RESUMEN
In this study an in vitro model was developed with the aim of investigating the modulatory effect of cholera toxin (CT) and its counterpart the heat labile toxin of Escherichia coli (LT) on TNF-alpha release induced by murine macrophages and primary human monocytes. Previous studies have demonstrated that the enzymatic activity of CT and LT molecules can inhibit TNF-alpha release by macrophages. The results obtained in this study showed that CT and LT are both, in a dose dependent manner, able either to induce or inhibit TNF-alpha release by murine macrophages and primary human monocytes. The results also showed that recombinant B subunits of CT and LT in the absence of their A subunit induce high levels of TNF-alpha release by macrophages and, in addition, increase the level of TNF-alpha release induced by LPS. The ability of both B subunits (CTB and LTB) in inducing TNF-alpha release by macrophages is not related to the level of LPS contamination, since direct measurements of LPS made in the samples employed in this study showed only traces of LPS (3.4 x 10(-8) EU/ml) which is in our system does not induce TNF-alpha release by macrophages. In contrast to the results obtained with the B subunits, incubation of cells with the A subunit of CT (CTA) inhibit TNF-alpha release induced by native CT, native LT, recombinant LTB and LPS. This inhibitory effect must be related to the activity of the A subunit since viability tests performed in terms of metabolic rate demonstrated that high concentrations of CTA are not toxic to the cells. The data presented herein demonstrate that the A subunits of CT and LT have an inhibitory effect on TNF-alpha release in macrophages, whereas their B subunits have a stimulatory effect on TNF-alpha. The results also suggest that the dose dependent bi-modal effect of native CT and native LT on TNF-alpha release by macrophages is a result of the combined effect of their individual A and B subunits.
Asunto(s)
Toxinas Bacterianas/farmacología , Toxina del Cólera/farmacología , Enterotoxinas/farmacología , Proteínas de Escherichia coli/farmacología , Escherichia coli/metabolismo , Macrófagos/efectos de los fármacos , Subunidades de Proteína/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células Cultivadas , Humanos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/farmacologíaRESUMEN
It is well known that Loxosceles venom induces local dermonecrosis in rabbits, guinea pigs and humans but not in mice, although, depending on the dose, Loxosceles venom can be lethal to mice. In this work we demonstrate that mice injected intradermally in the dorsal area of the back can survive a lethal dose of Loxosceles gaucho venom and also develop an inflammatory reaction (with infiltration of leukocytes shown by histological analysis) at the local injection site when the venom is co-administered with sphingomyelin. It was observed that more venom was retained for a longer period of time at the local injection site when venom was co-administered with sphingomyelin. The presence of exogenous sphingomyelin did not influence significantly the release of TNF-alpha induced by L. gaucho venom. These results suggest that the action of venom on sphingomyelin, producing ceramide phosphate, causes the development of an inflammatory reaction, which in turn traps the venom in the local area for a long period of time and does not allow it to disperse systemically in a dose sufficient to cause death. Our findings also indicate that the size and availability of the local sphingomyelin pool may be important in determining the outcome of Loxosceles envenoming in different mammalian species.
Asunto(s)
Inflamación/inducido químicamente , Hidrolasas Diéster Fosfóricas/toxicidad , Esfingomielinas/metabolismo , Venenos de Araña/toxicidad , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Ceramidas/administración & dosificación , Ceramidas/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Inyecciones Intradérmicas , Inyecciones Subcutáneas , Dosificación Letal Mediana , Liposomas , Masculino , Ratones , Ratones Endogámicos BALB C , Hidrolasas Diéster Fosfóricas/administración & dosificación , Hidrolasas Diéster Fosfóricas/inmunología , Esfingomielinas/administración & dosificación , Venenos de Araña/administración & dosificación , Venenos de Araña/inmunología , Arañas/metabolismo , Factores de TiempoRESUMEN
This study was performed to investigate whether the toxic effects of Loxosceles gaucho venom on cells might be exerted via stimulators of TNF-alpha release generated by sphingomyelinase D--a major component of the venom. It was demonstrated that L. gaucho venom alone is unable to induce TNF-alpha release by J774A.1 cells, while in the presence of exogenous sphingomyelin it induces a high level of TNF-alpha release which is significantly increased by incubation with non-inactivated serum. Ceramide phosphate also induces TNF-alpha release in J774A.1 cells, but (unlike sphingomyelin/sphingomyelinase) the level of release is not influenced by the presence or otherwise of non-inactivated serum. L. gaucho venom does not induce proliferation of J774A.1 cells and even at high concentrations it does not affect their viability. J774A.1 cells, which prior to venom treatment were elongated and clumped, round up after venom treatment, but, revert to their original morphology after incubation with fresh medium. TNF-alpha resistant MRC-5 cells and TNF-alpha sensitive MCF-7 cells are susceptible to the toxic effect of both L. gaucho venom and ceramide phosphate. The results obtained in this study demonstrate that exogenous sphingomyelin can modulate, in vitro, the release of TNF-alpha induced by L. gaucho venom in mouse macrophages. In addition, the results also indicate that ceramide phosphate and L. gaucho venom are toxic to several different cell types, via a variety of mechanisms, some, but not all, of which may involve TNF-alpha as an intermediary.
Asunto(s)
Ceramidas/metabolismo , Macrófagos/efectos de los fármacos , Hidrolasas Diéster Fosfóricas/toxicidad , Esfingomielinas/metabolismo , Venenos de Araña/toxicidad , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Línea Celular , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/efectos de los fármacosRESUMEN
1. Bothrops jararaca venom was detected by ELISA at different times in the skin, muscle, blood, liver, lung, heart, kidney and spleen of mice injected with venom i.m. or i.d. 2. The results showed that even 10 min after i.m. injection the venom is detected mostly in skin rather than in the muscle of the venom injection site. A small amount of venom was detected in the kidney up to 12 h after im venom injection, and none was detected in tissues of lung, heart, liver or spleen. 3. However, in mice injected i.d., the venom could be detected in the skin up to 24 h after injection. Local necrosis and haemorrhage could be neutralized by antivenom injected by the i.d. or i.v. routes only if the antivenom was given a short time after venom injection, even when antivenom is administered in high concentration. 4. In contrast, experiments performed in mice receiving venom i.d. and treated by i.d. or i.v. routes with antivenom injected at different times after envenoming showed that the effect of venom on blood coagulation could be counteracted by antivenom administered by either route up to 2 h after venom injection. 5. We suggest that a feasible amount of antivenom administered i.d. could be given as a first aid measure after a snake bite accident. However, further experimental studies using the i.d. route for antivenom administration are essential to confirm this possibility.
Asunto(s)
Antivenenos , Bothrops , Venenos de Crotálidos , Animales , Antivenenos/análisis , Venenos de Crotálidos/administración & dosificación , Venenos de Crotálidos/inmunología , Venenos de Crotálidos/aislamiento & purificación , Inyecciones Intradérmicas , Inyecciones Intravenosas , Riñón/química , Masculino , Ratones , Piel/química , Factores de TiempoRESUMEN
1. Bothrops jararaca venom was detected by ELISA at different times in the skin, muscle, blood, liver, lung, heart, kidney and spleen of mice injected with venom im or id. 2. The results showed that even 10 min after im injection the venom is detected mostly in skin rather than in the muscle of the venom injection site. A small amount of venom was detected in the kidney up to 12 h after im venom injection, and none was detected in tissues of lung, heart, liver or spleen. 3. However, in mice injected id, the venom could be detected in the skin up to 24 h after injection. Local necrosis and haemorrhage could be neutralized by antivenom injected by the id or iv routes only if the antivenom was given a short time after venom injection, even when antivenom is adminsitered in high concentration. 4. In contrast, experiments performed in mice receiving venom id and treated by id or iv routes with antivenom injected at different times after envenoming showed that the effect of venom on blood coagulation could be counteracted by antivenom administered by either route up to 2 h after venom injection 5. We suggest that a feasible amount of antivenom administered id could be given as a first aid measure after a snake bite accident. However, further experimental studies using the id route for antivenom administration are essential to confirm this possibility
Asunto(s)
Animales , Masculino , Ratones , Venenos de Crotálidos/administración & dosificación , Antivenenos/análisis , Bothrops , Inyecciones Intravenosas , Inyecciones Intradérmicas , Riñón/química , Piel/química , Factores de Tiempo , Venenos de Crotálidos/efectos adversos , Venenos de Crotálidos/inmunologíaRESUMEN
The isotype and specificity of antibodies produced by patients bitten by B. jararaca and submitted to serum therapy were studied. The IgG anti-B. jararaca antibodies have large individual dispersion, starting to appear 10 days after the first bite and increasing to at least 80 days after the bite. IgM antibodies appeared sooner than IgG antibodies but disappeared about 20 days after the bite. Secondary responses induced by an additional bite were characterized by a fast and higher IgG antibody response with no apparent change in the IgM antibody. The immunoblotting tests showed that the specificity of human anti-B. jararaca antibodies is heterogeneous, each patient recognizing different fractions in the B. jararaca venom.