Andrew Brittingham

Murine macrophages produce endothelin-1 after microbial stimulation.

Endothelin-1 (ET-1) was originally characterized as a potent vasoconstrictor secreted by the endothelium and participating in the regulation of vascular tone. Subsequent analysis has revealed ET-1 to be a multifunctional peptide produced by a wide variety of cells and tissues under normal and pathologic conditions. The importance of macrophages as a source of ET-1 during infection and inflammation is supported by clinical observations in humans and in animal models of inflammation. We hypothesize that the production of ET-1 is part of the characteristic macrophage response to infection, and have begun to investigate the ability of various classes of microbes or microbial products to induce macrophage ET-1 production. We report the production of ET-1 by murine macrophages in response to stimulation with both gram-positive and gram-negative bacteria. Stimulation of macrophages with yeast (Candida albicans or Saccharomyces cerevisiae) or the protozoan parasite Leishmania major, elicited no significant release of ET. The production of ET-1 in response to lipopolysaccharide (LPS) was dose and time dependent, and required the expression of a functional toll-like receptor 4 (TLR4). Pharmacologic inhibition of the transcription factor, nuclear factor-κB (NF-κB) suppressed LPS-induced ET-1 production. Our findings complement the growing body of literature implicating a role for macrophage-derived ET-1 in inflammatory pathologies. The production of ET-1 by macrophages during infection and inflammation has the potential to affect tissue perfusion, leukocyte extravasation, and immune cell function.

Mercury enhances susceptibility to murine leishmaniasis

The genetic background of mice infected with Leishmania major determines the response to infection, resulting in a resistant or susceptible phenotype. Susceptible mice develop a T‐helper type 2 (Th2)‐type immune response following infection distinguished by the development of interleukin (IL)‐4 secreting T cells in the lymph node and spleen. In SJL mice, which normally heal L. major lesions, subtoxic doses of mercury induce an autoimmune syndrome characterized by an expansion of Th2 cells. In this study, we examined the effect of mercury administration on the outcome of L. major infection in SJL mice. We show that subtoxic doses of mercuric chloride (HgCl2) exacerbate disease outcome in SJL mice resulting in increased footpad swelling and increased parasite burdens. Furthermore, the effects of HgCl2 treatment on resistance to L. major are time‐dependent. The nonhealing phenotype was observed only if mice had been treated with HgCl2 prior to L. major infection for at least 1 week, a timepoint at which mice treated with HgCl2 alone had increased splenocyte IL‐4 production. HgCl2 treatment also increased production of serum immunoglobulin (Ig)E and IgG1, two IL‐4 dependent isotypes. These results show that HgCl2 treatment enhances the susceptibility to L. major in SJL mice, consistent with the induction of host Th2 parameters. These findings have implications for the role of mercury contamination in areas of endemic leishmaniasis.

The antimicrobial effect of boric acid on Trichomonas vaginalis.

Background The treatment options for trichomoniasis are largely limited to nitroimidazole compounds (metronidazole and tinidazole). Few alternatives exist in cases of recalcitrant infections or in cases of nitroimidazole hypersensitivity. Recently, the intravaginal administration of boric acid has been advocated as an alternative treatment of trichomoniasis. However, no in vitro studies are available that directly assess the sensitivity of Trichomonas vaginalis to boric acid. Methods We examined the sensitivity of common laboratory strains and recent clinical isolates of T. vaginalis to boric acid. The effect of increasing concentrations of boric acid on parasite growth and viability was determined, and a minimal lethal concentration was reported. The effect of pH on boric acid toxicity was assessed and compared with that of lactic and acetic acid. Results Boric acid is microbicidal to T. vaginalis, and its antitrichomonal activity is independent of environmental acidification. Unlike acetic acid and lactic acid, boric acid exposure results in growth suppression and lethality over a wide range of pH (5–7) and under conditions that are normally permissible for growth in vitro. Conclusions The microbicidal effect of boric acid on T. vaginalis, coupled with its previous clinical use in treating vaginal candidiasis, supports the continued inclusion of boric acid in the therapeutic arsenal for treating trichomoniasis.  

Glycogen Accumulation and Degradation by the Trichomonads Trichomonas vaginalis and Trichomonas tenax

Several species of trichomonad have been shown to accumulate significant quantities of glycogen during growth, suggesting an important role for this compound in cell physiology. We provide the first analysis of the changes in glycogen content and glycogen phosphorylase activity that occur during in vitro growth of two trichomonad species: Trichomonas vaginalis and Trichomonas tenax. Both species accumulated glycogen following inoculation into fresh medium and utilized this compound during logarithmic growth. Glycogen phosphorylase activity also varied during growth in a species‐specific manner. The expression of phosphorylase genes in T. vaginalis remained constant during growth and thus transcriptional control did not explain the observed fluctuations in phosphorylase activity. After cloning, expression, and purification, two recombinant glycogen phosphorylases from T. vaginalis and one recombinant glycogen phosphorylase from T. tenax had robust activity and, in contrast to many other eukaryotic glycogen phosphorylases, did not appear to be regulated by reversible protein phosphorylation. Furthermore, allosteric regulation, if present, was not mediated by compounds known to impact the activity of better characterized phosphorylases.

Trichomonas vaginalis: a possible foe to prostate cancer.

Prostate cancer (PCA) is the most common malignancy in men in USA, and the role of Trichomonas vaginalis (T. vag) in the development of PCA is still controversial. Clonogenic assay, PCNA staining, TUNEL staining and caspase-3 activity assay were used to investigate the in vitro role of T. vag in human prostate cancer. We further investigated the possible molecular mechanisms using RT-PCR and immunohistochemical staining. Culture supernatant of T. vag inhibits growth of PC-3 prostate cancer cells, and this correlated with upregulation of p21. Culture supernatant of T. vag induced apoptosis of PC-3 cells, and this correlated with downregulation of Bcl-2. The growth inhibition effect of culture supernatant of T. vag is also demonstrated in another prostate cancer cell line DU145, suggesting that its effect is not specific to one prostate cancer cell line. Culture supernatant of T. vag inhibits growth of prostate cancer by inhibition of proliferation and promotion of apoptosis. Such a study might be helpful to address the association between PCA and infection of T. vag.

Expression and characterization of a β-fructofuranosidase from the parasitic protist Trichomonas vaginalis

BackgroundTrichomonas vaginalis, a flagellated protozoan, is the agent responsible for trichomoniasis, the most common nonviral sexually transmitted infection worldwide. A reported 200 million cases are documented each year with far more cases going unreported. However, T. vaginalis is disproportionality under studied, especially considering its basic metabolism. It has been reported that T. vaginalis does not grow on sucrose. Nevertheless, the T. vaginalis genome contains some 11 putative sucrose transporters and a putative β-fructofuranosidase (invertase). Thus, the machinery for both uptake and cleavage of sucrose appears to be present.ResultsWe amplified the β-fructofuranosidase from T. vaginalis cDNA and cloned it into an Escherichia coli expression system. The expressed, purified protein was found to behave similarly to other known β-fructofuranosidases. The enzyme exhibited maximum activity at pH close to 5.0, with activity falling off rapidly at increased or decreased pH. It had a similar Km and Vmax to previously characterized enzymes using sucrose as a substrate, was also active towards raffinose, but had no detectable activity towards inulin.ConclusionsT. vaginalis has the coding capacity to produce an active β-fructofuranosidase capable of hydrolyzing di- and trisaccharides containing a terminal, non-reducing fructose residue. Since we cloned this enzyme from cDNA, we know that the gene in question is transcribed. Furthermore, we could detect β-fructofuranosidase activity in T. vaginalis cell lysates. Therefore, the inability of the organism to utilize sucrose as a carbon source cannot be explained by an inability to degrade sucrose.

Growth-Phase Dependent Substrate Adhesion in Crithidia fasciculata

Abstract. Crithidia fasciculata is a trypanosomatid flagellate that parasitizes several species of mosquito. Within the alimentary tract of its host, C. fasciculata exists in two forms: one is a non‐motile form, attached in clusters to the lining of the gut, the other a more elongated form swimming freely in the gut lumen. We have developed an in vitro culture system that reproduces the appearance of these two distinct morphological forms. Using two different cultivation methods, shaking and stationary incubations, we have demonstrated that adherence phenotypes are growth‐phase dependent. Organisms in the logarithmic phase of growth possess the ability to adhere to substrates; this ability is lost when the organism enters a stationary growth phase. Parasite adherence was independent of cultivation method or substrate. Furthermore, adherent forms of Crithidia maintained their adhesive properties following their removal from substrates. Our data reveal a growth‐phase‐regulated process of cell attachment that may influence the transmission and dissemination of this parasitic flagellate.

Endothelin-1 production by the canine macrophage cell line DH82: Enhanced production in response to microbial challenge

Endothelin-1 (ET-1) is a potent vasoconstrictive peptide which plays an important role in regulating mammalian cardiovascular development and homeostasis. Originally identified as a factor released by vascular endothelial cells, ET-1 is now recognized as a product of numerous cells and tissues with demonstrated involvement in an array of physiological and pathological processes. An area of great interest is the production of ET-1 by mononuclear cells (monocytes and macrophages) and its role in inflammation. We report that the canine macrophage cell line, DH82, constitutively secretes both ET-1 and its biologically inactive precursor big ET-1. The production of both peptides was increased following stimulation with lipopolysaccharide (endotoxin) from gram-negative bacteria. ET-1 production was also increased in response to stimulation with intact and viable gram-positive and gram-negative bacteria. In addition to producing ET-1, DH82 cells express transcripts encoding two receptors for the ET-1 peptide (ET(A) and ET(B) receptors) and an enzyme involved in the conversion of big ET-1 to ET-1. The constitutive secretion of ET-1 and the expression of ET(A) and ET(B) receptors may be related to the malignant origin of this cell line. Our results are the first report of ET-1 production by a canine cell line and provide the basis for further investigation into the role of ET-1 during infection and inflammation.

Purification and identification of amylases released by the human pathogen Trichomonas vaginalis that are active towards glycogen

The parasitic protist Trichomonas vaginalis is the causative agent of the sexually transmitted infection trichomoniasis. In the laboratory, T. vaginalis is typically cultured in a serum-containing medium with maltose or glucose as the carbon source. The nature of the carbohydrates used by the organism in the environment of its host is unclear. However, the vagina contains substantial amounts of glycogen, which is believed to provide a growth substrate for the vaginal microbiota. We have shown previously that T. vaginalis releases glucosidases that are active towards glycogen into its environment. Here we purify and identifying these glucosidases. Using ammonium sulfate precipitation and precipitation with ethanol/glycogen, we purified glucosidase activity from conditioned growth medium, achieving over 300-fold enrichment. Maltose was released when glycogen was incubated with the glucosidase preparation, indicating that a β-amylase was present. However, after prolonged incubation, small quantities of larger products including maltotriose were obtained. Liquid chromatography and tandem mass spectrometry showed that the glucosidase preparation contained three proteins, the major component being a putative β-amylase encoded by the TVAG_080000 open reading frame. Lesser amounts of two putative α-amylases, encoded by the TVAG_178580 and TVAG_205920 open reading frames, were also present. We cloned and expressed the TVAG_080000 open reading frame and found that the recombinant protein was capable of digesting glycogen, releasing exclusively maltose. We conclude that T. vaginalis releases a variety of amylases into its growth environment and is well equipped to utilize the glycogen found in the vagina as a source of essential carbohydrates.

Glycogen Storage and Degradation During In Vitro Growth and Differentiation of Giardia intestinalis

Abstract: Giardia intestinalis is the causative agent of human giardiasis, a common diarrheal illness worldwide. Despite its global distribution and prevalence, many questions regarding its basic biology and metabolism remain unanswered. In this study, we examine the accumulation and degradation of glycogen, an important source of stored carbon and energy, during the in vitro growth and differentiation of G. intestinalis. We report that, as G. intestinalis progresses through its growth cycle, cultures of trophozoites accumulate glycogen during the lag and early logarithmic phases of growth and then utilize this compound during their remaining logarithmic growth. As cultures enter the stationary phase of growth, they re-accumulate glycogen stores. The activity of glycogen phosphorylase, an enzyme involved in glycogen metabolism, also varied throughout in vitro trophozoite growth. During the in vitro induction of trophozoite differentiation into water-resistant cyst forms, the cultures initially accumulated stores of glycogen which diminished throughout transition to the cyst form. This observation is suggestive of a role for glycogen in the differentiation process. These studies represent the first thorough analysis of changes in glycogen content and glycogen phosphorylase activity during G. intestinalis growth and differentiation.