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The 19 kDa carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (MSP119), an analog of the leading falciparum malaria vaccine candidate, induces protective immunity to challenge infection when formulated with complete/incomplete Freund’s adjuvant (CFA/IFA), an adjuvant unsuitable for use in humans. In this study, we investigate Montanide ISA51 and Montanide ISA720 as well as CpG oligodeoxynucleotide (ODN) as adjuvants for induction of immunity to MSP119. Mice immunized with MSP119 adjuvanted with Montanide ISA51 were protected even though some mice experienced low-grade parasitemia before resolving the infection. Mice immunized with MSP119 adjuvanted with Montanide ISA720 showed delayed patent parasitemia with all mice ultimately succumbing to infection. Interestingly, when the synthetic CpG ODN 1826 was included in either Montanide formulation, mice were completely protected with no parasites detected in the blood. MSP119-specific antibodies in MSP119-immunized mice adjuvanted with Montanide ISA51 or Montanide ISA720 showed predominantly IgG1 antibody and low levels of IgG2a. CpG ODN 1826 significantly enhanced both IgG1 and IgG2a antibody responses in Montanide ISA51-adjuvanted mice but significantly enhanced only the IgG2a antibody response in Montanide ISA720-adjuvanted mice. To investigate the relative roles of antibody and CD4+ T cells in protection, MSP119-immunized mice adjuvanted with Montanide ISA720 and CpG ODN 1826 were depleted of CD4+ T cells just prior to challenge. Results showed that three of nine immunized/T cell depleted mice died following infection. These results suggest that antibody and CD4+ T cells are critical for protection following immunization with MSP119 adjuvanted with Montanide and CpG ODN and that the formulation of a human malaria vaccine candidate in Montanide ISA720 or ISA51 together with human compatible CpG ODN would be useful for improving efficacy. [Copyright &y& Elsevier]

Flavivirus reporters provide a robust tool for viral pathogenesis studies, anti-viral drug screening, disease diagnosis and functional antibody assays. In this study, we generated a luciferase-secreting, single-round reporter virus by replacing the capsid coding region in a DENV-2 genome with the secretory form of Lucia luciferase gene to produce infectious viral particles in a stable capsid-expressing mosquito cell line. Replication of the reporter virus in trans-complementing mosquito cells was sustained for up to two weeks. There were strong correlations between the extracellular luciferase activity and infectious reporter virus inocula upon infection of mosquito and mammalian cell lines with graded quantities of the reporter virus. A set of anti-E and anti-prM monoclonal antibodies affected the infectivity of reporter virus with similar dose-effect relationships as the parent virus. This simplified version of DENV-2 reporter provides a rapid and reliable method for the detection of neutralizing and infection-enhancing antibodies against dengue virus., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The objectives of this study were to develop a method for concentrating rotavirus, to assess the detection rate, and to characterize the genotype of naturally occurring rotavirus in bivalve shellfish species; including oysters (Saccostrea forskali), cockles (Anadara nodifera), and mussels (Perna viridis). The results demonstrated that an adsorption-twice elution-extraction method was less-time consuming method of concentrating the spiked rotavirus, yielding high sensitivity of 1.14 genome copies/g of digestive tissues from all three shellfish species, as detected using an RT-nested PCR. In seeding experiments, rotavirus as low as 1.39 genome copies was able to be detected in 4 g of digestive tissues or per sample. In the period of August 2011 to July 2012, of the 300 bivalve shellfish samples collected and tested, 24 (8.0%) were found to be contaminated with rotavirus, the figures being: oysters, 13/100 samples; mussels, 10/100 samples; and cockles, 1/100 samples. By DNA sequencing of the RT-nested PCR products and phylogenetic analysis, the rotaviruses detected were classified into G1, lineage II (4 samples); G3 (10 samples): lineage I (3 samples), lineage IIIc (3 samples), lineage IIId (3 samples), lineage IV (1 sample); G9 (6 samples); and G12, lineage III (1 sample). These findings suggest that this virus concentration method provides high sensitivity for the detection of rotavirus from the three bivalve shellfish species. The prevalence of rotavirus and the identified genotypes contribute to the molecular epidemiology of rotavirus in different shellfish species., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

and young children, but rotavirus gastroenteritis in adults is uncommon. In this study, 260 stool samples collected in Thailand from January 2006 to February 2007 from patients, of all ages with acute gastroenteritis, were tested for group A rotavirus and compared with rotavirus infections in children and adults. Rota- virus was detected in 42% of the patients' samples, but children (< 18 years old) have a significantly higher prevalence (57%) of rotavirus infection than adults (≥ 18 years old) (27%) (OR 3.55; 95% CI: 2.11-5.96; p < 0.001). The highest attack rate was found in the age group of < 2 years old (14%), followed by 2-4 years of age (9%), 18-59 years of age (8%), 5-17 years of age (6%) and ≥ 60 years of age (5%). The dominant genotype was G1P[8] (27%), followed by G2P[4] (7%), G3P[8] (1%), and G9P[8] (1%). The rare genotypes identified were G1P[4], G1P[6], G2P[6], G2P[8], and G3P[6]. Mixed infections mostly occurred in children, comprising G1P[4]/P[8], G1P[4]/P[6], G1P[6]/P[8], G1/G2P[4], G1/G3P[4], and G1/G3P[4]/P[8]. Rotaviruses G3, G9, and P[4] were found only in children and genotype P[6] was found in adults (75%) at a higher frequency than in children (25%) (p < 0.001). The number of rotavirus in children was 1.99x10(8)/ml and in adult patients was 7.32x10(6)/ ml. The present study highlights the higher prevalence of rotavirus infection in children compared to adults and rotavirus genetic heterogeneity. Rotaviruses are the most important cause of severe diarrhea in infants

This study aimed to investigate the antibody responses in mice immunized with Gnathostoma spinigerum crude antigen (GsAg) incorporated with the combined adjuvant, a synthetic oligonucleotide containing unmethylated CpG motif (CpG ODN 1826) and a stable water in oil emulsion (Montanide ISA720). Mice immunized with GsAg and combined adjuvant produced all antibody classes and subclasses to GsAg except IgA. IgG2a/2b/3 but not IgG1 subclasses were enhanced by immunization with CpG ODN 1826 when compared with the control groups immunized with non-CpG ODN and Montanide ISA or only with Montanide ISA, suggesting a biased induction of a Th1-type response by CpG ODN. After challenge infection with live G. spinigerum larvae, the levels of IgG2a/2b/3 antibody subclasses decreased immediately and continuously, while the IgG1 subclass remained at high levels. This also corresponded to a continuous decrease of the IgG2a/IgG1 ratio after infection. Only IgM and IgG1 antibodies, but not IgG2a/2b/3, were significantly produced in adjuvant control groups after infection. These findings suggest that G. spinigerum infection potently induces a Th2-type biased response.

Objective: To investigate the efficacy of a vaccine formulation of the 19 kDa conserved carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (PyMSP1(19)) formulated with CpG ODN 1826 and Montanide ISA51 or ISA720 when used to immunize mice by a single injection., Methods: Groups of BALB/c mice were immunized parenterally with one, two or four injections with PBS or PyMSP1(19) formulated with CpG ODN in ISA51 or ISA720. Sera were collected weekly and assessed for total IgG and IgG subclass titers. Protection was tested by challenge infection with P. yoelii YM., Results: Interestingly, single injection immunization showed the same kinetics of antibody responses as two- or four-injection immunization. However, the peak antibody response induced by PyMSP1(19) in CpG ODN and ISA51 appeared earlier than that induced by PyMSP1(19) in CpG ODN and ISA720 (28 days vs 41 days). At day 63 after the first injection, the PyMSP1(19)-specific IgG antibody levels by single injection and four-injection immunizations were not different. However, the levels of the IgG2a antibody subclass were significantly lower by single injection immunization with PyMSP1(19) in CpG ODN and ISA720. The antibodies were sustained at high levels for at least 20 weeks. After challenge infection, all mice immunized by a single injection of PyMSP1(19) in CpG ODN and ISA51 survived with low-grade parasitemia, while 50% of mice immunized with PyMSP1(19) in CpG ODN and ISA720 died with high levels of parasitemia., Conclusion: These findings suggest that MSP1(19) immunization by a single injection can induce protective immunity, particularly when formulated with an appropriate strong adjuvant.

Development of a vaccine that targets blood-stage malaria parasites is imperative if we are to sustainably reduce the morbidity and mortality caused by this infection. Such a vaccine should elicit long-lasting immune responses against conserved determinants in the parasite population. Most blood-stage vaccines, however, induce protective antibodies against surface antigens, which tend to be polymorphic. Cell-mediated responses, on the other hand, offer the theoretical advantage of targeting internal antigens that are more likely to be conserved. Nonetheless, few of the current blood-stage vaccine candidates are able to harness vigorous T cell immunity. Here, we present what we believe to be a novel blood-stage whole-organism vaccine that, by combining low doses of killed parasite with CpG-oligodeoxynucleotide (CpG-ODN) adjuvant, was able to elicit strong and cross-reactive T cell responses in mice. Our data demonstrate that immunization of mice with 1,000 killed parasites in CpG-ODN engendered durable and cross-strain protection by inducing a vigorous response that was dependent on CD4+ T cells, IFN-gamma, and nitric oxide. If applicable to humans, this approach should facilitate the generation of robust, cross-reactive T cell responses against malaria as well as antigen availability for vaccine manufacture.

It is well known that exposure to one antigen can modulate the immune responses that develop following exposure to closely related antigens. It is also known that the composition of the repertoire can be skewed to favor epitopes shared between a current infection and a preceding one, a phenomenon referred to as "original antigenic sin." It was of interest, therefore, to investigate the antibody response that develops following exposure to the malaria vaccine candidate homologue Plasmodium yoelii MSP1(19) in mice that had previously experienced malaria infection and vice versa. In this study, preexposure of mice to Plasmodium yoelii elicited native anti-MSP1(19) antibody responses, which could be boosted by vaccination with recombinant MSP1(19). Likewise, infection of MSP1(19)-primed mice with P. yoelii led to an increase of anti-MSP1(19) antibodies. However, this increase was at the expense of antibodies to parasite determinants other than MSP1(19). This change in the balance of antibody specificities significantly affected the ability of mice to withstand a subsequent infection. These data have particular relevance to the possible outcome of malaria vaccination for those situations where the vaccine response is suboptimal and suggest that suboptimal vaccination may in fact render the ultimate acquisition of natural immunity more difficult.

Dendritic cells (DCs) initiate innate and adaptive immune responses including those against malaria. Although several studies have shown that DC function is normal during malaria, other studies have shown compromised function. To establish why these studies had different findings, we examined DCs from mice infected with two lethal species of parasite, Plasmodium berghei or P. vinckei, and compared them to DCs from nonlethal P. yoelii 17XNL or P. chabaudi infections. These studies found that DCs from only the lethal infections became uniformly mature 7 days after infection and were functionally impaired as they were unable to endocytose latex particles, secrete IL-12, or present OVA to transgenic OTII T cells. These changes coincided with a peak in levels of systemic TNF-alpha. Because TNF-alpha is known to mature DCs, we used TNF-KO mice to determine the role of this cytokine in the loss of DC function. In the TNF-KO mice, phenotype, Ag presentation, and IL-12 secretion by DCs were restored to normal following both lethal infections. This study shows that the systemic production of TNF-alpha contributes to poor DC function during lethal infections. These studies may explain, at least in part, immunosuppression that is associated with malaria.

Merozoite surface protein 1 (MSP1) is the major protein on the surface of the plasmodial merozoite, and its carboxy terminus, the 19-kDa fragment (MSP1(19)), is highly conserved and effective in induction of a protective immune response against malaria parasite infection in mice and monkeys. However, the duration of the immune response has not been elucidated. As such, we immunized BALB/c mice with a standard four-dose injection of recombinant Plasmodium yoelii MSP1(19) formulated with Montanide ISA51 and CpG oligodeoxynucleotide (ODN) and monitored the MSP1(19)-specific antibody levels for up to 12 months. The antibody titers persisted constantly over the period of time without significant waning, in contrast to the antibody levels induced by immunization with Freund's adjuvant, where the antibody levels gradually declined to significantly lower levels 12 months after immunization. Investigation of immunoglobulin G (IgG) subclass longevity revealed that only the IgG1 antibody level (Th2 type-driven response) decreased significantly by 6 months, while the IgG2a antibody level (Th1 type-driven response) did not change over the 12 months after immunization, but the boosting effect was seen in the IgG1 antibody responses but not in the IgG2a antibody responses. After challenge infection, all immunized mice survived with negligibly patent parasitemia. These findings suggest that protective immune responses to MSP1(19) following immunization using oil-based Montanide ISA51 and CpG ODN as an adjuvant are very long-lasting and encourage clinical trials for malaria vaccine development.

Naturally acquired immune response to C-terminal region of Plasmodium vivax merozoite surface protein1 (PvMSP1) in 200 individuals with recent clinical episodes of malaria from malaria endemic areas along Thai-Myanmar border in the west and Thai-Cambodia border in the east of Thailand was evaluated by enzyme-linked immunosorbent assay (ELISA). The anti-PvMSP1-IgG antibody was observed in 110 individuals (55%). Among IgG responders, IgG1 coexpressed with IgG3 were the predominant subclasses. The levels of anti-PvMSP1 total IgG, IgG1 and IgG3 antibody response seem to be increased with age although no detectable significant correlation was found (r = 0.004, p = 0.484 for total IgG; r = 0.035, p = 0.386 for IgG1; r = -0.600, p = 0.142 for IgG2; r = 0.077, p = 0.227 for IgG3; r = 0.664, p = 0.051 for IgG4). However, the mean level of specific total IgG was highest in the age group of >40 years. These levels of either specific total IgG or each IgG isotype did not vary among individuals with different malaria episodes. A higher level of specific total IgG, IgG1 and IgG3 antibody response related with the lower of parasitemia density was observed although no significant correlation was found. Our data indicate that individuals exposed to vivax malaria in Thailand developed antibodies to the potential candidate vaccine antigen, PvMSP1 (C-terminal).

Interaction between different bacterial plaque pathogens and dendritic cells may induce different types of T helper (Th) cell response, which is critical in the pathogenesis of periodontitis. In this study we investigated the effects of lipopolysaccharide (LPS) from Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans on human monocyte-derived dendritic cells (Mo-DCs) with respect to co-stimulatory molecule expression, cytokine production and Th cell differentiation. Unlike Escherichia coli and A. actinomycetemcomitans LPS, P. gingivalis LPS induced only low levels of CD40, CD80, HLA-DR and CD83 expression on Mo-DCs. LPS from both bacteria induced considerably lower TNF-alpha and IL-10 than did E. coli LPS. LPS from all three bacteria induced only negligible IL-12 production. In a human mixed-leukocyte reaction, and in an ovalbumin-specific T cell response assay in mice, both types of LPS suppressed IFN-gamma production. In conclusion, stimulation by P. gingivalis LPS and A. actinomycetemcomitans LPS appears to bias Mo-DCs towards Th2 production.

Protective cellular immune responses depend on MHC presentation of pathogen-derived Ag fragments. MHC diversity renders this process sensitive to point mutations coding for altered amino acid sequence of the short target Ag-derived peptides epitopes. Thus, in a given host, a pathogen with an altered epitope sequence will be more likely to escape detection and elimination by the immune system. At a population level, selection by immune pressure will increase the likelihood of polymorphism in important pathogen antigenic epitopes. This mechanism of immune evasion is found in viruses and other pathogens. The detection of polymorphic hot spots in an Ag is often taken as a strong indication of its role in protective immunity. We provide evidence that polymorphisms in the T cell epitopes of a malaria vaccine candidate are unlikely to have been selected by immune pressure in the human host.

Allelic variation in the Plasmodium falciparum circumsporozoite protein (CS) gene has been determined by sequencing the immunodominant T-cell epitopes, Th2R and Th3R, from 95 isolates from two malaria-endemic areas in the west of Thailand. Comparison with a reference sequence revealed only non-synonymous point mutations in the two epitope regions. Point mutations were found outside these epitopes in a minority of samples, and all but four were also non-synonymous. A relatively high number of variants, 11 Th2R and 9 Th3R, were detected and comprised some that had not been previously observed. However, the Th2R*05 and the Th3R*01 allelic variants predominated, as they were found in more than 70% of the 101 sequences obtained.

Although there is good evidence that immunity to the blood stages of malaria parasites can be mediated by different effector components of the adaptive immune system, target antigens for a principal component, effector CD4(+) T cells, have never been defined. We generated CD4(+) T cell lines to fractions of native antigens from the blood stages of the rodent parasite, Plasmodium yoelii, and identified fraction-specific T cells that had a Th1 phenotype (producing IL-2, IFN-gamma, and tumor necrosis factor-alpha, but not IL-4, after antigenic stimulation). These T cells could inhibit parasite growth in recipient severe combined immunodeficient mice. N-terminal sequencing of the fraction showed identity with hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT). Recombinant HGXPRT from the human malaria parasite, Plasmodium falciparum, activated the T cells in vitro, and immunization of normal mice with recombinant HGXPRT reduced parasite growth rates in all mice after challenge.

Background: The ability of T cells, acting independently of antibodies, to control malaria parasite growth in people has not been defined. If such was shown to be effective, an additional vaccine strategy could be pursued. Our aim was to ascertain whether or not development of cell-mediated immunity to Plasmodium falciparum blood-stage infection could be induced in human beings by exposure to malaria parasites in very low density., Methods: We enrolled five volunteers from the staff at our research institute who had never had malaria. We used a cryopreserved inoculum of red cells infected with P falciparum strain 3D7 to give them repeated subclinical infections of malaria that we then cured early with drugs, to induce cell-mediated immune responses. We tested for development of immunity by measurement of parasite concentrations in the blood of volunteers by PCR of the multicopy gene STEVOR and by following up the volunteers clinically, and by measuring antibody and cellular immune responses to the parasite., Findings: After challenge and a extended period without drug cure, volunteers were protected against malaria as indicated by absence of parasites or parasite DNA in the blood, and absence of clinical symptoms. Immunity was characterised by absence of detectable antibodies that bind the parasite or infected red cells, but by the presence of a proliferative T-cell response, involving CD4+ and CD8+ T cells, a cytokine response, consisting of interferon gamma but not interleukin 4 or interleukin 10, induction of high concentrations of nitric oxide synthase activity in peripheral blood mononuclear cells, and a drop in the number of peripheral natural killer T cells., Interpretation: People can be protected against the erythrocytic stage of malaria by a strong cell-mediated immune response, in the absence of detectable parasite-specific antibodies, suggesting an additional strategy for development of a malaria vaccine

Merozoite surface protein 1 (MSP1) of malaria parasites undergoes proteolytic processing at least twice before invasion into a new RBC. The 42-kDa fragment, a product of primary processing, is cleaved by proteolytic enzymes giving rise to MSP1(33), which is shed from the merozoite surface, and MSP1(19), which is the only fragment carried into a new RBC. In this study, we have identified T cell epitopes on MSP1(33) of Plasmodium yoelii and have examined their function in immunity to blood stage malaria. Peptides 20 aa in length, spanning the length of MSP1(33) and overlapping each other by 10 aa, were analyzed for their ability to induce T cell proliferation in immunized BALB/c and C57BL/6 mice. Multiple epitopes were recognized by these two strains of mice. Effector functions of the dominant epitopes were then investigated. Peptides Cm15 and Cm21 were of particular interest as they were able to induce effector T cells capable of delaying growth of lethal P. yoelii YM following adoptive transfer into immunodeficient mice without inducing detectable Ab responses. Homologs of these epitopes could be candidates for inclusion in a subunit vaccine.

Vaccination of mice with the leading malaria vaccine candidate homologue, the 19-kDa carboxyl terminus of merozoite surface protein-1 (MSP119), results in sterile immunity to Plasmodium yoelii, with no parasites detected in blood. Although such immunity depends upon high titer Abs at challenge, high doses of immune sera transferred into naive mice reduce parasitemia (and protect from death) but do not result in a similar degree of protection (with most mice experiencing high peak parasitemias); this finding suggests that ongoing parasite-specific immune responses postchallenge are essential. We analyzed this postchallenge response by transferring Abs into manipulated but malaria-naive mice and observed that Abs cannot protect SCID, nude, CD4+ T cell-depleted, or B cell knockout mice, with all mice dying. Thus, in addition to the Abs that develop following MSP119 vaccination, a continuing active immune response postchallenge is required for protection. MSP119-specific Abs can adoptively transfer protection to strains of mice that are not protected following vaccination with MSP119, suggesting that the Ags targeted by the immune response postchallenge include Ags apart from MSP119. These data have important implications for the development of a human malaria vaccine.

CD4+ T cells have been implicated in immunity to the blood stages of malaria and cytokines associated with both monocyte and T cell activation have been implicated in disease. To determine whether specific T cells capable of inhibiting parasite growth can also mediate pathology we have transfused populations of Plasmodium berghei-specific T cells into normal and immunodeficient naive mice. We observed that they could inhibit parasite growth but were unable to save the animals which exhibited significantly greater anaemia and weight loss than control infected animals receiving either no T cells or T cells specific for ovalbumin. T cell-dependent tomour necrosis factor (TNF)alpha was a critical component in both parasite killing and disease promotion. Experiments with blocking antibodies demonstrated that all T-cell mediated antiparasitic immunity and all T-cell mediated weight loss was TNF-dependent. Blocking TNF-alpha in mice that received parasite-specific T cells prolonged the survival of the mice. Nitric oxide demonstrated no antiparasite effect, but was involved in the regulation of T-cell mediated weight loss. The data thus show that while parasite-specific CD4+ T cells can significantly limit parasite growth, such an effect need not be beneficial to the host, and that TNF-alpha and nitric oxide are critical effector molecules operating downstream of parasite-specific T cells in both immunity and disease.

Variable protection against malaria blood-stage infection has been demonstrated in mice following parenteral immunization with the highly conserved 19 kD carboxylterminal fragment of the merozoite surface protein-1 (MSP119) using CFA/IFA and other adjuvants. Here we show that intranasal immunization of BALB/C mice with yeast expressed Plasmodium yoelii MSP119 plus a mixture of native and recombinant cholera toxin B subunit, could induce serum MSP119-specific antibodies at titres ranging from 20 000 to 2 560 000. The Ig subclass responses were predominantly G1 and G2b. Intranasal immunization led to protection following challenge (peak parasitaemia < 1%) in mice with the highest MSP119-specific titre (>/= 640 000). In two of the three protected mice, a peak parasitaemia of 0.1%-1% was followed by a boost of the antibody response whereas one of the three protected mice did not boost its antibody response after a peak parasitaemia of 0.02%. In unprotected mice, antibody levels rose, then fell, following the detection of parasites in the peripheral blood. CD4+ T cell-depletion abrogated the ability of the mice to boost their antibody response following challenge. These data demonstrate the potential for intranasal immunization with MSP119 to protect against malaria.

MSP1(19) is one of the leading malaria vaccine candidates. However, the mechanism of protection is not clear. To determine whether MSP1(19)-specific effector T cells can control parasitaemia, we analysed the specificity of T cells induced following immunization with recombinant forms of P. yoelii MSP1(19) and asked whether they could protect mice. There was no evidence that effector T cells were capable of protecting since: (1) immunization of mice with yMSP1(19), but not defined epitopes, was able to induce protection; and (2) long term MSP1(19)-specific CD4+ T cell lines were incapable of adoptively transferring protection. In contrast, priming mice with the T cell epitopes resulted in a rapid anamnestic antibody response to MSP1(19) after either challenge with MSP1(19) or parasite. Thus, MSP1(19) contains multiple T cell epitopes but such epitopes are the targets of helper T cells for antibody response but not of identified effector T cells capable of controlling parasitaemia.

The immune response to malaria parasites includes T cell responses that reduce parasites by effector T cell responses and by providing help for antibody responses. Some parasites are more sensitive to antibody and others are more sensitive to cell-mediated immunity. We demonstrate that cultured CD4(+) T cells that produce interferon gamma and interleukin 2, but not interleukin 4, in response to stimulation with the rodent parasite Plasmodium berghei can reduce but not eliminate parasites in vivo after adoptive transfer. Although cells can persist in vivo for up to 9 months in uninfected mice, infection results in elimination of up to 99% of specific T cells in different tissues, as judged by tracking T cells labeled with the fluorescent dye 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester. T cells specific for ovalbumin are unaffected. In vivo activation and division of transferred T cells per se are not responsible for deletion because T cells positive for 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester divide up to six times within 7 days in uninfected mice and are not deleted. Understanding the factors responsible for parasite-mediated specific deletion of T cells would enhance our knowledge of parasite immunity.

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.

Mouse-tobacco hybrid calli, and complete plants producing mouse gamma-3 heavy and lambda light chains, have been generated by somatic cell fusions of mouse spleen cells and tobacco mesophyll protoplasts. Both gamma 3 and lambda chains were detected in hybrid calli and complete plants by enzyme-linked immunosorbent assay, immunofluorescent staining, and Western blotting. When cellular DNA from hybrid tobacco mesophyll protoplasts was amplified by the polymerase chain reaction (PCR) using two pairs of gamma 3 chain DNA primers and one pair of lambda chain DNA primers, the PCR products contained gamma 3 and lambda chain DNAs, which could be detected by southern blotting and DNA hybridization, using specific synthetic oligonucleotide probes for gamma 3 and lambda respectively. In situ hybridization of hybrid tobacco mesophyll protoplasts with specific recombinant DNA probes of gamma 3 and lambda chains showed the presence of gamma 3 and lambda chain DNAs in the hybrid protoplasts.

An agar plating technique was developed for enumeration of IL-1-producing monocytes based on the principle that when IL-1-producing monocytes were cocultured with mouse thymocytes and PHA in semisolid agar medium in a plate, mouse thymocytes proliferated around IL-1-producing monocytes resulting in the clusters or colonies of cells. The IL-1-produced clusters or colonies of cells can be counted under a dissecting microscope. Optimal conditions were established for induction and development of IL-1-producing monocytes. The numbers of IL-1-producing monocytes ranged from 819 to 1930 cells/10(5) monocytes, with mean +/- SEM = 1344 +/- 182 cells/10(5) monocytes; the IL-1 activity ranged from 11.7 to 85.9 U/10(5) monocytes/ml, with mean +/- SEM = 42.8 +/- 11.2 U/10(5) monocytes/ml in seven normal subjects. The IL-1 activity per one monocyte ranged from 12.7 to 86.5 mU, with mean +/- SEM = 33.5 +/- 9.8 mU. The mean numbers of IL-1-producing monocytes and the mean IL-1 levels produced by monocytes from the same normal subjects were highly correlated (r = 0.981). The numbers of IL-1-produced colonies resulting from IL-1-producing monocytes could be completely abolished by incorporation of rabbit anti-human IL-1 in the semisolid agarose but not by rabbit anti-human IL-6 or anti-human TNF-alpha.

The capabilities of monocytes and lymphocytes in peripheral blood mononuclear leukocytes (PBML) to produce interleukin-1 (IL-1), IL-2, and interferon (IFN), respectively, were evaluated in various types and treatments of leprosy patients. IL-1 production in response to lipopolysaccharide was significantly lower in LL, BL, BB, and BT patients than in normal controls. However, there were no differences in IL-1 levels between TT patients and normal controls. The percentages of nonspecific-esterase-positive cells adhering to the plastic surfaces were not different in LL, BB and TT patients when compared to normal controls. However, they were significantly higher in BT and BL patients than in normal controls. When PBML from leprosy patients were stimulated with concanavalin-A (ConA) for IL-2 production, there were no differences in the IL-2 levels in treated BL/LL, untreated BL/LL, treated BT/TT, and untreated BT/TT patients compared to normal controls. Similar results were obtained when PBML were stimulated with phytohemagglutinin-P (PHA-P). However, when purified protein derivative (PPD) was used as the stimulating agent, there were significantly lower IL-2 levels in treated BL/LL, untreated BL/LL, treated BT/TT, and untreated BT/TT patients when compared to normal controls. There were also lower IL-2 levels in untreated BL/LL and BT/TT patients compared to treated BL/LL and BT/TT patients, respectively. PBML were stimulated with PHA-P or ConA for IFN production.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies of IgM, IgG and IgA classes against M.leprae specific antigens (PGL-I, ND-O-BSA, and NT-O-BSA) were determined in the sera of 80 leprosy patients (28 untreated, 34 treated lepromatous and 18 tuberculoid), 25 tuberculosis patients and 33 normal individuals of Northern Thailand. No strong distinction in reactivity could be found between the three antigens. The IgM antibody assay yielded more positive results than assays for IgG and IgA. It was found that the positivity rates of IgM antibodies to all three antigens were highest in untreated lepromatous leprosy (82%). In tuberculoid leprosy, the positivity rates of IgM, IgG and IgA to the antigens were more variable, ranging from 22 to 50 percent. Patients with tuberculosis and normal individuals did not produce IgM antibodies against the antigens. The results suggested that the determination of IgM against the three antigens is a more sensitive and specific test for active leprosy than those of IgG and IgA. The relationship between the duration of treatment and IgM antibody levels in lepromatous leprosy (LL) was studied. Untreated LL patients had significantly higher IgM and IgA antibody levels than treated patients. There was no difference in IgG antibody levels between the two groups, and the levels of both groups were higher than normal controls. Serial determination of IgM antibodies in 7 LL patients revealed that treatment was strongly associated with progressive decrease in IgM antibody levels against all three antigens.

An agar plating technique for detection and enumeration of IL-2 producing cells from human peripheral blood mononuclear leukocytes (PBML) has been developed. This method is based on the principle that PHA-stimulated PBML, as effector cells, secrete interleukin 2 (IL-2) into soft agar containing mouse 3-day Con A blasts as IL-2 dependent responder cells. The IL-2 dependent Con A blasts proliferating around the IL-2 producing cells form colonies or clusters of cells and are easily visualized under a dissecting microscope. The development of IL-2 producing cells was optimum when 1 X 10(6) cells/ml PBML were stimulated with 2 micrograms/ml PHA-P for 4 hours, and when 2.5 X 10(5) cells were co-cultured with 6 X 10(6) Con A blasts in soft agar for 5 days. The average number of IL-2 producing cells in 10 normal healthy controls were 754 +/- 94 (mean +/- S.E.M.) cells/10(6) PBML. The numbers of IL-2 producing cells and the levels of IL-2 produced were highly correlated (r = 0.929). The subpopulation of lymphocytes in the colonies was shown to be mostly murine T-cells, since they were mostly Thy 1.2 positive, CD3 negative and surface immunoglobulin negative. This technique is very simple to perform and provides an accurate and straightforward means to enumerate IL-2 producing cells from human PBML in a variety of human immunologic disorders.

A method for the enumeration of IL2-producing cells from rat spleen has been developed. Rat spleen cells were stimulated with concanavalin A (Con A), washed, then mixed with IL2-dependent cells (3 day Con A blasts) and plated in soft agar. Clusters of IL2-dependent cells formed around IL2-producing cells, giving colonies which were easy to count under a dissecting microscope. All experimental factors influencing development of colonies of IL2-producing cells surrounded by IL2-dependent cells were evaluated and set up. Optimum number of effector cells was 2.5 x 10(5) cells/culture, while optimum number of IL2-dependent cells was 6 x 10(6) cells/culture. Optimum concentration of Con A for IL2 stimulation was 40 micrograms/ml with an optimal stimulation time of 10 hours. Optimum incubation time for development of IL2-producing cell colonies was 5 days. The number of IL2-producing cells by this technique had a good correlation with the level of IL2 in the cell culture fluid (r = 0.885). When colonies were aspirated from agar and stained by Wright stain, a big purple stained cell at the center was surrounded by small cells in almost all colonies examined. All cells from colonies were fluoresed with anti-mouse Thy 1.2-fluorescein conjugate. However, they were negative with anti-mouse Ig-fluorescein conjugate. The number of IL2-producing cells was 816-2080 cells/10(6) of rat spleen cells with mean +/- S.E.M. = 1404 +/- 154/10(6) cells.