Forced swim stress exacerbates inflammation-induced hyperalgesia and oxidative stress in the rat trigeminal ganglia.

This study investigates the impact of combining psychophysical stress, induced by forced swim (FSS), with masseter inflammation on reactive oxygen species (ROS) production in trigeminal ganglia (TG), TRPA1 upregulation in TG, and mechanical hyperalgesia. In a rat model, we demonstrate that FSS potentiates and prolongs CFA-induced ROS upregulation within TG. The ROS levels in CFA combined with FSS group surpass those in the CFA-only group on days 4 and 28 post-treatment. FSS also enhances TRPA1 upregulation in TG, with prolonged expression compared to CFA alone. Furthermore, CFA-induced mechanical hyperalgesia is significantly prolonged by FSS, persisting up to day 28. PCR array analyses reveal distinct alterations in oxidative stress genes under CFA and CFA combined with FSS conditions, suggesting an intricate regulation of ROS within TG. Notably, genes like Nox4, Hba1, Gpx3, and Duox1 exhibit significant changes, providing potential targets for managing oxidative stress and inflammatory pain. Western blot and immunohistochemistry confirm DUOX1 protein upregulation and localization in TG neurons, indicating a role in ROS generation under inflammatory and stress conditions. This study underscores the complex interplay between psychophysical stress, inflammation, and oxidative stress in the trigeminal system, offering insights into novel therapeutic targets for pain management.

Intraganglionic reactive oxygen species mediate inflammatory pain and hyperalgesia through TRPA1 in the rat.

Reactive oxygen species (ROS) are generated in nociceptive pathways in response to inflammation and injury. ROS are accumulated within the sensory ganglia following peripheral inflammation, but the functional role of intraganlionic ROS in inflammatory pain is not clearly understood. The aims of this study were to investigate whether peripheral inflammation leads to prolonged ROS accumulation within the trigeminal ganglia (TG), whether intraganglionic ROS mediate pain hypersensitivity via activation of TRPA1, and whether TRPA1 expression is upregulated in TG during inflammatory conditions by ROS. We demonstrated that peripheral inflammation causes excess ROS production within TG during the period when inflammatory mechanical hyperalgesia is most prominent. Additionally, scavenging intraganglionic ROS attenuated inflammatory mechanical hyperalgesia and a pharmacological blockade of TRPA1 localized within TG also mitigated inflammatory mechanical hyperalgesia. Interestingly, exogenous administration of ROS into TG elicited mechanical hyperalgesia and spontaneous pain-like responses via TRPA1, and intraganglionic ROS induced TRPA1 upregulation in TG. These results collectively suggest that ROS accumulation in TG during peripheral inflammation contributes to pain and hyperalgesia in a TRPA1 dependent manner, and that ROS further exacerbate pathological pain responses by upregulating TRPA1 expression. Therefore, any conditions that exacerbate ROS accumulation within somatic sensory ganglia can aggravate pain responses and treatments reducing ganglionic ROS may help alleviate inflammatory pain.

Serum antibodies to surface proteins of Chlamydia trachomatis as candidate biomarkers of disease: results from the Baltimore Chlamydia Adolescent/Young Adult Reproductive Management (CHARM) cohort.

We previously observed that the nine-member family of autotransported polymorphic membrane proteins (Pmps) of Chlamydia trachomatis is variably expressed in cell culture. Additionally, C. trachomatis-infected patients display variable Pmp-specific serum antibody profiles indirectly suggesting expression of unique Pmp profiles is an adaptive response to host-specific stimuli during infection. Here, we propose that the host response to Pmps and other outer surface proteins may correlate with disease severity. This study tests this hypothesis using an ELISA that measures serum IgG antibodies specific for the nine C. trachomatis Pmp subtypes and four immunodominant antigens (MOMP, OmcB, Hsp60, ClpP) in 265 participants of the Chlamydia Adolescent/Young Adult Reproductive Management (CHARM) cohort. More C. trachomatis-infected females displayed high Pmp-specific antibody levels (cut-off Indexes) than males (35.9%-40.7% of females vs. 24.2%-30.0% of males), with statistical significance for PmpC, F and H (P < 0.05). Differences in Pmp-specific antibody profiles were not observed between C. trachomatis-infected females with a clinical diagnosis of pelvic inflammatory disease (PID) and those without. However, a statistically significant association between high levels of OmcB-specific antibody and a PID diagnosis (P< 0.05) was observed. Using antibody levels as an indirect measure of antigen expression, our results suggest that gender- and/or site-specific (cervix in females vs. urethra in males) stimuli may control pmp expression in infected patients. They also support the possible existence of immune biomarkers of chlamydial infection associated with disease and underline the need for high resolution screening in human serum.

Simultaneous transcriptional profiling of bacteria and their host cells.

We developed an RNA-Seq-based method to simultaneously capture prokaryotic and eukaryotic expression profiles of cells infected with intracellular bacteria. As proof of principle, this method was applied to Chlamydia trachomatis-infected epithelial cell monolayers in vitro, successfully obtaining transcriptomes of both C. trachomatis and the host cells at 1 and 24 hours post-infection. Chlamydiae are obligate intracellular bacterial pathogens that cause a range of mammalian diseases. In humans chlamydiae are responsible for the most common sexually transmitted bacterial infections and trachoma (infectious blindness). Disease arises by adverse host inflammatory reactions that induce tissue damage & scarring. However, little is known about the mechanisms underlying these outcomes. Chlamydia are genetically intractable as replication outside of the host cell is not yet possible and there are no practical tools for routine genetic manipulation, making genome-scale approaches critical. The early timeframe of infection is poorly understood and the host transcriptional response to chlamydial infection is not well defined. Our simultaneous RNA-Seq method was applied to a simplified in vitro model of chlamydial infection. We discovered a possible chlamydial strategy for early iron acquisition, putative immune dampening effects of chlamydial infection on the host cell, and present a hypothesis for Chlamydia-induced fibrotic scarring through runaway positive feedback loops. In general, simultaneous RNA-Seq helps to reveal the complex interplay between invading bacterial pathogens and their host mammalian cells and is immediately applicable to any bacteria/host cell interaction.

Variable expression of surface-exposed polymorphic membrane proteins in in vitro-grown Chlamydia trachomatis.

The hypothesized variable expression of polymorphic membrane proteins (PmpA-PmpI) in Chlamydia trachomatis-infected patients was tested by examination of the expression of each Pmp subtype in in vitro-grown C. trachomatis. A panel of monospecific polyclonal and monoclonal antibodies was used to demonstrate surface exposure of Pmps of each subtype by differential immunofluorescence (IF) with and without prior detergent permeabilization of paraformaldehyde-fixed inclusions and for selected Pmps by immunogold labelling. Although specific transcript was detected for each pmp gene late in development, IF experiments with Pmp subtype-specific antibodies reveal that a number of inclusions in a single infection do not express Pmps of a given subtype. Coexpression experiments suggest that pmp genes are shut off independently from one another in non-expressing inclusions, i.e. different inclusions are switched off for different Pmps. Overall, these studies establish the existence of an efficient shutoff mechanism independently affecting the expression of each member of the pmp gene family in in vitro-grown C. trachomatis. Like other paralogous gene families of bacterial pathogens, the pmp gene family of C. trachomatis may serve the critical dual function of a highly adaptable virulence factor also providing antigenic diversity in the face of the host adaptive immune response.

Chlamydia trachomatis-infected patients display variable antibody profiles against the nine-member polymorphic membrane protein family.

Genomic analysis of the Chlamydiaceae has revealed a multigene family encoding large, putatively autotransported polymorphic membrane proteins (Pmps) with nine members in the sexually transmitted pathogen Chlamydia trachomatis. While various pathogenesis-related functions are emerging for the Pmps, observed genotypic and phenotypic variation among several chlamydial Pmps in various Chlamydia species has led us to hypothesize that the pmp gene repertoire is the basis of a previously undetected mechanism of antigenic variation. To test this hypothesis, we chose to examine the serologic response of C. trachomatis-infected patients to each Pmp subtype. Immune serum samples were collected from four populations of patients with confirmed C. trachomatis genital infection: 40 women with pelvic inflammatory disease from Pittsburgh, PA; 27 and 34 adolescent/young females from Oakland, CA, and Little Rock, AR, respectively; and 58 adult male patients from Baltimore, MD. The Pmp-specific antibody response was obtained using immunoblot analysis against each of the nine recombinantly expressed Pmps and quantified by densitometry. Our results show that nearly all C. trachomatis-infected patients mount a strong serologic response against individual or multiple Pmp subtypes and that the antibody specificity profile varies between patients. Moreover, our analysis reveals differences in the strengths and specificities of the Pmp subtype-specific antibody reactivity relating to gender and clinical outcome. Overall, our results indicate that the Pmps elicit various serologic responses in C. trachomatis-infected patients and are consistent with the pmp gene family being the basis of a mechanism of antigenic variation.

Effect of Chlamydiaphage phiCPG1 on the course of conjunctival infection with "Chlamydia caviae" in guinea pigs.

Over the last several years, four different phages of chlamydiae, in addition to a phage associated with Chlamydia psittaci isolated from an ornithosis infection in ducks over 25 years ago, have been described and characterized. While these phages and their chlamydial host specificities have been characterized in vitro, there is virtually nothing known about the interaction of the phage with chlamydiae in their natural animal host. phiCPG1 is a lytic phage specific for "Chlamydia caviae," which is a natural parasite of the guinea pig. In this study, guinea pigs were inoculated in the conjunctiva with suspensions of phiCPG1 and C. caviae and the effect on the development of pathology and on the course of chlamydial infection in the conjunctiva was determined. The presence of phage delayed the appearance of the peak level of chlamydiae in the animal and decreased the pathological response. Evidence for replication of the phage in C. caviae in the conjunctival tissue was observed. Modifying the ratio of phage to chlamydiae altered the course of infection and affected phage replication. There was no evidence for the phage increasing the virulence of C. caviae infection.