RESUMEN
There are many methods for evaluating the cytotoxic effect of monoclonal antibodies (MAbs) against cancer cells. Most of these methods require either purified MAbs or biological solutions (e.g., cell culture supernatants, ascitic fluids) containing high concentrations of MAbs. This makes the primary screening of antibody-producing hybridomas for specific cytotoxic antibodies a challenging task. Addressing this issue, this work introduces a high throughput screening method, which enables the identification of cytotoxic antibodies using primary hybridoma populations without prior antibody concentration and/or purification. The method is comprised of a dual-chamber system, where antibody-producing hybridomas and target cancer cells are co-cultured but separated by a porous membrane in which the pore size is sufficient for the diffusion of antibody molecules. The MAbs produced in the system continuously diffuse through the membrane between the two chambers and interact with the target cells placed on the other side of a membrane, resulting in death or proliferation arrest of these cells, if MAbs are cytotoxic or cytostatic. The cytotoxic/cytostatic effect can be registered by measuring the viability of target cells. The advantage of this method is that purification or concentration of antibodies secreted by hybridomas is not required. In addition, this method does not require MAb-secreting hybridomas, which are subcloned or have a high level of MAb production. The method may serve as an effective primary high throughput screening for cytotoxic antibodies.
Asunto(s)
Anticuerpos Monoclonales/análisis , Antígenos de Neoplasias/inmunología , Citotoxicidad Inmunológica/inmunología , Hibridomas/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Neoplasias de la Mama/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Ensayos Analíticos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/inmunología , Membranas Artificiales , Neoplasias Ováricas/inmunología , Células Tumorales CultivadasRESUMEN
In vitro drug sensitivity to chloroquine (CQ), mefloquine (MQ) and quinine was investigated in 60 culture-adapted Plasmodium falciparum isolates from malaria patients in Padrecocha, a village in the Amazonian Department of Loreto, Peru. All isolates showed resistance to CQ, decreased susceptibility to quinine, and sensitivity to MQ. These isolates were examined for mutations in the P. falciparum multidrug resistance 1 (pfmdr1) and chloroquine resistance transporter (pfcrt) genes previously linked to CQ resistance. The mutations N86Y and D1246Y, two of the five mutations commonly observed in the pfmdr1 gene of CQ-resistant clones, were not found. The pfcrt mutation K76T, associated with CQ resistance, was identified in all the isolates tested. Sequence analysis of codons 72-76 in the pfcrt gene showed the haplotypes SVMNT and CVMNT.
Asunto(s)
Antimaláricos/farmacología , Cloroquina/farmacología , Genes MDR , Proteínas de la Membrana/genética , Plasmodium falciparum/efectos de los fármacos , Polimorfismo Genético , Animales , Resistencia a Múltiples Medicamentos , Genotipo , Mefloquina/farmacología , Proteínas de Transporte de Membrana , Mutación , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/genética , Proteínas Protozoarias , Quinina/farmacologíaRESUMEN
CD4(+) T cells co-expressing CD25 (CD4(+)CD25(+) T cells) have been identified as immunoregulatory suppressors modulating autoimmune response. Beside that, autoimmune response was supposed to be associated with malaria infection. Based on these data, we hypothesised that CD4(+)CD25(+) T cells may influence protective immunity to malaria parasites, while suppressing autoimmune response arising throughout the course of malarial infection. To test this possibility, we evaluated the kinetics of CD4(+)CD25(+) T cells during malaria infection and investigated the influence of CD25 depletion by anti-mouse CD25 monoclonal antibody (PC61) on the infection, using a mouse model of premunition to Plasmodium berghei NK65 malaria. The results showed that, during exacerbation of P. berghei NK65 infection, the proportion of CD4(+)CD25(+) T cells among CD4(+) T cells decreased, although that of CD4(+) T cells increased. CD25 depletion clearly delayed the growth of parasitaemia during parasite challenge, particularly in immunised mice. These findings demonstrated that CD4(+)CD25(+) T cells are able to influence protective immunity underlying premunition to P. berghei NK65 parasites.
Asunto(s)
Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/inmunología , Malaria/inmunología , Plasmodium berghei/inmunología , Receptores de Interleucina-2/inmunología , Animales , Antígenos de Protozoos/inmunología , Autoinmunidad , Recuento de Linfocito CD4 , Femenino , Inmunización , Malaria/prevención & control , Ratones , Ratones Endogámicos BALB C , Modelos AnimalesRESUMEN
Hypoglycaemia and lactic acidosis are potentially life-threatening, poorly understood sequelae of Plasmodium falciparum infections. We investigated relationships between clinical status, treatment, and glucose and lactate kinetics during management of falciparum malaria in 14 Vietnamese adults. Nine had severe malaria, of whom 4 were administered quinine (Group 1a) and 5 artesunate (Group 1b). Five uncomplicated cases received artesunate (Group 2). Glucose and lactate turnover were studied on 3 occasions: (i) immediately after initial antimalarial treatment, (ii) at parasite clearance a median of 3 days later, and (iii) at discharge from hospital a median of 9 days post-admission. Steady-state glucose and lactate kinetics were derived from plasma isotopic enrichment during a primed-continuous infusion of D-[6,6-D2]glucose and a parallel infusion of L-[1-13C]lactate. Group 1a patients had the lowest plasma glucose concentrations in the admission study (median [range] 3.9 [3.6-5.1] vs 6.3 [4.9-7.1] and 4.5 [4.3-5.5] mmol/L in Groups 1b and 2 respectively; P < 0.05 vs Group 1b), but glucose production rates and serum insulin concentrations that were similar to those in the other groups (P > 0.17). This was also the case at parasite clearance and suggested an inappropriate beta cell response. Group 1a patients had the highest admission lactate production (60 [36-77] vs 26 [21-47] and 22 [4-31] mumol/kg.min in Group 1b and 2 respectively; P < 0.05 vs Group 2). Amongst the 9 severe cases, there was an inverse association between plasma glucose and lactate production at admission and parasite clearance (P < 0.05), but no correlation between admission lactate production and serum bicarbonate (P = 0.73). The present data confirm previous studies showing that quinine depresses plasma glucose through stimulation of insulin secretion. It is hypothesized that the low plasma glucose activates Na+,K(+)-ATPase through increased plasma catecholamine concentrations, leading to accelerated glycolysis and increased lactate production in well-oxygenated tissues. In some severely ill patients with falciparum malaria, a raised plasma lactate on its own may, therefore, be an unreliable index of a developing acidosis.
Asunto(s)
Antimaláricos/uso terapéutico , Glucemia/metabolismo , Ácido Láctico/metabolismo , Malaria Falciparum/metabolismo , Adolescente , Adulto , Artemisininas/uso terapéutico , Artesunato , Femenino , Humanos , Malaria Falciparum/complicaciones , Malaria Falciparum/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Quinina/uso terapéutico , Sesquiterpenos/uso terapéuticoRESUMEN
Antimalarial treatments during primary Plasmodium berghei NK65 infection in BALB/c mice influenced the acquisition of protective immunity against reinfection. Among subcurative treatments, lower doses better enable mice to acquire protective immunity than do higher doses. Eradication of parasites from the start of infection did not promote protective immunity.