Effect of Phytochemical Compounds on Trichomonas tenax, an Oral Protozoan.

Trichomonas tenax is an oral protozoan with an estimated global pooled prevalence of 17% in the human population.1 Observational studies have demonstrated a significant statistical correlation between oral colonization by T. tenax and the progression of periodontal disease.2 Proposed pathogenic mechanisms for this protozoan include the production of tissue-damaging enzymes, induction of apoptosis in human cells, and dysbiosis of the oral microbiome.3 In patients for whom metronidazole (MTZ) is contraindicated, phytochemicals may offer a viable alternative for controlling T. tenax. Various plant extracts have shown promising in vitro activity against other trichomonads, such as T. vaginalis and Tritrichomonas foetus, as reviewed by Friedman et al.4.

Cytokine array analysis of mediators produced by human macrophages stimulated with Trichomonastenax.

The incidence of oral colonization by the protozoan Trichomonas tenax correlates with gingival inflammation and periodontitis in humans. To determine whether T. tenax might contribute to inflammation by eliciting cytokines from human cells, differentiated THP-1 (dTHP-1) macrophages were cultured with live or sonicated T. tenax trophozoites, and the conditioned media were assayed for 36 different mediators by a membrane-based cytokine array. Scanning densitometry of the membranes revealed that live T. tenax trophozoites stimulated secretion of interleukin-8 (IL-8), macrophage migration inhibitory factor (MIF), IL-1ß, intercellular adhesion molecule-1 (ICAM-1), and IL-1 receptor antagonist (IL-1ra) from dTHP-1 macrophages. T. tenax lysates stimulated release of IL-8, MIF, and IL-1ra. Despite often being classified as a commensal organism, T. tenax elicited a wider variety of cytokines than the human urogenital pathogen, T. vaginalis, which elicited only IL-8 and MIF production from dTHP-1 cells.

Implementation of Oral Case Presentations in an Immunology Course.

Implementation of oral case presentations (OCP) in the Immunology course at A.T. Still University-Kirksville College of Osteopathic Medicine has significantly improved written examination scores and student satisfaction with the course by enhancing its clinical relevance. With six faculty facilitators, an average class size of 172 students can complete the exercise in a single day. The exercise requires small group meeting rooms, each equipped with a computer and wall-mounted monitor, but no other physical resources.

Fine Epitope Mapping of Monoclonal Antibodies to the DNA Repair Protein, RadA.

Repair of DNA damage is vital to the health and survival of all organisms. In Escherichia coli, a protein known as RadA (or Sms) participates in recombinational repair, a process that uses an undamaged DNA strand in one DNA duplex to fill a gap in a homologous DNA strand in a sister DNA duplex. In a prior report, we described the production of monoclonal antibodies (MAbs) specific for RadA. Here, we investigated the epitopes recognized by two of the antibodies, MAbs 6F5 and 2A2. Premature stop codons (ochre mutations) were introduced into the radA gene at selected sites, and the truncated RadA proteins were probed by western blotting. Deletion of as few as four amino acids (457-460) from the C-terminus of RadA significantly increased the sensitivity of E. coli to ultraviolet (UV) radiation and abolished recognition of RadA by MAb 6F5. Single alanine substitutions made between positions 443-460 also adversely affected the ability of MAb 6F5 to bind to RadA, further supporting the idea that MAb 6F5 is specific for the RadA C-terminus. An ochre mutation at position 258 abolished the recognition of RadA by MAb 2A2, whereas an ochre mutation at position 279 did not, suggesting that MAb 2A2 binds to an epitope between residues 258 and 279. MAb 2A2 recognition of RadA was destroyed by endoproteinase glu-C cleavage of RadA at position 266, and by a single alanine substitution at position 265. In a competitive enzyme-linked immunosorbent assay (ELISA), a synthetic peptide comprising residues 263-273 of RadA blocked MAb 2A2 recognition of immobilized full-length RadA by more than 97%. We infer from our results that MAb 6F5 binds to the extreme C-terminus of RadA and that MAb 2A2 is specific for an epitope within positions 263-273.

Changes in Cytokines, Sensory Tests, and Self-reported Pain Levels After Manual Treatment of Low Back Pain.

STUDY DESIGN: Unbalanced 3-factor design with repeated measures on 1 factor. OBJECTIVE: To determine the effect of manual treatment (MT) on cytokine and pain sensations in those with and without low back pain (LBP). SUMMARY OF BACKGROUND DATA: Evidence suggests that MT reduces LBP but by unknown mechanisms. Certain cytokines have been elevated in patients with LBP and may be affected by MT. METHODS: Participants aged 20-60 years with chronic LBP or without LBP were recruited and randomly assigned to MT, sham ultrasound treatment, or no treatment groups. Venous blood samples were collected and pain levels assessed at baseline, 1 hour later, and 24 hours later. Blood was analyzed for interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, and C-reactive protein. Pain levels were measured by pressure pain threshold (PPT), mechanical detection threshold (MDT), dynamic mechanical allodynia, and self-report. RESULTS: Forty (30 women, age 36±11 y) participants completed the study, 33 with LBP (13 MT, 13 sham ultrasound treatment, and 7 no treatment) and 7 without LBP. Participants with or without LBP could not be differentiated on the basis of serum cytokine levels, PPT, or MDT (P≥0.08). There were no significant differences between the groups at 1 hour or 24 hours on serum cytokines, PPT, or MDT (P≥0.07). There was a significant decrease from baseline in IL-6 for the no treatment (LBP) group (P=0.04), in C-reactive protein for the sham ultrasound treatment group (P=0.03), in MDT for all 3 LBP groups (P≤0.02), and in self-reported pain for the MT and sham ultrasound treatment groups (P=0.03 and 0.01). CONCLUSIONS: Self-reported pain was reduced with MT and sham ultrasound treatment 24 hours after treatment, but inflammatory markers within venous circulation and quantitative sensory tests were unable to differentiate between study groups. Therefore, we were unable to characterize mechanisms underlying chronic LBP.

Cytokine response of human THP-1 macrophages to Trichomonas tenax.

Trichomonas tenax is a protozoan that inhabits the oral cavity of humans, most often those with poor oral hygiene. Although T. tenax is widely considered a commensal, recent studies have suggested a pathogenic role for the protozoan in persons with periodontitis. Here we investigated the capacity of T. tenax to induce pro-inflammatory cytokine secretion in human macrophages, with the idea that elicitation of inflammation may be one mechanism by which T. tenax contributes to oral pathology. Human THP-1 cells differentiated to the macrophage phenotype (dTHP-1) were incubated with live or sonicated T. tenax at trophozoite:dTHP-1 ratios of 1:5, 1:10, and 1:20. Culture media removed from the wells after 4, 8, and 16 h of stimulation were assayed by ELISA for tumor necrosis factor alpha, interleukin-1 beta, interleukin-8, and the immunoregulatory cytokine interleukin-10. Live T. tenax trophozoites failed to induce production of any of the cytokines tested, regardless of trophozoite:dTHP-1 cell ratio or length of co-incubation. T. tenax lysates stimulated interleukin-8 synthesis, but only after 16 h of incubation at the 1:5 trophozoite:dTHP-1 cell ratio. These results suggest that pro-inflammatory cytokine synthesis by human macrophages in direct response to T. tenax contributes little to oral pathology.

Monoclonal antibody 10A5 recognizes an antigen unique to the water-insoluble 25/45 membrane fraction of the rat ocular lens.

The water-insoluble 25/45 fraction and non-sedimenting membrane fraction (NSMF) are two membrane preparations isolated from the ocular lens. The fractions are postulated to represent distinct subdomains of the lens with unique functions. However, attempts to distinguish between the two fractions by detecting proteins present in one fraction but absent from other have been unsuccessful. In this study, we exploited the ability of the mouse immune system to detect antigenic differences between the 25/45 fraction and NSMF isolated from the lenses of 20-day-old rats. We generated a monoclonal antibody (MAb 10A5) that reacts with a ganglioside-like antigen that is present in the 25/45 fraction but absent from the NSMF. Restriction of the antigen to the 25/45 fraction in 20-day-old animals supports the hypothesis that the 25/45 fraction and NSMF represent different subdomains within the ocular lens.

Utilization of case presentations in medical microbiology to enhance relevance of basic science for medical students.

BACKGROUND: Small-group case presentation exercises (CPs) were created to increase course relevance for medical students taking Medical Microbiology (MM) and Infectious Diseases (ID) METHODS: Each student received a unique paper case and had 10 minutes to review patient history, physical exam data, and laboratory data. Students then had three minutes to orally present their case and defend why they ruled in or out each of the answer choices provided, followed by an additional three minutes to answer questions. RESULTS: Exam scores differed significantly between students who received the traditional lecture-laboratory curriculum (Group I) and students who participated in the CPs (Group II). In MM, median unit exam and final exam scores for Group I students were 84.4% and 77.8%, compared to 86.0% and 82.2% for Group II students (P<0.018; P<0.001; Mann-Whitney Rank Sum Test). Median unit and final ID exam scores for Group I students were 84.0% and 80.0%, compared to 88.0% and 86.7% for Group II students (P<0.001; P<0.001). CONCLUSION: Students felt that the CPs improved their critical thinking and presentation skills and helped to prepare them as future physicians.

Monoclonal antibodies against the Escherichia coli DNA repair protein RadA/Sms.

The RadA/Sms protein facilitates DNA repair in Escherichia coli cells damaged by UV radiation, X-rays, and chemical agents. However, the precise mechanism by which RadA/Sms aids DNA repair is unknown. Here we report the production of monoclonal antibodies (MAbs) specific for RadA/Sms for use in biochemical and physiological investigations. Histidine-tagged RadA/Sms (RadA-6xHis) was overproduced in E. coli BL21 cells transformed with the radA/sms coding region in plasmid pRSET A and purified by nickel affinity chromatography. Splenocytes from female BALB/c mice hyperimmunized with the purified protein were fused to SP2/0-Ag14 myeloma cells, and the resultant hybridomas were selected in HAT medium. MAbs were detected in hybridoma culture supernatants by indirect ELISA and Western blot analysis against purified RadA-6xHis. MAbs from four cell lines were further evaluated by Western blotting against peptide maps generated by endoproteinase Glu-C digestion of RadA-6xHis. Each of the four MAbs recognized a unique epitope on the fusion protein. Two of the MAbs (6F5 and 2A2) also detected wild-type (tagless) RadA/Sms produced from the pJS003 plasmid in E. coli K-12 cells. We anticipate that these antibodies will prove useful for the detection, isolation, and functional analysis of RadA/Sms.

A monoclonal antibody that inhibits translation in Sf21 cell lysates is specific for glyceraldehyde-3-phosphate dehydrogenase.

Monoclonal antibody (Mab) 8B7 was shown in a previous study to inhibit protein translation in lysates of Sf21 cells. The antibody was thought to be specific for a 60-kDa form of elongation factor-1 alpha (EF-1alpha), primarily because the antigen immunoprecipitated by Mab 8B7 cross-reacted with Mab CBP-KK1, an antibody generated to EF-1alpha from Trypanosoma brucei. The purpose of the current study was to investigate further the antigenic specificity of Mab 8B7. The concentration of the 60-kDa antigen relative to total cellular protein proved insufficient for its definitive identification. However, subcellular fractionation of Sf21 cells yielded an additional protein of 37 kDa in the cytosolic and microsomal fractions that was reactive with Mab 8B7. The 37-kDa protein could be easily visualized by colloidal Coomassie Blue G-250 staining as a series of pI 6.9-8.4 spots on two-dimensional gels. Excision of an abundant immunoreactive spot enabled identification of the protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and protein database searching. Subsequent immunoblotting of purified rabbit skeletal muscle GAPDH with Mab 8B7 confirmed the antibody's specificity for GAPDH. Besides the pivotal role GAPDH plays in glycolysis, the enzyme has a number of noncanonical functions, including binding to mRNA and tRNA. The ability of Mab 8B7 to disrupt these lesser-known functions of GAPDH may account for the antibody's inhibitory effect on in vitro translation.

Insertional inactivation of branched-chain alpha-keto acid dehydrogenase in Staphylococcus aureus leads to decreased branched-chain membrane fatty acid content and increased susceptibility to certain stresses.

Staphylococcus aureus is a major community and nosocomial pathogen. Its ability to withstand multiple stress conditions and quickly develop resistance to antibiotics complicates the control of staphylococcal infections. Adaptation to lower temperatures is a key for the survival of bacterial species outside the host. Branched-chain alpha-keto acid dehydrogenase (BKD) is an enzyme complex that catalyzes the early stages of branched-chain fatty acid (BCFA) production. In this study, BKD was inactivated, resulting in reduced levels of BCFAs in the membrane of S. aureus. Growth of the BKD-inactivated mutant was progressively more impaired than that of wild-type S. aureus with decreasing temperature, to the point that the mutant could not grow at 12 degrees C. The growth of the mutant was markedly stimulated by the inclusion of 2-methylbutyrate in the growth medium at all temperatures tested. 2-Methylbutyrate is a precursor of odd-numbered anteiso fatty acids and bypasses BKD. Interestingly, growth of wild-type S. aureus was also stimulated by including 2-methylbutyrate in the medium, especially at lower temperatures. The anteiso fatty acid content of the BKD-inactivated mutant was restored by the inclusion of 2-methylbutyrate in the medium. Fluorescence polarization measurements indicated that the membrane of the BKD-inactivated mutant was significantly less fluid than that of wild-type S. aureus. Consistent with this result, the mutant showed decreased toluene tolerance that could be increased by the inclusion of 2-methylbutyrate in the medium. The BKD-inactivated mutant was more susceptible to alkaline pH and oxidative stress conditions. Inactivation of the BKD enzyme complex in S. aureus also led to a reduction in adherence of the mutant to eukaryotic cells and its survival in a mouse host. In addition, the mutant offers a tool to study the role of membrane fluidity in the interaction of S. aureus with antimicrobial substances.

Salivary immunoglobulin A response to a collegiate rugby game.

Transient fluctuations in immune function after heavy exercise have been linked to an increased incidence of infection in athletes. Several parameters of immunity, including salivary immunoglobulin A (IgA), are affected by heavy exercise in the laboratory setting. However, few observations have been made during true competition. We tested the hypothesis that salivary IgA levels will be decreased after a collegiate rugby game. Saliva samples obtained from 16 men's college rugby players before and after an 80-minute regulation rugby game were analyzed for total volume, IgA, total protein content, and osmolality. Salivary IgA was expressed relative to secretion rate (s-IgA), osmolality (IgA-Osm), and total protein (IgA-Pro). No significant pregame-postgame changes in salivary IgA were observed (s-IgA: -13%, IgA-Osm: -16%, IgA-Pro: +10%). These data indicate that strenuous physical activity, such as a competitive rugby game, does not affect IgA levels. More study on the immune response to athletic competition is needed.

Distribution of elongation factor-1alpha in larval tissues of the fall armyworm, Spodoptera frugiperda.

Elongation factor-1alpha (EF-1alpha) promotes the delivery of aminoacyl-tRNA to the acceptor site of the ribosome during protein synthesis. The enzyme has a number of additional functions, including regulation of apoptosis and interaction with the cytoskeleton. We determined the distribution of EF-1alpha in larval tissues of the fall armyworm, Spodoptera frugiperda , with a monoclonal antibody generated to EF-1alpha from Sf21 cells, a cell line developed from ovarian tissue of S. frugiperda. Enzyme-linked immunosorbent assay showed that EF-1alpha comprised 1.9-9.9% of the total protein within the tissues that were examined, which included fat body, Malpighian tubules, midgut, muscle, salivary glands, trachea, and ventral nerve cord. To a certain extent, EF-1alpha concentrations reflected the expected metabolic activity level of each of the represented tissues. Closer examination by immunofluorescence microscopy revealed that EF-1alpha concentrations varied among different cell types within a given tissue, i.e. midgut columnar epithelial cells yielded strong signals, while goblet cells failed to react with the EF-1alpha-specific antibody.

Rheumatoid factor-like IgM in Plasmodium berghei (Apicomplexa: Haemosporida) infections of BALB/c mice.

Groups of female BALB/c mice infected by intravenous injection with 50 erythrocytes containing Plasmodium berghei Vincke et Lips, 1948 were sacrificed on days 3 through 12 after infection. Rheumatoid factor-like IgM (RF-IgM) and parasite-specific IgG levels were determined by enzyme-linked immunosorbent assay in serum specimens and in culture medium removed from spleen cell cultures established at sacrifice. All four mouse IgG subisotypes were recognized by RF-IgM molecules induced by Plasmodium berghei infection, and in this regard, the parasite-induced RF-IgM response resembled that induced by lipopolysaccharide polyclonal activation. Plasmodium berghei infection resulted in a biphasic RF-IgM response, with infected animals demonstrating significantly increased levels of RF-IgM early in the infection and significantly decreased levels late in the infection, compared to uninfected control mice. The decreased levels of RF-IgM observed late in infection correlated with increasing parasitaemia levels, and were primarily due to a decrease in RF-IgM specific for mouse IgG2a. Late infection levels of RF-IgM specific for IgGI, IgG2b, and IgG3 were not significantly different from those of control animals.