. We found a new FHbp variant, peptide allele identification no. 896 (ID 896), was highly expressed by an emerging meningococcal pathotype, the nonencapsulated urethritis clade (US_NmUC). This clade has been responsible for outbreaks of urethritis in multiple U.S. cities since 2015, other mucosal infections, and cases of invasive meningococcal disease. FHbp ID 896 is a member of the variant group 1 (subfamily B), bound protective anti-FHbp monoclonal antibodies, bound high levels of human FH, and enhanced the resistance of the clade to complement-mediated killing in low levels of human complement likely present at human mucosal surfaces. Interestingly, expression of FHbp ID 896 resulted in augmented killing of the clade by LL-37. FHbp ID 896 of the clade was recognized by antibodies elicited by FHbp in MenB vaccines.

Experimental Human Infection with Neisseria gonorrhoeae.

Experimental infection of male volunteers with Neisseria gonorrhoeae is safe and reproduces the clinical features of naturally acquired gonococcal urethritis. The human model is useful for testing the importance of putative gonococcal virulence factors for urethral infection in men and the model presents opportunities to examine host immune responses that may be exploited or improved in development and testing of gonococcal vaccines. In this chapter, we describe methods for production, characterization, and storage of N. gonorrhoeae stocks for experimental human challenge, preparation and delivery of inoculum suspensions, monitoring experimental infection, and statistical considerations for data analysis.

Chronically Elevated Levels of Short-Chain Fatty Acids Induce T Cell-Mediated Ureteritis and Hydronephrosis.

Short-chain fatty acids (SCFAs) are major products of gut microbial fermentation and profoundly affect host health and disease. SCFAs generate IL-10(+) regulatory T cells, which may promote immune tolerance. However, SCFAs can also induce Th1 and Th17 cells upon immunological challenges and, therefore, also have the potential to induce inflammatory responses. Because of the seemingly paradoxical SCFA activities in regulating T cells, we investigated, in depth, the impact of elevated SCFA levels on T cells and tissue inflammation in mice. Orally administered SCFAs induced effector (Th1 and Th17) and regulatory T cells in ureter and kidney tissues, and they induced T cell-mediated ureteritis, leading to kidney hydronephrosis (hereafter called acetate-induced renal disease, or C2RD). Kidney hydronephrosis in C2RD was caused by ureteral obstruction, which was, in turn, induced by SCFA-induced inflammation in the ureteropelvic junction and proximal ureter. Oral administration of all major SCFAs, such as acetate, propionate, and butyrate, induced the disease. We found that C2RD development is dependent on mammalian target of rapamycin activation, T cell-derived inflammatory cytokines such as IFN-γ and IL-17, and gut microbiota. Young or male animals were more susceptible than old or female animals, respectively. However, SCFA receptor (GPR41 or GPR43) deficiency did not affect C2RD development. Thus, SCFAs, when systemically administered at levels higher than physiological levels, cause dysregulated T cell responses and tissue inflammation in the renal system. The results provide insights into the immunological and pathological effects of chronically elevated SCFAs.

Molecular analysis of two novel Neisseria gonorrhoeae virulent components: the macrophage infectivity potentiator and the outer membrane protein A.

Molecular analyses of mip and ompA genes were performed on 20 Neisseria gonorrhoeae isolates. The genes were present with a high degree of conservation in all strains. Sera from patients with urethritis or disseminated gonococcal infections were able to recognize the purified Neisseria gonorrhoeae macrophage infectivity potentiator (Ng-MIP) and Neisseria gonorrhoeae outer membrane protein A (Ng-OmpA).

Local host response to chlamydial urethral infection in male guinea pigs.

Very little is known about the host response to chlamydial genital infection in the male, particularly about the nature of the local response in the urethra. In this study, the pathological and immunologic responses to urethral infection of the male guinea pig with Chlamydia caviae (Chlamydophila caviae) were characterized both during a primary infection and following a challenge infection. A dose-response experiment found that the 50% infectious dose for male urethral infection was 78 inclusion-forming units. The histopathologic response was similar to that of the female, with an initial acute inflammatory response followed by a chronic inflammatory response and plasma cell infiltration. Production of IgG and IgA antibodies in local urethral secretions developed following infection, and levels of both increased in a typical anamnestic response following a challenge infection. CD4 and CD8 T cells, as well as B cells, were observed in the local site by flow cytometry, with a slightly increased number of CD8 cells. Following challenge infection, the dominant anamnestic response was solely in the B-cell compartment, with only a minimal number of T cells. The T-cell response was clearly a Th1 response, as judged by increased levels of gamma interferon (IFN-gamma), interleukin-12 p40 (IL-12p40), and IL-2. The proinflammatory cytokines and chemokines IL-8, IL-1beta, tumor necrosis factor alpha (TNF-alpha), CCL2 (monocyte chemoattractant protein 1 [MCP-1]), and CCL5 (RANTES) were elicited in the urethra following primary infection, but only CCL5 showed increased levels upon challenge. This study represents the first comprehensive analysis of the local immune response in the male urethra to a chlamydial genital infection.

Clinical tetrad of arthritis, urethritis, conjunctivitis, and mucocutaneous lesions (HLA-B27-associated spondyloarthropathy, Reiter syndrome): report of a case.

A 21-year-old male patient with the clinical tetrad of arthritis, urethritis, conjunctivitis, and mucocutaneous lesions, commonly known as Reiter syndrome was presented. He was hospitalized in poor condition, with fever, bilateral conjunctivitis, swollen and painful knee and tarsal joints, low back pain, Achilles tendonitis, dactilitis, keratoderma blenorrhagica, purulent urethritis, circinate balanitis, and oral erosive lesions. Radiography and Computerized Axial Tomography (CAT) showed sacroileitis, spondilosis thoracalis, and arthritis of the feet. The laboratory studies revealed anemia, neutrophilic leukocytosis, elevated erythrocyte sedimentation rate (ESR), hypoalbuminemia, negative rheumatoid factor, pyuria, proteinuria, and the presence of HLA-B27. The microbiological examinations of samples from pustular lesions, throat, eyes, urethra, stool, and blood were sterile. Urethral smear was positive for Chlamydia trachomatis (PCR). The histopathological picture of skin lesions was consistent with pustular psoriasis. Systemic treatment with antibiotics, corticosteroids, and non-steroidal anti-inflammatory drugs produced clinical improvement. This clinical syndrome requires comprehensive evaluation and multidisciplinary management.

Male urethritis with and without discharge: relation to microbiological findings and polymorphonuclear counts.

OBJECTIVE: We studied the relation between the presence or absence of urethral discharge, urethral pathogens, and polymorphonuclear (PMN) counts on Gram stained urethral smears in men with symptomatic urethritis. METHODS: The study population was composed of 630 sexually active heterosexual men (aged 18-45 years) who had urethral symptoms and signs (discharge, dysuria or urethral discomfort). Participants were divided into two groups: the first (n=320) was comprised of men with urethral discharge confirmed on examination, while the other (n=310) was composed of patients with urethral symptoms but without discharge. Urethral swabs for Gram stained smears and microbiological analyses (N. gonorrhoeae, C. trachomatis, T. vaginalis and U. urealyticum) were taken from all study participants. Polymorphonuclear leukocytes (PMN) on Gram-stained urethral smears were counted in 5 oil immersion x1000 PMN per high power fields (phpf). Urethritis was defined as the presence of > or =5 PMN/hpf. RESULTS: N. gonorrhoeae was isolated only in men with urethritis accompanied by discharge. The prevalence of T. vaginalis, C. trachomatis and U. urealyticum was significantly higher (F=8.854, P<0.01) in urethral swabs of urethritis patients with discharge compared to patients with no discharge. The most common urethral pathogen in both groups of patients was T. vaginalis (31.56% and 26.45%, respectively). One or more microorganisms were isolated in 258 (81%) subjects with urethritis with discharge, and in 166 (53.5%) urethritis patients without discharge. There was a positive correlation between the significant number of PMN in Gram stained urethral smears and positive microbiological findings in men with urethritis both with and without urethral discharge (Spearmanns coefficients rho=0.986 and rho=0.993, respectively; P<0.01). CONCLUSIONS: The study found a relatively high prevalence of T. vaginalis among our men with urethritis irrespective of the presence or absence of urethral discharge, and showed that taking into account both discharge found on examination, and relevant PMN counts on Gram stained urethral smears fails to detect only 4.2% of oligosymptomatic urethritis patients who are infected with one of the strict urethral pathogens.

Identification and characterization of immunogenic proteins of mycoplasma genitalium.

Mycoplasma genitalium causes nonchlamydial nongonococcal urethritis. M. genitalium was detected by PCR in 17 urethral swabs obtained from 99 men with and without urethritis (J. S. Jensen, R. Orsum, B. Dohn, S. Uldum, A. M. Worm, and K. Lind, Genitourin. Med. 69:265-269, 1993), and later, four M. genitalium strains were isolated (J. S. Jensen, H. T. Hansen, and K. Lind, J. Clin. Microbiol. 34:286-291, 1996). The objective of this study was to characterize immunogenic proteins of M. genitalium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting by using a hyperimmune rabbit serum against M. genitalium G37, determine their identity by mass spectrometry, and develop an M. genitalium-specific enzyme-linked immunosorbent assay (ELISA) free from cross-reactivity with M. pneumoniae antibodies. Using recombinant fragments of the C-terminal part of MgPa (rMgPa), we developed a specific ELISA for detection of M. genitalium antibodies. This antigen did not bind M. pneumoniae antibodies. Using serum samples from the 99 men with and without urethritis, we found that 26 had immunoglobulin G (IgG) antibodies to M. genitalium. There was a strong statistically significant correlation between PCR and IgG antibodies to M. genitalium (odds ratio [OR], 5.9; 95% confidence interval [CI], 2.3 to 21.5; P = 0.002). Furthermore, men with recurrent urethritis were more likely to have antibodies to M. genitalium than were those without recurrent urethritis (OR, 4.0; 95% CI, 1.1 to 14.5; P = 0.0383) and they had significantly higher antibody titers. By use of the rMgPa ELISA, this study further substantiates the importance of M. genitalium as a cause of male urethritis.

Mucosal and systemic immune responses to plasmid protein pgp3 in patients with genital and ocular Chlamydia trachomatis infection.

The circulating and cervical B cell responses to Chlamydia trachomatis plasmid protein pgp3 were characterized in children and adults with ocular or genital chlamydial infection using the enzyme-linked immunospot assay (ELISPOT) and ELISA. No pgp3-specific ASCs were detected in healthy controls, but predominantly IgA ASCs were detected in UK adults with uncomplicated cervicitis or urethritis (P = 0.03, 0.019). In patients with extragenital complications or pelvic inflammatory disease a mixed response with more IgG and IgM ASCs was evident, suggesting a breach of mucosal immune compartmentalization with more extensive infection. In women with chlamydial cervicitis, ASCs secreting predominantly IgA, but also IgG, to pgp3 were present in cervix at presentation, with a frequency 30-50 times higher than blood. Cervical ASC numbers, especially IgG, fell markedly six weeks after antibiotic treatment. We detected principally IgA pgp3-specific antibody secreting cells (ASCs) in children resident in a Gambian endemic area, with a trend towards suppression of IgA responses during intense trachomatous inflammation (P = 0.06), as previously reported for other chlamydial antigens, and in keeping with the findings in genital disease. These data provide a rationale for further studies of immune responses to pgp3 in humans and animal models of chlamydia-induced disease, and its potential use in diagnostic assays and protective immunization strategies.

Antibodies to Chlamydia trachomatis heat-shock protein 60 kDa and detection of Mycoplasma genitalium and ureaplasma urealyticum are associated independently with chronic nongonococcal urethritis.

BACKGROUND: Chronic nongonococcal urethritis (NGU) is a well-recognized clinical problem in genitourinary medicine clinics, but its etiology and optimal management are poorly understood. GOAL: The authors showed previously that antibody to chlamydial hsp60 is associated with urethritis 30 to 92 days after treatment of acute NGU (chronic NGU) and that the detection of ureaplasmas or is associated with chronic NGU in which symptoms or signs are present. The aim was to determine whether these associations are independent of each other. STUDY DESIGN: This was a longitudinal prospective follow-up study over a 3-month period of 86 men with acute NGU. Men were tested for, ureaplasmas, and antibody to chlamydia hsp60 at presentation and during follow-up. RESULTS: The detection of either ureaplasmas or (OR, 29.45; 95% CI, 1.78-487) and the occurrence of hsp60 antibody at 30 to 92 days' follow-up (OR, 26.45; 95% CI, 1.34-523) were associated independently with the development of chronic NGU 30 to 92 days after treatment of acute NGU. The presence of chlamydial hsp60 antibody at 30 to 92 days was not associated with the development of chronic NGU in which symptoms or signs were present (P= 0.363). Chlamydial hsp60 antibody associated, however, with chronic NGU in which there were no symptoms or signs (P = 0.01). CONCLUSION: The results suggest that the immune response to chlamydial hsp60 may have a role in the etiology of chronic NGU in asymptomatic men who have no discharge on examination. The clinical relevance of this is unknown.

The effects of antiretroviral therapy on HIV-1 RNA loads in seminal plasma in HIV-positive patients with and without urethritis.

BACKGROUND: High seminal plasma HIV-1 RNA loads (SVL) have been reported during gonococcal, non-gonococcal and chlamydial urethritis in patients not taking antiretroviral therapy. OBJECTIVE: To examine if urethritis leads to increased SVL in HIV-positive patients taking antiretroviral therapy. METHODS: Men who had been taking therapy for at least 3 months were recruited: 24 had urethitis (PWU) and 16 were without urethritis (controls). At three visits, 1 week apart, blood plasma viral load (BVL) and SVL were assayed by quantitative polymerase chain reaction or the NASBA assay. RESULTS: Most subjects had undetectable SVL (18 PWU, 13 controls). Among those with undetectable BVL prior to first study visit, virus was undetectable in semen in 5/5 episodes of chlamydial urethritis, 6/7 episodes of non-gonococcal urethritis and 4/5 cases of gonococcal urethritis. Two PWU with undetectable BVL just prior to the first study visit had low to moderate SVL, which became undetectable by visit 2 following treatment. Of nine subjects with detectable SVL, eight had detectable BVL (3/3 controls and 5/6 PWU). Of these, 1/3 controls and 4/5 PWU (all with gonococcal urethritis) had poorly controlled BVL just prior to the first study visit. These four PWU had high SVL and one had higher levels in semen than in blood. This patient's SVL was reduced more than 20-fold following treatment for gonococcal urethritis. CONCLUSIONS: Effective antiretroviral therapy appeared to limit the effect of urethritis on SVL. When BVL was poorly controlled by antiretroviral therapy, high SVL occurred during gonococcal urethritis, increasing the potential risk of transmitting both wild type and drug resistant strains of HIV-1.

Recognition of the 60 kilodalton cysteine-rich outer membrane protein OMP2 by CD4(+) T cells from humans infected with Chlamydia trachomatis.

T cell-mediated immunity is important in the control of chlamydia infection but chlamydia-specific T cells are also implicated in the inflammation and tissue damage which characterize chlamydia associated diseases. To investigate target antigens of the T cell-mediated immune response to chlamydia infection, Chlamydia trachomatis-specific CD4+ T cell clones were isolated from a patient with chlamydia-induced reactive arthritis. T cell immunoblotting indicated that an antigen of approximately 60 kilodaltons molecular mass was recognized, and recombinant 60 kilodalton cysteine-rich outer membrane 2 (OMP2) proved to be stimulatory. By using deletion constructs and synthetic peptides an epitope presented by HLA-DRB1*0401 was defined and proved to contain the nonamer peptide within the OMP2 sequence predicted to have the greatest binding affinity for DRB1*0401 The sequence of the epitope is conserved in all C. trachomatis strains but not in C. pneumoniae. Investigation of patients with acute urethritis and additional patients with sexually acquired reactive arthritis showed that OMP2-reactive T cells were readily detectable in peripheral blood and synovial fluid. Thus OMP2 is a target antigen of the T cell-mediated immune response to CT infection.

Experimental gonococcal urethritis and reinfection with homologous gonococci in male volunteers.

BACKGROUND: Reinfection, a common occurrence with gonorrhea, may result from a lack of protective immune response, or from the tremendous gonococcal strain variation. GOAL: A two-phase study in human volunteers tested whether experimental infection with Neisseria gonorrhoeae MS11mkC would protect against reinfection with the same organisms. STUDY DESIGN: In phase 1, an intraurethral inoculum of 57,000 piliated, transparent (opacity protein-negative [Opa-]) MS11mkC N gonorrhoeae infected 14 of 15 (93%) volunteers. The volunteers were encouraged to delay treatment for at least 5 days. In phase 2, which began 2 weeks after treatment for the initial infection, volunteers were inoculated with 7,100 piliated, Opa- MS11mkC. RESULTS: The phase 2 challenge infected 6 of 14 (43%) previously infected volunteers and 5 of 10 (50%) naïve control subjects. Phase 1 volunteers who resisted reinfection were significantly more likely to have had a fourfold or greater increase in lipooligosaccharide immunoglobulin G during phase 1 than those who did not resist reinfection (P = 0.026). CONCLUSIONS: Although infection did not provide protection from reinfection under the conditions used, the results suggest that immunity to reinfection is more complex than anticipated by the experimental design.

Peripheral blood T cell proliferative response to chlamydial organisms in gonococcal and non-gonococcal urethritis and presumed pelvic inflammatory disease.

OBJECTIVE: To study peripheral blood mononuclear cell (PBMC) proliferative response to Chlamydia trachomatis elementary bodies in (a) controls, (b) various stages of gonococcal (c) and non-gonococcal urethritis, and (d) women with a clinical diagnosis of pelvic inflammatory disease (PID). METHODS: We categorised 102 men presenting to a GUM clinic with urethritis by organisms (C trachomatis (CT) or Neisseria gonorrhoeae (NG) (both by culture), and whether it was their first (urethritis naive) or subsequent (urethritis experienced) attack. 23 women presenting to the clinic with a clinical diagnosis of PID were also investigated. We measured PBMC proliferative responses to C trachomatis (DK20--an oculogenital strain, serovar E), lysate of McCoy cells (used to propagate chlamydiae), and the recall antigen PPD. Controls were 37 men and women without present or past history of urethritis or chlamydial infection. Results were expressed as the ratio of the stimulation index (SI) obtained with DK20 compared with McCoy cells (DK index), and the ratio of the SI obtained with DK20 compared with PPD (PPD index). RESULTS: The median SI to DK20 in the urethritis was 12.7 which was significantly higher than the controls (7.6, p < 0.003). The median SI to the recall antigen PPD was similar in the urethritis patients (17.4) and the controls (22.4). All urethritis patient subgroups had a significantly higher DK index and PPD index than the controls. There was no difference in the PPD and DK index between urethritis naive and urethritis experienced patients and between the culture positive and culture negative urethritis subgroups. In PID patients only the PPD index was significantly higher than the controls. CONCLUSION: Men presenting with urethritis and women presenting with PID both have significantly greater peripheral blood mononuclear cell proliferative responses to the DK20 strain of C trachomatis than controls. A similar T cell proliferative response pattern in urethritis naive patients with either gonococcal or non-gonococcal urethritis could be because low sensitivity of CT culture failed to diagnose some cases of C trachomatis. However, it may also signify earlier exposure of the patients to chlamydial antigens (for example, C pneumoniae), cross reacting antigens such as heat shock proteins from other microbial species, or a "bystander" activation of chlamydia specific memory T cells trafficking through mucosal lymphoid tissue during urethritis. These results suggest evidence of T cell mediated response to C trachomatis cannot be used as a diagnostic tool.

Isolation and characterisation of T lymphocytes from the urethra of patients with acute urethritis.

OBJECTIVES: To investigate local cellular immune responses in patients with acute urethritis. METHODS: We have established T cell lines from the urethral exudate and examined their phenotype by flow cytometry. As controls, T cell lines were cultured from first pass urine specimens of asymptomatic healthy individuals. RESULTS: Using interleukin 2 (IL-2) alone a T cell line was obtained on only one occasion. Following culture with IL-2, and subsequent expansion by a single stimulation with irradiated allogenic peripheral blood mononuclear cells (PBMC), phytohaemagglutinin (PHA), and IL-2, it was possible to establish T cell lines from 6/6 acute urethritis patients. T cell lines were also obtained from 4/12 controls subjects, but required repetitive rounds of stimulation with mitogen and allogeneic PBMC to produce sufficient cell numbers for analysis. Three of the patient T cell lines were dominated by T cells expressing the gamma delta receptor. CONCLUSION: The gamma delta T cell subset has been associated with immune responses at mucosal surfaces and has the ability to recognise certain bacterial antigens. The gamma delta T cell response may represent an important aspect of the immune response to organisms associated with acute urethritis.