Gamma delta T-cell function in pathogenesis of cerebral malaria in mice infected with Plasmodium berghei ANKA.

Mice depleted of gammadelta T cells by monoclonal antibody treatment and infected with Plasmodium berghei ANKA did not develop cerebral malaria (CM). In striking contrast, delta0/0 mice infected with P. berghei developed CM despite their gammadelta T-cell deficiency. gammadelta T cells appear to be essential for the pathogenesis of CM in mice having experienced normal ontogeny but not in mice genetically deprived of gammadelta T cells from the beginning of life.

The effects of interleukin-15 on human gammadelta T cell responses to Plasmodium falciparum in vitro.

We observed that the gammadelta T cell subset expands when human peripheral blood mononuclear cells (PBMC) from malaria-naive donors are cultured with Plasmodium falciparum lysate in the presence of IL-2 or IL-15, cytokines that utilize two common IL-2 receptor subunits. IL-15 induced the expansion of the gammadelta T cell subset at all levels tested, whereas IL-2 was not stimulatory at high levels. Flow cytometric analysis of apoptosis using the TUNEL assay indicated that the percentage and absolute number of gammadelta T cells undergoing apoptosis were greater in cultures stimulated with antigen and IL-2 than in cultures stimulated with either antigen and IL-15 or control erythrocyte lysate and IL-2. The ability of IL-15 to enhance gammadelta T cell function was also assessed; the results suggest that IL-15 can function with IL-2 to enhance the capacity of gammadelta T cells to inhibit parasite replication. Together these data indicate that IL-2 and IL-15, which both bind to IL-2Rbeta and IL-2R(gamma)c, enhance gammadelta T cell function, but they appear to have different effects on proliferation and survival.

Human gamma delta T cell subset-proliferative response to malarial antigen in vitro depends on CD4+ T cells or cytokines that signal through components of the IL-2R.

We examined the cellular and molecular basis of the proliferative response of human gamma delta T cells in cultures of PBMC stimulated with blood-stage Plasmodium falciparum malarial Ag. Flow cytometry revealed that maximal gamma delta T cell proliferation occurs after maximal CD4+ alpha beta T cell proliferation. Depletion of CD4+ T cells from PBMC before stimulation with malarial Ag markedly reduces the number of proliferating gamma delta T cells, which suggests that CD4+ T cells function in providing help to gamma delta T cells to respond to this parasite Ag. Removal of gamma delta T cells, however, did not alter the expansion of the CD4+ T cell subset. The addition of exogenous IL-2, IL-4, or IL-15 restored the capacity of gamma delta T cells to proliferate in Ag-stimulated cultures of PBMC depleted of CD4+ T cells. mAbs specific for the alpha- and beta-subunits of the IL-2 receptor inhibit the gamma delta T cell subset expansion in cultures stimulated with malarial Ag. Taken together, these findings suggest that the proliferation of gamma delta T cells in response to malarial Ag is dependent on the presence of CD4+ alpha beta T cells, but the requirement for CD4+ alpha beta T cells can be met by cytokines that use the IL-2R.

Participation of lymphocyte subpopulations in the pathogenesis of experimental murine cerebral malaria.

We determined the requirement for selected lymphocyte subsets and cytokines in the pathogenesis of experimental murine cerebral malaria (CM) by using gene-targeted knockout and mAb-suppressed mice. Plasmodium berghei ANKA infection induced CM in A 0/0 mice, which lack expression of surface MHC class II glycoproteins and consequently express a severe and chronic reduction in numbers of CD4+ T cells. However, when A 0/0 mice, which are on a C57BL/6 x 129 genetic background, or immune-intact C57BL/6 controls treated with anti-CD4 mAb were infected, none developed CM. The latter finding confirms an earlier report that CD4+ T cells are required for CM to occur and additionally indicates that the reduced numbers of CD4+ T cells present in A 0/0 mice are sufficient for CM development. Neither the recently described CD4+, NK1.1+ T cell subset shown to be present in A 0/0 mice nor traditional NK cells seem to be required for the induction of CM because A 0/0 and C57BL/6 mice severely depleted of both NK1.1+ populations with mAb developed CM as readily as did normal Ig-treated controls. Deficiency of Th1-associated cytokines (IFN-gamma or IL-2) in mice by gene-targeted disruptions completely inhibited CM development, whereas the lack of Th2-associated cytokines (IL-4 or IL-10) did not prevent this disease. Our observation that B cell-deficient JHD and microMT mice developed CM provides evidence that neither B cells, their products, nor B cell Ag presentation are a requisite for CM pathology. We further observed that neither beta 2m 0/0 knockout mice, which lack CD8+ alpha beta T cells, nor C57BL/6 mice depleted of CD8+ T cells with anti-CD8 mAb treatment developed CM, leading us to conclude that CD8+ T cells are also crucial for the development of CM.

Gamma delta T cells function in cell-mediated immunity to acute blood-stage Plasmodium chabaudi adami malaria.

To determine whether gamma delta T cells are essential for the resolution of acute Plasmodium chabaudi adami (P. c. adami) malaria, we depleted gamma delta T cells from C57BL/6 mice with hamster monoclonal anti-TCR gamma delta Ab treatment. During the period in which control mice that had received normal hamster IgG completely resolved infections, gamma delta T cell-depleted mice were unable to suppress their infections. Because the number of splenic CD4+ alpha beta T cells in these anti-TCR-gamma delta-treated mice with nonresolving malaria was similar to control mice, it appears that CD4+ alpha beta T cells alone cannot mediate early resolution even though they are known to play a critical role in immunity to blood-stage malaria. Mice treated with anti-CD4 mAb also failed to resolve P. c. adami malaria. Depletion of CD4+ alpha beta T cells from the spleens of infected mice resulted in minimal expansion of the splenic CD4- gamma delta T cell subset compared with infected control mice. Together, these findings indicate that activation of the gamma delta T cell subset, which requires the presence of CD4+ alpha beta T cells, is essential for resolution of acute P. c. adami malaria. To determine whether gamma delta T cells require either Abs or B cells to achieve their protective activity, B cell-deficient JHD mice were treated with the same depleting anti-TCR-gamma delta Abs. Whereas control JHD mice injected with hamster IgG resolved acute P. c. adami malaria, JHD mice depleted of gamma delta T cells failed to do so. We conclude that gamma delta T cells suppress P. c. adami parasitemia by mechanisms of immunity independent of Ab and B cells.

Inhibition of Plasmodium falciparum in vitro by human gamma delta T cells.

As assessed by flow cytometry, human gamma delta T cells were shown here to inhibit replication of blood-stage Plasmodium falciparum in vitro in a dose-dependent fashion; no other leukocyte population tested was suppressive. Replication of intraerythrocytic stages of the parasite (rings, trophozoites, and schizonts) was not affected by coculture with gamma delta T cells nor were erythrocytes damaged by this coculture, indicating that the targets recognized by gamma delta T cells are extracellular merozoites in transit to new host erythrocytes. Moreover, parasite inhibition requires contact between gamma delta T cells and merozoites. These findings suggest that gamma delta T cells may exert a protective effect in immunity to malaria.

The resolution of acute malaria in a definitive model of B cell deficiency, the JHD mouse.

Because the role of cell-mediated immunity (CMI) in the resolution of blood-stage malaria remains unclear, we examined the question of whether mice completely lacking Ab-mediated immunity (AMI) but possessing some CMI can resolve experimental malaria previously reported not to require AMI for resolution. Severe combined immunodeficient mice reconstituted with enriched immune T cells (< 0.5% B220+ cells) suppressed acute Plasmodium chabaudi adami parasitemia, suggesting that T, but not B, cells are required to clear this form of malaria. In addition, JHD mice, which are a definitive model of B cell deficiency, were also shown to resolve P. chabaudi adami, Plasmodium vinckei petteri and Plasmodium chabaudi chabaudi malaria. These observations collectively establish that CMI alone can mediate the clearance of acute malaria caused by these subspecies of Plasmodium. Moreover, the protective cell-mediated immune response involved depends upon CD4+ T cells because JHD mice treated with anti-CD4 mAb do not resolve their infections. These results suggest that evaluation of immunization regimens to activate CD4+ T cell dependent cell mediated immunity against Plasmodium falciparum may be appropriate.

Role of CD4+ T cells in the expansion of the CD4-, CD8- gamma delta T cell subset in the spleens of mice during blood-stage malaria.

The previously observed expansion of the splenic gamma delta T cell subset was examined during the course of murine malaria to determine whether CD4+ T cells are required. Flow cytometric analysis during the course of Plasmodium chabaudi adami malaria in both C57BL/6 and BALB/c mice revealed that the maximal percentage of CD4+ T cells that were blasts occurred during the period of ascending parasitemia, whereas the maximal numbers of gamma delta T cells and blast cells occurred during the period of descending parasitemia. Transfer of enriched populations of CD4+ cells (> 75%) containing < 0.9% gamma delta T cells from immune BALB/c donor to SCID mice led to a population of gamma delta T cells that constituted 37% of the splenic T cells in the recipients and allowed them to resolve their infections. Transfer of the CD4- fraction did not suppress parasitemia. These results suggest that CD4+ T cells are activated early during the infection and are required for the subsequent expansion of the gamma delta T cell population. Furthermore, the maximal gamma delta T cell blast response during the period of descending parasitemia and the detection of high levels of these cells only in models that resolved their infections suggest that these cells may function in the resolution of blood-stage malaria.

Resolution of blood-stage malarial infections in CD8+ cell-deficient beta 2-m0/0 mice.

We utilized a definitive model of CD8+ T cell deficiency, the beta 2-microglobulin-deficient (beta 2-m0/0) mouse, to determine whether CD8+ T cells are required in the resolution of blood-stage malaria. In a parallel experiment, C57Bl/6 mice treated with anti-CD8 mAb showed significantly higher levels of parasitemia than untreated C57Bl/6 control mice at several points during the infection. This finding suggests some role for CD8+ cells in containing malaria. However, the beta 2-m0/0 mice, which are genetically blocked from expressing MHC class I or class Ib glycoproteins and therefore have < 2.5% of the normal number of CD8+ T cells, nevertheless resolved infections with three virulence variants of murine Plasmodium. The resolution of Plasmodium chabaudi adami, Plasmodium yoelii yoelii 17X, and Plasmodium chabaudi chabaudi AS infections by beta 2-m0/0 mice in the virtual absence of CD8+ cells demonstrates that these cells are not required to suppress murine malaria and that the suppression mechanism is not MHC class I restricted. The similarity of the time-course for resolution of infection in beta 2-m0/0 and intact control mice with all three subspecies of Plasmodium further supports the lack of a requirement for CD8+ T cells in the suppression of malarial parasitemia.

Expansion of the CD4-, CD8- gamma delta T cell subset in the spleens of mice during non-lethal blood-stage malaria.

Splenic gamma delta T cells (CD4-, CD8-) increased more than 10-fold upon resolution of either Plasmodium chabaudi adami or P.c. chabaudi infections in C57BL/6 mice compared to controls. Similarly, a 10- to 20-fold expansion of the gamma delta T cell population was observed in beta 2-microglobulin deficient (beta 2-m0/0) mice that had resolved P.c. adami, P.c. chabaudi or P. yoelii yoelii infections. In contrast, increases in the number of splenic alpha beta T cells in these infected mice were only two to three-fold indicating a differential expansion of the gamma delta T cell subset during malaria. Because nucleated cells of beta 2-m0/0 mice lack surface expression of major histocompatibility complex class I and class Ib glycoproteins, our findings suggest that antigen presentation by these glycoproteins is not necessary for the increasing number of gamma delta T cells. Our observation that after resolution of P.c. adami malaria, C57BL/6 mice depleted of CD8+ cells by monoclonal antibody treatment had lower numbers of gamma delta T cells than untreated controls suggests that the demonstrated lack of CD8+ cells in beta 2-m0/0 mice does not contribute to the expansion of the gamma delta T cell population during non-lethal malaria.

Evaluation of Campylobacter jejuni colonization of the domestic ferret intestine as a model of proliferative colitis.

Forty 3- to 17-week old domestic ferrets, including 2 gnotobiotes, were inoculated orally and/or rectally with 10(6) to 10(9) colony-forming units of 1 or more of 4 strains of Campylobacter jejuni, 3 of mink and 1 of human origin. Feeding or gavage of any of the 4 strains, in milk or broth, with or without preinoculation sodium bicarbonate treatment to neutralize stomach acid, induced colonization in 38/40 ferrets; diarrhea lasted 2 to 4 days in conventional kits, 6 days in gnotobiotes. Bacteremia was detected in 4 of 18 tested, 2 to 5 days after inoculation. Two strains caused no more severe disease or prolonged colonization after 3 serial IV passages in kits than they did before passage. Multiple inoculations with a given strain resulted in progressively briefer colonization and milder disease, but subsequent inoculation with a different strain induced colonization and gastrointestinal disease similar to a primary infection. Five kits inoculated rectally after 4 previous homologous inoculations were resistant to colonization as well as to disease. Agglutinin titers of ferrets inoculated orally or rectally once were low or undetectable, but increased in response to repeated inoculation. Pretreatment with a 1% formalin enema caused mild colon irritation without clinical or histologic evidence of proliferative colitis in ferrets concurrently inoculated orally and/or rectally, whether or not they had preexisting antibodies to any strain of C jejuni. Histologic examination of tissues revealed leukocytic infiltration of intestinal lamina propria in 29 of 35 infected kits and 5 of 8 noninfected controls, and cryptosporidiosis in 5 infected kits plus 1 control.(ABSTRACT TRUNCATED AT 250 WORDS)

Reproductive failure in mink and ferrets after intravenous or oral inoculation of Campylobacter jejuni.

Four pregnant mink and seven pregnant ferrets, including five with previous exposure and specific antibody, were injected intravenously with 10(8)-10(10) colony-forming units of Campylobacter jejuni. All 11 pregnancies failed 1-16 days after infection, with results ranging from fetal resorption to expulsion of dead or premature living kits. In every case, uterine contents (placenta, uterine fluid and/or kits) were culture-positive for C. jejuni. Three pregnant mink and nine pregnant ferrets, including four with previous exposure and antibody, were fed 10(9)-10(11) C. jejuni. Two of the mink aborted; kits of all three were culture-positive, but those of one female survived. Seven of the nine ferrets aborted, with two having culture-positive uterine contents. None of 28 uninfected ferret control pregnancies ended in abortion. The most prominent histological feature observed was severe placentitis, which appears to be a more likely cause of Campylobacter-induced abortion than direct pathogenic effects on infected kits. These results suggest that infection of mink or ferrets with C. jejuni during pregnancy poses a serious risk of reproductive failure, even for previously exposed females.

Lack of detectable blood groups in domestic ferrets: implications for transfusion.

Evidence of blood groups in domestic ferrets was sought by testing serum samples for naturally acquired or experimentally induced erythrocyte antibodies. All sera were tested for ability to cause direct agglutination, antiglobulin-enhanced (Coombs test) agglutination, or lysis. Examination of 212 randomly paired combinations of ferret serum and erythrocytes produced no evidence of naturally acquired blood group antibodies. Six pairs of ferrets were reciprocally transfused twice, 34 days apart, with 6-ml quantities of anticoagulated blood. All were tested 21 days after the first transfusion, as well as 10 and 30 days after the second transfusion; erythrocyte antibodies were not detected. Four additional pairs of ferrets were reciprocally inoculated SC with a series of six 1.25-ml quantities of blood in Alsever solution, administered over a 3-week period, and tested 8 days after the last injection; again, erythrocyte antibodies could not be detected. These observations suggest that blood groups of the kind in human beings and other mammals either do not exist in domestic ferrets or represent antigen systems too weak to elicit measurable responses under the reported conditions. It appears, therefore, that transfusion in this species poses little clinical risk, even without crossmatching.

A domestic ferret model of immunity to Campylobacter jejuni-induced enteric disease.

Oral or intravenous inoculation of previously unexposed juvenile and adult ferrets with Campylobacter jejuni uniformly resulted in intestinal colonization lasting 2 to 12 days. Disease varied from mild to moderate diarrhea, which resolved in 2 to 3 days. Orally infected animals developed agglutinin titers of 8 to 256 within 3 weeks, while those infected intravenously developed titers of 256 to 2,048. Ferrets which had recovered from campylobacteriosis all developed high titers of agglutinating and bacterial antibodies but were readily colonized by subsequent oral inoculation with the same strain of C. jejuni. Orally infected ferret kits 3 to 6 weeks of age exhibited the same general pattern of infection and disease as adults, but diarrhea was somewhat more severe. Kits resolved their diarrhea in 1 to 6 days and developed agglutinin titers in serum of 16 to 32 within 3 weeks. A series of five oral or rectal inoculations of kits during the 5- to 9-week age interval resulted in progressively shorter clearance times and eventual strain-specific resistance against infection, as well as disease. Gnotobiotic adults showed the same pattern of strain-specific accelerated clearance and resistance to disease. Kits born to immune dams with high levels of whey antibodies had passively acquired serum agglutinin titers of 256 to 2,048. These kits showed no resistance to colonization with the homologous strain of C. jejuni but were completely refractory to diarrhea. These observations suggest that (i) some form(s) of specific immunity, rather than factors relating solely to age or normal flora, is responsible for resistance to C. jejuni colonization and disease production and (ii) humoral immunity at a level that does not prevent colonization can protect against enteric disease caused by this organism.

Prevalence of Campylobacter jejuni in ranch mink at pelting: Cultural, serological, and histological evidence of infection.

This survey of 500 mink on three Wisconsin ranches at pelting gives an estimate of the prevalence of Campylobacter jejuni in the feces of clinically normal animals. On ranches 1 and 2, which used wet feed, C. jejuni was isolated by colon content culture from 7% and 32% of mink one year, and 43% and 13% the next year; the 200 bile samples tested were culture-negative. On ranch 3, which fed a pelleted ration, the organism was never isolated. Among culture-positive mink tested, 22 of 55 had bacterial agglutination serum titers to homologous and/or heterologous Campylobacter isolates from the ranch of origin. Four of 23 culture-negative animals tested had titers. No histological evidence of inflammatory changes in the lower ileum and/or colon was found, although Campylobacter-like organisms were rarely seen in silver-stained sections from both culture-negative and culture-positive animals. We conclude that the presence of C. jejuni in the mink gut does not necessarily indicate a role in gastrointestinal disease.

Derivation of gnotobiotic ferrets: perinatal diet and hand-rearing requirements.

A procedure is described which has resulted in successful gnotobiotic derivation of the domestic ferret. The most critical element of this hand-rearing procedure was found to be diet, with ferret milk being required for at least the first 7 days. Puppy milk replacer was phased in during the next 10 days, and enriched cow's milk sufficed thereafter. Around-the-clock sip-feeding with fire-polished Pasteur pipettes was necessary at intervals gradually increasing from 1 to 1.5 hours at birth to 3 hours by day 21. Temperature regulation was accomplished with an electric heating pad placed eccentrically under towel bedding to provide a 30 degrees-40 degrees C gradient, along which the kits positioned themselves to their own comfort. Techniques are described for minimizing fatalities due to dehydration, milk-aspiration pneumonia, underfeeding, overfeeding, gut stasis and obstipation. Internal hemorrhage, the greatest single cause of mortality in this study, manifested at day 13 and involved all kits by day 17. Despite immediate vitamin K1 dietary supplementation, five of the seven remaining kits died of hemorrhage by day 19. Around day 50, the two surviving kits were weaned from milk to dry commercial cat and ferret diets supplemented with vitamins K, C, A, D, E and B-complex and were reared to adulthood on this diet.

Suppression of specific IgM, IgA and IgG responses in mice treated with anti-delta from birth.

In contrast to previous studies, we report that heterologous anti-delta antibodies can act as a powerful multi-class humoral immunosuppressant. Primary direct plaque-forming cell (IgM) responses of BALB/c mice injected from birth with a rabbit anti-delta antiserum were reduced to 3% of control levels against a T-dependent antigen (sheep red blood cells), and to 2% against a T-independent antigen (dextran); IgA responses against 2 intraduodenal injections of cholera toxin were reduced to 7% of control levels. Other secondary immune responses of anti-delta-suppressed mice were suppressed to a lesser degree. IgM plaque responses generated by 2 injections of sheep red cells, for example, were reduced to 50% of control levels, with reduction of the corresponding IgG response to 42%. The multi-class suppression observed indicates a clonal differentiation pathway in which the IgD+ cell develops into IgM-, IgA- and IgG-secreting cells. In light of reports that the IgD+ cell is antigen sensitive, our demonstration that IgD is capable of delivering the same sort of immunosuppressive signal in response to anti-heavy chain antibodies that IgM delivers also suggests, though it does not establish, an antigen-receptor role for IgD.

Comparison of coagglutination, latex agglutination, and enzyme-linked immunosorbent assay for detection of Haemophilus influenzae type b infection.

Two commercially available kits were compared to an enzyme-linked immunosorbent assay (ELISA) for detection of Haemophilus influenzae type b antigen polyribophosphate in clinical specimens with infections proved by culture. Methods employed by the kits were latex agglutination (LA) and staphylococcal coagglutination (COA). The COA kit detects H. influenzae type b and types a and c-f with a polyvalent antiserum, whereas the ELISA assay and the LA kit detect only type b. A total of 139 specimens (41 spinal fluid, 35 urine, 25 serum, and 38 sputum) were tested. All spinal fluid samples positive by culture were positive by all three procedures. Of urine specimens from patients with a variety of H. influenzae type b infections, 13 of 15 were positive by ELISA, 8 of 15 by COA, and 8 of 14 by LA. Of serum samples collected from the same patients at various times during their illness, 8 of 15 were positive by ELISA, 6 of 15 by COA, and 10 of 15 by LA. Of sputum samples positive by culture for H. influenzae type b, 14 of 17 were positive by ELISA, 9 of 17 by COA, and 4 of 16 by LA. The ability to detect additional serotypes of H. influenzae was shown by the COA kit, which detected H. influenzae type a in spinal fluid from a patient with type a meningitis proved by culture.