Human mast cells release metalloproteinase-9 on contact with activated T cells: juxtacrine regulation by TNF-alpha

Mast cells, essential effector cells in allergic inflammation, have been found to be activated in T cell-mediated inflammatory processes in accordance with their residence in close physical proximity to T cells. We have recently reported that mast cells release granule-associated mediators and TNF-alpha upon direct contact with activated T cells. This data suggested an unrecognized activation pathway, where mast cells may be activated during T cell-mediated inflammation. Herein, we show that this cell-cell contact results in the release of matrix metalloproteinase (MMP)-9 and the MMP inhibitor tissue inhibitor of metalloproteinase 1 from HMC-1 human mast cells or from mature peripheral blood-derived human mast cells. The expression and release of these mediators, as well as of beta-hexosaminidase and several cytokines, were also induced when mast cells were incubated with cell membranes isolated from activated, but not resting, T cells. Subcellular fractionation revealed that the mature form of MMP-9 cofractionated with histamine and tryptase, indicating its localization within the secretory granules. MMP-9 release was first detected at 6 h and peaked at 22 h of incubation with activated T cell membranes, while TNF-alpha release peaked after only 6 h. Anti-TNF-alpha mAb inhibited the T cell membrane-induced MMP-9 release, indicating a possible autocrine regulation of MMP release by mast cell TNF-alpha. This cascade of events, whereby mast cells are activated by T cells to release cytokines and MMP-9, which are known to be essential for leukocyte extravasation and recruitment to affected sites, points to an important immunoregulatory function of mast cells within the context of T cell-mediated inflammatory processes.     

Mast cells induce upregulation of P-selectin and intercellular adhesion molecule 1 on carotid endothelial cells in a new in vitro model of mast cell to endothelial cell communication

It is suggested that mast cells contribute to cell recruitment in inflammation through the upregulation of endothelial adhesion molecules. P-selectin and intercellular adhesion molecule(ICAM)-1 are two key adhesion molecules that have been associated indirectly with mast cell activity. The canine C2 mastocytoma cell line and primary cultures of canine carotid endothelial cells were used to establish a new in vitro model to help study the interaction between mast cells and endothelial cells. Carotid endothelial cells were incubated with mast cell mediators to uncover their effect on endothelial ICAM-1 and P-selectin expression. To assess the relative contributions of tumour necrosis factor (TNF)-alpha and histamine to such effect, an H1 antihistamine and a TNF-alpha blocking antibody were used. Prior to activation by mast cell mediators, P-selectin was expressed only within the cytoplasm, and ICAM-1 was constitutively expressed on the surface of the canine carotid endothelial cells. Both adhesion molecules were enhanced significantly and strongly upon mast cell activation at various time points. Unstored TNF-alpha was fully responsible for ICAM-1 upregulation. P-selectin was up-regulated by both preformed and newly synthesized mast cell mediators, but neither histamine nor TNF-alpha accounted for such an effect. Therefore,a new model is proposed in which the pro-inflammatory effect of mast cells on endothelial cells can be studied in vitro.In this model, it has been demonstrated that only TNF-alpha accounts for the overexpression of ICAM-1 induced by mast cells, and that mast cells up-regulate P-selectin expression through a histamine-independent mechanism.     

Identification of antigenic proteins in Trichomonas vaginalis

Trichomoniasis is a sexually transmitted disease due to infection with Trichomonas vaginalis, and it can cause serious consequences for women's health. To study the virulence factors of this pathogen, T. vaginalis surface proteins were investigated using polyclonal antibodies specific to the membrane fractions of T. vaginalis. The T. vaginalis expression library was constructed by cloning the cDNA derived from mRNA of T. vaginalis into a phage λ Uni-ZAP XR vector, and then used for immunoscreening with the anti-membrane proteins of T. vaginalis antibodies. The immunoreactive proteins identified included adhesion protein AP65-1, α-actinin, kinesin-associated protein, teneurin, and 2 independent hypothetical proteins. Immunofluorescence assays showed that AP65-1, one of the identified immunogenic clones, is prevalent in the whole body of T. vaginalis. This study led us to identify T. vaginalis proteins which may stimulate immune responses by human cells.     

A comparison of mediators released or generated by IFN-gamma-treated human mast cells following aggregation of Fc gamma RI or Fc epsilon RI

The high affinity receptor for IgG (Fc gamma RI, CD64) is expressed on human mast cells, where it is up-regulated by IFN-gamma and, thus, may allow mast cells to be recruited through IgG-dependent mechanisms in IFN-gamma-rich tissue inflammation. However, the mediators produced by human mast cells after aggregation of Fc gamma RI are incompletely described, and it is unknown whether these mediators are distinct from those produced after activation of human mast cells via Fc epsilon RI. Thus, we investigated the release of histamine and arachidonic acid metabolites and examined the chemokine and cytokine mRNA profiles of IFN-gamma-treated cultured human mast cells after Fc gamma RI or Fc epsilon RI aggregation. Aggregation of Fc gamma RI resulted in histamine release and PGD(2) and LTC(4) generation. These responses were qualitatively indistinguishable from responses stimulated via Fc epsilon RI. Aggregation of Fc epsilon RI or Fc gamma RI led to an induction or accumulation of 22 cytokine and chemokine mRNAs. Among them, seven cytokines (TNF-alpha, IL-1beta, IL-5, IL-6, IL-13, IL-1R antagonist, and GM-CSF) were significantly up-regulated via aggregation of Fc gamma RI compared with Fc epsilon RI. TNF-alpha mRNA data were confirmed by quantitative RT-PCR and ELISA. Furthermore, we confirmed histamine and TNF-alpha data using IFN-gamma-treated purified human lung mast cells. Thus, aggregation of Fc gamma RI on mast cells led to up-regulation and/or release of three important classes of mediators: biogenic amines, lipid mediators, and cytokines. Some cytokines, such as TNF-alpha, were released and generated to a greater degree after Fc gamma RI aggregation, suggesting that selected biologic responses of mast cells may be preferentially generated through Fc gamma RI in an IFN-gamma-rich environment.     

Trichomonas vaginalis and trichomoniasis in the Republic of Korea

Vaginal trichomoniasis, caused by Trichomonas vaginalis, is the most common sexually transmitted disease. More than 170 million people worldwide are annually infected by this protozoan. In the Republic of Korea, 10.4% of women complaining of vaginal symptoms and signs were found to be infected with T. vaginalis. However, despite its high prevalence, the pathogenesis of T. vaginalis infection has not been clearly characterized although neutrophil infiltration is considered to be primarily responsible for the cytologic changes associated with this infection. We hypothesized that trichomonads in the vagina sometime after an acute infection secrete proteins like excretory-secretory product that have a chemotactic effect on neutrophils, and that these neutrophils are further stimulated by T. vaginalis to produce chemokines like IL-8 and GRO-alpha, which further promote neutrophil recruitment and chemotaxis. Thus, neutrophil accumulation is believed to maintain or aggravate inflammation. However, enhanced neutrophil apoptosis induced by live T. vaginalis could contribute to resolution of inflammation. Macrophages may constitute an important component of host defense against T. vaginalis infection. For example, mouse macrophages alone and those activated by lymphokines or nitric oxide are known to be involved in the extracellular killing of T. vaginalis. In the host, T. vaginalis uses a capping phenomenon to cleave host immunoglobulins with proteinases and thus escape from host immune responses. Recently, we developed a highly sensitive and specific diagnostic polymerase chain reaction (PCR) technique using primers based on a repetitive sequence cloned from T. vaginalis (TV-E650), and found that the method enables the detection of T. vaginalis at concentrations as low as 1 cell per PCR mixture.     

Redox regulation of mast cell histamine release in thioredoxin-1 （TRX） transgenic mice

Thioredoxin-1 （TRX） is a stress-inducible redox-regulatory protein with antioxidative and anti-inflammatory effects. Here we show that the release of histamine from mast cells elicited by cross-linking of high-affinity receptor for IgE （FcεRI） was significantly suppressed in TRX transgenic （TRX-tg） mice compared to wild type （WT） mice. Intracellular reactive oxygen species （ROS） of mast cells stimulated by IgE and antigen was also reduced in TRX-tg mice compared to WT mice. Whereas there was no difference in the production ofcytokines （IL-6 and TNF-α） from mast cells in response to 2,4-dinitrophenylated bovine serum albumin （DNP-BSA） stimulation in TRX-tg and WT mice. Immunological status of TRX-tg mice inclined to T helper （Th） 2 dominant in primary immune response, although there was no difference in the population of dendritic cells （DCs） and regulatory T cells. We conclude that the histamine release from mast cells in TRX-tg mice is suppressed by inhibition of ROS generation. As ROS are involved in mast cell activation and facilitate mediator release, TRX may be a key signaling molecule regulating the early events in the IgE signaling in mast cells and the allergic inflammation.     

Box H/ACA snoRNAs are preferred substrates for the trimethylguanosine synthase in the divergent unicellular eukaryote Trichomonas vaginalis

The 2,2,7-trimethylguanosine caps of eukaryal snRNAs and snoRNA are formed by the enzyme Tgs1, which catalyzes sequential guanine-N2 methylations of m(7)G caps. Atypically, in the divergent unicellular eukaryote Trichomonas vaginalis, spliceosomal snRNAs lack a guanosine cap and the recombinant T. vaginalis trimethylguanosine synthase (TvTgs) produces only m(2,7)G in vitro. Here, we show by direct metabolic labeling that endogenous T. vaginalis RNAs contain m(7)G, m(2,7)G, and m(2,2,7)G caps. Immunodepletion of TvTgs from cell extracts and TvTgs add-back experiments demonstrate that TvTgs produces m(2,7)G and m(2,2,7)G caps. Expression of TvTgs in yeast tgs1Δ cells leads to the formation of m(2,7)G and m(2,2,7)G caps and complementation of the lethality of a tgs1Δ mud2Δ strain. Whereas TvTgs is present in the nucleus and cytosol of T. vaginalis cells, TMG-containing RNAs are localized primarily in the nucleolus. Molecular cloning of anti-TMG affinity-purified T. vaginalis RNAs identified 16 box H/ACA snoRNAs, which are implicated in guiding RNA pseudouridylation. The ensemble of new T. vaginalis H/ACA snoRNAs allowed us to predict and partially validate an extensive map of pseudouridines in T. vaginalis rRNA.     

Trichinella spiralis: histamine secretion induced by TSL-1 antigens from unsensitized mast cells

Mast cells' hyperplasia and activation are prominent features in Trichinella spiralis infection. Recently, it was shown that TSL-1 antigens from T. spiralis muscle larvae induce IL-4 and TNF release by unsensitized, normal mast cells (MC) involving an Ig-independent mechanism. In this study, we characterized histamine secretion induced by TSL-1 antigens from normal, unsensitized rat peritoneal MC. Maximum histamine secretion (30+/-5.3% SEM, n=13) was achieved with 30 ng/mL TSL-1 antigens. However, TSL-1 did not induce an increase in beta-hexosaminidase release or NADPH oxidase activity by MC. Interestingly, histamine secretion by TSL-1 was completed at 10s, and was inhibited by both Bordetella pertussis toxin and neuraminidase V, characteristics similar to those involved in substance P-induced histamine secretion. However, in contrast to substance P, TSL-1 induced histamine secretion in the absence of detectable changes in intracellular Ca(2+). We are investigating the molecular pathways involved in MC activation by TSL-1.     

Regulatory effects of mast cells on lymphoid cells: the role of histamine type 1 receptors in the interaction between mast cells, helper T cells and natural suppressor cells

We have investigated the role of mast cells as modulators of lymphocyte function because the mast cells are concentrated in the areas of lymphoid storage; they are dependent upon T-cell growth factor for their proliferation; and they appear to be the principle if not sole storage site for histamine. We have tested the influence of mast cells on the proliferation of alloreactive cloned helper T cells, mixed leukocyte reactions, and the suppressive capacity of natural suppressor cells. We used an IL-3-dependent mast cell line that at high numbers (greater than 10(5)) suppressed and at low numbers (10(3) to 6 X 10(4)) augmented the proliferation of TH cells. Addition of histamine to cocultures enhanced the mast cell mediated proliferation of TH cells without directly affecting the helper cells. The action of histamine appeared to be mediated with H1 type receptors on these mast cells. Pretreatment of natural suppressor cells with supernatants from mast cell enhanced their suppressive capability. Here too, histamines enhanced suppression by the NS cell via histamine type 1 receptors on the natural suppressor cells. Our data suggest that mast cells may be a major modulator of the lymphoid cell immune function and demonstrate a role of histamine type 1 receptors in the interaction between mast cells, helper T cells, and natural suppressor cells.     

Tumor necrosis factor-alpha dependent cytotoxicity of human skin mast cells is enhanced by anti-IgE antibodies

Mast cells dispersed from human skin and purified by density-gradient centrifugation were cytotoxic toward the mouse fibrosarcoma cell line WEHI-164. Skin mast cells were not cytotoxic toward the NK cell-sensitive cell line K562. Killing of WEHI-164 occurred over a prolonged (greater than 18 h) period of incubation with mast cells and was effectively inhibited by polyclonal antibodies and mAb against TNF-alpha suggesting that this cytokine plays an important role in mast cell-mediated cytotoxicity. Whereas lysates of rat peritoneal mast cells exhibited cytotoxicity toward WEHI-164, this was not found with lysates of unstimulated skin mast cells suggesting that TNF-alpha is not stored preformed in the latter. Killing of WEHI-164 cells by skin mast cells was enhanced by anti-IgE and there was a significant correlation between histamine release and cytotoxicity after activation with this stimulus. We conclude that human skin mast cells are a potential source of TNF-alpha and suggest that these cells, particularly after activation, might contribute to the synthesis of this multifunctional cytokine in inflammatory sites.     

PCR for diagnosis of male Trichomonas vaginalis infection with chronic prostatitis and urethritis

The aim of this study was to assess the usefulness of PCR for diagnosis of Trichomonas vaginalis infection among male patients with chronic recurrent prostatitis and urethritis. Between June 2001 and December 2003, a total of 33 patients visited the Department of Urology, Hanyang University Guri Hospital and were examined for T. vaginalis infection by PCR and culture in TYM medium. For the PCR, we used primers based on a repetitive sequence cloned from T. vaginalis (TV-E650). Voided bladder urine (VB1 and VB3) was sampled from 33 men with symptoms of lower urinary tract infection (urethral charge, residual urine sensation, and frequency). Culture failed to detect any T. vaginalis infection whereas PCR identified 7 cases of trichomoniasis (21.2%). Five of the 7 cases had been diagnosed with prostatitis and 2 with urethritis. PCR for the 5 prostatitis cases yielded a positive 330 bp band from bothVB1 and VB3, whereas positive results were only obtained from VB1 for the 2 urethritis patients. We showed that the PCR method could detect T. vaginalis when there was only 1 T. vaginalis cell per PCR mixture. Our results strongly support the usefulness of PCR on urine samples for detecting T. vaginalis in chronic prostatitis and urethritis patients.     

Receptor-mediated modulation of murine mast cell function by alpha-melanocyte stimulating hormone.

The proopiomelanocortin (POMC)-derived neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) is known to modulate some aspects of inflammation through direct effects on T cells, B cells, and monocytes. To determine whether alpha-MSH might similarly influence mast cell responsiveness, mast cells were examined to see if they expressed the receptor for alpha-MSH, melanocortin-1 (MC-1), and whether alpha-MSH altered mast cell function. We thus first identified MC-1 on bone marrow cultured murine mast cells (BMCMC) and a murine mast cell line (MCP-5) employing flow cytometry and through detection of specific binding. Subsequent treatment of mast cells with alpha-MSH increased the cAMP concentration in a characteristic biphasic pattern, demonstrating that alpha-MSH could affect intracellular processes. We next examined the effect of alpha-MSH on mediator release and cytokine expression. IgE/DNP-human serum albumin-stimulated histamine release from mast cells was inhibited by approximately 60% in the presence of alpha-MSH. Although activation of BMCMC induced the expression of mRNAs for the inflammatory cytokines IL-1beta, IL-4, IL-6, TNF-alpha, and the chemokine lymphotactin, mRNAs for IL-1beta, TNF-alpha, and lymphotactin were down-modulated in the presence of alpha-MSH. Finally, IL-3-dependent proliferative activity of BMCMC was slightly but significantly augmented by alpha-MSH. Taken together, these observations suggest that alpha-MSH may exert an inhibitory effect on the mast cell-dependent component of a specific inflammatory response.     

Inflammatory responses in the intestinal mucosa of gerbils and hamsters experimentally infected with the adult stage of Taenia solium

The inflammatory response in gerbils and hamsters harbouring experimental infections with Taenia solium adult parasites as well as worm burden and duration of infections were examined. For this purpose, non-suppressed or immunosuppressed rodents were infected with eight cysticerci and necropsied at different times up to 35 days post-infection. Cells in the mucosa surrounding the implantation site of T. solium scolices (duodenum-jejunum) and in ileum were counted in stained sections. A competitive enzyme linked immunosorbent assay was used to determine histamine concentration in intestinal fluid. In non-suppressed hosts, an inflammatory reaction developed with scarce macrophages, a slight increase of plasma cells, lymphocytes and fibroblasts, a moderate increase of eosinophils and neutrophils, and high numbers of goblet and mast cells. Goblet cells began to increase at 6 days post-infection and peaked at 13 days post-infection with a four-fold increase with respect to the control group. Mast cells only increased in gerbils starting at 9 days post-infection with an eight-fold increase when cells peaked between 11 and 19 days post-infection. Histamine concentration in intestinal fluid of gerbils had a similar behaviour to mast cells. Minimal increase of mast cells was seen in hamsters. The recovery of tapeworms was inversely related to the number of both cell types, which decreased when tapeworms were eliminated. Infections lasted up to 25 days in gerbils and up to 46 days in hamsters. Worms measured only 1-2 cm in gerbils and up to 40 cm in hamsters. When gerbils were suppressed with the steroid methyl predinisolone, tapeworms could be recovered up to 35 days post-infection and tapeworms measured up to 22 cm, a minor increase of goblet and mast cells was observed and histamine concentration was similar to that in non-infected animals. Our results suggest that expulsion of T. solium in gerbils and hamsters may be related to the increase of goblet cells and mast cells, but these cells may have different roles in each rodent model of taeniosis.     

The Tec family kinase, IL-2-inducible T cell kinase, differentially controls mast cell responses

The Tec family tyrosine kinase, IL-2-inducible T cell kinase (Itk), is expressed in T cells and mast cells. Mice lacking Itk exhibit impaired Th2 cytokine secretion; however, they have increased circulating serum IgE, but exhibit few immunological symptoms of allergic airway responses. We have examined the role of Itk in mast cell function and FcepsilonRI signaling. We report in this study that Itk null mice have reduced allergen/IgE-induced histamine release, as well as early airway hyperresponsiveness in vivo. This is due to the increased levels of IgE in the serum of these mice, because the transfer of Itk null bone marrow-derived cultured mast cells into mast cell-deficient W/W(v) animals is able to fully rescue histamine release in the W/W(v) mice. Further analysis of Itk null bone marrow-derived cultured mast cells in vitro revealed that whereas they have normal degranulation responses, they secrete elevated levels of cytokines, including IL-13 and TNF-alpha, particularly in response to unliganded IgE. Analysis of biochemical events downstream of the FcepsilonRI revealed little difference in overall tyrosine phosphorylation of specific substrates or calcium responses; however, these cells express elevated levels of NFAT, which was largely nuclear. Our results suggest that the reduced mast cell response in vivo in Itk null mice is due to elevated levels of IgE in these mice. Our results also suggest that Itk differentially modulates mast cell degranulation and cytokine production in part by regulating expression and activation of NFAT proteins in these cells.     

Mast cells,neuropeptides, histamine,and prostaglandins in UV-induced systemic immunosuppression

There is a direct correlation between dermal mast cell prevalence in dorsal skin of different mouse strains and susceptibility to UVB-induced systemic immunosuppression; highly UV-susceptible C57BL/6 mice have a high dermal mast cell prevalence while BALB/c mice, which require considerable UV radiation for 50% immunosuppression, have a low mast cell prevalence. There is also a functional link between the prevalence of dermal mast cells and susceptibility to UVB- and cis-urocanic acid (UCA)-induced systemic immunosuppression. Mast cell-depleted mice are unresponsive to UVB or cis-UCA for systemic immunosuppression unless they are previously reconstituted at the irradiated or cis-UCA-administered site with bone marrow-derived mast cell precursors. cis-UCA does not stimulate mast cell degranulation directly. Instead, in support of studies showing that neither UVB nor cis-UCA was immunosuppressive in capsaicin-treated, neuropeptide-depleted mice, cis-UCA-stimulated neuropeptide release from sensory c-fibers which, in turn, could efficiently degranulate mast cells. Studies in mice suggested that histamine, and not tumor necrosis factor alpha (TNF-alpha), was the product from mast cells that stimulated downstream immunosuppression. Histamine receptor antagonists reduced by approximately 60% UVB and cis-UCA-induced systemic immunosuppression. Indomethacin administration to mice had a similar effect which was not cumulative with the histamine receptor antagonists. Histamine can stimulate keratinocyte prostanoid production. We propose that both histamine and prostaglandin E(2) are important in downstream immunosuppression; both are regulatory molecules supporting the development of T helper 2 cells and reduced expression of type 1 immune responses such as a contact hypersensitivity reaction.     

Trichomonas vaginalis inhibits proinflammatory cytokine production in macrophages by suppressing NF-kappaB activation

Activation of NF-kappaB leads to the production of proinflammatory cytokines such as IL-12 and TNF-alpha that are involved in innate and adaptive immunity. We determined whether T. vaginalis-induced inflammatory response in macrophages associated with NF-kappaB. T. vaginalis adhesion led to transient NF-kappaB activation at 6 h but activation declined dramatically by 8 h. Super-shift assays showed that the gel-shifted complexes consisted of p65 (Rel A) and p50 (NF-kappaB1). NF-kappaB activation was accompanied by IkappaB-alpha degradation, and was inhibited by blocking T. vaginalis adhesion, indicating that the early NF-kappaB activation by T. vaginalis depends on IkappaB-alpha degradation. Quantitative real-time RT-PCR analyses revealed that the expression of TNF-alpha and IL-12 mRNA in T. vaginalis-adhesive cells was rapidly suppressed in comparison with LPS stimulation. We also observed that the parasite inhibited the nuclear translocation of NF-kappaB at 8 h, and diminished IL-12 and TNF-alpha production in response to LPS. In addition, inhibition of IkappaB-alpha degradation by MG-132 resulted in apoptosis. These results demonstrate that effects of T. vaginalis on NF-kappaB regulation are critical for cytokine production and the survival of macrophages. We suggest that there exist inhibitory mechanisms induced by T. vaginalis to evade host immunity.     

Involvement of p38 and JNK MAPKs pathways in Substance P-induced production of TNF-alpha by peritoneal mast cells

Mast cells play a central role in both inflammation and immediate allergic reactions. We have previously shown that Substance P (SP) stimulates TNF-alpha mRNA and protein expression in rat peritoneal mast cells (PMC). In the present paper, we investigated whether the induction of TNF-alpha production by the mast cells agonist involves MAPKs signalling pathways. We found that as early as 5 min after PMC exposure to SP, phosphorylation of p38 MAPK and JNK was induced. On the contrary, phosphorylation of p42/44 MAPK occurred only after a 30 min exposure to SP and did not correlate with SP-induced TNF-alpha production. The highly specific p38 MAPK inhibitor SB203580 and the blocker of PI-3K wortmannin, abolished SP-induced increase in TNF-alpha mRNA and protein levels and showed to reduce the SP-mediated histamine secretion. In addition, wortmannin reduced SP-mediated JNK phosphorylation. The results reveal that the induction of TNF-alpha expression and histamine exocytosis by exposure of rat PMC to substance P requires the activation of p38 and JNK MAPKs pathways. Moreover, they suggest PI-3K as a possible upstream component of JNK pathway in SP-induced inflammatory reactions.     

IL-4 modulates the histamine content of mast cells in a mast cell/fibroblast co-culture through a Stat6 signaling pathway in fibroblasts

IL-4 plays a crucial role in the pathogenesis of allergic diseases, such as the induction of IgE synthesis and the development of mast cells. To further understand the effect of IL-4 on mast cells in skin, we utilized a mast cell/fibroblast co-culture system as an in vitro model of dermal mast cells. IL-4 induced mast cell growth in the culture with fibroblasts. Immunoblot analysis revealed that IL-4 activated Stat6 in both mast cells and fibroblasts. The over-expression of dominant-negative Stat6 in fibroblasts in the presence of IL-4 decreased the histamine content per mast cell, but not the number of mast cells. In contrast, the over-expression of constitutively-active Stat6 in fibroblasts increased the histamine content per mast cell, indicating that the activation of Stat6 in fibroblasts supports the maturation of mast cells co-cultured with fibroblasts.     

Spliceosomal snRNAs in the unicellular eukaryote Trichomonas vaginalis are structurally conserved but lack a 5'-cap structure

Few genes in the divergent eukaryote Trichomonas vaginalis have introns, despite the unusually large gene repertoire of this human-infective parasite. These introns are characterized by extended conserved regulatory motifs at the 5' and 3' boundaries, a feature shared with another divergent eukaryote, Giardia lamblia, but not with metazoan introns. This unusual characteristic of T. vaginalis introns led us to examine spliceosomal small nuclear RNAs (snRNAs) predicted to mediate splicing reactions via interaction with intron motifs. Here we identify T. vaginalis U1, U2, U4, U5, and U6 snRNAs, present predictions of their secondary structures, and provide evidence for interaction between the U2/U6 snRNA complex and a T. vaginalis intron. Structural models predict that T. vaginalis snRNAs contain conserved sequences and motifs similar to those found in other examined eukaryotes. These data indicate that mechanisms of intron recognition as well as coordination of the two catalytic steps of splicing have been conserved throughout eukaryotic evolution. Unexpectedly, we found that T. vaginalis spliceosomal snRNAs lack the 5' trimethylguanosine cap typical of snRNAs and appear to possess unmodified 5' ends. Despite the lack of a cap structure, U1, U2, U4, and U5 genes are transcribed by RNA polymerase II, whereas the U6 gene is transcribed by RNA polymerase III.     

Activation and inhibition of mast cells degranulation affect their morphometric parameters

Activation of mast cells, the key cells of allergic inflammation, causes typical morphological changes associated with an increase in volume, that is a function of area and perimeter. The purpose of this study was to evaluate the effect of mast cell activation to degranulate, carried out by the secretagogue Compound 48/80, and of inhibition of this activation carried out by Nedocromil sodium, a mast cell stabilizing drug, on mast cell area, perimeter and shape factor by a computerized image analyzer. Mast cells were isolated and purified by peritoneal lavage of rats (purity >98%) and co-cultured with mouse 3T3 fibroblasts to which they adhere. Cultures were incubated for 10 min at 37 degrees C with culture medium alone (Enriched Medium) or Enriched Medium containing either Nedocromil (10(-4) M) or Compound 48/80 (0.3 microg/ml) or Compound 48/80 and Nedocromil (0.3 microg/ml and 10(-4) M respectively). Supernatants were then assessed for histamine release, as a marker of mast cell activation and the cell monolayers were fixed and stained with an alcoholic-acidic toluidine blue solution and examined with a computerized image analyzer connected with a light microscope. Mast cells incubated in Enriched Medium or Nedocromil possessed similar morphometric parameters. Mast cells activated with Compound 48/80 (70% histamine release) had a significant increase in area and perimeter and a decrease in shape factor in comparison to mast cells in Enriched Medium alone. Simultaneous incubation of mast cells with Compound 48/80 and Nedocromil significantly inhibited their histamine release (36% histamine release) and the increase in area and perimeter, but did not affect significantly their shape factor, in comparison with mast cells incubated with Compound 48/80 alone. These data clearly show that there is a relationship between mast cell activation, consequent histamine release and changes in cell area, perimeter and shape factor and that Nedocromil not only inhibits mast cell histamine release but also the activation induced morphometric changes in mast cells.