Morphologic analysis correlates with gene expression changes in cultured F344 rat mesothelial cells.

The gene expression pattern of mesothelial cells in vitro was determined after 4 or 12 h exposure to the rat mesothelial, kidney, and thyroid carcinogen and oxidative stressor potassium bromate (KBrO(3)). Gene expression changes observed using cDNA arrays indicated oxidative stress, mitotic arrest, and apoptosis in treated immortalized rat peritoneal mesothelial cells. Increases occurred in oxidative stress responsive genes HO-1, QR, HSP70, GADD45, GADD153, p21(WAF1/CIP16), GST's, GAPDH, TPX, and GPX-1(0); transcriptional regulators c-jun, c-fos, jun B, c-myc, and IkappaB; protein repair components Rdelta, RC10-II, C3, RC-7, HR6B ubiquitin-conjugating enzyme and ubiquitin; DNA repair components PCNA, msh2, and O-6 methylguanine DNA methyltransferase; lipid peroxide excision enzyme PLA2; and apoptogenic components TNFalpha, iNOS1 and FasL. Decreases occurred in bcl-2 (antiapoptotic), bax alpha, bad, and bok (proapoptotic) and cell cycle control elements (cyclins). Cyclin G and p14ink4b (which inhibit entry into cell cycle) were increased. Numerous signal transduction, cell membrane transport, membrane-associated receptor, and fatty acid biosynthesis and repair components were altered. Morphologic endpoints examined were number of mitotic figures, number of apoptotic cells, and antibody-specific localization of HO-1 (which demonstrated increased HO-1 protein expression). PCR analysis confirmed HO-1, p21(waf1/cip1), HSP70, GPX1, GADD45, QR, mdr1, PGHS, and cyclin D1 changes. A model for KBrO(3)-induced carcinogenicity in the F344 rat mesothelium is proposed, whereby KBrO(3) generates a redox signal that activates p53 and results in transcriptional activation of oxidative stress and repair genes, dysregulation of growth control, and imperfect DNA repair leading to carcinogenesis.

Construction and use of a multi-competitor gene for quantitative RT-PCR using existing primer sets.

We describe a simple and inexpensive method for the construction of multi-competitor molecules for use as internal standards in quantitative RT-PCR. The construction involves the linking and annealing of 20mer PCR primers with complementary 40mers using either a step-wise or bulk process. The entire construct is then ligated and amplified by PCR prior to cloning. Using this approach, we have constructed a gene containing priming sites for 18 different products of immunological interest, including murine cytokines and cell surface markers, as well as murine beta-actin and T. cruzi rRNA. The cost of production of the competitor is minimized by use of a high-throughput multi-oligonucleotide synthesizer for production of the individual components of the synthetic gene, and by use of the same oligonucleotides in gene construction and as primers for the RT-PCR reactions. This procedure can be applied to the production of other polycompetitor molecules as well as to the construction of other types of synthetic genes.

Differentiation of trypanosomatid species by hybridization to selected rRNA probes.

Oligonucleotides with sequences complementary to selected regions of the Trypanosoma cruzi large sub-unit ribosomal RNA (rRNA) were used to specifically detect and quantify T. cruzi and other kinetoplastids. By selecting sequences with varying homologies with Crithida fasciculata, another kinetoplastid for which this sequence was known, probes which hybridized to T. cruzi alone or T. cruzi and T. rangeli, various Leishmania species or C. fasciculata were identified. This identification was possible even though the sequences of the large sub-unit (LSU) rRNA of T. rangeli and Leishmania are not known. None of the probes hybridized with rRNA from mouse or human cell lines, and all could quantitatively detect T. cruzi in tissue culture cells. Probing of replicate membranes with these different oligonucleotides allowed discrimination between these species. The functional application of rRNA-specific probes in diagnosis was demonstrated by identification of unknown trypanosomatids in hemocultures of wild-captured owl and squirrel monkeys using a combination of oligonucleotides. Therefore, these probes should be useful in diagnosis and identification of T. cruzi and related parasites.

Differences in the regulation of humoral responses between mice infected with Trypanosoma cruzi and mice administered T. cruzi-induced suppressor substance.

In the present study, the effects of Trypanosoma cruzi and the T. cruzi-induced serum suppressor substance (SSS) on antibody responses were compared. Although infection with T. cruzi led to an alteration in T cell helper activity and a reduced specific B cell precursor frequency, SSS did not have a similar effect on either of these cell populations. The characteristics of the altered T cell helper activity was further investigated, and it was found that helper activity appeared earlier in infected mice than in normal or SSS-suppressed mice, and less antigen was required for optimal elicitation of T helper cells in infected mice. The potency of T cell helper activity also was shown to differ, and in the order T. cruzi-infected 6E normal 6E SSS-suppressed mice. It was found that spleen cells from T. cruzi-infected mice elaborated more potent specific helper factors than spleen cells from normal or SSS-suppressed mice, but did not produce a detectable nonspecific helper factor in vitro. Finally, the addition of B cells from low-dose primed, T. cruzi-infected mice to cultures of normal spleen cells resulted in subnormal responses to the priming antigen (sheep erythrocytes) but not to another unrelated antigen (trinitrophenyl-haptenated Brucella abortus), whereas similarly sensitized B cells from normal or SSS-suppressed mice caused no such effect.

Suppression of mitogen-induced blastogenesis by the Trypanosoma cruzi-induced suppressor substance.

Serum from mice infected with Trypanosoma cruzi-suppressed (=SSS) lipopolysaccharide (LPS)-, phytohemagglutinin (PHA)-, and concanavalin A (Con A)-induced lymphoblast transformation when added to cultures of spleen cells or lymph node cells. This serum maximally suppressed blastogenic responses in spleen cell and lymph node cell cultures that contained supportive fetal bovine serum concentrations of 2% and 4%, respectively. Preincubation of lymphoid cells with SSS for 18 to 48 hr prior to initiation of the blastogenesis assay led to suppression of LPS-, PHA-, and Con A-induced proliferation at the optimal concentration of supportive fetal bovine serum (5%), whereas adsorption of lymphoid cells with SSS at 4 C for 30 min before stimulation with mitogen led to suppression of LPS-induced proliferation in spleen cells only. There was a close temporal correspondence between the induction and manifestation of suppression to the T-cell mitogens (PHA and Con A), but the manifestation of suppression preceded the induction of suppression to the B-cell mitogen (LPS) by approximately 12 hr. The SSS-induced suppression of proliferative responses, except in spleen cell cultures stimulated with LPS, was shown to be dependent on the presence of macrophages during the preincubation and stimulation phases of the assay system. The combined results of experiments in which macrophages were preincubated with SSS, or in which macrophages from the spleen were cultured with lymphocytes from the lymph nodes and vice versa (before and after preincubation with SSS), clearly demonstrated the presence of SSS-activated suppressor cells in the spleen, but not in the lymph nodes. Furthermore, the activation of these suppressor macrophages was reliant upon interactions with splenic lymphocytes.