RESUMO
Mosquito larvae soluble fractions obtained by molecular exclusion chromatography altered the mitotic rate of several epithelial cell populations in hepatectomised mice, as well as the proliferation of human mononuclear cells (MNC), stimulating or inhibiting them depending on the fraction and dose applied. The effect was also thermolabile, suggesting a proteic nature of the compounds involved. Analysis of cell viability after culture indicated that the extract did not have lethal toxic effects. One fraction with a molecular weight ranging between 12-80 kDa caused only an inhibitory effect. In the present study, we performed further characterisation of this fraction by assaying the effect of new fractions obtained from this one, by the use of a column with a lower molecular weight exclusion range. Assays were performed on the proliferation of adult human MNCs. Our results showed that two out of four of the sub-fractions analysed, with a MW of about 70 and 17 kDa, caused a dose-dependent response, either inhibiting or stimulating MNC proliferation respectively.
Assuntos
DNA/biossíntese , Larva/metabolismo , Leucócitos Mononucleares/metabolismo , Análise de Variância , Animais , Divisão Celular , Sobrevivência Celular , Concanavalina A/farmacologia , Culicidae , Relação Dose-Resposta a Droga , Humanos , Camundongos , Fatores de TempoRESUMO
In the cat ventricle angiotensin II exerts a positive inotropic effect produced by an increase in intracellular calcium associated with a prolongation of relaxation. The signaling cascades involved in these effects as well as the subcellular mechanisms of the negative lusitropic effect are still not clearly defined. The present study was directed to investigate these issues in cat papillary muscles and isolated myocytes. The functional suppression of the sarcoplasmic reticulum (SR) with either 0.5 microm ryanodine or 0.5 microm ryanodine plus 1 microm thapsigargin or the preincubation of the myocytes with the specific inhibitor of the inositol 1,4,5-triphosphate (IP3) receptors [diphenylborinic acid, ethanolamine ester (2-APB), 5-50 microm] did not prevent the positive inotropic effect and the increment in Ca2+ transient produced by 1 microm angiotensin II. In contrast, protein kinase C (PKC) inhibitors, chelerythrine (20 microm) and calphostin C (1 microm) completely inhibited both, the angiotensin II-induced increase in L-type calcium current and positive inotropic effect. The prolongation of half relaxation time produced by 0.5 microm angiotensin II [207+/-15.4 msec (control) to 235+/-19.98 msec (angiotensin II), P<0.05] was completely blunted by PKC inhibition. This antirelaxant effect, which was independent of intracellular pH changes, was associated with a prolongation of the action potential duration and was preserved after either the inhibition of the SR and the SR Ca2+ ATPase (ryanodine plus thapsigargin) or of the reverse mode of the Na+/Ca2+ exchanger (KB-R7943, 5 microm). We conclude that in feline myocardium the positive inotropic and negative lusitropic effects of angiotensin II are both entirely mediated by PKC without any significant participation of the IP3 limb of the phosphatidylinositol/phospholipase C cascade. The results suggest that the antirelaxant effect of angiotensin II might be determined by the decrease in Ca2+ efflux through the Na+/Ca2+ exchanger produced by the angiotensin II-induced prolongation of the action potential duration.
Assuntos
Angiotensina II/farmacologia , Cardiotônicos/farmacologia , Angiotensina II/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Gatos , Colagenases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Indóis/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Microscopia de Fluorescência , Miocárdio/citologia , Naftalenos/farmacologia , Músculos Papilares/metabolismo , Técnicas de Patch-Clamp , Fosforilação , Proteína Quinase C/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Rianodina/farmacologia , Retículo Sarcoplasmático/metabolismo , Transdução de Sinais , Tapsigargina/farmacologia , Fatores de TempoRESUMO
1. Cat ventricular myocytes loaded with [Ca2+]i- and pHi-sensitive probes were used to examine the subcellular mechanism(s) of the Ang II-induced positive inotropic effect. Ang II (1 microM) produced parallel increases in contraction and Ca2+ transient amplitudes and a slowly developing intracellular alkalisation. Maximal increases in contraction amplitude and Ca2+ transient amplitude were 163 +/- 22 and 43 +/- 8 %, respectively, and occurred between 5 and 7 min after Ang II administration, whereas pHi increase (0.06 +/- 0.03 pH units) became significant only 15 min after the addition of Ang II. Furthermore, the inotropic effect of Ang II was preserved in the presence of Na+-H+ exchanger blockade. These results indicate that the positive inotropic effect of Ang II is independent of changes in pHi. 2. Similar increases in contractility produced by either elevating extracellular [Ca2+] or by Ang II application produced similar increases in peak systolic Ca2+ indicating that an increase in myofilament responsiveness to Ca2+ does not participate in the Ang II-induced positive inotropic effect. 3. Ang II significantly increased the L-type Ca2+ current, as assessed by using the perforated patch-clamp technique (peak current recorded at 0 mV: -1.88 +/- 0.16 pA pF-1 in control vs. -3.03 +/- 0.20 pA pF-1 after 6-8 min of administration of Ang II to the bath solution). 4. The positive inotropic effect of Ang II was not modified in the presence of either KB-R7943, a specific blocker of the Na+-Ca2+ exchanger, or ryanodine plus thapsigargin, used to block the sarcoplasmic reticulum function. 5. The above results allow us to conclude that in the cat ventricle the Ang II-induced positive inotropic effect is due to an increase in the intracellular Ca2+ transient, an enhancement of the L-type Ca2+ current being the dominant mechanism underlying this increase.
Assuntos
Angiotensina II/farmacologia , Cardiotônicos/farmacologia , Coração/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Gatos , Eletrofisiologia , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Indóis , Potenciais da Membrana/fisiologia , Miocárdio/ultraestrutura , Técnicas de Patch-Clamp , Retículo Sarcoplasmático/fisiologia , Trocador de Sódio e Cálcio/metabolismoRESUMO
A crude mosquito larvae and dialysed extract alters the mitotic rate of several epithelial cell populations in normal young and adult hepatectomized mice. A crude extract also showed a biphasic effect on the proliferation of human mononuclear cells (MNCs), either stimulating or inhibiting them depending on the dose applied. In the present paper, we assayed the effect of the dialysed mosquito larvae extract and two different protein fractions on human MNCs. Analysis of cell viability after culture indicated that the extract did not have toxic effects. Our results show a dual response of the MNCs to the dialysed, as well as to the protein fraction, with the highest molecular weight inhibiting or stimulating proliferation, depending on the dose applied. The protein fraction with the lowest molecular weight (range between 12-80 kDa) showed only an inhibitory effect on cell proliferation.
Assuntos
Culex/metabolismo , DNA/biossíntese , Leucócitos Mononucleares/metabolismo , Adulto , Animais , Divisão Celular , Sobrevivência Celular , Células Cultivadas , Cromatografia em Gel/métodos , Diálise , Humanos , Larva , Leucócitos Mononucleares/citologia , Soluções , UltracentrifugaçãoRESUMO
Mosquito larvae crude extract have been found to alter the mitotic rate of several mouse epithelial cell populations such as enterocytes and tongue keratinocytes. Also, the dialysed fraction inhibits hepatocyte proliferation in hepatectomized males. These experiments suggested an inhibitory effect on the G1/S interphase. Consequently, we suggested the presence of some molecule or molecules related to the TGF-beta superfamily. In the present paper, we have assayed the crude extract on human mononuclear cells and the dialysed fraction of the extract on tongue keratinocyte proliferation. Furthermore, different protein fractions obtained using a molecular exclusion chromatographic column were assayed on hepatocyte proliferation of hepatectomized mice. Three groups of proteins have been isolated. Results show a dose-dependent effect of crude extract on mononuclear cell proliferation and the dialysed extract caused an inhibitory effect on tongue keratinocyte proliferation. With regard to the hepatocyte mitotic rate, an inhibitory effect appeared only in animals receiving the fraction with lower molecular weight. These results suggest the presence in mosquito larvae of some peptidic molecule or molecules resembling the activity of members of the TGF-beta superfamily.
Assuntos
Culicidae , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Substâncias de Crescimento/farmacologia , Fígado/efeitos dos fármacos , Fígado/patologia , Animais , Divisão Celular/efeitos dos fármacos , Humanos , Larva , Camundongos , Fator de Crescimento Transformador beta/farmacologiaRESUMO
The aim of this review is to update the knowledge on dendritic cells (CD), as potent antigen-presenting cells (APC) expressing class II major histocompatibility (MHC) antigen. The different types of DC are derived from a common bone marrow precursor. They differentiate and migrate to lymphoid and non-lymphoid tissues under the influence of diverse stimuli. After binding antigen in their periphery they move to the lymph node activating T cells. Depending on the microenvironment, DC express several surface markers and secrete cytokines such as IL-12, Il-1 and TNF alpha. DC play a role in the pathogenesis of autoimmune and viral diseases being relevant in AIDS. These cells also infiltrate human tumors where they could be involved in the induction of anti-tumor immune response. The immunostimulatory properties of DC are currently applied in DC-based therapies of melanoma and lymphoma patients.
Assuntos
Apresentação de Antígeno/fisiologia , Células Dendríticas/fisiologia , Complexo Principal de Histocompatibilidade/imunologia , Neoplasias/imunologia , Síndrome da Imunodeficiência Adquirida/imunologia , Apresentação de Antígeno/imunologia , Células Dendríticas/imunologia , Células Dendríticas/ultraestrutura , Humanos , Células de Langerhans/imunologia , Células de Langerhans/fisiologiaRESUMO
A growing body of evidence suggests that corticotrophin releasing hormone (CRH) may exert direct modulatory effects on immune cells. In the present study we assessed the effects of its precursor molecule, proCRH, on interleukin-6 (IL-6) release by human peripheral blood mononuclear cells (MNC). Human MNC were incubated with the corresponding stimuli for 24 hr. The supernatants were collected and IL-6 measured by ELISA. Conditioned medium from CHO-K1 cells stably transfected with the recombinant plasmid pEE14/rat pre-proCRH cDNA was used as the source of proCRH. Western blot analysis of this medium, using an antibody specific for the intact precursor, showed that no proCRH degradation products were present. The proCRH had an inhibitory effect on basal and LPS-stimulated release of IL-6. These results suggest that the full length CRH precursor may possess immunomodulatory properties.
Assuntos
Hormônio Liberador da Corticotropina/farmacologia , Interleucina-6/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Precursores de Proteínas/farmacologia , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/farmacologia , Adulto , Animais , Células CHO , Hormônio Liberador da Corticotropina/química , Hormônio Liberador da Corticotropina/genética , Cricetinae , Meios de Cultivo Condicionados , Humanos , Técnicas In Vitro , Conformação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Ratos , TransfecçãoRESUMO
Preincubation of peripheral blood lymphocytes (PBL) from drug-free, healthy volunteers with either the protein tyrosine kinase inhibitor genistein (GNT, n = 10, final concentration 200 microM) or the protein kinase A activator dybutiryl-cyclic-AMP (cAMP, n = 11, final concentration 10 microM), resulted in a significant inhibition of natural killer cell activity (NKCA, expressed as percentage of specific chromium release). With the exception of 4 out of the 11 cAMP-treated samples, individual values for NKCA in the drug preincubated specimens were at least 20% below the same subject baseline activity; furthermore, NKC lytic function was non-detectable in 4 out of the 10 and in 1 out of the 11 samples pretreated with either GNT or cAMP, respectively. PBL preincubation with glutaraldehyde-fixed Gram-negative bacteria (GNB, n = 13, final GNB-to-effector cell ratio of 50 : 1) resulted in a statistically significant increase in NKCA (baseline (x +/- SD) of 21.6 +/- 16.4 and bacteria treated samples of 41.5 +/- 24.6, respectively, Student's paired t-test p < 0.05). At least a 20% increase in NKC lytic function over its own baseline value was recorded for 11 out of the 13 samples tested (Table 1). Preincubation with GNB and GNT (5 samples) not only blocked the immunostimulant effects of GNB (Student's paired t-test p < 0.05), but in most cases individual values for NKCA were similar to those recorded for GNT-only treated samples. Use of cAMP instead of GNT also blocked, but to a smaller extent, the GNB-produced increases in NKC lytic function (paired Student's t-test < 0.05). PBL preincubation with lipopolysaccharide (LPS, n = 11, final concentration 50 micrograms/ml) resulted in a statistically significant increase in NKCA (baseline (x +/- SD) of 20.7 +/- 14.1 and LPS treated samples of 39.2 +/- 18.5, respectively, Student's paired t-test < 0.05). At least a 20% increase in NKCA over its own baseline value was observed for each and everyone of the 11 samples studied (Table 2). Addition of LPS and GNT to the incubation mixture resulted not only in inhibition of the NKCA upmodulating LPS effects (Student's paired t-test p < 0.05), but each and everyone of the individual samples' NKCA were, in fact, significantly lower than their corresponding control baseline values and similar to those recorded for GNT-only treated samples. However, the use of LPS and cAMP (Table 2) produced less dramatic results, significant inhibition of LPS effect were recorded in only 2 samples (Nos 8 and 10), and individual NKCA in the remaining 3 specimens was significantly higher than the corresponding baseline value. Whereas experimental results obtained with GNT support the involvement of PTK-dependent pathways in the stimulation of human NKCA produced by GNB and LPS, cAMP experiments suggest modulation of PKA-dependent pathways as responsible for the decrease in NK lytic function produced by a number of chemicals involved in the pathophysiology associated with certain forms of stress, including septic shock. Further research in this area could help in the rational design of pharmacological approaches for the treatment of these conditions.
Assuntos
Bactérias/química , Proteínas de Escherichia coli , Células Matadoras Naturais/fisiologia , Lipopolissacarídeos/farmacologia , Proteínas Tirosina Quinases/metabolismo , Adulto , Toxinas Bacterianas/farmacologia , Bucladesina/farmacologia , Enterotoxinas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Escherichia coli/química , Feminino , Genisteína , Humanos , Isoflavonas/farmacologia , Masculino , Pessoa de Meia-Idade , Proteínas Tirosina Quinases/antagonistas & inibidores , Salmonella typhimurium/químicaRESUMO
Preincubation of peripheral blood lymphocytes with the microtubule disturbing agents estramustine (ETMN; n = 7, final conc. 20 microM) or taxol (TX; n = 13, final conc. 10 microM), resulted in a statistically significant inhibition of natural killer cell activity [(NKCA); baseline (x +/- SD; expressed as percentage of specific chromium release) of 32.2 +/- 30.5 and 34.4 +/- 27.7 and drug treated samples of 13.9 +/- 19.9 and 12.5 +/- 20.8, respectively; Student's paired t-test p < 0.005]. Furthermore, most individual values for NKCA in the drug preincubated samples were at least 20% below the same subject baseline lytic function (except for TX sample No.1), and NKCA was non detectable in 4 out of 7 and 5 out of 13 samples (pretreatment with either ETMN or TX< respectively). The use of other concentrations and different preincubation times for these chemotherapeutic agents also produced NKCA inhibition, which was time and dose dependent. Preincubation with lipopolysaccharide (LPS; n = 16, final conc. 50 micrograms/ml), an endotoxin prominently involved in the etiology of septic shock, resulted in a statistically significant enhancement of NKCA [baseline (x +/- SD; expressed as percentage of specific chromium release) of 25.4 +/- 20.4 and LPS treated sample of 36.6 +/- 17.4, respectively; Student's t paired t-test p < 0.005]. At least a 20% increase in NKC lytic function over its own baseline value was recorded for each and everyone of the samples tested with LPS.2+ the pathophysiology associated with septic shock.