Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein.

Immunization with irradiated sporozoites protects animals and humans against malaria, and the circumsporozoite protein is a target of this protective immunity. We now report that adjuvant-free intramuscular injection of mice with plasmid DNA encoding the Plasmodium yoelii circumsporozoite protein induced higher levels of antibodies and cytotoxic T lymphocytes against the P. yoelii circumsporozoite protein than did immunization with irradiated sporozoites. Mice immunized with this vaccine had an 86% reduction in liver-stage parasite burden after challenge with 5 x 10(5) sporozoites (> 10(5) median infectious doses). Eighteen (68%) of 28 mice that received two or three doses of vaccine were protected against challenge with 10(2) sporozoites, and the protection was dependent on CD8+ T cells. These studies demonstrate the utility of plasmid DNA immunization against a nonviral infection. By obviating the requirement for peptide synthesis, expression and purification of recombinant proteins, and adjuvants, this method of immunization provides an important alternative for rapid identification of protective B- and T-cell epitopes and for construction of vaccines to prevent malaria and other infectious diseases.     

Oral tolerance in myelin basic protein T-cell receptor transgenic mice: suppression of autoimmune encephalomyelitis and dose-dependent induction of regulatory cells.

Orally administered antigens induce a state of immunologic hyporesponsiveness termed oral tolerance. Different mechanisms are involved in mediating oral tolerance depending on the dose fed. Low doses of antigen generate cytokine-secreting regulatory cells, whereas high doses induce anergy or deletion. We used mice transgenic for a T-cell receptor (TCR) derived from an encephalitogenic T-cell clone specific for the acetylated N-terminal peptide of myelin basic protein (MBP) Ac-1-11 plus I-Au to test whether a regulatory T cell could be generated from the same precursor cell as that of an encephalitogenic Th1 cell and whether the induction was dose dependent. The MBP TCR transgenic mice primarily have T cells of a precursor phenotype that produce interleukin 2 (IL-2) with little interferon gamma (IFN-gamma), IL-4, or transforming growth factor beta (TGF-beta). We fed transgenic animals a low-dose (1 mg x 5) or high-dose (25 mg x 1) regimen of mouse MBP and without further immunization spleen cells were tested for cytokine production. Low-dose feeding induced prominent secretion of IL-4, IL-10, and TGF-beta, whereas minimal secretion of these cytokines was observed with high-dose feeding. Little or no change was seen in proliferation or IL-2/IFN-gamma secretion in fed animals irrespective of the dose. To demonstrate in vivo functional activity of the cytokine-secreting cells generated by oral antigen, spleen cells from low-dose-fed animals were adoptively transferred into naive (PLJ x SJL)F1 mice that were then immunized for the development of experimental autoimmune encephalomyelitis (EAE). Marked suppression of EAE was observed when T cells were transferred from MBP-fed transgenic animals but not from animals that were not fed. In contrast to oral tolerization, s.c. immunization of transgenic animals with MBP in complete Freund's adjuvant induced IFN-gamma-secreting Th1 cells in vitro and experimental encephalomyelitis in vivo. Despite the large number of cells reactive to MBP in the transgenic animals, EAE was also suppressed by low-dose feeding of MBP prior to immunization. These results demonstrate that MBP-specific T cells can differentiate in vivo into encephalitogenic or regulatory T cells depending upon the context by which they are exposed to antigen.     

Protective immunity in the rat model of congenital toxoplasmosis and the potential of excreted-secreted antigens as vaccine components

Toxoplasma infection is a major cause of severe foetal pathology both in humans and in domestic animals, particularly sheep. We have previously reported the development of an experimental model to study congenital toxoplasmosis in the rat. Here we demonstrate that, as in humans, total protection against congenital toxoplasmosis can be achieved regardless of the strain of Toxoplasma gondii used to infect rats, or when initial and challenge infections were carried out with different strains. Chronic infection is associated with a highly specific immunity that involves both B-and T-cell responses beginning at day 10 postinfection. The antibody isotype analysis revealed that whereas immunoglobulin (Ig)G2b is the major elicited isotype, no IgG1 antibodies are detected. T cell proliferation was assayed using crude Toxoplasma extracts or excretory-secretory antigens (ESA). The analysis of T cell supernatants showed the specific secretion of both interleukin-2 and interferon-gamma by activated T cells. Immunization of rats before pregnancy with either crude Toxoplasma extracts or with ESA elicited a B cell response that included antibodies of the IgG1 isotype and conferred on the newborns high levels of protection. Preliminary experiments of immunization using two HPLC-purified ESA, GRA2 and GRA5, conferred, a significant protection although to a lesser extent. This experimental model represents an attractive model for the identification of future vaccine candidates against congenital toxoplasmosis.     

Induction of anergy or active suppression following oral tolerance is determined by antigen dosage.

Oral tolerance was generated to hen egg white lysozyme in the mouse or to guinea pig myelin basic protein in the rat by a low-dose (1 mg) or a high-dose (5-20 mg) feeding regimen. High doses of antigen induced tolerance characterized by anergy with little or no active suppression and increased secretion of interleukin 4 (IL-4). Anergy was shown by an increase in frequency of IL-2-secreting cells following culture in recombinant IL-2. Low doses of antigen induced tolerance characterized by antigen-driven active suppression with increased secretion of transforming growth factor beta (TGF-beta) and IL-4 and minimal anergy. Without further immunization, spleen cells from animals orally tolerized by both regimens secreted increased levels of IL-4 and TGF-beta in an antigen-specific manner. Animals fed high doses secreted more IL-4 and less TGF-beta, whereas those fed low doses secreted more TGF-beta and less IL-4. These results demonstrate that the two feeding regimens induced cell populations that differed in their cytokine secretion profile and their capacity to actively suppress in vitro and to induce anergy. Our results provide a basis for distinguishing different forms of antigen-driven peripheral tolerance and have important implications for orally induced antigen-specific modulation of human autoimmune diseases.     

Adaptive Immune Response to Vaccinia Virus LIVP Infection of BALB/c Mice and Protection against Lethal Reinfection with Cowpox Virus

Mass vaccination has played a critical role in the global eradication of smallpox. Various vaccinia virus (VACV) strains, whose origin has not been clearly documented in most cases, have been used as live vaccines in different countries. These VACV strains differed in pathogenicity towards various laboratory animals and in reactogenicity exhibited upon vaccination of humans. In this work, we studied the development of humoral and cellular immune responses in BALB/c mice inoculated intranasally (i.n.) or intradermally (i.d.) with the VACV LIVP strain at a dose of 10⁵ PFU/mouse, which was used in Russia as the first generation smallpox vaccine. Active synthesis of VACV-specific IgM in the mice occurred on day 7 after inoculation, reached a maximum on day 14, and decreased by day 29. Synthesis of virus-specific IgG was detected only from day 14, and the level increased significantly by day 29 after infection of the mice. Immunization (i.n.) resulted in significantly higher production of VACV-specific antibodies compared to that upon i.d. inoculation of LIVP. There were no significant differences in the levels of the T cell response in mice after i.n. or i.d. VACV administration at any time point. The maximum level of VACV-specific T-cells was detected on day 14. By day 29 of the experiment, the level of VACV-specific T-lymphocytes in the spleen of mice significantly decreased for both immunization procedures. On day 30 after immunization with LIVP, mice were infected with the cowpox virus at a dose of 46 LD₅₀. The i.n. immunized mice were resistant to this infection, while 33% of i.d. immunized mice died. Our findings indicate that the level of the humoral immune response to vaccination may play a decisive role in protection of animals from orthopoxvirus reinfection.     

Pilot study of recombinant human interleukin 2 for chronic type B hepatitis

Recombinant human interleukin 2 was administered to 10 patients with chronic type B hepatitis as a part of a pilot study to evaluate its antiviral activity. Patients received 1 to 3 x 10(5) units per day of interleukin 2 for 21 to 28 days, and all completed the treatment schedule. During therapy, serum values of DNA polymerase decreased in 6 and became negative in four patients. However, when therapy was discontinued, DNA polymerase levels increased to pretreatment levels in most cases. Serum HBeAg levels did not change during treatment. Serum aminotransferase levels transiently increased in 6 of the 10 patients during therapy; but once therapy was stopped, levels fell markedly. Side effects of interleukin 2 therapy included fever, chills, anorexia and fatigue. After 1 year of follow-up, three treated patients had lost HBeAg and had marked improvement in aminotransferase levels. These serologic and biochemical improvements occurred 1.5 to 11 months after therapy was stopped. Whether a 3- to 4-week course of interleukin 2 therapy leads to an increased rate of seroconversion from HBeAg to antibody in chronic type B hepatitis deserves further evaluation in prospectively randomized, controlled trials.     

Induction of synthesis of the cytolytic C9 (ninth component of complement)-related protein in human peripheral mononuclear cells by monoclonal antibody OKT3 or interleukin 2: correlation with cytotoxicity and lymphocyte phenotype.

Synthesis of the cytolytic C9-related protein (C9RP) was induced by activation of resting human peripheral T lymphocytes with the anti-CD3 antibody OKT3 or interleukin 2. Comparison of cellular cytotoxicity and C9RP content at various times during activation yielded a coefficient of correlation r = 0.92. During OKT3 stimulation of peripheral mononuclear cells, maximal C9RP content and cytotoxicity were observed by day 2 or 3, with subsequent decline to baseline values by day 5, whereas during interleukin 2 stimulation, both parameters reached the maximal level at days 3-5. After fluorescence-activated cell sorting, C9RP and cytotoxicity were quantitated in CD4+, CD8+, and Leu-19+ subsets. In OKT3-activated CD8+ cells, C9RP increased to approximately 3 X 10(6) molecules per cell, with a corresponding increase in lysis of human melanoma cells mediated by anti-CD3-anti-melanoma monoclonal antibody conjugates. Interleukin 2-stimulated CD8+ cells showed similar increases, but cytotoxicity was conjugate-independent. Activated CD4+ cells showed minimal increase in C9RP content. Leu-19+ cells, which exhibit natural killer cell activity, had a high C9RP content (approximately 2.5 X 10(6) molecules per cell) before stimulation.     

Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization.

We compared the antigen-specific antibody isotypes and lymphokine secretion by CD4+ T cells in BALB/c mice immunized intradermally with either Escherichia coli beta-galactosidase (beta-gal) or plasmid DNA (pDNA) encoding beta-gal in a cytomegalovirus-based expression vector (pCMV-LacZ). pCMV-LacZ induced mainly IgG2a, whereas beta-gal in saline or alum induced IgG1 and IgE beta-gal-specific antibodies. In addition, splenic CD4+ T helper (Th) cells isolated from pDNA-immunized mice secreted interferon-gamma but not interleukin (IL)-4 and IL-5, whereas Th cells from beta-gal-injected mice secreted IL-4 and IL-5 but not interferon-gamma after in vitro stimulation with antigen. Together these data demonstrate that pDNA immunization induced a T helper type 1 (Th1) response, whereas protein immunization induced a T helper type 2 (Th2) response to the same antigen. Interestingly, priming of mice with pCMV-LacZ prevented IgE antibody formation to a subsequent i.p. beta-gal in alum injection. This effect was antigen-specific, because priming with pCMV-LacZ did not inhibit IgE anti-ovalbumin antibody formation. Most importantly, intradermal immunization with pCMV-LacZ (but not pCMV-OVA) of beta-gal in alum-primed mice caused a 66-75% reduction of the IgE anti-beta-gal titer in 6 weeks. Also, pCMV-LacZ induced specific IgG2a antibody titers and interferon-gamma secretion by Th cells in the beta-gal in alum-primed mice. The data demonstrate that gene immunization induces a Th1 response that dominates over an ongoing protein-induced Th2 response in an antigen-specific manner. This suggests that immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases.     

Oxidative/reductive conjugation of mannan to antigen selects for T1 or T2 immune responses.

The induction of CD8+ cytotoxic T lymphocytes (CTLs) is desirable for immunization against many diseases, and recombinant-synthetic peptide antigens are now favored agents to use. However, a major problem is how to induce CTLs, which requires a T1-type response to such synthetic antigens. We report that T1-type (generating high CTL, low antibody) or T2-type (the reciprocal) responses can be induced by conjugation of the antigen to the carbohydrate polymer mannan: T1 responses are selected by using oxidizing conditions; T2 responses are selected by using reducing conditions for the conjugation. Using human MUC1 as a model antigen in mice, immunization with oxidized mannan-MUC1 fusion protein (ox-M-FP) led to complete tumor protection (challenge up to 5 x 10(7) MUC1+ tumor cells), CTLs, and a high CTL precursor (CTLp) frequency (1/6900), whereas immunization with reduced mannan-MUC1 FP (red-M-FP) led to poor protection after challenge with only 10(6) MUC1+ tumor cells, no CTLs, and a low CTLp frequency (1/87,800). Ox-M-FP selects for a T1 response (mediated here by CD8+ cells) with high interferon gamma (IFN-gamma) secretion, no interleukin 4 (IL-4), and a predominant IgG2a antibody response; red-M-FP selects for a T2-type response with IL-4 production and a high predominant IgG1 antibody response but no IFN-gamma.     

Growth hormone (GH) hypersecretion and GH receptor resistance in streptozotocin diabetic mice in response to a GH secretagogue

The growth hormone (GH) and insulin-like growth factor I (IGF-I) axis were studied in streptozotocin (STZ) diabetic and nondiabetic female mice following intravenous (IV) injection of the GH secretagogue (GHS) ipamorelin or saline. On day 14, blood samples were obtained before and 10 minutes after the injection. Livers were removed and frozen for determination of the mRNA expressions of the GH receptor, GH-binding protein, and IGF-I, and hepatic IGF-I peptide. Serum samples were analyzed for GH and IGF-I. Following ipamorelin injection, the GH levels were found to be 150 +/- 35 microg/L and 62 +/- 11 microg/L in the diabetic compared to the nondiabetic mice (P <.05). Serum IGF-I levels were lower in diabetic than in nondiabetic animals, and rose after stimulation only in the nondiabetic animals. Furthermore, hepatic GH resistance and IGF-I mRNA levels and IGF-I peptide were increased in nondiabetic animals in response to GH stimulation, whereas the low levels per se of all these parameters in diabetic mice were unaffected. The study shows that STZ diabetic mice demonstrate a substantial part of the clinical features of type 1 diabetes in humans, including GH hypersecretion and GH resistance. Accordingly, it is proposed that STZ diabetic mice may be a better model of the perturbations of the GH/IGF-I axis in diabetes than STZ diabetic rats.     

Standardization of a technique for analysing the frequency of parasite-specific cytotoxic T lymphocyte precursors in cattle immunized with Theileria parva

Summary A limiting dilution microculture system was optimized to quantify the frequency of Theileria parva-specific cytotoxic T lymphocyte precursors (CTLp) in peripheral blood mononuclear cells (PBMC) from immune cattle. Optimal results were obtained with responder cell input levels ranging from 2 × 10⁴/well to 6.25 × 10²/well, along with 1–5 × 10³/well stimulator cells in standard supplemented RPMI 1640 medium containing 2.5–5% T cell growth factors. Thirty-six microtitre wells were established at each responder input level. Cultures were incubated for 7 days at 38°C, at the end of which time individual wells were screened for cytotoxic activity in a 4-h ¹¹¹indium oxine-release assay. Analysis of the cytotoxicity data, by a computer-programmed maximum likelihood estimation method indicated that they conformed to the Poisson model of single-hit kinetics. Estimates of frequencies ranged from 1:3600 to 1:5275 CTLp in PBMC of eight cattle between 1 and 24 months after immunization with T. parva. By contrast, no CTLp were detected in six naïve animals analysed to a responder cell input of 10⁵/well. Split-well analysis of individual microwells showed that the CTL clones generated under limiting dilution conditions displayed exquisite specificity for parasitized cells, were genetically restricted and in some animals were parasite strain-specific.     

Soluble interleukin 2 receptor in acute viral hepatitis and chronic liver disease

Serum levels of soluble interleukin 2 receptor were determined in patients with acute viral hepatitis and patients with various chronic liver diseases. In addition, the ability of peripheral blood mononuclear cells of patients with alcoholic cirrhosis to generate soluble interleukin 2 receptor following mitogenic stimulation was studied in vitro. Serum soluble interleukin 2 receptor concentrations in all patients with acute viral hepatitis were found to be significantly elevated (1,319 +/- 527 units per ml) during the first week after onset of disease, as compared to healthy control individuals (375 +/- 102 units per ml; p less than 0.0005) and declined toward normal levels during the course of the illness. Similarly, patients suffering from chronic liver disease such as alcoholic liver cirrhosis (1,172 +/- 507 units per ml), primary biliary cirrhosis (619 +/- 190 units per ml) or chronic active HBsAg+ hepatitis (941 +/- 357 units per ml) showed increased serum soluble interleukin 2 receptor concentrations (p less than 0.0005 vs. controls, respectively). In vitro mitogen stimulation of peripheral mononuclear cells derived from patients with alcoholic cirrhosis resulted in a soluble interleukin 2 receptor production not different from that seen in healthy individuals, suggesting that elevated soluble interleukin 2 receptor serum levels seen in this disease are not the result of an increased synthesis by circulating lymphocytes. Due to the ability of soluble interleukin 2 receptor to bind free interleukin 2--thus making it a potential immunoregulatory molecule--its high serum levels could explain some of the immunologic abnormalities observed in acute and chronic liver disease.     

Expansion of hematopoietic stem cells in the developing liver of a mouse embryo

The activity of hematopoietic stem cells in the developing liver of a C57BL/6 mouse embryo was quantified by a competitive repopulation assay. Different doses of fetal liver cells at days 11 to 18 of gestation were transplanted into irradiated mice together with 2 x 10(5) adult bone marrow cells. A long-term repopulation in myeloid-, B-cell, and T-cell lineage by fetal liver cells was evaluated at 20 weeks after transplantation. At day 12 of gestation multilineage repopulating activity was first detected in the liver as 50 repopulating units (RU) per liver. The number of RU per liver increased 10-fold and 33-fold by day 14 and day 16 of gestation, and decreased thereafter, suggesting a single wave of stem cell development in the fetal liver. A limiting dilution analysis revealed that the frequency of competitive repopulating units (CRU) in fetal liver cells at day 12 of gestation was similar to that at day 16 of gestation. Because of an increase of total fetal liver cell number, the absolute number of CRU per liver from days 12 to 16 of gestation increased 38-fold. Hence, the mean activity of stem cells (MAS) that is given by RU per CRU remained constant from days 12 to 16 of gestation. From these data we conclude that hematopoietic stem cells expand in the fetal liver maintaining their level of repopulating potential.     

Incidence of intussusception as studied from a hospital-based retrospective survey over a 10-year period (2001-2010) in Akita Prefecture, Japan

One concern about rotavirus vaccines is its possible association with intussusception. Thus, it is necessary to determine the baseline incidence for intussusception in the first year of life in places where rotavirus vaccines are introduced. However, few safety data exist for the period at which the first dose of Rotarix and RotaTeq are allowed to administer in Japan. The first dose of Rotarix is scheduled to administer at 6-20 weeks of age and that of RotaTeq is scheduled to administer at 6-24 weeks of age; the upper limits for these vaccines is later than the upper limit recommended by the World Health Organization by 5 and 9 weeks, respectively. We performed a retrospective cross-sectional study by reviewing medical charts of all hospitals that provided pediatric beds in Akita Prefecture, Japan, and identifying the cases of intussusception that met the Brighton criteria level 1 in these hospitals between January 2001 and December 2010. During this 10-year period, 122 children younger than 1 year of age were diagnosed with intussusception. The incidence of intussusception was estimated at 158 per 100,000 person-years among children younger than 1 year (95% confidence interval, 131-188), 10 per 100,000 person-years for children aged 0-2 months, 165 for children aged 3-5 months, and 300 for children aged 6-8 months. This rapid and substantial increase in the incidence of intussusception during the first year of life should be considered when formulating the immunization schedule for administering rotavirus vaccines in Japan.     

Analysis of interleukin 2 and various effector cell populations in adoptive immunotherapy of 9L rat gliosarcoma: allogeneic cytotoxic T lymphocytes prevent tumor take.

Recombinant interleukin 2 (rIL-2) and various effector cell populations were used for adoptive immunotherapy in the Fischer strain 9L rat gliosarcoma model. The in vivo cytotoxicities of nonspecifically activated lymphocytes and specifically activated cytotoxic T lymphocytes (CTLs) were assessed in a modified in vivo neutralization (Winn) assay. Effector cells (10(6)) and 9L tumor cells (10(5] were combined with 10(4) units of rIL-2 and stereotactically implanted into the right frontal centrum semiovale of the Fischer (F344) rat. At 7 and 14 days, additional effector cells (10(6] and rIL-2 (10(4) units) were administered through the same burr hole. Nonspecifically activated splenocytes were lymphokine-activated killer (LAK) cells, both plastic-adherent and nonadherent, whereas specifically activated CTLs were either syngeneic (genetically identical) or allogeneic (genetically dissimilar). Syngeneic CTLs were T lymphocytes from Fischer rats primed in vivo with 9L cells and restimulated in vitro. Allogeneic CTLs were generated by exposing DA rat lymphocytes either to irradiated Fischer lymph node cells or to 9L Fisher tumor cells in vitro. Control groups included rats bearing 9L tumor who were untreated, those who received peripheral (i.p. or s.c.) administration of rIL-2, or those who received syngeneic unstimulated T lymphocytes and rIL-2. For a set of animals given the same inoculum of 9L tumor, significantly improved survival was shown for groups treated with nonadherent or adherent LAK cells (P less than or equal to 0.0003), syngeneic CTLs (P = 0.0327), or allogeneic CTLs (P = 0.0025) over untreated control animals by using Mantel-Haenzel nonparametric logrank equations. Only treatment with allogeneic CTLs prevented tumor take.     

Behaviorally conditioned suppression of a graft-versus-host response.

Cyclophosphamide (CY), previously used to condition suppression of humoral immune responses, was used to condition suppression of a graft-versus-host response (GvHR). Female (Lewis x Brown Norway) F1 rats were conditioned by pairing consumption of a saccharin solution with an intraperitoneal injection of CY at 50 mg/kg of body weight 48 days before immunization. On day 0, all animals were injected with a suspension of splenic leukocytes (2 x 10(7) cells per footpad) obtained from female Lewis donors. The regional GvHR was assessed on day 5 by weighing popliteal nodes. Conditioned animals given a single low-dose injection of CY and reexposed to conditioned stimuli had lymph node weights significantly lower than control groups and did not differ from animals given three injections of CY during the ongoing GvHR. The results suggest that conditioned immunosuppression, previously demonstrated in thymus-dependent and thymus-independent humoral immune responses, also affects the popliteal GvHR, a cellular immune response.     

Mode of gonadotropin secretion in infantile female rats and the role of estrogen in feedback regulation.

The temporal aspects of gonadotropin release were studied in infantile (14-day-old) female rats. Blood samples were collected using vena cava cannulae at 10-, 15-, 20-, or 30-min intervals and assayed for either LH alone or for both LH and FSH. Within individual animals, blood LH levels exhibited a degree of variability suggestive of a pulsatile release of the hormone. Wide fluctuations in circulating FSH were also observed, indicating that FSH might also be secreted in a pulsatile manner. Peak values for both gonadotropins were 2- to 5-fold higher than baseline levels. The majority of pups exhibited a nonrhythmic release of hormones. In some animals, LH and FSH appeared to be secreted in a temporally coincident manner. Administration of LHRH to cannulated pups evoked a simultaneous discharge of both gonadotropins. To determine whether endogenous 17 beta-estradiol (E2) plays a physiological role in the control of LH secretion in infantile rats, circulating LH levels were monitored in cannulated pups which had been treated twice daily with anti-E2 serum for 4 days. Passive immunization to E2 did not affect pulsatile LH secretion. Therefore, the results of this study demonstrate that in infantile female rats, LH is secreted in a pulsatile manner which is independent of E2 feedback regulation.     

DNA immunization circumvents deficient induction of T helper type 1 and cytotoxic T lymphocyte responses in neonates and during early life

The relative deficiency of T helper type 1 (Th1) and cytotoxic T lymphocyte (CTL) responses in early life is associated with an increased susceptibility to infections by intracellular microorganisms. This is likely to reflect a preferential polarization of immature CD4 T cells toward a Th2 rather than a Th1 pattern upon immunization with conventional vaccines. In this report, it is shown that a single immunization within the first week of life with DNA plasmids encoding viral (measles virus hemagglutinin, Sendai virus nucleoprotein) or bacterial (C fragment of tetanus toxin) vaccine antigens can induce adult-like Th1 or mixed Th1/Th2 responses indicated by production of IgG2a vaccine-specific antibodies and preferential secretion of interferon-γ (IFN-γ) compared with interleukin (IL)-5 by antigen-specific T cells, as well as significant CTL responses. However, in spite of this potent Th1-driving capacity, subsequent DNA immunization was not capable of reverting the Th2-biased responses induced after early priming with a recombinant measles canarypox vector. Thus, DNA vaccination represents a novel strategy capable of inducing Th1 or mixed Th1/Th2 and CTL responses in neonates and early life, providing it is performed prior to exposure to Th2-driving conventional vaccine antigens.     

Steady state analysis of hypothalamic GnRH mRNA levels in male Syrian hamsters: influences of photoperiod and androgen.

An in situ hybridization assay, utilizing a free floating technique was used to estimate the steady state levels of hypothalamic luteinizing hormone-releasing hormone (GnRH) mRNA levels in the brains of male golden hamsters maintained in different photoperiods. In situ histochemistry was performed using a 32P-labelled 66-nucleotide long oligomer complementary to the sequence of the human GnRH mRNA coding region. The oligonucleotide hybridized specifically to mRNA encoding the GnRH precursor as suggested by the distribution of labelled neurons and as shown by an RNAse protection assay on septal and preoptic-hypothalamic mRNA from gonadally regressed hamsters. To test the hypothesis that short-day photoperiods reduce GnRH synthesis, intact male hamsters or castrated males bearing subcutaneously inserted testosterone implants were exposed to long-day (14 h light:10 h dark) or short-day (10 h light 14 h dark) photoperiods for 4 weeks. Exposure to short day lengths never caused a decrease in GnRH expressing neurons and actually was associated with an increase in the number of radiolabelled cells specifically in the diagonal band of Broca/medial septum in the gonadally intact group. The mean number of grains per labelled cell for the short day animals similarly was not reduced from that seen in long day animals. The results are consistent with previous studies on photoperiod and GnRH content in the same brain regions and support the notion that the suppression of the synthesis of GnRH does not accompany the low levels of LH secretion observed during the early stages of reproductive quiescence in this species.     

Adaptation of islets of Langerhans to pregnancy: increased islet cell proliferation and insulin secretion correlates with the onset of placental lactogen secretion.

To elucidate the temporal profile of adaptive changes of the islets of Langerhans to the increased insulin demands of pregnancy, we have studied islet cell proliferation and insulin secretion during gestation in the rat. 5-Bromo-2'-deoxyuridine incorporation into dividing islet cells was significantly (P less than 0.05) increased over age-matched controls by day 10, rose continuously to a peak at day 14, and then returned to control levels by day 18. By day 20, cell division was significantly inhibited (P less than 0.05). The pattern of changes in insulin secretory profiles observed with perfused pancreata of pregnant animals was similar to that obtained for islet cell proliferation. Both the threshold of glucose-stimulated insulin secretion and the amount of above threshold insulin secretion began to diverge from controls by day 10. By day 12, the glucose-stimulation threshold was significantly decreased from 5.7 mM glucose to 3.3 mM (P less than 0.05), remained at this low level through day 15, and returned toward normal by day 20. Concomitant with the increased sensitivity of B cells to glucose, the above threshold insulin secretion was significantly increased by day 12 (P less than 0.05), peaked at day 15, and returned to control levels by day 20. This insulin secretory data demonstrates that the increased sensitivity of B cells to glucose is an important component of the adaptation of islets during pregnancy to the increased demand for insulin at physiological concentrations of plasma glucose. To correlate the above changes in islet cell proliferation and insulin secretion with levels of placental lactogen (PL), serum lactogenic hormone activity was measured by Nb2 lymphoma cell replication assays. This analysis revealed the expected biphasic pattern: a midpregnancy peak at day 12, followed by a nadir at day 14, and then continuously elevated levels until term. The bioassay data agreed with the known secretory profiles of rat (r) PL-I (midpregnancy) and rPL-II (late pregnancy). Our results provide the first systematic evaluation of changes in islet function during pregnancy in the rat. In addition, they provide evidence that rPL-I may be the critical hormonal signal which triggers the primary adaptive changes in islet function characteristic of pregnancy. The return to normal values of insulin secretion and inhibition of cell division observed at day 20 in the presence of high concentrations of rPL-II suggests that other inhibitory influences become dominant in the later stages of rat pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)