The presence and phenotype of apoptotic lymphocytes was studied in spleen cell suspensions taken from CB6F1 mice infected with Plasmodium chabaudi chabaudi AS. High levels of apoptotic cells were found, associated with high parasitaemias and splenomegaly. This was also accompanied by expansion and disarray of spleen white pulp. Apoptosis levels lowered when parasitaemia was cleared, but were still higher than in normal mice. At this time, the spleen was diminishing in size and the white pulp was contracting and rearranging. When parasitaemia was patent, the cells most affected by apoptosis were CD4+ T cells followed by CD8+ T cells, and to a lesser extent B220+ B cells. When parasitaemia was cleared, CD8+ T cells and B220+ B cells returned to basal levels of apoptosis, while CD4+ T cells still had higher apoptosis levels than normal mice. A similar pattern of lymphocyte subpopulation apoptosis was found in infected BALB/c mice, despite the fact that, for this mouse model, it has been reported that B cells are the cells that are most affected by apoptosis. We consider that the high levels of apoptosis in CD4+ T cells when parasitaemias are still high are not easily explained by a normal mechanism of down regulation of the immune response.

A group of 9 Mexican lepromatous leprosy patients was studied at the beginning of a type II reaction (erythema nodosum leprosum, ENL) and after 1 or 2 months of thalidomide treatment. ENL patients at the onset of the reaction had slightly higher amounts of anti-Mycobacterium leprae IgG1 and IgG2 antibodies, compared to similar lepromatous patients that did not develop ENL. Neither these antibody levels nor IgM and the other IgG subclasses were importantly modified after thalidomide treatment. Serum TNF was significantly higher in the patients that developed ENL compared to those that did not develop the reaction. TNF levels were slightly decreased after 1 month of thalidomide treatment and significantly decreased after 2 months of treatment. Serum IFN-γ was significantly lower in patients at the onset of ENL and was increased after 1 and 2 months of thalidomide treatment.

Assessment of cytokine expression has become crucial to understand host responses to infections as well as autoimmunity. Several approaches including Northern blot, RNase protection assay and enzyme-linked immunosorbent assay have been used for this purpose, but they are time consuming, labour intense, and relatively large quantity of the samples is usually required. Recently, a technique termed real-time reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to determine genetic expression with great sensitivity and specificity; however, specialized instrumentation and costly reagents are usually needed. We aimed at using low-cost reagents for real-time PCR. This was achieved by adapting a conventional RT-PCR protocol to the quantitative real-time format, by the addition of the SYBR 1 Green I reagent. We validated the approach by assessing the cytokine gene expression of murine splenocytes upon stimulation with phorbol 12-myristate 12-acetate (PMA)-ionomycin. The results using this technique were compared with those obtained with the well-established gene array method. We conclude that the use of the SYBR 1 Green I reagent during real-time RT-PCR provides a highly specific and sensitive method to quantify cytokine expression with accuracy and no post-PCR manipulation.

A number of reports have suggested that the spleen plays a key role in the regulation of immunity to malaria but the role, if any, of other tissues is less clear. Furthermore, numerous functional changes occur in the spleen following malaria infection and it is not known whether the spleen's role relates primarily to its content of malaria-specific lymphocytes or to the altered structure and function that has occurred. To address these issues we have generated splenic chimeras by transplanting spleens between Plasmodium berghei-immune and naive rats. In the absence of a functional spleen, specific immune responses from both isolated splenic and non-splenic cells can partially control infection. However, an immune spleen in a naive rat can solidly protect the animal from malaria and a normal spleen in an otherwise immune rat can provide enhanced protection over the non-splenic state. Thus, in the presence of functional splenic architecture both splenic and non-splenic malaria-specific lymphocytes operate more effectively. However, these studies do demonstrate an important role for non-splenic tissue in immunity at least for P. berghei in the rat. The study could have important implications for induction of protective immune responses by vaccination and suggests that malaria-specific lymphocyte responses induced in the periphery following vaccination could interact with parasites in both spleen-dependent and spleen-independent ways.

The transfer of spleen cells from CBA/Ca mice recovered from a P. c. chabaudi AS primary infection into irradiated syngeneic recipients conferred very poor protection. Neither elimination of Ly2 cells from immune spleen cells nor reinfection of the donors some days before transfer improved protection significantly. Significant protection was transferred with spleen cells from donors which had been infected 7 times prior to cell transfer. Transferred protection was reduced or eliminated by pretreatment of cells with anti-Thy-1 or anti-L3T4 monoclonal antibodies but not with anti-Ly2.

Thalidomide is a drug that is being used in several diseases with an immunological component, but the effects on the different immune functions have only been studied partially. Therefore, we studied the effect of thalidomide on PPD- or Con-A-induced proliferation of human mononuclear cells. We found no direct effect of thalidomide at up to 50 μg/ml on the cultures. Cells taken from subjects 6 h after ingestion of 200 mg of thalidomide proliferated equally well to PPD and Con-A than cells taken prior to drug administration. Plasma taken from subjects that ingested 200 mg of thalidomide 6 h before did not affect the proliferative response of their own cells when added to the culutures. Plasma from rabbits that were injected with doses 5 or 15 times higher than the dose given to humans did not diminish the proliferative response of human mononuclear cells to PPD. We conclude that neither thalidomide nor its metabolites affect the proliferative response of human mononuclear cells.

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