Effect of the increase of steroid binding plasma levels after passive immunization against testosterone on the control of luteinizing hormone (LH) secretion in ovariectomized underfed dairy heifers.

The ability of passive immunization against testosterone to increase sex steroid binding levels in plasma and thus to overcome the negative feedback of oestradiol-17 beta (E2) on LH secretion in underfed heifers was investigated. Dairy heifers were ovariectomized and divided in 3 groups: high energy diet (H group, n = 4), low energy diet (L group, n = 3) and low energy diet + E2 implants (LE2 group, n = 4). Twenty-four h before injection of bovine immunoglobulins, the mean concentrations of LH were not different between H and L groups. LH baseline was lower (0.8 vs 1.1 ng/ml, P less than 0.03) and the median number of LH pulses was higher (10 vs 5, P less than 0.03) in H than in L group. E2 markedly decreased (P less than 0.01) the mean and basal concentrations of LH (0.27 ng/ml), and number of LH pulses (0) in the LE2 group (P less than 0.05). After injection of anti-testosterone immunoglobulins in the L group, mean and basal LH concentrations tended to decrease. The median number of LH pulses in the L group rose 8 days after immunization (5 vs 7 on day -1 and day +8, P less than or equal to 0.05). Amplitude of pulses tended to decrease after injection (P = 0.08). In the LE2 group, the mean concentration and baseline of LH were not affected by passive immunization against testosterone, while pulses of LH appeared at day +1 and rose (P = 0.07) at day +8 after immunization with 3.5 pulses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oral immunization with recombinant Lactococcus lactis confers protection against respiratory pneumococcal infection

In the present work, we evaluated if oral immunization with the pneumococcal protective protein A (PppA), expressed in the cell wall of Lactococcus lactis (L. lactis PppA+), was able to confer protective immunity against Streptococcus pneumoniae. Mice were immunized orally with L. lactis PppA+ for 5 consecutive days. Vaccination was performed one (nonboosted group) or 2 times with a 2 week interval between each immunization (boosted group). Oral priming with L. lactis PppA+ induced the production of anti-PppA IgM, IgG, and IgA antibodies in serum and in bronchoalveolar (BAL) and intestinal (IF) lavage fluids. Boosting with L. lactis PppA+ increased the levels of mucosal and systemic immunoglobulins. Moreover, the avidity and the opsonophagocytic activity of anti-PppA antibodies were significantly improved in the boosted group. The presence of both IgG1 and IgG2a anti-PppA antibodies in serum and BAL and the production of both interferon gamma and interleukin-4 by spleen cells from immunized mice indicated that L. lactis PppA+ stimulated a mixture of Th1 and Th2 responses. The ability of L. lactis PppA+ to confer cross-protective immunity was evaluated using challenge assays with serotypes 3, 6B, 14, and 23F. Lung bacterial cell counts and hemocultures showed that immunization with L. lactis PppA+ improved resistance against all the serotypes assessed, including serotype 3, which was highly virulent in our experimental animal model. To our knowledge, this is the first demonstration of protection against respiratory pneumococcal infection induced by oral administration of a recombinant lactococcal vaccine.     

Circulating immunoglobulins and complement in mice with Hymenolepis nana infection

Changes in the circulating immunoglobulins and complement in ddY mice were assayed at various times after immunizing and challenge infections with Hymenolepis nana eggs. The levels of IgG1 and IgG2a consistently increased during 3–4 weeks after immunizing infection. The increase of these immunoglobulins after challenge infection was quicker and more intense than that following immunization. It was not possible to correlate increased levels of IgG1 and IgG2a with the onset of destruction of challenge larvae in immunized mice. IgM concentrations increased slightly during 4 days after immunization but challenge infection did not further increase IgM levels. IgA and IgG2b levels showed no significant change during the course of the infection. Serum C3 levels showed no discernible change after either immunizing or challenge infections. An attempt to specifically suppress the acquisition of resistance by administration of the complement-depleting agent, cobra venom factor (CoF), before immunization failed and depletion of complement activity with CoF that was administered just before challenge infection also failed to affect resistance. These results suggest that complement has no critical role in either induction of the response nor in the anamnestic response to H. nana infection in mice.     

Affinity and distribution of subpopulations of antibody-producing cells in protein-restricted C57BL/6 mice

To study the effect of protein restriction on the affinity of antibodies produced by plaque-forming cells (PFC), C57BL/6 mice were fed diets containing 4% (R4%), 8% (R8%), or 27% (N) casein for 2 (short-term) or 12 (long-term) weeks and immunized with dinitrophenyl (DNP) bovine gamma-globulin in complete Freund's adjuvant. Affinity was assessed by inhibition of plaque formation in the presence of free hapten. Anti-DNP PFC per 10(7) spleen cells were not diminished in short- and long-term R8% mice, and were increased in the former group at certain times after immunization. Affinity of indirect PFC was increased at Days 14 and 21 after immunization in short-term R8% mice and at Day 7 in R4% mice, and was similar in long-term R8% and N animals. No limitation in the heterogeneity of PFC affinities was observed in the restricted groups. Short-term restricted mice showed a rise of the high-affinity PFC subpopulation. The number of mice with hapten-augmentable PFC was diminished in the short-term R8% group at 7 days after immunization and in long-term restricted mice at 14 days, suggesting depressed levels of auto-anti-idiotypic antibodies in protein restriction.     

Fluorescence emission properties of S-Layer enhanced green fluorescent fusion protein as a function of temperature, pH conditions, and guanidine hydrochloride concentration

The fluorescent properties of the S-layer enhanced green fluorescent fusion protein (rSbpA31-1068/EGFP) were investigated as a function of temperature, pH conditions, and guanidine hydrochloride concentration. These results were compared to the fluorescent properties of the recombinant enhanced green fluorescent protein (EGFP) and an equimolar mixture of the S-layer protein rSbpA and EGFP. The intensity of the fluorescence emission of the EGFP at 510 nm, after excitation at 490 nm, is not affected by the presence of rSbpA, either as a fusion partner or as a free protein in solution. In each of the three protein systems, the emission intensity at 510 nm reaches its maximum value between pH 7 and 9 at 20 degrees C and at 0 M guanidine hydrochloride. No fluorescence could be measured at pH 4 and 6 M guanidine hydrochloride. These results show that the S-layer fusion protein (rSbpA31-1068/EGFP) is a suitable candidate for future applications in nanobiotechonology at a wide range of pH, temperature, and guanidine hydrochloride concentrations.     

Construction of EGFP-tagged rBCG of E.tenella and distribution in chickens

Chicken coccidiosis is a major parasitic disease with substantial economic burden to the poultry industry. Enhanced green fluorescent protein (EGFP) tagged recombinant Bacille Calmette-Guerin (rBCG), as a fusion protein with coccidian rhomboid antigen was constructed to track rBCG in vivo in chickens in this study. Immunization of chickens with one dose of rBCG pMV361-Rho/EGFP induced humoral immune response. The colonization of rBCG in liver, spleen, lung, kidney and caecum was observed by laser confocal microscopy. Real-time quantitative RT-PCR showed a rise expression level of rhomboid protein on the 7th day and a peak on the 14th day and disappearance on the 28th day after immunization. These results have significant implications for the development of rBCG vaccines against avian coccidiosis. Supported by Grants from the Foundation of the Jilin Provincial Science and Technology Department of China (Grant No. 20050211-2) and the National Natural Science Foundation of China (Grant No.30170696, 30500370 and 30671580).     

The simultaneous production of two classes of cytoplasmic immunoglobulins by single cells in Xenopus laevis

The classes of cytoplasmic immunoglobulins of individual lymphoid cells from the spleen and peripheral blood of normal and immunized Xenopus laevis were investigated. Immunofluorescence microscopy and simultaneous double staining of the cytoplasm, using a mixture of class-specific TRITC-anti-19S Ig and FITC-anti-7S Ig conjugated antisera, showed that 70% of the immunoglobulin producing cells are “double producers” and contain the two classes of 19S and 7S immunoglobulins. The remaining 30% are composed of “single producers.” The proportion of splenic cells containing either 19S Ig or 7S Ig, respectively, was unequal, and reversed between two groups of animals, one examined at the primary immune response (29% 19S Ig and 4% 7S Ig) and the second group examined at the secondary immune response (8% 19S Ig and 30% 7S Ig). A spontaneous lymphoid tumor with an increased serum level of 19S Ig was examined in the same way. The tumor cells appear to produce 19S Ig exclusively.     

Production of murine collagen-induced arthritis using Klebsiella pneumoniae O3 lipopolysaccharide as a potent immunological adjuvant.

Collagen-induced arthritis (CIA) was produced in mice with non H-2q and H-2r haplotypes by repeated immunization of porcine type-II collagen (CII) together with Klebsiella O3 lipopolysaccharide (KO3 LPS) as an immunological adjuvant. Histological changes that appeared in joints of repeatedly immunized mice were characterized by destruction of normal joint structure, synovial hyperplasia with proliferation of synovial cells, and infiltration of inflammatory cells. No such lesions were produced in mice receiving repeated injections of CII alone or KO3 LPS alone. Development of the humoral antibody and the delayed-type hypersensitivity to CII was exclusively found in mice immunized with the mixture of CII and KO3 LPS. It was therefore suggested that arthritis lesions induced by repeated immunization with the mixture of CII and KO3 LPS might be caused by an autoimmune mechanism, and that the experimental model might be useful for characterization of human rheumatoid arthritis (RA).     

Testicular development, ultrasonographic and histological appearance of the testis in ram lambs immunized against recombinant LHRH fusion proteins

Sixteen native ram lambs weaned at 10 wk of age were divided into two groups. Eight animals were immunized against LHRH with a mixture of two fusion proteins: ovalbumin-LHRH-7 and thioredoxin-LHRH-7. The immunized lambs received a primary immunization plus two booster immunizations at 4 and 12 wks. Animals in the control group (n=8) were not treated. Scrotal measurements and blood samples were taken at 2-week intervals. Beginning at 25 wk of age, semen was collected and sexual behaviour was evaluated on a weekly basis. At 35 and 37 wk of age testes and accessory glands of all animals were subjected to ultrasound scanning. At 37 wk of age animals were slaughtered and testes were evaluated histologically. Serum LHRH antibodies (P<0.01) were detected in animals of the immunized group which had reduced serum testosterone concentrations (P<0.01). Testicular development was suppressed in the immunized animals (P<0.01). Immunized animals exhibited mounting activity 5 wks later than control animals. No mature spermatozoa containing ejaculates were collected from immunized animals. Control animals had moderately echogenic ultrasonographic appearance at 37 wk age, whereas immunized animals had hypoechogenic images. Mean seminiferous tubule diameter in immunized lambs was significantly smaller than that in control lambs. Basal membrane was thickened and hyalinized; there was an increase in peritubular connective tissue. No proliferating spermatogonia or mature spermatozoa were present in the tubules in these animals. There were no differences in the ultrasonographic appearance of prostate and vesicular gland between control and immunized animals. The LHRH recombinant fusion proteins were effective in immunological castration in ram lambs when started at 10 wk of age as noted by differences in serum testosterone, testicular histology and ultrasonographic appearance of testis and weight of accessory sex glands. Determining the effects of immunization on ultrasonographic appearance of the testis related to time after immunization requires further investigations.     

Titration of tetanus antitoxin by passive hemagglutination. II. Serological characteristics of antitoxin production in rabbits and monkeys.

1) Production of tetanus antitoxin in rabbits and monkeys was followed by passive hemagglutination (HA) and toxin-neutralization (TN) tests. The HA activity was observed in both IgM and IgG in both animal species. 2) In rabbits, IgM antitoxin was detected as early as in 7 days, reached the maximum titer in 10--14 days, and disappeared in 3 weeks after the primary immunization. Antitoxin of IgG class was detected in 10 days, and increased gradually. The ratio of HA/TN titers ("serum ratio") was high at an early stage of primary immunization and approached the unity in 3--4 weeks. Unlike the case of guinea pigs, IgM was found to contribute greatly to this high level of ratio. Besides, most rabbits produced IgG antitoxin of high ratios at early stages of immunization. 3) The immune response of monkeys showed a pattern very similar to that of rabbits except a few days' delay in the time course of antitoxin titers. No IgG antitoxin with a high serum ratio was demonstrated. Therefore, the high serum ratio of early sera could be accounted for mainly by IgM. 4) In response to the secondary immunization, no IgM antitoxin was detected in either animal species. 5) No definite correlation between serum ratio and avidity in terms of "dilution ratio" was demonstrated. However, both the dilution ratio and serum ratio were high at an early stage of immunization and gradually decreased, though the magnitudes of the ratios were variable depending on individual animals.     

花生45S rDNA和5S rDNA的染色体定位研究

对四粒红和蜀花四号花生材料进行了核型分析,四粒红为2B核型,核型公式为2n=4x=40=38m＋2sm（4SAT）;蜀花四号为1B核型,核型公式为2n=4x=40=40 m（2SAT）。利用双色荧光原位杂交技术,对45S rDNA和5S rDNA这两个材料有丝分裂中期染色体上的物理位置进行了定位分析。定位结果表明,四粒红有6对45S rDNA位点,位于A2L、A7S、A9L、B3L、B7S、B8L（A和B分别代表基因组A和基因组B,L和S代表长臂和短臂,数字代表染色体序号,下同）;2对5S rDNA位点,位于A3S和B3S;蜀花四号有5对45S rDNA位点,位于A2L、A9L、B3L、B7S、B9L;2对5S rDNA位点,位于A3S和B3S。花生的45S rDNA位点具有可变性,5S rDNA则相对保守。     

Serum levels of immunoglobulins, allergen-specific IgE antibodies, and interleukin-4 in cosmonauts before and after short flights on the International Space Station

Serum levels of immunoglobulins G, A, M, and E (IgG, IgA, IgM, and IgE, respectively); allergenspecific IgE antibodies; and interleukin-4 (IL-4) were measured in ten cosmonauts before and after flights of 7–11 days on the International Space Station. These relatively short spaceflights did not cause any significant changes in the parameters tested. No linear correlation was found between the serum levels of total IgE and IL-4 for either the pre-or the postflight period.     

Hematologic values and serum enzymes in horses inoculated with snake venoms for the production of antivenins in Costa Rica]

Blood components were studied in six horses immunized with snake venoms for the production of polyvalent antivenom in Costa Rica. No significant changes in hemoglobin or hematocrit throughout the immunization period were observed, whereas a significant increment in total serum proteins occurred in the second half of the immunization process, probably due to an increased synthesis of immunoglobulins. There were no significant changes in creatine kinase, but a slight increment was detected in both transaminases, although they did not exceed normal limits. These findings suggest the absence of relevant tissue damage in skeletal muscle, cardiac muscle and liver. In agreement with these results, horses did not develop signs of systemic poisoning, presenting only minor alterations at the site of venom injection, such as oedema, abscesses and fistulas. The development of anti-phospholipase A2 antibody response showed a prominent individual variability, as previously described.     

A screening of phage displayed peptides for the recognition of fullerene (C60)

A novel approach to develop a peptide, that can recognize fullerene (C60) is described for affinity selection of phage displayed peptides from a combinatorial peptide library. Biopanning was performed using cyclic 7-mer peptide library against C60 films deposited on silicon (Si) substrate, and eluted phages with organic solvent. The phage, that recognized C60 films deposited on Si substrate, were obtained from biopanning. The nucleotides of the phage, coding a cyclic 7-mer peptide, were sequenced by standard method. Seventeen kinds of peptide displayed phages were obtained. One kind of peptide (peptide No. 4) displayed phage recognized the C60 films deposited on Si substrate. Peptide No. 4 displayed no affinity towards the Si substrate. The recognition event was monitored by a fluorescent immunoassay. Additionally, peptide No. 4 phage could recognize C60 in powder form, but not the graphite powder. This recognition event in powder form was also observed by a fluorescent immunoassay.     

Nature of Hamster Complement-fixing Antibody to Adenovirus 12 Tumor Antigen

The presence of complement-fixing (CF) antibody reactive with T antigen and with viral C antigen in hamsters bearing adenovirus 12-induced tumors has been confirmed. Antibody activity in serum obtained at a time when the host was bearing large tumors was found to be associated exclusively with 7S immunoglobulins. Two populations of 7S immunoglobulins showing CF reactivity were distinguished by electrical charge, as determined by diethylaminoethyl cellulose chromatography and immunoelectrophoresis. No antibodies of the 19S IgM type were detected in the serum of hamsters bearing large tumors.     

Structural organization of the antigen-binding receptors of immunocompetent cells]

It was demonstrated in this work that rabbit antimouse serum against the aggregated immunoglobulins (RAAS) and mouse serum against the aggregated mouse immunoglobulins (MAAS) inhibited the rosette-forming B-cells (RFC) on the 5th day after the immunization of mice CBA with SRBC in a dose of 5 X 10(-8) cells in vitro in 1:20--1:80, and 1:10--1:40 dilutions in 83--55 and 72--39%, respectively. In difference from RAAS, MAAS in a dilution of 1:20 induced a statistically significant suppression of the antigen-binding receptors of RFC of-B type in the intact animals, and on the 8th--9th day after their immunization with SRBC. In vivo MAAS induced inactivation of the antigen-binding receptors of B-lymphocytes only. Results of the work carried out served as a confirmation of the fact that immunoglobulins in the form of an antigen-antibody complex (functioning in the capacity of the antigen-binding receptors) were sorbed on B-lymphocytes of the spleen.     

Virus-encoded b7-2 costimulation molecules enhance the protective capacity of a replication-defective herpes simplex virus type 2 vaccine in immunocompetent mice

Herpes simplex virus 2 (HSV-2) and, to a lesser extent, HSV-1 cause the majority of sexually transmitted genital ulcerative disease. No effective prophylactic vaccine is currently available. Replication-defective HSV stimulates immune responses in animals but produces no progeny virus, making it potentially useful as a safe form of live vaccine against HSV. Because it does not replicate and spread in the host, however, replication-defective virus may have relatively limited capacity to solicit professional antigen presentation. We previously demonstrated that in mice devoid of B7-1 and B7-2 costimulation molecules, replication-defective HSV-2 encoding B7-1 or B7-2 induces stronger immune responses and protection against HSV-2 challenge than immunization with replication-defective virus alone. Here, we vaccinated wild-type mice fully competent to express endogenous B7 costimulation molecules with replication-defective HSV-2 or replication-defective virus encoding B7-2 and compared their capacities to protect against vaginal HSV-2 infection and disease. Replication-defective virus encoding B7-2 induced more IFN-γ-producing CD4 T cells than did replication-defective virus alone. Immunization with B7-2-expressing virus decreased challenge virus replication in the vaginal mucosa, genital and neurological disease, and mortality more effectively than did immunization with the parental replication-defective virus. Prior immunization with B7-expressing, replication-defective virus also effectively suppressed infection of the nervous system compared to immunization with the parental virus. Thus, B7 costimulation molecules expressed at the site of HSV infection can enhance vaccine efficacy even in a fully immunocompetent host.     

Long‐term regulation of local cytokine production following immunization in mice

Vaccines based on pathogen components require adjuvants to enhance the antigen‐specific adaptive immune response. Intramuscular injection of adjuvanted‐vaccines induces inflammatory cytokines and inflammatory nodules at the injection site within 48 hr after injection (Vaccine 2014; 32 : 3393–401). In the present study, long‐term regulation of cytokine production was investigated at 3, 6, 24, and 48 hr, 5 and 7 days, and 2 and 4 weeks after immunization with human papilloma virus (HPV), diphtheria and tetanus toxoids combined with acellular pertussis (DTaP), Haemophilus influenza type B (Hib), and pneumococcal conjugated (PCV) vaccines in mouse models. The second dose was given 4 weeks later, and cytokine profiles were investigated 2, 5, and 7 days after re‐immunization. IL‐1β, IL‐6, granulocyte‐colony stimulating factor (G‐CSF), and MCP‐1 were produced from 3 hr and peaked at 48 hr after immunization with Cervarix in mice. IL‐4, MCP‐1, and TNF‐α peaked at 5 or 7 days after immunization with Gardasil. These cytokines decreased 7 days after immunization with Cervarix and Gardasil. After the second dose, similar responses were observed. Both vaccines induced neutrophil extracellular traps (NET) in inflammatory nodules. The peak amount of IL‐1β, IL‐6, G‐CSF, and MCP‐1 was observed on day 5 of immunization and that of IL‐4 on days 5‐7 of immunization with DTaP, but no increase in IL‐6 and G‐CSF was observed after re‐immunization. A similar response was noted after immunization with PCV13. An inflammatory response is essential for the development of adaptive immunity through the production of inflammatory cytokines.

     

Evolution of mouse intestinal and blood immunoglobulins after oral route vaccination]

The authors have studied the mice immunoglobulins level after vaccination by oral route with a killed-pathogenic strain of Salmonella typhimurium and an avirulent mutant of the same bacteria. The obtained results show an increase of the intestinal IgA and IgG1 levels and a slighter one of sera IgG between the 10 th and 30 th day following immunization. No correlation was observed concerning the IgM, IgG and IgA levels and the mice protection against a challenge of pathogenic bacteria.     

A New Method for Quantitative Determination of Steroid Metabolites of Cytochrome P450-Dependent Reactions Using Fluorescent Spectroscopy

A method for determination of hydroxylase activity of cytochrome P450 3A4 (CYP3A4) towards its substrate hydrocortisone using fluorescent analysis of the product was developed. 6β-hydroxycortisol, formed during CYP3A4-dependent electrocatalysis, has a characteristic fluorescent peak at λ = 427 ± 2 nm after treating with the sulfuric acid : ethanol (3 : 1) mixture and excitation at λ = 365 nm, which is different from the substrate (hydrocortisone) fluorescence (λ = 525 ± 2 nm). The limit of detection of 6β-hydroxycortisol was 0.32 μM. The developed analytical approach was used to determine the kinetic parameters of CYP3A4-dependent hydrocortisone hydroxylation.