First Report and Characterization of a Plasmid-Encoded blaSFO-1 in a Multi-Drug-Resistant Aeromonas hydrophila Clinical Isolate.

Antibiotic resistance remains one of the most pressing public health issues facing the world today. At the forefront of this battle lies the ever-increasing identification of extended-spectrum beta-lactamases and carbapenemases within human pathogens, conferring resistance towards broad-spectrum and last-resort antimicrobials. This study was prompted due to the identification of a pathogenic Aeromonas hydrophila isolate (strain MAH-4) collected from abdominal fluid, which presented a robust resistance pattern against second-, third-, and fourth-generation cephalosporins, ertapenem, ciprofloxacin, gentamicin, levofloxacin and moxifloxacin, and beta lactam/beta-lactamase inhibitor combinations. Whole genome sequencing was performed and identified a 328 kb plasmid (pMAH4) encoding 10 antibiotic resistance genes, including blaSFO-1, blaTEM-1, and blaOXA-1 of A. hydrophia MAH-4. This is the first report of beta-lactamase SFO-1 within a clinical strain of Aeromonas. Due to the remarkable sequence identity of pMAH4 to plasmids associated with Enterobacterales genera like Klebsiella and the extensive capabilities of Aeromonas for horizontal gene transfer, our identification of a clinical isolate encoding SFO-1 on a plasmid suggests antibiotic resistance gene mobility between Enterobacterales and non-Enterobacterales species.

One Health and Global Health View of Antimicrobial Susceptibility through the "Eye" of Aeromonas: Systematic Review and Meta-Analysis.

Antimicrobial resistance (AMR) is one of the most pressing public health concerns; therefore, it is imperative to advance our understanding of the factors influencing AMR from Global and One Health perspectives. To address this, Aeromonas populations were identified using 16S rRNA gene libraries among human, agriculture, aquaculture, drinking water, surface water, and wastewater samples, supporting its use as indicator bacteria to study AMR. A systematic review and meta-analysis was then performed from Global and One Health perspectives, including data from 221 articles describing 15 891 isolates from 57 countries. The interconnectedness of different environments was evident as minimal differences were identified between sectors among 21 different antimicrobials. However, resistance to critically important antibiotics (aztreonam and cefepime) was significantly higher among wastewater populations compared with clinical isolates. Additionally, isolates from untreated wastewater typically exhibited increased AMR compared with those from treated wastewater. Furthermore, aquaculture was associated with increased AMR to ciprofloxacin and tetracycline compared with wild-caught seafood. Using the World Health Organization AWaRe classifications, countries with lower consumption of "Access" compared to "Watch" drugs from 2000 to 2015 demonstrated higher AMR levels. The current analysis revealed negative correlations between AMR and anthropogenic factors, such as environmental performance indices and socioeconomic standing. Environmental health and sanitation were two of the environmental factors most strongly correlated with AMR. The current analysis highlights the negative impacts of "Watch" drug overconsumption, anthropogenic activity, absence of wastewater infrastructure, and aquaculture on AMR, thus stressing the need for proper infrastructure and global regulations to combat this growing problem.

Similarities in Virulence and Extended Spectrum Beta-Lactamase Gene Profiles among Cefotaxime-Resistant Escherichia coli Wastewater and Clinical Isolates.

The World Health Organization has identified antibiotic resistance as one of the largest threats to human health and food security. In this study, we compared antibiotic resistance patterns between ESBL-producing Escherichia coli from human clinical diseases and cefotaxime-resistant environmental strains, as well as their potential to be pathogenic. Antibiotic susceptibility was tested amongst clinical isolates (n = 11), hospital wastewater (n = 22), and urban wastewater (n = 36, both influent and treated effluents). Multi-drug resistance predominated (>70%) among hospitalwastewater and urban wastewater influent isolates. Interestingly, isolates from clinical and urban treated effluents showed similar multi-drug resistance rates (~50%). Most hospital wastewater isolates were Phylogroup A, while clinical isolates were predominately B2, with a more diverse phylogroup population in urban wastewater. ESBL characterization of cefotaxime-resistant populations identified blaCTX-M-1 subgroup as the most common, whereby blaKPC was more associated with ceftazidime and ertapenem resistance. Whole-genome sequencing of a carbapenemase-producing hospital wastewater E. coli strain revealed plasmid-mediated blaKPC-2. Among cefotaxime-resistant populations, over 60% of clinical and 30% of treated effluent E. coli encoded three or more virulence genes exhibiting a pathogenic potential. Together, the similarity among treated effluent E. coli populations and clinical strains suggest effluents could serve as a reservoir for future multi-drug resistant E. coli clinical infections.

Antibiotic resistance patterns of Escherichia coli isolates from the clinic through the wastewater pathway.

Antimicrobial resistance (AMR) remains one of the leading global health threats. This study compared antimicrobial resistance patterns among E. coli isolates from clinical uropathogenic Escherichia coli (UPEC) to hospital wastewater populations and throughout an urban wastewater treatment facility - influent, pre- and post-chlorinated effluents. Antibiotic susceptibility of 201 isolates were analyzed against eleven different antibiotics, and the presence of twelve antibiotic resistant genes and type 1 integrase were identified. AMR exhibited the following pattern: UPEC (46.8%) > hospital wastewater (37.8%) > urban post-chlorinated effluent (27.6%) > pre-chlorinated effluent (21.4%) > urban influent wastewater (13.3%). However, multi-drug resistance against three or more antimicrobial classes was more prevalent among hospital wastewater populations (29.7%) compared to other sources. E. coli from wastewaters disinfected with chlorine were significantly correlated with increased trimethoprim-sulfamethoxazole resistance in E. coli compared to raw and treated wastewater populations. blaCTX-M-1 group was the most common extended spectrum beta-lactamase in E. coli from hospital wastewater (90%), although UPEC strains also encoded blaCTX-M-1 group (50%) and blaTEM (100%) genes. Among tetracycline-resistant populations, tetA and tetB were the only resistance genes identified throughout wastewater populations that were associated with increased phenotypic resistance. Further characterization of the E. coli populations identified phylogroup B2 predominating among clinical UPEC populations and correlated with the highest AMR, whereas the elevated rate of multi-drug resistance among hospital wastewater was mostly phylogroup A. Together, our findings highlight hospital wastewater as a rich source of AMR and multi-drug resistant bacterial populations.

A Comparison of Porphyrin Photosensitizers in Photodynamic Inactivation of RNA and DNA Bacteriophages.

Effective broad-spectrum antiviral treatments are in dire need as disinfectants and therapeutic alternatives. One such method of disinfection is photodynamic inactivation, which involves the production of reactive oxygen species from dissolved oxygen in response to light-stimulated photosensitizers. This study evaluated the efficacy of functionalized porphyrin compounds for photodynamic inactivation of bacteriophages as human virus surrogates. A blue-light light emitting diode (LED) lamp was used to activate porphyrin compounds in aqueous solution (phosphate buffer). The DNA bacteriophages ΦX174 and P22 were more resistant to porphyrin TMPyP photodynamic inactivation than RNA bacteriophage fr, with increasing rates of inactivation in the order: ΦX174 << P22 << fr. Bacteriophage ΦX174 was therefore considered a resistant virus suitable for the evaluation of three additional porphyrins. These porphyrins were synthesized from TMPyP by inclusion of a central palladium ion (PdT4) and/or the addition of a hydrophobic C14 chain (PdC14 or C14). While the inactivation rate of bacteriophage ΦX174 via TMPyP was similar to previous reports of resistant viruses, ΦX174 inactivation increased by a factor of approximately 2.5 using the metalloporphyrins PdT4 and PdC14. The order of porphyrin effectiveness was TMPyP < C14 < PdT4 < PdC14, indicating that both Pd2+ ligation and C14 functionalization aided virus inactivation.

Survivor microbial populations in post-chlorinated wastewater are strongly associated with untreated hospital sewage and include ceftazidime and meropenem resistant populations.

Wastewater treatment plant (WWTP) effluent has been implicated in the spread of antibiotic resistant bacteria (ARB), including pathogens, as the WWTP environment contains multiple selective pressures that may increase mutation rates, pathogen survivability, and induce gene transfer between bacteria. In WWTPs receiving hospital sewage, this selective effect may be more pronounced due to increased concentrations of antibiotics, ARB, and clinical pathogens from hospital sewage. To determine the extent to which hospital sewage contributes to the microbial community of disinfected wastewater which is released into the environment, we used 16S rRNA sequencing of hospital sewage, WWTP influent, primary effluent, Post-Chlorinated Effluent, and receiving sediments in a combined sewage system to track changes in microbial community composition. We also sequenced the culturable survivor community resistant to ß-lactam antibiotics within disinfected effluent. Using molecular source tracking, we found that the hospital sewage microbiome contributes an average of 11.49% of the microbial community in Post-Chlorinated Effluents, suggesting microorganisms identified within hospital sewage can survive or are enriched by the chlorination disinfection process. Additionally, we identified 28 potential pathogens to the species level, seven of which remained detectable in Post-Chlorinated Effluent and environmental sediments. When Post-Chlorinated Effluents were cultured on media containing ß-lactam antibiotics ceftazidime and meropenem, a diverse antibiotic resistant survivor community was identified including potential human pathogens Bacillus cereus, Bacillus pumilus, and Chryseobacterium indologenes. Together, these results indicate that although wastewater treatment does significantly reduce pathogenic loads and ARBs, their continual presence in disinfected wastewater and receiving sediments suggests additional treatment and microbial tracking systems are needed to reduce human and animal health risks.

Metalloporphyrin Pd(T4) Exhibits Oncolytic Activity and Cumulative Effects with 5-ALA Photodynamic Treatment against C918 Cells.

Photodynamic therapy is a non-invasive method where light activates a photosensitizer bound to cancer cells, generating reactive oxygen species and resulting in cell death. This study assessed the oncolytic potential of photodynamic therapy, comparing European Medicines Agency and United States Food and Drug Administration-approved 5-aminolevulinic acid (5-ALA) to a metalloporphyrin, Pd(T4), against a highly invasive uveal melanoma cell line (C918) in two- and three-dimensional models in vitro. Epithelial monolayer studies displayed strong oncolytic effects (>70%) when utilizing Pd(T4) at a fraction of the concentration, and reduced pre-illumination time compared to 5-ALA post-405 nm irradiance. When analyzed at sub-optimal concentrations, application of Pd(T4) and 5-ALA with 405 nm displayed cumulative effects. Lethality from Pd(T4)-photodynamic therapy was maintained within a three-dimensional model, including the more resilient vasculogenic mimicry-forming cells, though at lower rates. At high concentrations, modality of cell death exhibited necrosis partially dependent on reactive oxygen species. However, sub-optimal concentrations of photosensitizer exhibited an apoptotic protein expression profile characterized by increased Bax/Bcl-2 ratio and endoplasmic stress-related proteins, along with downregulation of apoptotic inhibitors CIAP-1 and -2. Together, our results indicate Pd(T4) as a strong photosensitizer alone and in combination with 5-ALA against C918 cells.

Prevalence of Potentially Pathogenic Antibiotic-Resistant Aeromonas spp. in Treated Urban Wastewater Effluents versus Recipient Riverine Populations: a 3-Year Comparative Study.

Antibiotic resistance continues to be an emerging threat both in clinical and environmental settings. Among the many causes, the impact of postchlorinated human wastewater on antibiotic resistance has not been well studied. Our study compared antibiotic susceptibility among Aeromonas spp. in postchlorinated effluents to that of the recipient riverine populations for three consecutive years against 12 antibiotics. Aeromonas veronii and Aeromonas hydrophila predominated among both aquatic environments, although greater species diversity was evident in treated wastewater. Overall, treated wastewater contained a higher prevalence of nalidixic acid-, trimethoprim-sulfamethoxazole (SXT)-, and tetracycline-resistant isolates, as well as multidrug-resistant (MDR) isolates compared to upstream surface water. After selecting for tetracycline-resistant strains, 34.8% of wastewater isolates compared to 8.3% of surface water isolates were multidrug resistant, with nalidixic acid, streptomycin, and SXT being the most common. Among tetracycline-resistant isolates, efflux pump genes tetE and tetA were the most prevalent, though stronger resistance correlated with tetA. Over 50% of river and treated wastewater isolates exhibited cytotoxicity that was significantly correlated with serine protease activity, suggesting many MDR strains from effluent have the potential to be pathogenic. These findings highlight that conventionally treated wastewater remains a reservoir of resistant, potentially pathogenic bacterial populations being introduced into aquatic systems that could pose a threat to both the environment and public health.IMPORTANCE Aeromonads are Gram-negative, asporogenous rod-shaped bacteria that are autochthonous in fresh and brackish waters. Their pathogenic nature in poikilotherms and mammals, including humans, pose serious environmental and public health concerns especially with rising levels of antibiotic resistance. Wastewater treatment facilities serve as major reservoirs for the dissemination of antibiotic resistance genes (ARGs) and resistant bacterial populations and are, thus, a potential major contributor to resistant populations in aquatic ecosystems. However, few longitudinal studies exist analyzing resistance among human wastewater effluents and their recipient aquatic environments. In this study, considering their ubiquitous nature in aquatic environments, we used Aeromonas spp. as bacterial indicators of environmental antimicrobial resistance, comparing it to that in postchlorinated wastewater effluents over 3 years. Furthermore, we assessed the potential of these resistant populations to be pathogenic, thus elaborating on their potential public health threat.

Photodynamic therapy using pheophorbide and 670nm LEDs exhibits anti-cancer effects in-vitro in androgen dependent prostate cancer.

Prostate cancer (PCa) is the third leading cause of death in men in the United States and its treatment options include surgery, anti-hormonal drugs for androgen sensitive tumors, and radiotherapy. An alternative treatment is the use of photodynamic therapy (PDT), which involves the activation of a photosensitizer by a defined wavelength of light in the presence of oxygen, generating transient concentrations of reactive oxygen species (ROS). In this study, we explored the anti-cancer potential and mechanism of action of PDT using pheophorbide (Pheo) as a photosensitizer in combination with 670nm LEDs. Our hypothesis was that Pheo-PDT combination will demonstrate anti-cancer activity against androgen dependent PCa (ADPC), suggesting an alternative and less toxic cancer treatment. Pheo-PDT demonstrated significant anti-cancer activity against ADPC in as little as 5J/cm2 with increased effects, in a Pheo concentration dependent manner. We also observed in vitro inhibition in the clonogenic potential, as well as significant inhibition of invasion and migration, implicating the anti-metastatic activity of Pheo-PDT on PCa. Furthermore, Pheo-PDT caused G0/G1 cell cycle arrest. The oncolytic activity of PDT involves the generation of ROS, and we demonstrated immediate ROS formation subsequent to Pheo-PDT as well. Mechanistic analysis suggested that the anti-cancer activity of Pheo-PDT is via ER stress, along with inhibition of important cell growth and proliferation markers. Hence, we conclude that Pheo-PDT proves to be a potential therapeutic strategy against ADPC.

Photosensitizers in prostate cancer therapy.

The search for new therapeutics for the treatment of prostate cancer is ongoing with a focus on the balance between the harms and benefits of treatment. New therapies are being constantly developed to offer treatments similar to radical therapies, with limited side effects. Photodynamic therapy (PDT) is a promising strategy in delivering focal treatment in primary as well as post radiotherapy prostate cancer. PDT involves activation of a photosensitizer (PS) by appropriate wavelength of light, generating transient levels of reactive oxygen species (ROS). Several photosensitizers have been developed with a focus on treating prostate cancer like mTHPC, motexafin lutetium, padoporfin and so on. This article will review newly developed photosensitizers under clinical trials for the treatment of prostate cancer, along with the potential advantages and disadvantages in delivering focal therapy.

Photodynamic inactivation of methicillin-resistant Staphylococcus aureus and Escherichia coli: A metalloporphyrin comparison.

Increasing rates of antibiotic resistance coupled with the lack of novel antibiotics threatens proper clinical treatment and jeopardizes their use in prevention. A photodynamic approach appears to be an innovative treatment option, even for multi-drug resistant strains of bacteria. Three components are utilized in photodynamic inactivation: a photosensitizer, light source, and oxygen. Variations in photosensitizers strongly influence microbial binding and bactericidal activity. In this study, four different cationic metalloporphyrins (Cu2+, Fe2+, Pd2+, Zn2+) were compared to the free-base ligand 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin regarding their electronic properties and generation of reactive oxygen species upon subsequent 405nm violet-blue irradiation. Staphylococcus aureus and Escherichia coli were used as representatives of Gram-positive and -negative, respectively, to assess bactericidal effects by the photodynamic process. Bacterial cultures were pre-incubated with porphyrins and exposed to varying doses of 405nm irradiation (0-30J/cm2). Metalloporphyrins containing Cu2+ and Fe2+ demonstrated minimal effects on viability. Pronounced bactericidal activity was evident with free-base ligand, Zn2+, and Pd2+; though significantly stronger effects were apparent with Pd2+. Photodynamic killing was directly proportional to reactive oxygen species production post-illumination. These data provide new insight into the influence of metal chelation on photosensitizer activity on bactericidal singlet oxygen production. The strong anti-microbial photodynamic action through the use of a portable light-emitting diode over short time intervals (seconds) provides support for its potential use in self-treatment.

Aeromonas hydrophila and Aeromonas veronii predominate among potentially pathogenic ciprofloxacin- and tetracycline-resistant aeromonas isolates from Lake Erie.

Members of the genus Aeromonas are ubiquitous in nature and have increasingly been implicated in numerous diseases of humans and other animal taxa. Although some species of aeromonads are human pathogens, their presence, density, and relative abundance are rarely considered in assessing water quality. The objectives of this study were to identify Aeromonas species within Lake Erie, determine their antibiotic resistance patterns, and assess their potential pathogenicity. Aeromonas strains were isolated from Lake Erie water by use of Aeromonas selective agar with and without tetracycline and ciprofloxacin. All isolates were analyzed for hemolytic ability and cytotoxicity against human epithelial cells and were identified to the species level by using 16S rRNA gene restriction fragment length polymorphisms and phylogenetic analysis based on gyrB gene sequences. A molecular virulence profile was identified for each isolate, using multiplex PCR analysis of six virulence genes. We demonstrated that Aeromonas comprised 16% of all culturable bacteria from Lake Erie. Among 119 Aeromonas isolates, six species were identified, though only two species (Aeromonas hydrophila and A. veronii) predominated among tetracycline- and ciprofloxacin-resistant isolates. Additionally, both of these species demonstrated pathogenic phenotypes in vitro. Virulence gene profiles demonstrated a high prevalence of aerolysin and serine protease genes among A. hydrophila and A. veronii isolates, a genetic profile which corresponded with pathogenic phenotypes. Together, our findings demonstrate increased antibiotic resistance among potentially pathogenic strains of aeromonads, illustrating an emerging potential health concern.

Inhibitory effects of 405 nm irradiation on Chlamydia trachomatis growth and characterization of the ensuing inflammatory response in HeLa cells.

BACKGROUND: Chlamydia trachomatis is an intracellular bacterium that resides in the conjunctival and reproductive tract mucosae and is responsible for an array of acute and chronic diseases. A percentage of these infections persist even after use of antibiotics, suggesting the need for alternative treatments. Previous studies have demonstrated anti-bacterial effects using different wavelengths of visible light at varying energy densities, though only against extracellular bacteria. We investigated the effects of visible light (405 and 670 nm) irradiation via light emitting diode (LEDs) on chlamydial growth in endocervical epithelial cells, HeLa, during active and penicillin-induced persistent infections. Furthermore, we analyzed the effect of this photo treatment on the ensuing secretion of IL-6 and CCL2, two pro-inflammatory cytokines that have previously been identified as immunopathologic components associated with trichiasis in vivo. RESULTS: C. trachomatis-infected HeLa cells were treated with 405 or 670 nm irradiation at varying energy densities (0 - 20 J/cm2). Bacterial growth was assessed by quantitative real-time PCR analyzing the 16S: GAPDH ratio, while cell-free supernatants were examined for IL-6 and monocyte chemoattractant protein-1 (CCL2) production. Our results demonstrated a significant dose-dependent inhibitory effect on chlamydial growth during both active and persistent infections following 405 nm irradiation. Diminished bacterial load corresponded to lower IL-6 concentrations, but was not related to CCL2 levels. In vitro modeling of a persistent C. trachomatis infection induced by penicillin demonstrated significantly elevated IL-6 levels compared to C. trachomatis infection alone, though 405 nm irradiation had a minimal effect on this production. CONCLUSION: Together these results identify novel inhibitory effects of 405 nm violet light on the bacterial growth of intracellular bacterium C. trachomatis in vitro, which also coincides with diminished levels of the pro-inflammatory cytokine IL-6.

Characterization of humoral immune responses to chlamydial HSP60, CPAF, and CT795 in inflammatory and severe trachoma.

PURPOSE: Chlamydia trachomatis (Ct) remains the leading global cause of preventable blindness. There are limited data on humoral immune responses in trachoma. Evaluating these responses is important for understanding host-pathogen interactions and informing vaccine design. Antibodies to chlamydial heat shock protein 60 (cHSP60) have been associated with infertility and trachomatous scarring. Other proteins, including chlamydial protease-associated factor (CPAF) and a hypothetical protein unique to the family Chlamydiaceae, CT795, elicit strong immune responses in urogenital infections, but their role in trachomatous disease is unknown. METHODS: This study was conducted to expand on previous cHSP60 findings and evaluate the association of CPAF and CT795 antibodies with ocular Ct infection and disease. Clinical trachoma grading was performed, and conjunctival samples were obtained from individuals with trachomatous trichiasis (TT; one or more inturned eyelashes) or inflammatory trachoma without trichiasis and control subjects without disease, all of whom resided in trachoma-endemic regions of Nepal. Ct infection was determined using commercial PCR. IgG and IgA tear antibodies against cHSP60, CT795, and CPAF fusion proteins were measured by quantitative ELISA. RESULTS: Significantly higher IgG antibody levels were found against cHSP60, CPAF, and CT795 in the inflammatory cases compared with levels in the controls (P < 0.005 for all three). Ct infection was independently associated with IgG antibodies against all three immunogens in the inflammatory cases but not in the controls (P = 0.025, P = 0.03 and P = 0.017, respectively). Only IgG antibodies against CPAF were significantly elevated among the TT cases (P = 0.013). CONCLUSIONS: Among individuals with trachoma, IgG antibody responses to CPAF are likely to be both a marker and risk factor for inflammatory trachoma and severe trachomatous disease.

Identification of novel single nucleotide polymorphisms in inflammatory genes as risk factors associated with trachomatous trichiasis.

BACKGROUND: Trachoma is the leading preventable cause of global blindness. A balanced Th1/Th2/Th3 immune response is critical for resolving Chlamydia trachomatis infection, the primary cause of trachoma. Despite control programs that include mass antibiotic treatment, reinfection and recurrence of trachoma are common after treatment cessation. Furthermore, a subset of infected individuals develop inflammation and are at greater risk for developing the severe sequela of trachoma known as trachomatous trichiasis (TT). While there are a number of environmental and behavioral risk factors for trachoma, genetic factors that influence inflammation and TT risk remain ill defined. METHODOLOGY/FINDINGS: We identified single nucleotide polymorphisms (SNP) in 36 candidate inflammatory genes and interactions among these SNPs that likely play a role in the overall risk for TT. We conducted a case control study of 538 individuals of Tharu ethnicity residing in an endemic region of Nepal. Trachoma was graded according to World Health Organization guidelines. A linear array was used to genotype 51 biallelic SNPs in the 36 genes. Analyses were performed using logic regression modeling, which controls for multiple comparisons. We present, to our knowledge, the first significant association of TNFA (-308GA), LTA (252A), VCAM1 (-1594TC), and IL9 (T113M) polymorphisms, synergistic SNPs and risk of TT. TT risk decreased 5 times [odds ratio = 0.2 (95% confidence interval 0.11.-0.33), p = 0.001] with the combination of TNFA (-308A), LTA (252A), VCAM1 (-1594C), SCYA 11 (23T) minor allele, and the combination of TNFA (-308A), IL9 (113M), IL1B (5'UTR-T), and VCAM1 (-1594C). However, TT risk increased 13.5 times [odds ratio = 13.5 (95% confidence interval 3.3-22), p = 0.001] with the combination of TNFA (-308G), VDR (intron G), IL4R (50V), and ICAM1 (56M) minor allele. CONCLUSIONS: Evaluating genetic risk factors for trachoma will advance our understanding of disease pathogenesis, and should be considered in the context of designing global control programs.

Role of secreted conjunctival mucosal cytokine and chemokine proteins in different stages of trachomatous disease.

BACKGROUND: Chlamydia trachomatis is responsible for trachoma, the primary cause of preventable blindness worldwide. Plans to eradicate trachoma using the World Health Organization's SAFE program (Surgery, Antibiotics, Facial Cleanliness and Environment Improvement) have resulted in recurrence of infection and disease following cessation of treatment in many endemic countries, suggesting the need for a vaccine to control infection and trachomatous disease. Vaccine development requires, in part, knowledge of the mucosal host immune responses in both healthy and trachomatous conjuctivae-an area of research that remains insufficiently studied. METHODOLOGY/PRINCIPAL FINDINGS: We characterized 25 secreted cytokines and chemokines from the conjunctival mucosa of individuals residing in a trachoma endemic region of Nepal using Luminex X100 multiplexing technology. Immunomodulating effects of concurrent C. trachomatis infection were also examined. We found that proinflammatory cytokines IL-1beta (r = 0.259, P = 0.001) and TNFalpha (r = 0.168, P<0.05) were significantly associated with trachomatous disease and concurrent C. trachomatis infection compared with age and sex matched controls from the same region who did not have trachoma. In support of these findings, anti-inflammatory cytokine IL-1 receptor antagonist (IL-1Ra) was negatively associated with chronic scarring trachoma (r = -0.249, P = 0.001). Additional cytokines (Th1, IL-12p40 [r = -0.212, P<0.01], and Th2, IL-4 and IL-13 [r = -0.165 and -0.189, respectively, P<0.05 for both]) were negatively associated with chronic scarring trachoma, suggesting a protective role. Conversely, a pathogenic role for the Th3/Tr1 cytokine IL-10 (r = 0.180, P<0.05) was evident with increased levels for all trachoma grades. New risk factors for chronic scarring trachoma included IL-6 and IL-15 (r = 0.259 and 0.292, respectively, P<0.005 for both) with increased levels for concurrent C. trachomatis infections (r = 0.206, P<0.05, and r = 0.304, P<0.005, respectively). Chemokine protein levels for CCL11 (Eotaxin), CXCL8 (IL-8), CXCL9 (MIG), and CCL2 (MCP-1) were elevated in chronic scarring trachoma compared with age and sex matched controls (P<0.05, for all). CONCLUSIONS/SIGNIFICANCE: Our quantitative detection of previously uncharacterized and partially characterized cytokines, a soluble cytokine receptor, and chemokines for each trachoma grade and associations with C. trachomatis infections provide, to date, the most comprehensive immunologic evaluation of trachoma. These findings highlight novel pathologic and protective factors involved in trachomatous disease, which will aid in designing immunomodulating therapeutics and a vaccine.

Altered inflammatory responses following transforming growth factor-beta neutralization in experimental guinea pig tuberculous pleurisy.

The predominant extrapulmonary form of tuberculosis, which develops in 10% of diseased individuals, is pleurisy. The immune response mounted against Mycobacterium tuberculosis in the pleural cavity is one that is sufficient for clearing the organism without therapeutic intervention. Thus, examining the role of immune constituents in this context will provide understanding of the vital role they play in controlling tuberculosis. In this study, experimental tuberculous pleurisy was induced in guinea pigs, and anti-TGF-beta was administered intrapleurally to the guinea pigs daily throughout the study (8 days). Neutralizing TGF-beta resulted in a significant reduction in the percentage of lymphocytes and CD8+ cells present in the pleural exudate, decreased proliferative responses of pleural cells to ConA and PPD, and decreased mRNA expression of IFN-gamma and CCL5 in pleural effusion cells. Conversely, the percentage of neutrophils was significantly increased in anti-TGF-beta-treated guinea pigs, along with upregulated mRNA expression of CXCL8. The percentage of macrophages in the pleural exudate, TNF-alpha and IL-12p40 mRNA expression, and the histopathological response were not significantly altered. While TGF-beta is generally thought of as an immunosuppressive cytokine, the results of this study demonstrate its importance in promoting an inflammatory response, and highlight its bipolar nature.

BCG vaccination of guinea pigs modulates Mycobacterium tuberculosis-induced CCL5 (RANTES) production in vitro and in vivo.

CCL5 can attract and activate macrophages and Th1 lymphocytes, which are involved in eliciting a protective immune response against tuberculosis. In this study, the effects of BCG vaccination on CCL5 production in vitro and in vivo in the guinea pig model were examined. Splenocytes, alveolar, and resident peritoneal macrophages obtained from naïve and BCG-vaccinated animals were infected with Mycobacterium tuberculosis in vitro at various time points and analyzed for CCL5 mRNA and protein levels. All three leukocyte populations harvested from BCG-vaccinated guinea pigs and infected with M. tuberculosis produced elevated CCL5 mRNA and protein compared to infected cells from naïve animals. The kinetics of CCL5 production in vivo was evaluated by inducing tuberculous pleurisy in BCG-vaccinated guinea pigs and analyzing CCL5 in pleural effusions at daily intervals. Both CCL5 mRNA and protein levels increased to maximum levels at day 4 post-pleurisy induction. These data suggest that BCG-vaccination enhances CCL5 production in vitro and in vivo. The effect of neutralizing CCL5 with polyclonal anti-CCL5 IgG in vivo during tuberculous pleurisy resulted in a trend toward diminished levels of pro-inflammatory cytokine mRNA, although neutralizing CCL5 in vivo did not appear to alter the intensity of the histopathological response.

Recombinant guinea pig CCL5 (RANTES) differentially modulates cytokine production in alveolar and peritoneal macrophages.

The CC chemokine ligand 5 (CCL5; regulated on activation, normal T expressed and secreted) is known to recruit and activate leukocytes; however, its role in altering the responses of host cells to a subsequent encounter with a microbial pathogen has rarely been studied. Recombinant guinea pig (rgp)CCL5 was prepared, and its influence on peritoneal and alveolar macrophage activation was examined by measuring cytokine and chemokine mRNA expression in cells stimulated with rgpCCL5 alone or exposed to rgpCCL5 prior to lipopolysaccharide (LPS) stimulation. Levels of mRNA for guinea pig tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, CCL2 (monocyte chemoattractant protein-1), and CXC chemokine ligand 8 (IL-8) were analyzed by reverse transcription followed by real-time polymerase chain reaction analysis using SYBR Green. Bioactive TNF-alpha protein concentration was measured using the L929 bioassay. Both macrophage populations displayed significant enhancement of all the genes and TNF-alpha protein levels when stimulated with rgpCCL5, except for CCL2 in alveolar macrophages. When peritoneal or alveolar macrophages were pretreated with rgpCCL5 for 2 h and then exposed to low concentrations of LPS, diminished cytokine and chemokine mRNA levels were apparent at 6 h compared with LPS alone. At the protein level, there was a reduction in TNF-alpha protein at 6 h in the CCL5-pretreated cells compared with LPS alone. These results further support a role for CCL5 in macrophage activation in addition to chemotactic properties and suggest a role in regulating the inflammatory response to LPS in the guinea pig by modulating the production of proinflammatory cytokines by macrophages.