Venkatachalam Udhayakumar

A simple perfusion technique for isolation of maternal intervillous blood mononuclear cells from human placentae

A noninvasive perfusion method for the recovery of maternal placental (intervillous) blood for use in immunologic assays is described. 60% of the perfused blood samples tested for fetal red blood cell (RBC) contamination were found to be pure maternal blood; in the remainder, fetal RBC contamination, with a single exception, was less than 6%. The intervillous mononuclear cells (IVBMC) isolated from this blood were of predominantly maternal origin as demonstrated by a polymerase chain reaction-based DNA typing technique. The number of IVBMC obtained was within the range of 9 to 55 X 10(6) cells. Phenotypic analysis of IVBMC surface antigens revealed that 61% of the cells were CD3 + T-cells and 18% were CD19 + B-cells. The CD4 + and CD8 + T-lymphocyte subsets accounted for 28 and 26% of the IVBMC, respectively. The IVBMC were functionally competent as evidenced by in vitro lymphoproliferation and cytokine production in response to mitogen and PPD stimulation. This technique allows for rapid and safe isolation of large numbers of IVBMC which are functionally active up to 12 h post-delivery, thus representing a significant improvement over previously described methods. It should facilitate more vigorous research in the study of uteroplacental immunity and infectious disease research, particularly in field settings where sample collection and laboratory facilities are distant.

Immunity to placental malaria. IV. Placental malaria is associated with up-regulation of macrophage migration inhibitory factor in intervillous blood.

Macrophage migration inhibitory factor (MIF) may play a role in immune responses to malaria during pregnancy by virtue of its ability to activate macrophages and to overcome the immunosuppressive effect of glucocorticoids. The present study investigated whether plasma MIF levels are altered in pregnant women with placental malaria (PM) and/or human immunodeficiency virus (HIV) infection. For the first time it is demonstrated that MIF levels in the intervillous blood (IVB) plasma were significantly elevated, compared with that in both peripheral plasma ( approximately 500-fold) and cord plasma (4.6-fold; P<.01). IVB mononuclear cells also produced significantly higher levels of MIF, compared with that of peripheral blood mononuclear cells. PM was associated with increased levels of MIF in the IVB plasma (P<.02). Primigravid and secundigravid women had significantly higher levels of MIF in their IVB plasma than did multigravid women (P<.05). HIV infection did not significantly alter MIF levels in any site examined.

Immunity to Placental Malaria. II. Placental Antigen—Specific Cytokine Responses Are Impaired in Human Immunodeficiency Virus—Infected Women

An association was demonstrated recently between elevated in vitro production of interferon (IFN)-gamma by intervillous blood mononuclear cells (IVBMCs) and protection against placental malaria (PM). Because human immunodeficiency virus (HIV)-infected pregnant women have increased susceptibility to PM, loss of the IFN-gamma response in these women may impair their ability to control PM. Measurement of cytokines in culture supernatants by ELISA revealed that IFN-gamma responses by HIV-positive IVBMCs were impaired, especially after malarial antigen stimulation. Interleukin (IL)-4 and IL-10 responses also were reduced in HIV-positive persons, the latter more so in HIV-positive, PM-positive persons. In contrast, tumor necrosis factor-alpha production generally was enhanced in PM-positive and HIV-positive persons. Overall, cytokine production was reduced in HIV-positive persons with CD4 T cell counts <500/microL, particularly in response to malarial antigen. Thus, HIV-mediated cytokine dysregulation and impairment of the protective IFN-gamma response may contribute to the increased susceptibility of HIV-positive pregnant women to malaria.

Monopalmitic acid-peptide conjugates induce cytotoxic T cell responses against malarial epitopes : importance of spacer amino acids

Cytolytic T cells (CTL) play a critical role in providing protection against the liver stage of malaria infection. Previous investigations have shown that induction of CTL against peptide or proteins can be achieved by attachment of lipids. In the present study, we used the Plasmodium berghei circumsporozoite protein CTL epitope (SYIPSAEKI (PL76)). This peptide with cysteine-serine (CS) as spacer amino acids was coupled to palmitic acid (PA). The same CTL epitope containing only an extra serine was linked to S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-cysteine (tripam-C). Inbred mice [(BALB/c x C57BL/6)F1] were immunized intravenously with the lipopeptides. Both types of lipopeptides induced significant CTL responses after one injection. Immunization of the monopalmitic acid-peptide conjugate intraperitoneally emulsified in Freunds complete adjuvant also induced a significant CTL response, but the magnitude was lower as compared to the intravenous route. The major advantages of the use of the simple monopalmitic acid-peptide conjugates are: (i) low costs of the fatty acid; (ii) coupling of lipid to peptide can be performed using the peptide synthesizer during standard peptide synthesis, and (iii) standard peptide methodology can be used for purification. To investigate whether a spacer amino acid sequence between the actual CTL epitope and PA is required for induction of an optimal CTL response, we prepared monopalmitic acid-peptide conjugates with different spacer amino acids. A lipopeptide without a spacer amino acid and another one containing the CS spacer sequence both induced a CTL response, whereas a lipopeptide with a serine as spacer failed to induce CTL. These results indicate that the amino acid spacer sequences influence the immunological properties of the palmitic acid-peptide conjugates.

Gonadotropin-Releasing Hormone Alters the T Helper Cytokine Balance in the Pregnant Rat

Abstract The interactions between immune-endocrine and reproductive systems are heightened during pregnancy as an adaptive mechanism, and are regulated by a complex array of hormones and cytokines that control the survival of a semiallogeneic conceptus. GnRH can exert direct effects on the immune system via its receptor (GnRH-R) on lymphoid cells. In the present study, we employed in vitro, ex vivo, and in vivo approaches to investigate the role of GnRH in the modulation of T helper cytokines in pregnant rats undergoing termination of pregnancy. Day 8 pregnant rats were infused with a GnRH agonist (GnRH-Ag) for 24 h using an osmotic minipump. Sham control rats were infused with the vehicle, saline. Lymphocytes were isolated from sham and treated rats and polyclonally stimulated with immobilized anti-CD3 antibody. The levels of the signature T helper 1 (Th-1) cytokines (interferon-γ [IFN-γ] and interleukin-2 [IL-2]) and Th-2 cytokines (IL-4 and IL-10) were measured in culture supernatants. Using immunoflourescence confocal microscopy, we demonstrated for the first time the spatial localization of GnRH-R protein on the surface of lymphocytes. We observed a marked increase in IFN-γ and inhibition of IL-4 production from lymphocytes of pregnant rats treated in vitro with different doses of GnRH-Ag. Further, the responsiveness of lymphocytes to produce IFN-γ was markedly increased in cells cultured ex vivo from GnRH-Ag infused rats, whereas the capacity of lymphocytes to produce IL-4 was significantly inhibited. In addition, GnRH-Ag infusion in pregnant rats induced a shift toward Th-1 cytokines in the serum. We did not observe any significant difference in IL-2 and IL-10 production in response to GnRH-Ag. Our results suggest an additional function for GnRH as a Th-1 inducer and Th-2 inhibitor. GnRH can thus skew the cytokine balance to predominantly Th-1 type in pregnancy, leading to the termination of pregnancy in rats.

Sequence variations in the non-repetitive regions of the liver stage-specific antigen-1 (LSA-1) of Plasmodium falciparum from field isolates☆☆☆

Liver-stage-specific antigen-l (LSA-1) of Phsmodium falciparum is a 200-kDa protein localized in the parasitophorous vacuole space of liver-stage parasites [1,2]. It is composed of a large central repetitive region and two flanking short non-repetitive Nand C-terminal regions [3]. This antigen is of considerable interest in vaccine development, because two epitopes (Ls6, Ls8) of ISA-1 have been shown to generate cytotoxic T-cell (CTL) responses in HLA-B53 and HLA-B35 positive individuals 141,