Constitutive expression of interferon gamma-inducible protein 10 in lymphoid organs and inducible expression in T cells and thymocytes.

Interferon gamma-inducible protein 10 (IP-10), a member of a family of small proinflammatory chemotactic polypeptides, is expressed in interferon gamma-stimulated keratinocytes, macrophages, fibroblasts, and endothelial cells. Here we report that IP-10 is also expressed by activated but not resting T hybridoma cells, normal T cells, and thymocytes. Although resting lymphocytes did not synthesize IP-10, surprisingly high levels of IP-10 transcripts were found in lymphoid organs (spleen, thymus, and lymph nodes). Thymic and splenic stromal cells were found to express constitutively high levels of both IP-10 mRNA and protein, accounting for the high level of spontaneous expression in lymphoid tissue. Therefore, in addition to its role as a proinflammatory cytokine, IP-10 may participate in T cell effector function and perhaps T cell development.

Oleanolic acid inhibits the activity of the multidrug resistance protein ABCC1 (MRP1) but not of the ABCB1 (P-glycoprotein): possible use in cancer chemotherapy.

The effects of oleanolic acid (OA) on ABCB1 and ABCC1 activities were studied in a cell line constitutively expressing both proteins. It was observed that OA did not alter ABCB1 activity, but inhibited the activity of ABCC1 protein. This inhibition was reversible and only occurred in the presence of OA. In addition, OA did not alter the expression of ABCC1 mRNA. These results suggest that OA could be a good choice in the treatment of MDR tumours, either as a chemotherapic itself in tumours bearing ABCB1, or as an adjuvant in the chemotherapy of ABCC1 expressing tumours.

Ca-2+-dependent inhibitory effects of Na+ and K+ on Ca-2+ transport in sarcoplasmic reticulum vesicles.

Effects of Na+ and K+ on Ca-2+ transport by sarcoplasmic reticulum vesicles were studied in a medium containing high Mg-2+ and ATP (2mM) and low Ca-2+ (0.44muM) concentrations. Under these conditions, Na+ and K+ inhibit Ca-2+ uptake, ATPase activity and membrane phosphorylation by ATP. Since the concentrations of ATP and Ca-2+ used are consistent with relaxation in vivo, the results suggest that under physiological resting conditions the Ca-2+ pump of the sarcoplasmic reticulum operates below its maximal capacity.

Acute Trypanosoma cruzi infection differentially affects CD3 and Thy-1 mediated T cell activation.

Monoclonal antibodies directed against the Thy-1 molecule or the CD3 complex were used to analyze the activation of T cells from mice acutely infected with Trypanosoma cruzi. When stimulated with G7, a mitogenic anti-Thy-1 monoclonal antibody, spleen cells from infected mice showed a markedly reduced or absent response that could not be restored by varying the culture time or the antibody concentration. However, cells from acutely infected animals proliferated to 145-2C11, an anti-CD3 monoclonal antibody. Flow cytometric analysis showed that the impaired response to G7 could not be attributed to a lack of expression of Thy-1 or CD3. Indeed, G7 seemed to deliver a positive signal to the cells since the proliferative response was completely restored by the addition of PMA. Moreover, purified T cells from infected mice responded to G7 in the presence of accessory cells from uninfected animals. These results suggest that a defective co-stimulatory cell function could be involved in the immunosuppression. In addition, our data present evidence against a generalized T cell anergy in the acute phase of the disease, since CD3-mediated activation was normal.

Specific inhibition of OKT8 binding to peripheral blood mononuclear cells by jacalin.

Human T-specific monoclonal antibodies were used to study the interactions between the binding of jacalin to peripheral blood mononuclear cells (PBMC) and the immunoregulatory molecules displayed at the surface of T cells. Jacalin inhibits the binding of OKT8 (anti-CD8) to both fresh PBMC and jacalin-induced T cell blasts. In both cases the binding of anti-CD3 (OKT3) or anti-CD4 (OKT4) was not affected by the lectin. The effect of jacalin on OKT8 binding is abolished by 1-O-alpha-D-methylgalactopyranoside, suggesting its mediation by the lectin saccharide combining sites. Preincubation experiments indicated that the inhibitory effect of jacalin is due to a competition between the lectin and the monoclonal antibody. The effect of the lectin could also be reversed by increasing concentrations of the monoclonal antibody. Taken together this data demonstrates a specific inhibition of OKT8 (anti-CD8) binding by jacalin. This effect is mediated by the binding of the lectin to structures on the cell surface, perhaps the CD8 antigen. The data also points to the discovery of a new mitogen that could be useful for studying the physiological role of CD8 on T cell responses.

Lymphocyte subpopulations in chronic Chagas' disease.

Monoclonal antibodies of the Orthoclone series were used to identify total T lymphocytes (OKT3) and their helper-inducer (OKT4) and cytotoxic-suppressor (OKT8) subsets in 25 patients with chronic Chagas' disease and 25 healthy controls. No significant difference in the number of total T cells (OKT3+) circulating in the peripheral blood of patients and controls was found. However, in contrast to normal subjects, chagasic patients show quantitative alterations in both helper (OKT4+) and suppressor (OKT8+) T cell subsets. The chagasic patients have abnormal helper/suppressor ratios, with low and high values scattered around the mean. Surprisingly, high ratios were found within females while almost all males have low ratio values. These findings suggest that the putative immunoregulatory disfunctions in patients with chronic chagasic cardiomyopathy may involve both helper and suppressor T cell subsets.

Antibody response in mice immunized with a plasmid DNA encoding the colonization factor antigen I of enterotoxigenic Escherichia coli.

The colonization factor antigen I (CFA/I) is one of the most epidemiologically relevant enterotoxigenic Escherichia coli (ETEC) fimbrial adhesins, which mediates the binding to human small intestine epithelium. A recombinant eukaryotic expression plasmid, pRECFA, encoding the CFA/I protein fused to the glycoprotein D of herpes simplex type 1 virus, was used to generate an antibody response in a murine model following intramuscular inoculation of purified DNA. Eukaryotic cells (BHK-21) transfected with pRECFA expressed the CFA/I protein in vitro, as revealed by Western blot and immunofluorescence microscopy. Administration of a single pRECFA 100-microg dose induced a long-term CFA/I-specific antibody response in BALB/c mice composed mainly of IgG and, to a lesser extent, IgA isotypes. The major CFA/I-specific IgG subclass was IgG2a, suggesting a Th-1-type immune response. A second dose with the same amount of purified DNA, given 2 weeks later, caused a booster effect on the immunoglobulin levels, but did not qualitatively alter the isotypes and subclasses of the induced antibody response. Immunization with different amounts of purified DNA and/or number of doses showed that maximal transient CFA/I-specific antibody levels could be obtained after two 100-microg doses of pRECFA given 2 weeks apart, but long-term antibody levels were similar.

Trypanosoma cruzi: compromise of reproductive system in acute murine infection.

Infection of isolated organs of the reproductive system by Trypanosoma cruzi has been described since Chagas' disease was first studied. A detailed histopathological analysis of mice acutely infected with T. cruzi CL strain showed colonization of male (preputial glands and skin, penis, testicular albuginea, epididymis, vas deferens, seminal vesicles, prostate, coagulative, bulbo urethral and urethral glands) and female (vagina, uterus, oviduct, ovary, mesovary, clitoris and mammary glands) structures of the reproductive system. The results presented herein demonstrated invasion of epithelial cells, pronounced colonization of the epididymis and male genital adnexa, but absence of parasitism in penile corpora cavernosa.

Analysis of cell populations in crescentic glomerulonephritis.

The cell types present in the crescents were studied in 5 human patients with crescentic glomerulonephritis: two cases of systemic lupus erythematosus, one case of hemolytic uremic syndrome and two cases of rapidly progressive glomerulonephritis. Frozen sections of renal biopsies were studied by immunofluorescence, using murine monoclonal antibodies (orthoclones) against specific antigens on the membrane of human peripheral blood cells, and by histochemical methods. Monocytes (OKM1+, OKIa+ cells) but no lymphocytes (OKT+ cells), were detected in the crescentic glomeruli. Subsets of T lymphocytes (inducer-helper and cytotoxic-suppressor) were detected in the interstitium. Non-specific esterase-positive cells were observed in the glomeruli and in small numbers in the crescents. Fibrinogen deposits were present in the crescents of four of the five cases studied. No immunoglobulins (IgG, IgM, IgA) or complement (C1q, C3) deposits were detected in the crescents. Fibrinogen, immunoglobulins and complement were present in the glomerular tufts.

[Distribution of Langerhans cells in the skin of patients with Hansen's disease]. / Distribuição de células de Langerhans na epiderme de pacientes com hanseníase.

Intra-epidermal OKT+ and OKla+ cells were identified and counted in 25 cases of Leprosy (7 lepromatous, 7 bordeline, 8 tuberculoid and 3 indeterminate). No significant variations were found in the number of Langerhans cells (OKT6+) in the clinical forms of the disease, nor marked variations were determined in the relation of OKT6+/OKla+ intra-epidermal cells. Nevertheless significant variation in the number of Langerhans cells were noted in several cases independently of the clinical form of the disease.

The husk fiber of Cocos nucifera L. (Palmae) is a source of anti-neoplastic activity.

In the present study, we investigated the in vitro anti-tumoral activities of fractions from aqueous extracts of the husk fiber of the typical A and common varieties of Cocos nucifera (Palmae). Cytotoxicity against leukemia cells was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Cells (2 x 10(4)/well) were incubated with 0, 5, 50 or 500 microg/mL high- or low-molecular weight fractions for 48 h, treated with MTT and absorbance was measured with an ELISA reader. The results showed that both varieties have almost similar antitumoral activity against the leukemia cell line K562 (60.1 +/- 8.5 and 47.5 +/- 11.9% for the typical A and common varieties, respectively). Separation of the crude extracts with Amicon membranes yielded fractions with molecular weights ranging in size from 1-3 kDa (fraction A) to 3-10 kDa (fraction B) and to more than 10 kDa (fraction C). Cells were treated with 500 microg/mL of these fractions and cytotoxicity was evaluated by MTT. Fractions ranging in molecular weight from 1-10 kDa had higher cytotoxicity. Interestingly, C. nucifera extracts were also active against Lucena 1, a multidrug-resistant leukemia cell line. Their cytotoxicity against this cell line was about 50% (51.9 +/- 3.2 and 56.3 +/- 2.9 for varieties typical A and common, respectively). Since the common C. nucifera variety is extensively cultured in Brazil and the husk fiber is its industrial by-product, the results obtained in the present study suggest that it might be a very inexpensive source of new antineoplastic and anti-multidrug resistant drugs that warrants further investigation.

Pomolic acid-induced apoptosis in cells from patients with chronic myeloid leukemia exhibiting different drug resistance profile.

Pomolic acid (PA) is a pentacyclic triterpene which has been previously described as active in inhibiting the growth of K562 cell line-originated from chronic myeloid leukemia (CML) in blast crisis-and its vincristine-resistant derivative K562-Lucena1. In this work, cells from CML patients were treated with PA and the apoptotic index was compared with the multidrug resistance (MDR) profile and clinical status of the patients. Our findings show that PA 12.5 microg/ml at 24 h (p = 0.000), at 48 h (p = 0.012) and at 72 h (p = 0.005) has a potent apoptotic index in CML cells as compared to mononuclear cells from healthy donors. PA was capable to induce apoptosis in cells from CML patients exhibiting functional MDR phenotype but not in P-glycoprotein expression. In addition, PA was effective in chronic as well as in blast phase of CML. Moreover, similar apoptotic index induced by PA was observed in low, intermediate and high-risk Sokal score as well as in samples from the group of patients with clinical resistance to interferon and/or imatinib and non-treated patients. These results suggest that PA may be an effective agent for the treatment of CML.

Negative tissue parasitism in mice injected with a noninfective clone of Trypanosoma cruzi.

Trypanosoma cruzi is a heterogeneous population of parasites as shown by differences between strains and cloned stock from the same strain. Herein we present evidence of the noninfectivity of CL-14, a clone derived from the CL strain of T. cruzi. In a previous paper we reported the absence of parasitemia and mortality in mice injected with metacyclic trypomastigotes of this clone. To investigate further this lack of infectivity we did and extensive histopathological analysis in mice at different intervals after i.p. (5 and 15 days as well as 1, 4, and 12 months) or i.v. (5 and 30 days) injection of trypomastigotes. In spite of a systematic search in all tissues and organs of the animals, no parasite or significant pathological change was detected in any of the tissue sections. These data suggest the inability of this clone to mediate infection and/or cause pathological alterations in vivo.

High expression of a functional cruzipain by a non-infective and non-pathogenic Trypanosoma cruzi clone.

We compared a Trypanosoma cruzi clone unable to infect or induce pathology in mice (CL-14), with virulent T. cruzi (Y and CL strains) in terms of cruzipain expression, subcellular distribution and functional activity. Our results showed that (1) intracellular Y amastigotes expressed R1 (carboxy-terminal) and R2 (catalytic) domains concentrated in cytoplasmic vesicles, while CL-14 presented R1 labelling on membrane clusters and R2 in intracellular compartments, (2) CL-14-trypomastigotes presented R1 and R2 staining preferentially on flagellar and cellular membranes, similar to CL, but different from Y strain intracellular labelling pattern, (3) flow-cytometry revealed higher expression of R1 by CL-14-trypomastigotes than virulent strains, but R2 staining similar to CL-trypomastigotes, (4) CL-14-trypomastigotes presented normal cruzipain activity in gelatin gels, but different banding patterns were found in CL-14 versus CL and Y strains. Our data rule out failure in cruzipain expression, activity or subcellular distribution as an explanation for CL-14 biological behaviour, but suggest the expression of a different isoform. These results also cast doubt on the putative role of cruzipain as a target of immunopathological responses, since high levels of functional cruzipain are expressed by a non-pathogenic T. cruzi.

Trypanosoma cruzi: properties of a clone isolated from CL strain.

BALB/c mice injected i.p. with 2 x 10(6) metacyclic forms of CL-14, a clone isolated from the CL strain of Trypanosoma cruzi, did not show parasitemia as evaluated by direct blood microscopy examination, hemoculture and xenodiagnosis. Moreover, new-born mice (1-2 days old) injected with culture- or insect-derived CL-14 trypomastigotes also displayed negative parasitemia. No mortality was observed in either group of animals. However, despite this apparent non-infectivity, mice injected with clone 14 developed high resistance against a lethal challenge with virulent trypomastigotes. All challenged mice survived and the parasitemia was negative. These results indicate that clone 14 is a very good antigen for the study of acquired immunity in T. cruzi infection and, therefore, a potential candidate for the development of a vaccine against this parasite.

Trypanosoma cruzi: IgG1 and IgG2b are the main immunoglobulins produced by vaccinated mice.

Mice vaccinated with CL-14, a non-infective and non-pathogenic clone isolated from Trypanosoma cruzi CL strain, become protected against lethal challenge by infective trypomastigotes. It has been shown that animals infected with T. cruzi show polyclonal activation of B lymphocytes with an early production of several non-specific immunoglobulins. Vaccinated mice, however, have an early production of antigen-specific IgG1 and IgG2b. Considering the lack of infectivity of CL-14, our data strongly suggest a role for IgG1 and IgG2b in protection to T. cruzi.

Trypanosoma cruzi: protective response of vaccinated mice is mediated by CD8+ cells, prevents signs of polyclonal T lymphocyte activation, and allows restoration of a resting immune state after challenge.

Currently, there is no vaccine available against Chagas' disease. Immune abnormalities induced by T. cruzi pose particular difficulties for vaccine development, since immunological memory must be able to overcome them to prevent spread of infection/sequelae. We have previously demonstrated that experimental vaccination with live CL-14 trypomastigotes does not induce polyclonal lymphocyte activation, immunosuppression, or pathology and efficiently immunizes against virulent T. cruzi. Herein we show that: (1) expansion of CD4+ and CD8+ subsets peaks 2 weeks after infective challenge in both challenged-vaccinated mice and infected controls, but the former exhibit a smaller increase in blastogenesis and in the numbers of activated CD11a(hi)CD4+ and CD11a(hi)CD8+ cells; (2) in long-term-vaccinated mice, expansion of activated subsets (CD62Llo/- and CD11a(hi)) is accelerated among CD8+ PBL 1 week after challenge; (3) challenged-vaccinated mice retract the CD8+-activated subset 5 weeks after challenge, different from infected controls; (4) protection conferred by CL-14 immunization can be adoptively transferred to naïve recipients with lymphocyte suspensions, and prior depletion of CD8+ (but not of CD4+) cells abolishes protective immunity. Our findings indicate that protective immunity generated by CL-14 immunization involves a transient CD8+ recall response and is capable of preventing the signs of polyclonal lymphocyte activation induced by virulent challenge.

Trypanosoma cruzi: paninfectivity of CL strain during murine acute infection.

A systematic study of the distribution of intracellular parasites in the organs and tissues of mice acutely infected (15 days) with the CL strain of Trypanosoma cruzi was performed. Almost all tissues and organs were parasitized with different intensities, including several epithelial cell types. In addition to striated, cardiac, and smooth muscles a very high parasitism of fat cells, pancreas, and genital adnexa was observed. A smaller number of parasites was found in all other structures studied except in highly vascularized structures such as in the penile corpora cavernosa, pulmonary and renal parenchyma, islets of Langerhans, hepatic sinusoids, and in atrial endothelium. This paper also shows, for the first time in the literature, the parasitism of milky spots, cornea epithelium, cornea stroma, retroorbital fibroblasts, seminal vesicles, and coagulative, Cowper's, urethral, preputial, sebaceous anal, and clitoris glands. The results indicated that CL strain is highly invasive, being able to infect cells derived from the three embryonic layers (ectoderm, mesoderm, and endoderm), suggesting that the paninfectivity may influence the outcome of immunological and pathological events.

Do self-heart-reactive T cells expand in Trypanosoma cruzi-immune hosts?

Anti-heart T-cell activity was evaluated by a lymph node cell proliferative assay in isogenic strains of mice immunized with several Trypanosoma cruzi epimastigote and trypomastigote antigenic preparations. In addition, chronically infected animals were boosted with trypomastigote antigens and their lymph node cells were tested by in vitro proliferative responses. Our results indicated that (i) use of allogeneic sources of heart antigens may induce alloreactive responses in T. cruzi-immune T cells, (ii) specific autoimmune T-cell reactivity against self-heart constituents could not be demonstrated after immunization of the host with T. cruzi, and (iii) a proportion of chronically infected mice showed a small but detectable level of auto-anti-heart T-cell reactivity. These results argue against the notion that T. cruzi epitopes cross-reactive with self-heart tissue play a role in initiating T-cell-mediated autoimmunity. Anti-heart autoreactive T cells, generated in a proportion of the animals, may result from heart lesions associated with the infection process.