Immunosuppression of mice injected with heterologous anti-immunoglobulin heavy chain antisera.

Neonatal injection of mice with rabbit anti-micro antiserum has been shown to produce complete loss of direct and indirect plaque-forming responses to sheep erythrocytes as well as loss of serum IgM and severe depressions of all other serum immunoglobulins. Similar injection of anti-gamma1gamma2 or anti-gamma1 antibodies effects a loss of the indirect response but induces relatively minor alterations in serum Ig levels. Delaying initiation of anti-micro treatment until young adulthood results in a somewhat diminished effect on plaque-forming responses and serum Ig levels but triggers the release of high serum levels of an aberrant micro-bearing protein. Anti-micro suppression of genetically thymusless mice indicates that at least part of the target cells for suppression are bone marrow derived. A working hypothesis for the maturation of humoral antibody-producing cell lines as it relates to these data is discussed.

Suppression of reaginic antibody (IgE) formation in mice by treatment with anti-mu antiserum.

Neonatally initiated injection of anti-mu antiserum in mice has been shown to suppress the formation of reaginic antibodies in response to infection with the intestinal nematode, Nippostrongylus brasiliensis. This observation supports the hypothesis that IgE-producing cells arise from IgM-bearing precursors.

Maintenance of skin xenografts of widely divergent phylogenetic origin of congenitally athymic (nude) mice.

Congenitally athymic (nude) mice accepted for their lifetime intact skin grafts from distantly related mammals (cat, human) and birds (chicken). They also failed to immunologically reject skin grafts from reptiles (lizards) and amphibians (tree frog), although the skin in these grafts underwent varying degrees of disorganization. A definitive role for the thymic defect in this failure to reject xenografts was established by showing that thymus implantation into nude mice enabled them to reject such foreign skin.

Transplantation of cultured thymic fragments. II. Results in nude mice.

Nine nude mice were transplanted with cultured thymic fragments derived from syngeneic (three recipients) or allogeneic (six recipients) sources. All transplanted mice survived for periods of up to 8-10 mo thereafter, at which time they were sacrificed. Weight gain had been progressive and the animals were in excellent health. Four nontransplanted littermates housed in the same cages died at the age of 4 mo. In the nontransplanted mice, the usual deficits of T and B cells were observed. In transplanted mice, normalization of IgG1 and IgA levels as well as IgG antibodies to sheep erythrocytes and precipitating antibodies to rabbit serum occurred. Lymphocyte counts and Thy-1 bearing cells increased to approximately 50% of normal values. Proliferative responses to phytohemagglutinin and concanavalin A, mixed leukocyte reactivity, and cell-mediated lympholysis were variably restored from approximately 10-100% of normal. Attained responses were the same in recipients of syngeneic or allogeneic tissues and these, in turn, were equal or superior to responses measured in animals transplanted with whole noncultured thymuses. Skin grafts from third party donors were vigorously rejected, whereas those derived from second party (allogeneic thymus donor strain) may have been accepted or slowly rejected. Cultured thymic fragments, consisting primarily of epithelial elements, can effectively repair the thymic deficiency of nude mice. Experiments to date do not indicate that syngeneic tissues enjoy an advantage over allogeneic grafts in this restoration procedure.

Long-term maintenance of psoriatic human skin on congenitally athymic (nude) mice.

Transplantation of involved psoriatic and nonpsoriatic human skin onto congenitally athymic (nude) mice has been performed successfully. Although biopsies at selected intervals demonstrate that the excess glycogen deposition normally seen in psoriasis is no longer consistently present, the psoriatic grafts did retain the usual characteristic histologic differences throughout the life of the animal, up to 11 weeks. This grafting procedure potentially represents a useful method whereby the study of psoriasis can be made in a nonhuman, living system.

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.

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.

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.

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)

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.

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.

Complete humoral immunosuppression of mice by rabbit anti-mu antibodies passing the murine placenta.

Female BALB/c mice were immunosuppressed with a series of neonatally initiated rabbit anti-mu serum injections, which eliminated serum IgM and greatly delayed production of antibodies against normal rabbit serum (anti-NRS). Females thus prepared maintained circulating anti-mu levels for several months. Study of the progeny from pregnancies completed during this maintenance period revealed that rabbit anti-mu antibodies readily cross the murine placenta but are not passed in murine colostrum at levels detectable by the technique used. Anti-NRS antibodies actively produced in NRS-injected control females do cross the placenta, but do so only irregularly and poorly; these antibodies may, however, be detected consistently at relatively low levels in colostrum. Suppression of humoral immunoglobulin synthesis in most mice prenatally exposed to anti-mu antibodies by transplacental passage appeared complete, even including loss of the remnant IgG levels which are consistently seen in mice first exposed to anti-mu at birth. The appearance of serum IgG and anti-NRS antibodies along with the complete absence of serum IgM in mice recovering from suppression suggests that active IgM synthesis and secretion may not be a prerequisite for the IgM to IgG "switch". Immune recovery occurred even in completely immunosuppressed mice after anti-mu injections were discontinued; the mechanism of recovery is not certain.

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.

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.

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.

Indirect sandwich enzyme-linked immunosorbent assay for rapid detection of Streptococcus pneumoniae type 3 antigen.

An indirect sandwich enzyme-linked immunosorbent assay for the detection of the polysaccharide antigen of type 3 pneumococcus (SSS-III) is reported. Polystyrene balls with attached anti-SSS-III antibody serve as the solid phase, and antigen is detected using an alkaline phosphatase-labeled antiglobulin conjugate. The reaction is quantitated by spectrophotometry. Concentrations of antigen are estimated by comparison with standard curves prepared with purified SSS-III. For this assay, the practical lower limit of detection of SSS-III is approximately 2 to 3 ng/ml, thus making the test sensitivity about 25 times that reported for counterimmunoelectrophoresis. In preliminary tests with simulated human clinical specimens, none of the body fluids tested (spinal fluid, serum, urine, and sputum) interfered with detection of antigen, nor did they produce false-positive or false-negative results. Streptococcus pneumoniae type 3 whole organisms were readily detected in simulated clinically positive sputum specimens. Cross-reactions were not observed with Haemophilus influenzae type b, group B Streptococcus, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.

Indirect sandwich enzyme-linked immunosorbent assay for rapid detection of Haemophilus influenzae type b infection.

We report the development and testing of an enzyme-linked immunosorbent assay with excellent sensitivity for the detection of Haemophilus influenzae type b (HI(b)) antigen in clinical specimens from patients with HI(b) meningitis. The assay, an indirect sandwich technique, uses polystyrene balls as a solid phase and an alkaline phosphatase-labeled goat anti-rabbit globulin conjugate. Specimens are incubated with polystyrene balls armed with burro anti-HI(b) antiserum, and recognition antibody is visualized by addition of alkaline phosphatase-labeled anti-globulin, together with the enzyme substrate p-nitrophenyl phosphate. Concentrations of antigen are determined from standard curves prepared by using purified HI(b) capsular antigen polyribophosphate. The assay reproducibly detects polyribophosphate at concentrations between 1 and 5 ng/ml. Cross-reactions have not as yet been encountered in simulated and authentic clinical specimens containing other species including Escherichia coli, Klebsiella pneumoniae, group B Streptococcus, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria meningitidis, and Listeria monocytogenes. In preliminary tests with 11 spinal fluid specimens, 2 serum specimens, and 5 urine specimens from patients with culture-proved HI(b) meningitis, antigen was detected in all specimens in concentrations ranging from 1 to 7,000 ng/ml. Antigen was not detected in any of 62 clinical specimens which were culture negative for HI(b), including 11 spinal fluid specimens from patients with bacterial meningitis caused by microorganisms other than HI(b). The enzyme-linked immunosorbent assay technique described here is considerably simpler than radioimmunoassay and, based on concurrent tests with 14 positive clinical specimens, may be more sensitive than counterimmunoelectrophoresis. It seems, therefore, to hold considerable promise for clinical use in rapid detection of systemic HI(b) infections.

Expulsion of Nippostrongylus brasiliensis from mice lacking antibody production potential.

Expulsion of the intestinal nematode of rodents, Nippostrongylus brasiliensis, was assessed in mice experiencing the immunosuppressive effects of anti-micron antibodies. Anti-micron treatment resulted in complete elimination of IgM and severe reduction of IgG1, IgG2 and IgA serum immunoglobulin levels. Specific antibody responses to sheep erythrocytes were virtually eliminated in anti-micron-treated mice as determined by direct and indirect plaque-forming-cell responses, haemagglutination and haemolytic assays. Using passive cutaneous anaphylaxis (PCA) and indirect haemagglutination (IHA) assays, antibodies against N. brasiliensis were not detectable in sera of anti-micron-treated mice; control mice, however, generated strong PCA and IHA responses. The kinetics of worm egg production coupled with adult worm recoveries at necropsy indicate that anti-micron treatment of mice had little or no effect on the capacity of mice to expel N. brasiliensis even though antibody production potential had been eliminated in these mice. Thus, anti-worm antibodies may not be requisite in the mechanism of N. brasiliensis expulsion from mice.

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.