Parasitaemia levels in Plasmodium chabaudi infected-mice modify IFN-gamma and IL-10 expression after a homologous or heterologous challenge.

CB6F1 mice infected with the nonlethal Plasmodium chabaudi chabaudi AS suffer parasitaemia levels up to 40% (full parasitaemia, FP) and develop both homologous and heterologous (against the lethal Plasmodium yoelii 17XL) protective immunity. However, if mice are treated with anti-malarial drug when parasitaemia is below 10% (low parasitaemia, LP), they only develop homologous immunity. For the better understanding of this interesting dissociation related to the degree of parasitaemia, in this work, we studied the genetic expression of some cytokines. We found that during primary parasitaemia both FP and LP mice showed at first a TNF-alpha, IL-2 and IFN-gamma response which is followed by an IL-4 and IL-10 response. When FP and LP mice were challenged with either the homologous (FP + AS and LP + AS mice) or the heterologous parasite (FP + 17XL and LP + 17XL mice), we observed that LP + 17XL mice, which failed to develop heterologous immunity and succumbed to the challenge, showed a stronger IFN-gamma and a weaker IL-10 expression than FP + 17XL mice, which developed heterologous immunity and survived the challenge. The importance and the possible implications of these findings are discussed.

Quantification of cytokine gene expression using an economical real-time polymerase chain reaction method based on SYBR Green I.

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 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 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.

Apoptosis in lymph nodes and changes in lymphocyte subpopulations in peripheral blood of pigs infected with porcine rubulavirus.

In a first experiment, five pigs were inoculated intranasally with porcine rubulavirus (PoRV) at 5 days of age and killed 7 days post-infection (pi). In a second experiment, four pigs were infected with the same virus at 17 days of age and killed at 9 or 15 days pi. Control piglets in each experiment received uninfected cell culture supernate. All PoRV-infected pigs developed respiratory and nervous signs, and histological lesions of non-suppurative encephalitis and interstitial pneumonia. All control pigs remained clinically normal and did not have histological lesions. Significantly increased numbers of apoptotic cells were detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) in tonsil and lymph nodes of the pigs infected at 7 days of age and killed at 7 days pi. Significantly increased percentages of CD2(+) and CD8(+) T lymphocytes were also found in peripheral blood of these animals at this time, while the percentages of CD4(+) and MHC class II lymphocytes were significantly reduced. Significantly increased numbers of apoptotic cells were detected in lymphoid tissues of the pigs infected at 17 days of age and killed at 9 days pi. The percentages of CD2(+), CD8(+) and MHC class II lymphocytes in peripheral blood were also significantly increased at this time; the percentage of MHC class II lymphocytes remained elevated at 15 days pi. These results indicate that induction of apoptosis is an important mechanism in the pathogenesis of PoRV infection in young pigs, and that this virus induces changes in lymphocyte subpopulations in peripheral blood.

Mouse splenic CD4+ and CD8+ T cells undergo extensive apoptosis during a Plasmodium chabaudi chabaudi AS infection.

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.

Plasmodium chabaudi chabaudi: effect of low parasitemias on immunity in CB6F1 mice.

We examined the effect that low parasitemias have on the immune response of CB6F1 mice infected with Plasmodium chabaudi chabaudi AS. Ascending parasitemias were stopped by chloroquine treatment when they were between 1.6 and 9.4%. Mice that suffered low parasitemias developed good immunity to homologous reinfection but, contrary to what happened in mice that suffered full parasitemias, they did not develop immunity to heterologous reinfection with Plasmodium yoelii 17XL. Total IgG antiparasite antibody responses were similar in mice that suffered low or full parasitemia, both in primary infection and after reinfection. At the level of isotypes, IgM, IgG1, IgG2b, and IgG3 responses were similar in mice that suffered low or full parasitemias, but after reinfection, mice that suffered low parasitemias responded with higher levels of IgG2a than mice that suffered full parasitemias. Mice that suffered low parasitemias did not have splenomegaly but their immunity to homologous reinfection was diminished after splenectomy in a manner similar to that of splenectomized mice that suffered full parasitemia. CB6F1 mice can develop homologous immunity even if exposed to low parasitemias but cannot develop heterologous immunity unless exposed to high parasite loads.

IgG antibody subclasses, tumor necrosis factor and IFN-gamma levels in patients with type II lepra reaction on thalidomide treatment.

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-gamma was significantly lower in patients at the onset of ENL and was increased after 1 and 2 months of thalidomide treatment.

The spleen, IgG antibody subsets and immunity to Plasmodium berghei in rats.

The development of IgG subclass-specific antibody responses to Plasmodium berghei in spleen-chimeric rats were monitored to determine if there was any relationship between IgG subset profiles and resistance. Strongly immune eusplenic rats respond to challenge with P. berghei by producing high levels of parasite-specific IgG2a, IgG2b and IgG2c but only modest levels of IgG1. Splenectomy profoundly affects the antibody response to infection. Thus, in splenectomized immunized rats, which harbour a chronic parasitaemia of 1%, the IgG2a, IgG2b and IgG2c responses peak 1 week later than in eusplenic immunized rats although the size of the peak is similar. More marked effects are apparent in the IgG1 response, the magnitude of which is far greater in splenectomized immunized rats than eusplenic immunized rats. Similar antibody profiles are seen in splenectomized immunized rats transplanted with a naive spleen. In contrast, splenectomized naive rats receiving either a transplant of a spleen from an immune rat or a transfer of immune spleen cells have high levels of IgG2a, IgG2b and IgG2c but modest levels of IgG1. However, only the former group of rats completely clears the parasite, the latter maintaining a chronic 1% parasitaemia. Thus, although complete resistance to P. berghei is always associated with high levels of parasite-specific IgG2a, IgG2b and IgG2c plus modest levels of IgG1, this is not a sufficient set of conditions to guarantee complete immunity. The IgG subset profile may be related to cytokine production; IFN-gamma was detected in the sera of rats receiving spleens from rats immune to P. berghei (modest IgG1 responses) but not in rats receiving spleens from naive animals (pronounced IgG1 responses).

Seroepidemiological studies of cutaneous leishmaniasis in the Campeche state of México.

Seroepidemiological studies of cutaneous leishmaniasis were carried out in 169 individuals in a rural area of the Campeche state of México. Fifty showed cutaneous lesions suggestive of leishmaniasis, 70% were parasite positive and 96% skin test positive. An overall 40% positivity to skin test with Montenegro's antigen was found. Most of the affected individuals were males from 11 to 30 years-old. Antibodies were determined by immunofluorescent antibody test (IFA) and by Western blot. Two antigen preparations were used, one from a Leishmania mexicana strain which produced localized cutaneous leishmaniasis (LCL) and the other from a diffuse cutaneous leishmaniasis (DCL). In the general population from the area of study 19% gave positive IFA tests with DCL antigen and 20% with LCL antigen while for the patients 67% gave positive IFA tests with DCL and 71% with LCL. By Western blot analysis most of the patients recognized more antigens in the DCL than in the LCL strain. In the DCL strain 78% of patients recognized a 105 kDa, 34% a 139 kDa, 28% a 117 kDa and 26% a 205 kDa MW antigen. In the LCL strain 40% of patients recognized a 205 kDa and 22% a 175 kDa antigens.

Thalidomide and its metabolites have no effect on human lymphocyte proliferation.

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 micrograms/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 cultures. 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.

Protection of rats against malaria by a transplanted immune spleen.

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.

Antibody formation in the transplanted spleen: a simple method of splenic transplantation in the rat using the cuff technique.

To study whether transplanted spleens produce antibodies or not, a simplified model of spleen transplantation using the cuff technique was developed. The whole spleen with vascular pedicles was implanted in the syngeneic DA (RT1a) rat combination, using the cuff technique applied to the renal artery and vein of the recipient. The functions of the grafted spleen were tested by antibody formation-titers of antibody in the serum and plaque-forming cells in the graft spleen against xenogeneic [BALB/c (H-2d) mouse] antigen. The grafted spleens included a number of plaque-forming cells, as did the host spleen. This report shows direct evidence that vascularized splenic grafts produced antibodies. The technique is described in detail.

Infection of BALB/c, C57B1/6 mice and F1 hybrid CB6F1 mice with strains of Leishmania mexicana isolated from Mexican patients with localized or diffuse cutaneous leishmaniasis.

Mice from the syngeneic strains BALB/c, C57B1/6 and (BALB/cxC57B1/6)F1 hybrids (CB6F1) were infected in the footpad with six different strains of Leishmania mexicana mexicana isolated from Mexican patients. Three Leishmania strains were isolated from patients with localized cutaneous leishmaniasis (LCL, the benign form of the disease) and three from patients with diffuse cutaneous leishmaniasis (DCL, the malignant form of the disease). In BALB/c mice, four Leishmania strains showed a sustained fast growth from 4 to 5 weeks postinfection until the end of the experiment (15 weeks), and the other two grew slowly up to 10 or 12 weeks after infection and then started to grow faster. In C57B1/6 mice four Leishmania strains showed a limited to moderate growth up to 6 to 11 weeks postinfection and then started to decrease. One strain showed a moderate growth during the entire experiment and one strain grew as fast as in BALB/c mice up to 11 weeks postinfection and then started to decrease. The CB6F1 hybrid behaved like the C57B1/6 parent strain with five Leishmania strains but was much more resistant to one Leishmania strain than the C57B1/6 mice. Sex of the mouse did not influence the outcome of infection. One important purpose of this work was to see if the Leishmania strains that cause DCL are intrinsically more virulent than those that cause the benign form (LCL). Although important variations in virulence among the Leishmania strains were observed, especially in BALB/c mice, they were not correlated with the type of disease caused in humans.

Infection of BALB/c, C57Bl/6 mice and F1 hybrid CB6F1 mice with strains of leishmania mexicana isolated from Mexican patients with localized or diffuse cutaneous leishmaniasis

Mice from the syngeneic strains BALB/c, C57Bl/6 and (BALB/cxC57Bl/6) F1 hybrids (CB6F1) were infected in the fottpad with six different stains of Leishmania mexicana mexicana isolated from Mexican patients. Three Leishmania strains were isolated from patients with localized cutaneous leishmaniasis (LCL, the benign form of the disease and three from patients with diffuse cutaneous leishmaniasis (DCL, the malignant form of the disease). In BALB/c mice, four Leishmania strains showed a sustained fast growth from 4 to 5 weeks postinfection until the end of the experiment (15 weeks), and the other two grew slowly up to 10 or 12 weeks after infection and then started to grow faster. In C57Bl/6 mice four Leismania strains showed a limited to moderate growth up to 6 to 11 weeks postinfection and then started to decrease. One strain showed a moderate growth during the entire experiment and one strain grew as fast as in BLB/c mice up to 11 weeks postinfection and then started to decrease. The CB6F1 hybrid behaved like the C57Bl/6 parent strain with five Leishmania strains but was much more resistant to one Leishmania strain than the C57Bl/6 mice. Sex of the mouse did not influence the outcome of infection. One important purpose of this work was to see if the Leishmania strains that cause DCL are intrinsically more virulent than those that cause the benign form (LCL). Although important variations in virulence among the Leishmania strains were observed, especially in BALB/c mice, they were not correlated with the type of disease caused in humans

Anti-Thy-1 treated and irradiated spleen cells from (BALB/c x C57Bl/6) F1 mice infected with Plasmodium chabaudi chabaudi can transfer protection into irradiated hosts.

The transfer of spleen cells from (BALB/c x C57Bl/6) F1 mice recovered from a Plasmodium chabaudi chabaudi AS infection into irradiated syngeneic recipients conferred protection. Neither elimination of Thy-1+ cells nor in vitro irradiation of immune cells before transfer affected protection while both anti-Thy-1 treatment and irradiation abolished the appearance of anti-P. c. chabaudi antibodies in the recipients. Superinfection of immune spleen cell donors did not improve their capability to transfer protection which was also unaffected by anti-Thy-1 treatment. The serum of mice after one infection was only marginally protective when transferred into irradiated recipients and a second infection improved the protective activity of serum which was not further improved by six infections. The co-transfer of immune serum and immune cells did not result in any synergistic effect. On the other hand, when P. c. chabaudi AS (BALB/c x C57Bl/6) F1 infected mice were challenged with a high dose of Plasmodium yoelii 17XL at crisis, the mice were unable to control the heterologous parasite. When mice were challenged with P. yoelii 17XL several weeks after infection with P. c. chabaudi AS, a good degree of cross-protection was observed.

Human leukocyte migration inhibition factor (LIF) increases polymorphonuclear cell endocytosis.

We prepared supernatants of Concanavalin-A activated human lymphocytes containing high titers of leukocyte migration inhibition factor (LIF). A pool of these supernatants was filtered thorough sephadex 6-100 as well as a pool of supernatants from parallel non activated cultures. A migration assay was carried out for each activated fraction, using as control migration the same fraction from non activated supernatants. In this way we found a fraction from activated supernatants with high LIF activity. We assayed the effect of this LIF containing fraction on a yeast endocytosis assay by polymorphonuclear (PMN) cells. We found that the LIF containing fraction increased the number of endocytic PMN in about 80%. This effect was absent from control supernatant and from other fractions from activated supernatant but without LIF activity. The LIF containing fraction did not increase the average number of endocytosed yeast per cell nor the ability to reduce NBT. The endocytosis enhancing effect was blocked by the specific LIF blocker N-acetyl-D-glucosamine. We conclude that LIF can increase the endocytic activity of PMN cells.

The adoptive transfer of T-cell dependent immunity to Plasmodium chabaudi chabaudi in CBA/Ca mice is achieved only after superinfection of immune spleen cell donors.

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.

Production of leukocyte migration inhibition factor (LIF) and migration stimulation factor (MStF) by CD4+ and CD8+ human lymphocytes subsets and T-cell clones.

The production of the lymphokines leukocyte migration inhibition factor (LIF) and migration stimulation factor (MStF) at the level of CD4+ and CD8+ human lymphocyte subsets was investigated. In a first series of experiments, anti-CD4 and anti-CD8 monoclonal antibodies capable of inhibiting the activation by concanavalin-A (Con-A) of the respective T-cell subset were used. It was observed that when CD8+ cell activation was blocked, LIF was always produced after Con-A activation. When CD4+ cell activation was blocked, MStF was produced in five out of nine experiments (no activity in the other four). The addition of N-acetyl-D-glucosamine to block LIF in supernatants of anti-CD8 treated cells was unable to show evidence of masked MStF activity. In a second series of experiments, T-cell clones were established from continuous growing T-lymphocyte cell lines developed from cultures of Con-A activated normal human leukocyte cultures. The phenotype of 22 clones was determined and their ability to produce LIF or MStF investigated. Four clones produced MStF after Con-A activation and all of them were CD3+, CD4-, CD8+. Three clones produced LIF after Con-A activation and all of them were CD3+, CD4+, CD8-. We conclude that LIF is produced by CD4+ cells and MStF by CD8+ cells.

Human lymphocytes produce both leukocyte migration inhibition (LIF) and simulation (MStF) factors after mitogen, alloantigens or antigenb activation

Utilizando un sistema desarrollado para producir factor inhibidor de la migración de leucocitos (LIF) en altos títulos, hemos encontrado que cuando activamos linfocitos humanos con concanavalina-A, obtenemos LIF en 55% de los experimentos y factor estimulador de la migración en 38%. Cuando los linfocitos fueron activados por medio de cultivos mixtos de linfocitos encontramos resultados muy parecidos. Se seleccionaron sobrenadantes de linfocitos humanos activados con concanavalina-A que tuvieron una alta actividad de LIF. Al bloquear el LIF con N-acetil-D-glucosamina se pudo observar que estos sobrenadantes contenían también factor estimulador de la migración. En pruebas de LIF directo usando como antígeno a la estreptocinasa-estreptodornasa, encontramos que cuando el ensayo se realiza en presencia de N-acetil-D-glucosamina el valor de índice de migración se incrementa de manera importante. Concluimos que cuando los linfocitos humanos son estimulados ya sea por mitógenos, aloantígenos o antígenos, ellos producen tanto factor inhibidor como estimulador de la migración de leucocitos. Finalmente se discute la importancia de este hallazgo en la determinación de immunidad celular in vitro en humanos por medio de la prueba de LIF

Some studies on acquired immunity to plasmodium chabaudi and plasmodium yoelii in mice

En este trabajo se estudió la duración de la inmunidad adquirida por ratones que curaron espontáneamente después de infectarlos con P. chabaudi y con la cepa no letal (17 X NL) de P. yoelii. Para ésto, se inocularon ratones con P. chabaudi y grupos de ellos fueron retados a diferentes tiempos después de la inoculación inical; unos con P. chabaudi y otros con la cepa letal (17 X L) de P. yoelii. En otros experimentos se inocularon ratones con la cepa 17 X NL de P. yoelii los cuales fueron retados con la cepa 17 X L de P. yoelii. En todas las combinaciones que fueron probadas se encontró un buen nivel de inmunidad aun cuando los ratones se retaron alrededor de 170 días despsués de la primera inoculación. Sin embargo, también se observó en todas las combinaciones que después de 50 o 60 días de la primera inoculación, algunos ratones tienden a perder inmunidad sólida. Se intentó, sin éxito, correlacionar esta pérdida de inmunidad con los niveles de anticuerpos anti-plasmodio. En el sistema de P. chabaudi, no se detectaron parásitos persistentes 180 días después de la inoculación en ninguno de los siguentes órganos: sangre, médula ósea, hígado, bazo o riñón