Differences in disease frequency between Europeans and Polynesians: directions for future research into genetic risk factors.

The purpose of this review is to identify complex genetic diseases that might be common in Polynesian ethnic groups because of a high frequency of susceptibility genes. Since a number of Polynesian ethnic groups are descended from recent founder populations, they may be especially suitable for studies designed to identify these genes. We have reviewed the epidemiological literature looking for diseases that i) have a higher frequency in at least two Polynesian groups than in Europeans living in the same geographic areas, ii) are not at high frequency in Polynesia entirely because of high levels of known environmental risk factors, and iii) are known to be inherited in other ethnic groups. Twenty-one diseases fulfilling these three criteria were identified. It may be possible to design studies to identify the genes that cause these diseases in Polynesian ethnic groups.

Defective antigen-induced lymphocyte proliferation in the X-linked hyper-IgM syndrome.

We examined T-cell proliferation in five patients with X-linked hyper-IgM syndrome (XHIM), using a panel of antigens and lectins. All patients had impaired antigen-induced proliferation, whereas their lectin responses were normal. Thus, in addition to severely depressed antibody responses, patients with XHIM have a defect in antigen-specific T-cell proliferation, which may explain their susceptibility to pathogens such as Pneumocystis carinii.

Molecular pathology of the X-linked hyper-immunoglobulin M syndrome: detection of wild-type transcripts in a patient with a complex splicing defect of the CD40 ligand.

The X-linked hyper-immunoglobulin M syndrome (XHIM) is a primary immune deficiency disorder characterized by an inability to produce immunoglobulin isotypes other than immunoglobulin M (IgM) and IgD. Recently, a B-cell surface antigen (CD40) and its conjugate T-cell counterstructure (CD40 ligand) were shown to mediate immunoglobulin isotype switching in the presence of cytokines such as interleukin 4. Most patients with XHIM have been shown to have mutations of the extracellular domain of the CD40 ligand. Here we describe a novel point mutation of an intronic splice acceptor site which results in a complex splicing defect of the CD40 ligand in a patient with XHIM. In addition to two species of deleted transcripts, wild-type transcripts were also detected in this individual. The demonstration of wild-type CD40 ligand transcripts may be an explanation for previous observations suggesting that some XHIM patients are able to undergo immunoglobulin isotype switching in vivo.

The influence of a recombinant-HBsAg vaccine on the repertoire of transcribed T-cell receptor beta chain variable region genes in peripheral blood in humans.

The purpose of this study was to determine whether in vivo changes in the repertoire of Tcr beta chain variable region (V beta) genes could be detected in peripheral blood mononuclear cells after immunization of humans with recombinant hepatitis B virus envelope protein (rHBsAg). We measured the percentage of Tcr RNA transcripts carrying each of 20 V beta genes in human PBMC before and after immunization with rHBsAg in Polynesians (8 non-immunized controls, 26 immunized subjects) and Europeans (9 non-immunized controls, 11 immunized subjects). The per cent of RNA transcripts containing V beta 7.4 family genes was increased in immunized vs control Polynesian (+ 1.6 +/- 0.5%) vs -1.1 +/- 0.3%, P = 0.0002) and European (+1.6 +/- 0.6% vs -0.1 +/- 0.5%, P = 0.05) subjects at 48 h and 28 h post-immunization, respectively. No changes in V beta repertoire were found after 48 h in either race. Thus, there is a transient increase in frequency of T cells with Tcr containing V beta 7.4 family genes within 48 h of an immunization containing rHBsAg in humans. There are a number of explanations for this finding, including the possibility that V beta 7.4 gene family products may be preferentially involved in the primary immune response to HBsAg.

The influence of a maternal chronic hepatitis B virus infection on the repertoire of transcribed T-cell receptor beta chain variable region genes in human cord blood.

We used an anchor polymerase chain reaction method to compare the repertoires of transcribed T-cell receptor beta chain variable region (V beta) genes in cord blood T cells from neonates of hepatitis B surface antigen (HBsAg) positive (n = 40) and HBsAg negative (n = 40) women. Fifteen of the HBsAg positive women were hepatitis B e antigen (HBeAg) positive, and 25 were HBeAg negative. The percentage of V beta 7.4 transcripts was lower in cord blood T cells from neonates of HBsAg-positive relative to HBsAg-negative women (9.7% +/- 0.5% vs. 12.7% +/- 0.6%, P = .002). The percent of V beta 5.1 transcripts was higher in cord blood T cells from neonates of HBeAg-positive relative to HBeAg-negative women (9.3% +/- 0.7% vs. 7.0% +/- 0.3%, P < .001). There were no correlations between neonatal maturity at birth and V beta repertoire. In summary, a maternal chronic hepatitis B virus (HBV) infection is associated with changes in the repertoire of transcribed T-cell receptor genes in neonatal cord blood T cells. It is possible that the T-cell response to the HBV is associated with a limited repertoire of V beta genes. The mechanism of vertical chronic HBV infection in human neonates may involve changes in the T-cell response to the virus that are induced in utero.

A high efficiency method for purification and assay of bee venom phospholipase A2.

Phospholipase A2 (PLA2) is the main allergen of hymenopteran venoms. We describe a highly efficient reverse phase high performance liquid chromatographic method (HPLC) for isolating PLA2 from crude bee venom. This method removes all detectable contaminants such as melittin from PLA2 while preserving the hemolytic action of PLA2. In addition we describe a simple functional assay of PLA2 based on its propensity to cause hemolysis of guinea pig red blood cells. These techniques are particularly well suited to the isolation and assessment of PLA2 of venoms which are available in limited quantities.

T cell receptor polymorphisms in Caucasians and Polynesians.

The purpose of this study was to find genetic polymorphisms that might be useful in studies of Polynesian-Caucasian racial admixture and Polynesian disease susceptibility. The allele frequencies of six T cell receptor locus RFLP were measured in 73 Caucasians and two Polynesian ethnic groups comprising 86 Maoris and 95 Samoans. The RFLP studied were (locus/enzyme/probe): C alpha/Taq1/Y14, V alpha/Taq1/Y14, C beta/BglII/Y35, C gamma/Pvu II/HGP02, V beta 7/BamHI/V beta 7.4 and V beta 8/Bam HI/V beta 8.1. Racial differences in allele frequency were present with all six RFLP (P < 0.001). The allele frequencies of the V alpha/Taq1/Y14 and the V beta 7/BamHI/7.4 RFLP were similar in the two Polynesian groups, both of which differed from the Caucasians. The 1.4 kb allele of the V alpha/Taq1/Y14 RFLP and the 8.0 kb allele of the V beta 7/BamHI/7.4 RFLP were present in low frequency in both Polynesian groups compared to the Caucasian group, consistent with a gene flow effect. These alleles may be useful in studies of Caucasian-Polynesian racial admixture.

Specificity variants in monoclonal antibodies reactive with peptide epitopes of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum.

It has been suggested that repeat sequence antigens of Plasmodium falciparum may serve the parasite in immune evasion by modifying the host antibody response and impairing the development of protective immunity. According to this proposal networks of cross-reactive, repeat sequence malarial antigens have the ability to stimulate a high proportion of all somatically mutated B cells with altered antibody specificity, and thus to hinder the normal process of antibody affinity maturation. To determine the rate at which immunoglobulin mutations produce new reactivities with repeat sequence antigens, hybridoma cell lines specific for the ring-infected erythrocyte surface antigen (RESA) were examined for the incidence of specificity variants that arose naturally or as a result of treatment with the chemical mutagen ethylmethane sulphonate (EMS). From one of the cell lines variants were readily isolated having reactivity towards a very closely related repeat sequence epitope within the same RESA antigen. However, the other hybridoma/antigen combinations revealed no variants. In general, mutations giving rise to antibodies with altered specificity for related repetitive antigens were not readily induced and only limited support of the hypothesis was obtained.

Hierarchy of cytotoxic T cell clones. II. Intraclonal triggering of lysis by hapten-specific CTL.

Cells from clones of anti-hapten murine cytotoxic T lymphocytes (CTL) can act as both target and effector cells, but will not lyse members of the same clone. The effect of haptenation on the cytolytic activity of anti-fluorescein (FL) and anti-trinitrophenol (TNP) CTL clones was examined. Treatment of anti-FL clones with fluorescein isothiocyanate or anti-TNP clones with trinitrobenzene sulphonic acid induces these clones to kill in an antigen-independent fashion. Targets killed by the haptenated CTL included syngeneic and allogeneic B lymphocyte blast cells, P815, YAC-1 and in one case human GM 4072 tumor cells. The importance of CD8 and T cell receptor (TCR) occupancy is demonstrated by the ability to block autotriggering by antibody directed against Ly 2 and the TCR. The results demonstrate that effects other than antigen recognition of the target play a role in the final outcome of effector-target cell interactions and provide a mechanism which could lead to autodestruction and immunosuppression particularly in some types of viral infection.

Hierarchy of cytotoxic T cell clones. I. Reversal of resistance to lysis and killing between anti-hapten cytotoxic T cells.

Cells from clones of anti-hapten cytotoxic T lymphocytes (CTL) can act as both effector cells and, when treated with the specific hapten, as target cells. Individual clones can kill haptenated cells only from other clones that are less efficient killers. Clones specific for both fluorescein and trinitrophenol could be ordered in a single hierarchy in which resistance to lysis correlated with lytic efficiency. When the killing efficiency was reduced with phorbol myristate acetate (PMA) or the colchicine analogue, Colcemid, the degree of resistance to lysis was also reduced. The use of PMA-treated fluoresceinated targets greatly enhanced intraclonal killing and similarly lead to a repositioning of clones within the hierarchy of normal cells. By the haptenation of appropriate clones, efficient CTL could kill cells from other clones in a direction apparently opposite to recognition. The results demonstrate that effects other than antigen recognition of the target cell may result in variations in the nature of T cell immune responses.

Development of thymocytes in organ culture: migrant cells with natural killer cell characteristics.

During organ culture of foetal thymic lobes, up to 5% of the thymic cells migrate out of the lobes and a majority of these have phenotypic characteristics of thymocytes or macrophages. Interleukin-2 (IL-2) increases the number of these migrant cells, and cytotoxic cells can be detected which display natural killer cell surface markers. In contrast, although cytotoxic activity can be detected in cells from adult thymic fragments cultured with IL-2, the cytotoxic activity is not detected with natural killer sensitive target cells. The appearance of natural killer-like cells during the culture of foetal thymic lobes suggests that they are involved in differentiation events which occur at this time in the thymus.

The effect of prostaglandins and indomethacin on cytotoxic T-lymphocytes and their precursors.

The effect of prostaglandins of the E and F series on the generation of cytotoxic T-lymphocytes (CTL) was investigated. High concentrations (10(-6)-10(-7) M) of prostaglandin E2(PGE2) inhibited the generation of both antigen-specific CTL and "activated" killers from precursor cells. Virtually no effect on the growth and cytotoxic activity of antigen-specific CTL clones was detectable. As with prostaglandins of the F series, lower concentrations of the E series (10(-8)-10(-9) M) enhanced the generation of antigen-specific CTL but inhibited the generation of lymphokine and spontaneously-activated CTL. When endogenous PG production was blocked with indomethacin, the generation of antigen-specific CTL was enhanced in bulk culture. Indomethacin also increased the frequency of allo-reactive CTL precursors and in anti-fluorescein responses it created conditions in which suppression was overridden. These results indicate that prostaglandins can exert both negative and positive regulatory effects on an antigen-specific cytotoxic response and can influence the apparent specificity of the cytotoxic cells that are generated.

Effect of interleukin 2 on fetal thymocytes in organ cultures: generation of lymphokine-activated killer cells.

Cells with cytolytic activity can be detected in mouse fetal thymic lobes cultured in the presence of interleukin 2 for 6 days. The lymphokine-activated killer cells from 14-day fetal thymic lobes are relatively resistant to treatment with anti-Ly-2 antibody and complement (CD8-) but sensitive to anti-Thy-1 and complement treatment (Thy-1+). They display major histocompatibility complex-unrestricted killing, lysing both syngeneic and allogeneic tumor cells, but will not lyse human xenogenic target cells. Low levels of cytotoxic activity can be detected in thymic lobes from Day 12-13 embryos and this activity increases with embryonic age. While the events which lead to the inhibition of normal maturation of fetal thymocytes by inclusion of IL-2 in fetal thymus organ cultures are unknown, the appearance of cytotoxic cells raises the question of whether they are involved in the normal intrathymic cell death process.

Development of fetal thymocytes in organ cultures. Effect of interleukin 2.

Most fetal thymocytes from 14-d mouse embryos are Thy-1+, L3T4-, Ly-2-, and express the receptor for interleukin 2 (IL-2). The development of thymocytes has been followed in fetal thymus organ cultures. When fetal thymus from 14-d embryos were cultured for a 6-d period, thymocytes increased in number 20-40-fold, and 95% became Thy-1+, L3T4+, Ly-2+. The addition of IL-2 to organ cultures of 14-d fetal thymus inhibited, in a dose-dependent manner, cell proliferation and the appearance of Thy-1+, L3T4+, Ly-2+ thymocytes. The addition of IL-2 also resulted in the appearance of a population of cells that were cytotoxic for syngeneic and allogeneic fetal thymocytes and syngeneic tumour targets. While the events that lead to the expression of the IL-2 receptor on 14-d fetal thymocytes are unknown, IL-2 in fetal thymus organ cultures inhibits the normal maturation of fetal thymocytes and raises the question of whether the cytotoxic cells that appear reflect selection through an alternative pathway of development.

Effects of in vivo modulation of splenic natural killer cell activity on the growth of spleen-seeking tumour variants.

A novel tumour system has been used to study the effect of natural killer cells on tumour growth by using agents which modify natural killer cell activity. The tumour cells are hybridoma cells which secrete antibody specific for red blood cells so that tumour growth can be quantitated by a haemolytic plaque assay. Spleen-seeking variants have been derived from original hybrids which are sensitive to natural killer cells. Treatment of mice with polyinosinic-polycytidylic acid substantially enhanced natural killer cell activity and correlated closely with a reduction in the growth of the hybridoma tumour cells in the spleen and life extension. Conversely, a single injection of anti-asialo GM, antibody resulted in a substantial increase in the number of plaque forming splenic tumour cells and virtual elimination of natural killer cell activity. These data demonstrate the important role of natural killer cells in constraining the growth of a tumour of B cell origin and establishes the usefulness of this tumour model in studying the biology of effects on tumour growth.

The analysis of an anti-fluorescein response at limit dilution: the effect of lymphokine preparations on the apparent specificity.

The specificity of cytotoxic T cells in an anti-fluorescein response has been analysed at limit dilution. According to the ability to discriminate between fluoresceinated and non-fluoresceinated targets, the apparent specificity of CTL depends on the quality and quantity of the lymphokine preparations used to support the response. An acid-labile component of the factor preparations contributes to the generation of non-specific CTL. No cytotoxicity against NK-sensitive target cells was detected in the 'non-specific' component of the responses.

The use of hybridoma cells as a murine model to study tumor immunity.

The secretion of antibody by hybridoma cells allows the growth of individual cells to be measured by a hemolytic plaque assay. Similarly, plaque reduction assays were sensitive indicators of tumor immunity. A series of strains of hybridoma have been isolated from an original isolate which is sensitive to cytotoxic T cells and NK cells. In the derivation of a spleen-seeking variant, the cells appear to have lost immunogenic but not antigenic characteristics. This model lends itself to the quantitative study of immune constraints on tumor growth in vivo. Combined with the selection of tumor variants, the model is of wide application to many fields of tumor biology, particularly where sensitive measurement of tumor cell number and viability is crucial.