Evidence for multiple B- and T-cell epitopes in Plasmodium falciparum liver-stage antigen 3.

Liver-stage antigen 3 (LSA-3) is a new vaccine candidate that can induce protection against Plasmodium falciparum sporozoite challenge. Using a series of long synthetic peptides (LSP) encompassing most of the 210-kDa LSA-3 protein, a study of the antigenicity of this protein was carried out in 203 inhabitants from the villages of Dielmo (n = 143) and Ndiop (n = 60) in Senegal (the level of malaria transmission differs in these two villages). Lymphocyte responses to each individual LSA-3 peptide were recorded, some at high prevalences (up to 43%). Antibodies were also detected to each of the 20 peptides, many at high prevalence (up to 84% of responders), and were directed to both nonrepeat and repeat regions. Immune responses to LSA-3 were detectable even in individuals of less than 5 years of age and increased with age and hence exposure to malaria, although they were not directly related to the level of malaria transmission. Thus, several valuable T- and B-cell epitopes were characterized all along the LSA-3 protein, supporting the antigenicity of this P. falciparum vaccine candidate. Finally, antibodies specific for peptide LSP10 located in a nonrepeat region of LSA-3 were found significantly associated with a lower risk of malaria attack over 1 year of daily clinical follow-up in children between the ages of 7 and 15 years, but not in older individuals.

On the mechanism of protective action of cold acclimatization against carbon tetrachloride - and ethionine-induced fatty liver

In this study the hepatic lipoprotein lipase (LPL), activity was evaluated in adult female mice acclimatized at 5-C and submitted to carbon tetrachloride (CCI) or ethionine, in order to determine the possible role of this enzuyme in the fatty liver. The results were compared with those obtained in mice kept at room temperature (27-C) that the same hepatoesteatosis inducing agent. In contrast to animals kept at room temperature, in cold aclimatized mice neither the enhancement of the LPL-liver activity by the action of CCI or ethionine occurred nor the development of fatty infiltration in the liver was observed. We conclude that the low temperature induced a protective effect against CCI or ethionine-induced fatty liver that was correlated with the no-increase of the hepatic LPL activity.

Tumor antigen-specific FOXP3+ CD4 T cells identified in human metastatic melanoma: peptide vaccination results in selective expansion of Th1-like counterparts.

We have previously shown that vaccination of HLA-A2 metastatic melanoma patients with the analogue Melan-A(26-35(A27L)) peptide emulsified in a mineral oil induces ex vivo detectable specific CD8 T cells. These are further enhanced when a TLR9 agonist is codelivered in the same vaccine formulation. Interestingly, the same peptide can be efficiently recognized by HLA-DQ6-restricted CD4 T cells. We used HLA-DQ6 multimers to assess the specific CD4 T-cell response in both healthy individuals and melanoma patients. We report that the majority of melanoma patients carry high frequencies of naturally circulating HLA-DQ6-restricted Melan-A-specific CD4 T cells, a high proportion of which express FOXP3 and proliferate poorly in response to the cognate peptide. Upon vaccination, the relative frequency of multimer+ CD4 T cells did not change significantly. In contrast, we found a marked shift to FOXP3-negative CD4 T cells, accompanied by robust CD4 T-cell proliferation upon in vitro stimulation with cognate peptide. A concomitant reduction in TCR diversity was also observed. This is the first report on direct ex vivo identification of antigen-specific FOXP3+ T cells by multimer labeling in cancer patients and on the direct assessment of the impact of peptide vaccination on immunoregulatory T cells.

Protective effects of hypercapnic acidosis on ventilator-induced lung injury.

To investigate whether respiratory acidosis modulates ventilator-induced lung injury (VILI), we perfused (constant flow) 21 isolated sets of normal rabbit lungs, ventilated them for 20 min (pressure controlled ventilation [PCV] = 15 cm H(2)O) (Baseline) with an inspired CO(2) fraction adjusted for the partial pressure of CO(2) in the perfusate (PCO(2) approximately equal to 40 mm Hg), and then randomized them into three groups. Group A (control: n = 7) was ventilated with PCV = 15 cm H(2)O for three consecutive 20-min periods (T1, T2, T3). In Group B (high PCV/normocapnia; n = 7), PCV was given at 20 (T1), 25 (T2), and 30 (T3) cm H(2)O. The targeted PCO(2) was 40 mm Hg in Groups A and B. Group C (high PCV/hypercapnia; n = 7) was ventilated in the same way as Group B, but the targeted PCO(2) was approximately equal to 70 to 100 mm Hg. The changes (from Baseline to T3) in weight gain (Delta WG: g) and in the ultrafiltration coefficient (Delta K(f) = gr/min/ cm H(2)O/100g) and the protein and hemoglobin concentrations in bronchoalveolar lavage fluid (BALF) were used to assess injury. Group B experienced a significantly greater Delta WG (14.85 +/- 5.49 [mean +/- SEM] g) and Delta K(f) (1.40 +/- 0.49 g/min/cm H(2)O/100 g) than did either Group A (Delta WG = 0.70 +/- 0.43; Delta K(f) = 0.01 +/- 0.03) or Group C (Delta WG = 5.27 +/- 2.03 g; Delta K(f) = 0.25 +/- 0.12 g/min/cm H(2)O/ 100 g). BALF protein and hemoglobin concentrations (g/L) were higher in Group B (11.98 +/- 3.78 g/L and 1.82 +/- 0.40 g/L, respectively) than in Group A (2.92 +/- 0.75 g/L and 0.38 +/- 0.15 g/L) or Group C (5.71 +/- 1.88 g/L and 1.19 +/- 0.32 g/L). We conclude that respiratory acidosis decreases the severity of VILI in this model.

Exploitation of parasite derived antigen in therapeutic success of human cutaneous leishmaniasis in Brazil

In a complete study in 25 patients with American cutaneous leishmaniasis, caused by Leishmania braziliensis complex, immunotherapeutic efficacy of parasite derived antigen (94-67 KD) has been compared to antimonial therapy. Additionally, to delineate the mechanism of therapeutic success, microscopical features of immune response in active lesions and healed or non-healed lesions following therapy were analyzed. The results showed that cure rates in immunotherapy and chemoterapy were equal (>83 por cento). The immunohistochemical changes in two therapeutic groups were also largely similar. The analysis of humoral and cellular immune response suggest that appropriate stimulation of T helper cells in the lesion site, in association with one or more cytokines, play a key role in the healing process.

Amino acid identity and/or position determines the proteasomal cleavage of the HLA-A*0201-restricted peptide tumor antigen MAGE-3271-279.

The proteasome plays a crucial role in the proteolytic processing of antigens presented to T cells in the context of major histocompatibility complex class I molecules. However, the rules governing the specificity of cleavage sites are still largely unknown. We have previously shown that a cytolytic T lymphocyte-defined antigenic peptide derived from the MAGE-3 tumor-associated antigen (MAGE-3(271-279), FLWGPRALV in one-letter code) is not presented at the surface of melanoma cell lines expressing the MAGE-3 protein. By using purified proteasome and MAGE-3(271-279) peptides extended at the C terminus by 6 amino acids, we identified predominant cleavages after residues 278 and 280 but no detectable cleavage after residue Val(279), the C terminus of the antigenic peptide. In the present study, we have investigated the influence of Pro(275), Leu(278), and Glu(280) on the proteasomal digestion of MAGE-3(271-285) substituted at these positions. We show that positions 278 and 280 are major proteasomal cleavage sites because they tolerate most amino acid substitutions. In contrast, the peptide bond after Val(279) is a minor cleavage site, influenced by both distal and proximal amino acid residues.

Constitutive expression of a chimeric receptor that delivers IL-2/IL-15 signals allows antigen-independent proliferation of CD8+CD44high but not other T cells.

We have prepared transgenic mice whose T cells constitutively express a chimeric receptor combining extracellular human IL-4R and intracellular IL-2Rbeta segments. This receptor can transmit IL-2/IL-15-like signals in response to human, but not mouse, IL-4. We used these animals to explore to what extent functional IL-2R/IL-15R expression controls the capacity of T cells to proliferate in response to IL-2/IL-15-like signals. After activation with Con A, naive transgenic CD8+ and CD4+ T cells respond to human IL-4 as well as to IL-2. Without prior activation, they failed to proliferate in response to human IL-4, although human IL-4 did prolong their survival. Thus, IL-2-induced proliferation of activated T cells requires at least one other Ag-induced change apart from the induction of a functional IL-2R. However, a fraction of CD8+CD44high T cells proliferate in human IL-4 without antigenic stimulation or syngeneic feeder cells. In contrast, CD4+CD44high T cells are not constitutively responsive to human IL-4. We conclude that although all transgenic T cells express a functional chimeric receptor, only some CD8+CD44high T cells contain all molecules required for entry into the cell cycle in response to human IL-4 or IL-15.

Attempted isolation of the gene encoding the 21 Kd Plasmodium berghei ookinete transmission blocking antigen from Plasmodium yoelli and Plasmodium vivax

The 21kD ookinete antigen of Plasmodium berghei (Pbs 21) has been shown to elicit an effective and long lasting transmission blocking immune response in mice. Having cloned and sequenced this antigen (Paton et al. 1993) the sequence was compared to the genes of the same family previously identified in P. falciparum, P. gallinaceum (Kaslow et al. 1989) and P. reichenowi (Lal et al. 1990). Four conserved areas were identified in this comparison, to which degenerate oligonucleotides were designed. PCR amplification and screening of genomic libraries was then carried out using these oligonucleotides. The P. yoelii gene was successfully cloned and a number of novel P. vivax genes identified but the P. vivax homologue of Pbs21 remains elusive.

Aspects of immunity for the AMA-1 family of molecules in humans and non-human primates malarias

The apical membrane antigen (AMA-1) family of malaria merozoite proteins is characterised by a high degree of inter-species conservation. Evidence that the protein (PK66/AMA-1) from the simian parasite Plasmodium knowlesi was protective in rhesus monkeys suggested that the 83kDa P. falciparum equivalent (PF83/AMA-1) should be investigated for protective effects in humans. Here we briefly review pertinent comparative data, and describe the use of an eukaryotic full length recombinant PF83/AMA-1 molecule to develop a sensitive ELISA for the determination of serological responses in endemic populations. The assay has revealed surprisingly high levels of humoral response to this quantitatively minor antigen. We also show that PK66/AMA-1 inhibitory mAb's are active against merozoites subsequent to release from schizont-infected red cells, further implicating AMA-1 molecules in red cell invasion.

Immunogenicity of multiple antigen peptides containing Plasmodium vivax CS epitopes in BALB/c mice

Multiple antigen peptide systems (MAPs) allow the incorporation of various epitopes in to a single synthetic peptide immunogen. We have characterized the immune response of BALB/c mice to a series of MAPs assembled with different B and T cell epitopes derived from the Plasmodium vivax circumsporozoite (CS) protein. A B-cell epitope from the central repeat domain and two T-cell epitopes from the amino and carboxyl flanking regions were used to assembled eight different MAPs. An additional universal T cell epitope (ptt-30) from tetanus toxin protein was included. Immunogenicity in terms of antibody responses and in vitro T lymphocyte proliferation was evaluated. MAPs containing B and T cell epitopes induced high titers of anti-peptides antibodies, which recognized the native protein on sporozoites as determined by IFAT. The antibody specificity was also determined by a competitive inhibition assay with different MAPs. A MAP containing the B cell epitope (p11) and the universal epitope ptt-30 together with another composed of p11 and the promiscuous T cell epitope (p25) proved to be the most immunogenic. The strong antibody response and specificity for the cognate protein indicates that further studies designed to assess the potential of these proteins as human malaria vaccine candidates are warranted.

A novel merozoite surface antigen of Plasmodium falciparum (MSP-3) identified by cellular-antibody cooperative mechanism antigenicity and biological activity of antibodies

We report the identification of a 48kDa antigen targeted by antibodies which inhibit Plasmodium falciparum in vitro growth by cooperation with blood monocytes in an ADCI assay correlated to the naturally acquired protection. This protein is located on the surface of the merozoite stage of P. falciparum, and is detectable in all isolates tested. Epidemiological studies demonstrated that peptides derived from the amino acid sequence of MSP-3 contain potent B and T-cell epitopes recognized by a majority of individuals living in endemic areas. Moreover human antibodies either purified on the recombinant protein, or on the synthetic peptide MSP-3b, as well as antibodies raised in mice, were all found to promote parasite killing mediated by monocytes.

Prognostic value of postoperative carcinoembryonic antigen concentration and extent of invasion of resection margins after hepatic resection for colorectal metastases.

OBJECTIVE: To evaluate the prognostic value of postoperative concentration of carcinoembryonic antigen (CEA) and extent of surgical margins after resection of liver metastases from colorectal cancer. DESIGN: Retrospective study. SETTING: Teaching hospital, Switzerland. SUBJECTS: 49 patients with hepatic metastases after primary colorectal cancer. INTERVENTIONS: Resection of hepatic metastases MAIN OUTCOME MEASURES: Assessment of prognostic value of variables by univariate and multivariate analysis. RESULTS: Median survival was 24 months (range 5-86 months). Resection margins were clear (> 1-cm) in 10, close (< 1-cm) in 25 and invaded in 9 patients. On univariate analysis, a postoperative concentration of CEA of <4ng/ml was correlated with prolonged survival (p < 0.001), but the width of the resection margin was not of prognostic importance. There was no correlation between width of resection margins and postoperative concentration of CEA (p = 0.5). On multivariate analysis, postoperative concentrations of CEA of 4 ng/ml or more were associated with increased risk of death (relative risk 7.3; 95% confidence interval (CI) 2.8-18.7, p < 0.001). CONCLUSION: Postoperative CEA offers better prognostic discrimination than the width of resection margins after resection of liver metastases from colorectal tumours. Some patients with invaded resection margins did survive for 3 years, but no patient did whose CEA concentration was 4 ng/ml or more. The definition of a potentially curative hepatic resection should include a postoperative CEA concentration of <4 ng/ml (within the reference range).

A protective cocktail vaccine against murine cutaneous leishmaniasis with DNA encoding cysteine proteinases of Leishmania major.

The protection elicited by the intramuscular injection of two plasmid DNAs encoding Leishmania major cysteine proteinase type I (CPb) and type II (CPa) was evaluated in a murine model of experimental cutaneous leishmaniasis. BALB/c mice were immunized either separately or with a cocktail of the two plasmids expressing CPa or CPb. It was only when the cpa and cpb genes were co-injected that long lasting protection against parasite challenge was achieved. Similar protection was also observed when animals were first immunized with cpa/cpb DNA followed by recombinant CPa/CPb boost. Analysis of the immune response showed that protected animals developed a specific Th1 immune response, which was associated with an increase of IFN-gamma production. This is the first report demonstrating that co-injection of two genes expressing different antigens induces a long lasting protective response, whereas the separate injection of cysteine proteases genes is not protective.