Diverse Host Immune Responses of Different Geographical Populations of the Coconut Rhinoceros Beetle to Oryctes Rhinoceros Nudivirus (OrNV) Infection.

Incursions of the coconut rhinoceros beetle (CRB), Oryctes rhinoceros, into different islands in the South Pacific have been detected in recent years. It has been suggested that this range expansion is related to an O. rhinoceros haplotype reported to show reduced susceptibility to the well-established classical biocontrol agent, Oryctes rhinoceros nudivirus (OrNV). Our understanding of the genetic characteristics which distinguish the population of O. rhinoceros that has recently established in Solomon Islands from other well-established populations across the region is very limited. Here, we hypothesized that the recently established O. rhinoceros population should have greater innate immune responses when challenged by OrNV than those of well-established and native O. rhinoceros populations. We used the RNA sequencing (RNA-Seq) approach to generate gene expression profiles of midgut tissue from OrNV-infected and noninfected individuals collected in the Solomon Islands (recent incursion), Papua New Guinea and Fiji (previously established), and the Philippines (within the native range). The collections included individuals from each of the three major mitochondrial lineages (CRB-G, CRB-PNG, and CRB-S) known to the region, allowing us to explore the specific responses of each haplotype to infection. Although insects from the Philippines and Solomon Islands that were tested belong to the same mitochondrial lineage (CRB-G), their overall responses to infection were different. The number of differentially expressed genes between OrNV-infected and noninfected wild-caught individuals from the four different locations varied from 148 to 252. Persistent OrNV infection caused a high level of induced antimicrobial activity and immune responses in O. rhinoceros, but the direction and magnitude of the responses were population specific. The insects tested from the Solomon Islands displayed extremely high expression of genes which are known to be involved in immune responses (e.g. coleoptericin, cecropin, and serpin). These variations in the host immune system among insects from different geographical regions might be driven by variations in the virulence of OrNV isolates, and this requires further investigation. Overall, our current findings support the importance of immunity in insect pest incursion and an expansion of the pest's geographic range. IMPORTANCE Oryctes rhinoceros nudivirus (OrNV) is a double-stranded DNA (dsDNA) virus which has been used as a biocontrol agent to suppress coconut rhinoceros beetle (CRB) in the Pacific Islands. Recently a new wave of CRB incursions in Oceania is thought to be related to the presence of low-virulence isolates of OrNV or virus-tolerant haplotypes of beetles (CRB-G). Our comparative analysis of OrNV-infected and noninfected CRBs revealed that specific sets of genes were induced by viral infection in the beetles. This induction was much stronger in beetles collected from the Solomon Islands, a newly invaded country, than in individuals collected from within the beetle's native range (the Philippines) or from longer-established populations in its exotic range (Fiji and Papua New Guinea [PNG]). Beetles from the Philippines and the Solomon Islands that were tested in this study all belonged to the CRB-G haplotype, but the country-specific responses of the beetles to OrNV infection were different.

Retrospective anti-tetanus antibody responses of zoo-based Asian elephants (Elephas maximus) and rhinoceros (Rhinocerotidae).

Tetanus toxoids (TT) commercially available for use in horses and livestock are commonly used to vaccinate elephants and rhinoceros that are in human care. Although recommendations for booster intervals have changed in human and horse protocols to reduce the risks associated with hyper-immunity (i.e. B-cell anergy and hypersensitivity reactions) these have generally not been adopted in zoo protocols. Additionally, there is no evidence to demonstrate commercial TT immunogenicity in rhinoceros. In this study, a preliminary analysis of rhinoceros antibody responses to TT was conducted, in addition to an exploration of the impact of various booster frequencies on antibody responses in elephant. Retrospective analysis of archived serum samples was conducted for 9 Asian elephants (Elephas maximus), 7 southern black (Diceros bicornis minor), one southern white (Ceratotherium simum simum), and two greater one-horned (Rhinoceros unicornis) rhinoceros. Pre-vaccination (baseline) samples and those following priming vaccination (rhinoceros only), annual and non-annual boosters were targeted. A commercially available competitive ELISA kit was used to quantify serum anti-TT antibodies. Average baseline and post-vaccination anti-tetanus antibody concentrations were greater in elephant (92 mg/L ± 42, n = 3, N = 3; 125 ± 76, n = 82, N = 9) than in rhinoceros (47 mg/L ± 39, n = 8, N = 8; 44 mg/L ± 37, n = 16, N = 7). Rhinoceros antibody concentrations did not differ markedly following vaccinations from their naturally acquired high pre-vaccination concentrations. Eight elephants demonstrated antibody maintenance for 3-5 years without a tetanus booster. Additionally, although five out of nine elephants developed local reactions consistent with delayed type IV hypersensitivity following some boosters, there was no association between high antibody concentrations and increased incidence of adverse reactions. In addition, no decrease in antibody concentrations was detected as a result of annual vaccination in elephants, though this does not entirely rule out potential for B-cell anergy.

Serologic response to West Nile virus vaccination in the greater one-horned rhinoceros (Rhinoceros unicornis).

Vaccination has been an important component of preventative health care programs of North American zoologic institutions in their protection of valuable species against West Nile virus (WNV) infection since its detection in 1999. Although approved only for horses, commercial WNV vaccine has been used for the purpose of protection of nondomestic species, including avian, equid, and rhinoceros species. Currently, there are two commercial equine vaccines available, a killed vaccine and a recombinant viral-vectored vaccine. Both products have been used for the vaccination of Greater One-horned rhinoceroses (Rhinoceros unicornis) held in North American zoologic institutions. In this study, the efficacy of these vaccines was evaluated in Greater One-horned rhinoceroses based on the humoral immune response stimulated by vaccine administration. Five rhinoceroses were vaccinated in 2005 by using the killed equine vaccine and four received boosters in 2006 by using the recombinant vaccine. Rhinoceroses were evaluated for differences in pre- and postvaccination neutralizing antibody titer and gamma and beta globulins on serum protein electrophoresis. No changes were observed after administration of the killed vaccine; however, antibody titers were observed in two of four rhinoceroses after administration of the recombinant vaccine. No significant changes were observed in the serum protein electrophoresis after either vaccine. Based on these findings, the WNV recombinant vaccine appeared to induce a more measurable humoral immune response than the killed product in the Greater One-horned rhinoceros. However, further investigation of both vaccines is warranted to evaluate whether changes in the frequency of administration, dosage, or adjuvant might stimulate an improved humoral response in these animals.

Cloning, sequencing and expression of white rhinoceros (Ceratotherium simum) interferon-gamma (IFN-gamma) and the production of rhinoceros IFN-gamma specific antibodies.

Bovine tuberculosis (BTB) is endemic in African buffalo (Syncerus caffer) in the Kruger National Park (KNP). In addition to buffalo, Mycobacterium bovis has been found in at least 14 other mammalian species in South Africa, including kudu (Tragelaphus strepsiceros), Chacma baboon (Papio ursinus) and lion (Panthera leo). This has raised concern about the spillover into other potentially susceptible species like rhinoceros, thus jeopardising breeding and relocation projects aiming at the conservation of biodiversity. Hence, procedures to screen for and diagnose BTB in black rhinoceros (Diceros bicornis) and white rhinoceros (Ceratotherium simum) need to be in place. The Interferon-gamma (IFN-gamma) assay is used as a routine diagnostic tool to determine infection of cattle and recently African buffalo, with M. bovis and other mycobacteria. The aim of the present work was to develop reagents to set up a rhinoceros IFN-gamma (RhIFN-gamma) assay. The white rhinoceros IFN-gamma gene was cloned, sequenced and expressed as a mature protein. Amino acid (aa) sequence analysis revealed that RhIFN-gamma shares a homology of 90% with equine IFN-gamma. Monoclonal antibodies, as well as polyclonal chicken antibodies (Yolk Immunoglobulin-IgY) with specificity for recombinant RhIFN-gamma were produced. Using the monoclonals as capture antibodies and the polyclonal IgY for detection, it was shown that recombinant as well as native white rhinoceros IFN-gamma was recognised. This preliminary IFN-gamma enzyme-linked immunosorbent assay (ELISA), has the potential to be developed into a diagnostic assay for M. bovis infection in rhinoceros.

Corticosteroid-induced suppression of in vitro lymphocyte proliferation in four captive rhinoceros species.

Captive African black rhinoceroses (Diceros bicornis) are unusually susceptible to several diseases not commonly observed in any of the other three rhinoceros species maintained in captivity. The potential role of corticosteroids (either endogenously produced or exogenously administered) in the development of these sometimes fatal diseases has been questioned. In this study, the suppressive effects of two therapeutic corticosteroids (dexamethasone and hydrocortisone) on in vitro lymphocyte proliferation was examined in four rhinoceros species, including the Sumatran rhinoceros (Dicerorhinus sumatrensis, n = 3), Indian rhinoceros (Rhinoceros unicornis, n = 4), African black rhinoceros (n = 10), and African white rhinoceros (Ceratotherium simum, n = 5). Three blood samples collected from each rhinoceros 1 mo to 1 yr apart provided replicates for the study. Both dexamethasone and hydrocortisone suppressed (P < 0.05) lymphocyte proliferation stimulated by B-cell mitogens (pokeweed and lipopolysaccharide) and T-cell mitogens (phytohemagglutinin and concanavalin A). Suppressive effects of the glucocorticoids differed (P < 0.05) depending on the mitogen used to stimulate the lymphocytes. Overall, dexamethasone was a more potent suppressor of cell proliferation when compared with hydrocortisone (P < 0.05). However, black rhinoceros cell proliferation in response to any of the four mitogens was never completely suppressed, even in cultures containing the highest steroid concentration tested (10(-3) M). The effect of the two corticosteroids differed slightly among the rhinoceros species and subspecies tested, but there was no evidence that eastern or southern black rhinoceros lymphocytes were more sensitive to the suppressive effects of corticosteroids than the other rhinoceros species.

Comparative studies of mitogen- and antigen-induced lymphocyte proliferation in four captive rhinoceros species.

Cellular immune function in four rhinoceros species was evaluated by way of in vitro lymphocyte proliferation responses to mitogenic and antigenic stimuli to establish normative data on white blood cell activity for each species and to identify species-specific differences that might help explain the predisposition of black rhinoceroses (Diceros bicornis) to disease. A cross section of the U.S. rhinoceros population encompassing all four captive species was sampled, including the Sumatran rhinoceros (Dicerorhinus sumatrensis) (n = 3); Indian rhinoceros (Rhinoceros unicornis) (n = 4); African black rhinoceros (n = 16); and African white rhinoceros (Ceratotherium simum) (n = 10). Of the four species evaluated, African black rhinoceroses exhibited the weakest (P < 0.05) lymphocyte proliferative responses to the mitogens: pokeweed (0.1 microg/ml), phytohemagglutinin (0.3 microg/ml), and concanavalin A (5.0 microg/ml). Total cell density at the end of culture was only 70% of that achieved with lymphocytes isolated from African white rhinoceroses, Indian rhinoceroses, and Sumatran rhinoceroses. However, lymphocyte response to bacterial endotoxin lipopolysaccharide was similar (P > 0.05) across species. Antigenic stimulation produced much weaker responses than mitogenic stimulation. No differences (P > 0.05) were observed among rhinoceros species in response to 1 and 10 microg/ml of Leptospira icterohemorrhagiae or Leptospira gryppotyphosa. Lymphocytes from African white rhinoceroses proliferated weakly in the presence of Aspergillus fumigatus filtrate, whereas lymphocytes from the southern black rhinoceros subspecies appeared slightly suppressed in the presence of increasing doses (0.1, 1, and 10 microg/ml) of Aspergillus filtrate. This comparative data set characterizing lymphocyte proliferation in the rhinoceros reveals several differences in immune cell responses among rhinoceros species and provides some evidence that lymphocytes of captive African black rhinoceroses are less vigorous than those of the other rhinoceros species.

Evolutionary history of MHC class I genes in the mammalian order Perissodactyla.

We carried out an analysis of partial sequences from expressed major histocompatibility complex (MHC) class I genes isolated from a range of equid species and more distantly related members of the mammalian order Perissodactyla. Phylogenetic analysis revealed a minimum of six groups, five of which contained genes and alleles that are found in equid species and one group specific to the rhinoceros. Four of the groups contained only one, or very few sequences, indicating the presence of relatively nonpolymorphic loci, while another group contained the majority of the equid sequences identified. These data suggest that a diversification of MHC genes took place after the split between the Equidae and the Rhinocerotidae yet before the speciation events within the genus Equus.

Characterization of a red blood cell antigen in donkeys and mules associated with neonatal isoerythrolysis.

A red cell antigen of donkeys and mules was identified using antibodies in serum from a mare which produced a mule foal affected with neonatal isoerythrolysis (NI). Subsequently antibodies with similar activity were identified in the sera of other mares which had produced mule foals and were produced by immunization of horses with blood from donkeys. The antigen detected by these antibodies does not correspond to any recognized horse red cell alloantigen. This may be a xenoantigen since all donkeys (and mules) tested have shared this antigen and all horses tested have lacked the antigen. The results suggest that all mule pregnancies (donkey sire x horse dam) are incompatible with regard to this factor and a potential for neonatal isoerythrolysis exists in all cases.

Comparison of five tests for the serologic diagnosis of myiasis by Gasterophilus spp. larvae (Diptera: Gasterophilidae) in horses and donkeys: a preliminary study.

Sera from 41 horses and 159 donkeys, from twelve States of México, were tested to ascertain anti-Gasterophilus circulating antibodies by double immunodiffusion (DD), counterimmunoelectrophoresis (CIE), indirect haemagglutination (IH), thin layer immunoassay (TIA) and diffusion-in-gel ELISA (DIG-ELISA) methods using crude somatic antigen from third instar larvae of G. intestinalis (DeGeer). At necropsy, 33/41 horses and 24/159 donkeys were found to be parasitized by G. intestinalis and/or G. nasalis (L.). Gasterophilus intestinalis was the species most commonly found in the equines. Analysis of the sera from the infected animals by DD showed positive results of 21.2% in horses and of 8% in donkeys. Screening the sera with CIE gave sensitivities of 69.7% in horses and of 32% in donkeys. Examination of the sera by IH showed positive results of 87.9% and of 48% in horses and donkeys, respectively. Testing the sera with TIA gave sensitivities of 93.9% in horses and of 96% in donkeys. Analysis of horses' sera by DIG-ELISA showed a sensitivity of 93.9%.

Circulation of African horsesickness virus in zebra (Equus burchelli) in the Kruger National Park, South Africa, as measured by the prevalence of type specific antibodies.

In the Kruger National Park 75% of zebra foals are born in October-March and they lose their passive immunity against African horsesickness virus (AHSV) when they are 5-6 months old. One month later infection with different serotypes of AHSV amounts to 31% and thereafter infections increase rapidly to almost 100% before the foals are 12 months old. The capability of zebra to maintain AHSV is clearly illustrated by the continuing infections during every month of the year with a peak period in winter. This peak is ascribed to the presence of large numbers of susceptible foals in the presence of active Culicoides species.

Immunokinetics of equine herpesvirus 1 in donkey mares: suppression of secondary cell-mediated response.

To study the immunokinetics of equine herpesvirus 1 (EHV1), donkey mares were immunised with a laboratory strain of EHV1, or with recommended doses of Pneumabort-K vaccine (EHV1 Army 183 strain, formalin-inactivated, with an oil adjuvant) and a booster was given after three months. Humoral immune responses were studied by employing a virus neutralisation (VN) test. A leucocyte migration inhibition test (LMIT) was employed for the assay of cellular immune responses. The VN antibody titre reached 1:64 or 1:128 after primary immunisation and showed a marginal increase (1:256) after secondary immunisation with either of the immunogens. After the primary dose of immunogen, there was a gradual increase in host cellular response which persisted for up to three months. However, on secondary immunisation, cell-mediated immune response was short-lived and weak compared to the primary response with both immunogens. This could be one possible explanation for breakdown of anti-EHV1 immunity leading to abortion in immunised mares.

Antibodies in horses, mules and donkeys following monovalent vaccination against African horse sickness.

A total of 256 sera collected from three species of domesticated equidae in four different Spanish provinces were examined 1-4 months after the administration of attenuated monovalent African horse sickness virus (AHSV) serotype 4 vaccine. Approximately 10% of the sera were negative by ELISA, virus neutralization, agar gel immuno-diffusion and complement fixation tests. Similar negative reactions were recorded with sera from two ponies after experimental primary vaccination. The rapid rise in antibodies in sera from these two ponies, after a second dose of vaccine, suggested they would probably have been immune to challenge. It is therefore suggested that the apparent absence of antibodies against AHSV in some animals after primary vaccination may not necessarily indicate a total lack of protection.

Expression of major histocompatibility complex (MHC) class 1 molecules on early trophoblast.

The expression of class I Major Histocompatibility Complex (MHC) molecules by early trophoblast of intraspecific horse and donkey, interspecific mule and extraspecific donkey-in-horse conceptuses was determined using a rat monoclonal antibody (MAC 291) in a peroxidase anti-peroxidase immunohistochemical technique. Most non-invasive allantochorion of horse, donkey and mule conceptuses did not express class I MHC molecules at any stage of gestation except in small isolated patches of pseudostratified trophoblast lying adjacent to the openings of endometrial glands. In contrast, MHC class I molecules were expressed strongly on horse chorionic girdle cells at Days 33 and 34 of gestation, just prior to their invasion. However, class I MHC was down-regulated with the differentiation of these girdle cells into mature gonadotrophin-secreting endometrial cup cells between Days 40 and 45 so that by Days 55-65, class I molecules were no longer detected on endometrial cups. Similarly, all endometrial cups originating from 3 intraspecific donkey conceptuses at Days 41, 59 and 82 and 2 interspecific mule conceptuses at Days 46 and 47 were negative for class I molecules. A total of 7 extraspecific donkey-in-horse pregnancies, in which no endometrial cups from and implantation is abnormal, were established by embryo transfer. The chorionic girdle recovered from a single donkey-in-horse conceptus at Day 35 of gestation stained strongly for MHC class I molecules. Later in gestation (Days 73-91) and in contrast to the other forms of equine pregnancy examined, most unimplanted, failing donkey allantochorion was strongly stained for MHC class I molecules and had large numbers of lymphocytes in the adjacent endometrial stroma. The hypothesis is raised that the mechanisms that normally suppress the expression of MHC class I molecules by the epithelial trophoblast layer of the equine placenta can only function if the apical surface of the cells is in close and stable contact with other tissues such as the endometrial epithelium.

Antibodies to some pathogenic agents in free-living wild species in Tanzania.

A total of 535 sera from eight species of wildlife were collected from different game areas in Tanzania between 1987 and 1989. These sera were tested for antibodies against foot-and-mouth disease, bovine herpes virus types 1 and 2, lumpy skin disease, bovine viral diarrhoea, Akabane, bovine ephemeral fever, bluetongue, enzootic bovine leucosis, African horse sickness and African swine fever viruses and Brucella abortus based on the expected species susceptibility. Sera from buffalo Syncerus caffer, wildebeest Connochaetes taurinus and topi Damaliscus korrigum contained antibodies against the majority of the pathogens tested. Antibodies to fewer pathogens were detected in sera from the other species. No antibodies to lumpy skin disease virus were detected in any of the sera examined. African horse sickness antibodies were detected in sera from Zebra and African swine fever antibodies were detected in wart hog. The occurrence of antibodies to these agents suggests that wild species act as reservoirs of infection for some of these pathogens. However, until the susceptibility of individual species is proven by isolation of the aetiological agents their role must remain speculative.

Suppression of lymphocyte proliferation by a greater than 30,000 molecular weight factor in horse conceptus-conditioned medium.

In this experiment we have identified and partially characterized the immunosuppressive activity of preimplantation horse conceptus-conditioned medium (HCCM). Horse conceptuses were nonsurgically flushed from mares at Days 9-10 (n = 6), 15-16 (n = 3), and 25-26 (n = 3). After incubating the conceptuses for 24 h in RPMI-1640 supplemented with 15% fetal calf serum (FCS) and 1% penicillin/streptomycin, HCCM was obtained from cultures and tested for immunosuppressive activity in lymphocyte proliferation assays. Peripheral blood lymphocytes obtained from randomly selected mares were stimulated with mitogens (pokeweed mitogen [PWM], concanavalin A [Con A], and phytohemagglutinin [PHA]) in cultures supplemented with 0%, 25%, or 50% HCCM. HCCM from all cultures suppressed lymphocyte proliferation induced by all three mitogens (p less than 0.001). After being subjected to various treatments (heating, freeze-thawing, and nitrocellulose filtration), HCCM maintained its full biological suppressor activity. Amicon microconcentrators with 10,000 and 30,000 molecular weight (MW) exclusion filter membranes were used to fractionate HCCM by molecular weight. The suppressor factor was found to be in the greater than 30,000 MW fraction. HCCM was further tested interspecifically on donkey and goat lymphocytes stimulated with PWM. HCCM did suppress proliferation of interspecific lymphocytes (p less than 0.01); however, the suppressive capacity of HCCM in caprine lymphocyte cultures was less (p less than 0.05) than that observed in equine cultures. These data support the hypothesis that the horse conceptus produces an immunoregulatory factor. This factor is extremely stabile and appears to exhibit some degree of species-specificity. The production and immunosuppressive effectiveness of such a factor may play an important role in maintaining the fetal allograft throughout gestation.

Feedback by early and late primary antisera on the primary and secondary adoptive immune responses of mice to burro erythrocytes.

Experiments on antibody feedback inhibition of the immune response have confirmed that control is more effective against a primary response than against a secondary response. The cells producing antibodies in primary and secondary responses are different both in terms of number of IgFC and amount of antibody produced by individual IgFC (plaque size). Late primary anti-burro RBC sera (greater than 200 days), despite low titers, are, on a volume for volume basis, feedback inhibitors at least as good as early (8-12 days) primary antisera on primary responses but are more effective in suppressing secondary responses (B memory cells). Late primary antisera, due to the process of affinity maturation, have a high affinity for antigen. The suppressive effect of early and late antisera is equally removable by absorption with burro erythrocytes: a result which it is thought, decreases the likelihood of feedback by anti-idiotype being involved in the observed suppression. It is suggested that feedback antibody acts (a) in competition with receptors, inter alia removing antigen into immunologically irrelevant pathways, (b) by a process involving the linking of antigen to Fc receptors, and (c) as a blocking antibody coating B cells (Bm) or APC which are already binding epitopes, thus preventing their cooperation with specific helper or other accessory cells.

Use of high-performance liquid chromatography for the purification of antibodies and antibody conjugates and the study of antibody-antigen interactions.

The purification and characterization of polyclonal and monoclonal antibodies and antibody-enzyme conjugates by high-performance liquid chromatography (HPLC) using a Protein Pak 300 SW column and a Protein Pak DEAE anion-exchange column is described. The following polyclonal antibodies were examined: (i) donkey anti-mouse immunoglobulin G (IgG), (ii) mouse IgG, (iii) sheep anti-human IgG and (IV) goat anti-human factor VIII. HPLC was used to analyse the purity of horseradish peroxidase conjugates of rabbit anti-mouse IgG and donkey anti-mouse IgG. In the case of donkey anti-mouse IgG, each stage of the production of the purified antibody and antibody-enzyme conjugate was monitored by HPLC. HPLC was used to examine monoclonal mouse anti-human T cell and mouse anti-human apoliproprotein B antibodies. The presence of antibody in ascites fluid from mice bearing Landschütz ascites tumour cells was also detected. The antigen-antibody reaction between human serum albumin and anti-human serum albumin was demonstrated using HPLC and this procedure should offer a novel method for studying antigen-antibody interaction.

Anti-antibody enhancement of iodine-131 anti-CEA radioimmunodetection in experimental and clinical studies.

Imaging of tumors with radiolabeled antibodies, especially when located in the blood-rich visceral organs, may be improved through administration of a second antibody directed against the primary tumor-associated antibody. In hamsters bearing a human colonic carcinoma xenograft producing carcinoembryonic antigen (CEA), we injected donkey anti-goat IgG 24 hr after administration of 131I-labeled goat anti-CEA IgG and achieved enhanced tumor imaging 24-48 hr later, with a significant relative decrease of radioactivity in blood and all major organs except the spleen. In seven of nine patients, this method of anti-antibody clearance of nontargeted radioactive murine monoclonal antibodies revealed sites of cancer, including liver metastases. Characterization of radioactivity in the plasma before and after administration of the second antibody confirmed that complexes were quickly formed between primary and secondary antibodies, and imaging of the patients revealed a rapid uptake of radioactivity in the liver at 2 hr that dissipated within 24 hr. Radioactivity in the spleen gradually increased over time. The method of anti-antibody immunological enhancement of cancer imaging is feasible and may reveal tumor sites missed by conventional imaging.

A cascade of T-T interactions, mediated by the linked recognition of antigen, in the induction of T cells able to help delayed-type hypersensitivity responses.

Previous work has shown that specific helper T cells are required for the primary induction of delayed-type hypersensitivity (DTH). Conditions are defined here under which the primary induction by antigen of precursor helper T cells only occurs in the presence of specific, irradiated effector T cells, demonstrating that the induction of helper T cells requires T-T cooperation. The interaction between precursor and effector helper T cells is mediated by the recognition of epitopes that must be physically linked to one another. In more detail, hapten-Ficoll conjugates and xenogeneic red blood cells induce medium-density but not low-density cultures of unprimed murine spleen cells to express antigen-specific DTH. Low-density cultures do not support the induction of DTH unless they are supplemented with specific irradiated helper T cells. These helper T cells are themselves induced when antigen is added to medium-density but not low-density cultures. Precursor helper T cells in low-density cultures are only induced by antigen in the presence of additional specific irradiated T cells. Further experiments were directed at analyzing the nature of this T-T interaction. Irradiated hapten-primed T cells help the induction of precursor helper T cells specific for burro red blood cells (BRBC) in the presence of haptenated BRBC and chicken red blood cells (CRBC), but do not help in the presence of haptenated CRBC and BRBC. These experiments demonstrate that the interaction between precursor and effector T cells is mediated by the linked recognition of antigen. These findings show that the induction of precursor cells for both DTH reactivity, and those T cells able to help in the induction of DTH, require specific helper T cells. It is further shown that the induction of T cells able to help in the induction of helper precursor cells takes place in medium-density but not low-density cultures. In order words, antigen, when added to medium-density cultures of normal spleen cells, induces T cells able to mediate DTH, and T cells able to help in the induction of these helper T cells, whereas antigen induces none of these T cells when added to low-density cultures unless appropriate specific helper T cells are added.(ABSTRACT TRUNCATED AT 400 WORDS)

A soluble class I molecule analogous to mouse Q10 in the horse and related species.

Horse serum is shown to contain a soluble class I molecule analogous to the secreted Q10 molecule in the mouse. This molecule has several similarities to the recently described mouse Q10 molecule: it is smaller than membrane-bound equine class I molecules; it occurs in a high molecular mass complex of 200-300 kd in serum; and the serum levels of the equine molecule are similar to that of the Q10 molecule (about 30 micrograms/ml). A soluble molecule is also detected in the sera of species related to the horse; it has in fact been found in all the wild members of the order Perissodactyla so far tested. However, it was not detected in the serum of members of the orders Carnivora, Sirenia, Proboscidea, Artiodactyla, and Primates that were tested, nor in the serum of members of the order Rodentia other than in that of the genus Mus.