RESUMEN
Survivin has received attention as a potential target for cancer immunotherapy because of its crucial role in oncogenesis. We undertook this study to evaluate the immunotherapeutic potential of combination of recombinant survivin along with adjuvant alum and immune modulator Mycobacterium indicus pranii (MIP). In vivo efficacy of the combination was studied in an invasive murine breast cancer model. Recombinant survivin protein was purified from Escherichia coli based expression system and characterized by western blotting. Purified survivin protein was combined with alum and MIP and was used for immunization of Balb/c mice. Antigen-primed animals were then challenged with syngeneic mammary tumor cells known as 4T-1. Balb/c mice spontaneously develop tumor when inoculated with 4T-1 cells. Antigen and adjuvant combination was immunogenic and significantly suppressed tumor growth in mice immunized with combination of recombinant survivin (10 µg), alum, and MIP. This is the first report that describes a combination immunotherapy approach using recombinant survivin, alum, and MIP in highly metastatic murine breast cancer model and holds promise for development of new biotherapeutics for cancer.
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Adyuvantes Inmunológicos/uso terapéutico , Compuestos de Alumbre/uso terapéutico , Neoplasias de la Mama/prevención & control , Vacunas contra el Cáncer/uso terapéutico , Proteínas Inhibidoras de la Apoptosis/uso terapéutico , Proteínas Represoras/uso terapéutico , Animales , Neoplasias de la Mama/patología , Modelos Animales de Enfermedad , Femenino , Inmunización , Inmunoterapia , Ratones Endogámicos BALB C , Proteínas Recombinantes/uso terapéutico , SurvivinRESUMEN
LHRH based vaccines are promising candidates for therapy of androgen and estrogen dependent cancers. We report in this communication development of a novel recombinant protein vaccine candidate against LHRH. A synthetic gene was designed in which the codon sequence in the LHRH decapeptide was modified by substituting the codon for 6-glycine with that of l-leucine. Further the LHRH(6leu) gene was linked to heat-labile enterotoxin of E. coli (LTB) as carrier. This LHRH(6leu)-LTB gene was cloned into a prokaryotic expression vector under the control of inducible and strong bacteriophage T7 promoter to over-express LHRH(leu) fused to LTB as recombinant protein in E. coli. Recombinant LHRH(leu)-LTB protein of â¼14 kDa size, was purified from inclusion bodies using in-situ refolding on the column and Ni-NTA based immobilized affinity chromatography. Western blot confirmed the immunoreactivity of purified LHRH(leu)-LTB fusion protein with anti-LHRH monoclonal antibody. The vaccine protein was further characterized by mass spectroscopy, circular dichroism and fluorescence spectroscopy. This communication reports a recombinant LHRH fusion protein with potential for blocking of sex hormones production for eventual therapy of sex hormones dependent neoplasms.
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Andrógenos , Vacunas contra el Cáncer , Estrógenos , Hormona Liberadora de Gonadotropina , Linfotoxina beta , Neoplasias/terapia , Proteínas Recombinantes de Fusión , Vacunas contra el Cáncer/biosíntesis , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/uso terapéutico , Hormona Liberadora de Gonadotropina/biosíntesis , Hormona Liberadora de Gonadotropina/genética , Hormona Liberadora de Gonadotropina/uso terapéutico , Humanos , Linfotoxina beta/biosíntesis , Linfotoxina beta/uso terapéutico , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/uso terapéuticoRESUMEN
Survivin is the smallest member of the Inhibitor of apoptosis (IAP) family of proteins, involved in inhibition of apoptosis and regulation of cell cycle. These functional attributes make Survivin a unique protein exhibiting divergent functions i.e. regulating cell proliferation and cell death. Expression pattern of Survivin is also distinctive; it is prominently expressed during embryonal development, absent in most normal, terminally differentiated tissues but upregulated in a variety of human cancers. Expression of Survivin in tumours correlates with not only inhibition of apoptosis and a decreased rate of cell death, but also resistance to chemotherapy and aggressiveness of tumours. Therefore, Survivin is an important target for cancer vaccines and therapeutics. Survivin has also been found to be prominently expressed on both human and embryonic stem cells and many somatic stem cell types indicating its yet unexplored role in stem cell generation and maintenance. Overall, Survivin emerges as a molecule with much wider role in cellular homeostasis. This review will discuss various aspects of Survivin biology and its role in regulation of apoptosis, cell division, chemo-resistance and tumour progression. Various molecular and immunotherapeutic approaches targeting Survivin will also be discussed.
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The effect of pituitary growth hormone on the biosynthesis of DNA in the thymus and other lymphoid organs, as well as the ability of the rat to respond immunologically to sheep red blood cells, has been evaluated. There is a marked reduction in plaque-forming cells, hemagglutination titers, and DNA synthesis in animals when examined at 15 wk after hypophysectomy. Administration of bovine growth hormone (BGH) leads to the enhancement of DNA synthesis in lymphoid organs and recovery of the immune response. Similar effects of the hormone are observed in plateaued rats. Injection of rabbit anti-BGH globulins, in contrast to normal rabbit globulins, over 5 days causes a drop in the weight of the thymus and in the rate of DNA synthesis in this organ. The thymus is also the organ in which stimulation of DNA synthesis is observed at a time period earlier than the spleen and lymph nodes after a single injection of BGH. The hormone stimulates not only the incorporation of thymidine-(3)H into DNA in the cortical cells, but also the incorporation of sodium sulfate-(35)S into TCA-insoluble biopolymers reported to be elaborated in the medullary area of the thymus. An in vitro system for the action of BGH on the thymus has been described. There is an obligatory requirement for calcium, but not for fetal calf serum in the medium for the hormone effect. An early action of the hormone is the enhanced incorporation of uridine-G-(3)H into RNA in thymocytes which is followed by a stimulation of the synthesis of proteins and DNA. The stimulatory action of growth hormone on RNA synthesis is not because of a facilitated uptake of the radioactive uridine by the cells under hormonal influence, a mechanism by which insulin is observed to increase RNA synthesis in thymocytes in vitro. The action of growth hormone on thymocytes is specific, since thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and heat-inactivated growth hormone are not effective. BGH has also a beneficial action on the regeneration of the thymus and spleen in starved rats.
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Formación de Anticuerpos/efectos de los fármacos , ADN/biosíntesis , Eritrocitos/inmunología , Hormona del Crecimiento/farmacología , Timo/metabolismo , Animales , Hemaglutinación/efectos de los fármacos , Hipofisectomía , Sueros Inmunes/farmacología , Técnicas In Vitro , Linfocitos/efectos de los fármacos , Masculino , Ratas , Ovinos , Estimulación Química , Sulfatos/metabolismo , Isótopos de Azufre , Timidina/metabolismo , Timo/citología , Timo/efectos de los fármacos , Tritio , Uridina/metabolismoRESUMEN
While 60% of women experiencing recurring episodes of bacterial vaginosis (BV) with vaginal pH >or= 5 are depleted of resident probiotic lactobacilli, the remainder carry one or more strains of lactobacilli. Their ability to make D-lactic acid is, however, low (3.94 +/- 0.72 mM/L) compared to the D-lactic acid produced by strains from healthy vagina with vaginal pH approximately 4 (8.04 +/- 1.07 mM/L) culture supernatant of 0.5 McFarland concentration (P < 0.001).
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Ácido Láctico/metabolismo , Lactobacillus/metabolismo , Vagina/química , Vagina/microbiología , Vaginosis Bacteriana/microbiología , Adulto , Femenino , Humanos , Concentración de Iones de Hidrógeno , Lactobacillus/aislamiento & purificación , Recurrencia , Adulto JovenRESUMEN
OBJECTIVE: A variety of cancers ectopically express human chorionic gonadotropin beta (hCGbeta). Patients harboring such cancers have poor prognosis and adverse survival. A recombinant chimeric antibody, cPiPP, exhibiting high affinity and specificity for hCGbeta/hCG was engineered. This study was designed to determine whether this antibody alone or conjugated to curcumin can selectively kill tumor cells expressing hCGbeta. EXPERIMENTAL DESIGN: The study was carried out on MOLT-4 and U-937 cells expressing hCGbeta and on peripheral blood leukocytes of acute myeloid leukemia (AML) patients. The anticancerous compound curcumin was conjugated to cPiPP. The binding of cPiPP and cPiPP-curcumin conjugate to cells was studied by flow cytometry and cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), FACS with propidium iodide staining, trypan blue exclusion assay and microscopy. RESULTS: The antibody did not impair the growth of MOLT-4 and U-937 cells in culture. Its conjugate with curcumin, however, was lethal to both cell lines. The immunoconjugate killed tumor cells bearing the CD33 marker of an AML patient expressing hCGbeta but did not have a similar action on cells of another AML patient with the CD13 marker but who was negative for hCGbeta. CONCLUSION: A humanized antibody against hCGbeta linked to curcumin has potential for therapy of hCGbeta-expressing tumors.
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Gonadotropina Coriónica Humana de Subunidad beta/metabolismo , Curcumina/metabolismo , Leucemia/tratamiento farmacológico , Linfoma/tratamiento farmacológico , Anciano , Antígenos CD/biosíntesis , Antígenos de Diferenciación Mielomonocítica/biosíntesis , Separación Celular , Gonadotropina Coriónica Humana de Subunidad beta/química , Diseño de Fármacos , Femenino , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Lectina 3 Similar a Ig de Unión al Ácido Siálico , Sales de Tetrazolio/farmacología , Tiazoles/farmacología , Células U937RESUMEN
BACKGROUND & OBJECTIVE: Lactobacilli are depleted in vagina of women suffering from recurring episodes of bacterial vaginosis with vaginal pH >or=5. With the objective of making available probiotic lactobacilli for replenishment in such women, a study was undertaken to isolate and characterize the Lactobacilli present in women with eco-healthy vagina in Delhi. No information is so far available on the species of Lactobacilli resident in vagina of women in India. METHODS: Vaginal swabs were taken from 80 women with informed consent after ethical approval and grown in MRS broth. Gram-positive, catalase-negative bacilli generating about 200 bp amplicon by PCR with Lactobacillus genus specific primers were further characterized by employing species specific primers followed by sequencing of 16S rDNA. Isolates of the same species were differentiated by random amplified polymorphic DNA (RAPD) profiles. RESULTS: The predominant species isolated were L. reuteri present in 26 (32.5%) women, L. fermentum in 20 (25%), and L. salivarius in 13 (16.25%) women. Sequencing of 16S rDNA of 20 isolates showed that except for two isolates of L. plantarum, sequences of the remaining agreed well with PCR identification. None of the isolates had similar RAPD profile. INTERPRETATION & CONCLUSION: Our findings showed lactobacilli species present in healthy vagina of women in India differ from those reported from other countries. This information would be useful to development of probiotic tablets seeking to replenish the missing lactobacilli for reproductive health of women in India.
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Lactobacillus , Probióticos/uso terapéutico , Vagina/microbiología , Vaginosis Bacteriana/microbiología , Adolescente , Adulto , Técnicas de Tipificación Bacteriana , ADN Bacteriano/análisis , Femenino , Humanos , India , Lactobacillus/clasificación , Lactobacillus/genética , Lactobacillus/aislamiento & purificación , Persona de Mediana Edad , Datos de Secuencia Molecular , ARN Ribosómico 16S/genética , Vaginosis Bacteriana/terapia , Adulto JovenRESUMEN
The effect of a novel polyherbal formulation BASANT on Chlamydia trachomatis was studied. In vitro sensitivity testing was done by direct exposure of C. trachomatis (pre-infection incubation with BASANT) and exposure of C. trachomatis within HeLa 229 cells (post-infection incubation with BASANT). Pre-infection incubation of standard serovar D/UW-3/Cx with BASANT showed complete inhibition after 60, 30 and 15 min of incubation at concentrations of 12, 30 and 60 microg/mL, respectively. In the post-infection incubation, 8-10 microg/mL of BASANT showed complete inhibition of standard serovar D/UW-3/Cx as well as of five clinical isolates of C. trachomatis after 48 h of incubation. BASANT also inhibited a clinical isolate obtained from a doxycycline treatment failure patient at a concentration of 30 microg/mL. Both assays with standard and clinical isolates showed that BASANT has antimicrobial activity against C. trachomatis, suggesting the potential clinical utility of BASANT for the prevention of C. trachomatis infection by the sexual route.
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Antibacterianos/farmacología , Chlamydia trachomatis/efectos de los fármacos , Extractos Vegetales/farmacología , Recuento de Colonia Microbiana , Células Epiteliales/microbiología , Femenino , Células HeLa , Humanos , Pruebas de Sensibilidad Microbiana , Plantas Medicinales , Factores de TiempoRESUMEN
A polyherbal cream (Basant) has been formulated using diferuloylmethane (curcumin), purified extracts of Emblica officinalis (Amla), purified saponins from Sapindus mukorossi, Aloe vera and rose water along with pharmacopoeially approved excipients and preservatives. Basant inhibits the growth of WHO strains and clinical isolates of Neisseria gonorrhoeae, including those resistant to penicillin, tetracycline, nalidixic acid and ciprofloxacin. It has pronounced inhibitory action against Candida glabrata, Candida albicans and Candida tropicalis isolated from women with vulvovaginal candidiasis, including three isolates resistant to azole drugs and amphotericin B. Basant displayed a high virucidal action against human immunodeficiency virus HIV-1NL4.3 in CEM-GFP reporter T and P4 (Hela-CD4-LTR-betaGal) cell lines with a 50% effective concentration (EC50) of 1:20000 dilution and nearly complete (98-99%) inhibition at 1:1000 dilution. It also prevented the entry of HIV-1(IIIB) virus into P4-CCR5 cells (EC50 approximately 1:2492). Two ingredients, Aloe and Amla, inhibited the transduction of human papillomavirus type 16 (HPV-16) pseudovirus in HeLa cells at concentrations far below those that are cytotoxic and those used in the formulation. Basant was found to be totally safe according to pre-clinical toxicology carried out on rabbit vagina after application for 7 consecutive days or twice daily for 3 weeks. Basant has the potential of regressing vulvovaginal candidiasis and preventing N. gonorrhoeae, HIV and HPV infections.
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Antiinfecciosos/farmacología , Candida/efectos de los fármacos , VIH-1/efectos de los fármacos , Papillomavirus Humano 16/efectos de los fármacos , Neisseria gonorrhoeae/efectos de los fármacos , Extractos Vegetales/farmacología , Aloe/química , Animales , Antiinfecciosos/toxicidad , Curcumina/química , Femenino , Enfermedades de los Genitales Femeninos/microbiología , Enfermedades de los Genitales Femeninos/virología , Células HeLa , Humanos , Phyllanthus emblica/química , Fitoterapia , Extractos Vegetales/toxicidad , Conejos , Sapindus/química , Cremas, Espumas y Geles VaginalesRESUMEN
AIM: To investigate the therapeutic potential of two recombinant proteins, Survivin and luteinizing hormone-releasing hormone (LHRH) fusion protein [LHRH(6leu)-LTB] for immunotherapy of breast cancer. METHODS: Murine 4T-1 breast cancer model was used to evaluate the efficacy of recombinant proteins in vivo. Twenty four Balb/c mice were divided into 4 groups of 6 mice each. Recombinant Survivin and LHRH fusion protein, alone or in combination, were administered along with immunomodulator Mycobacterium indicus pranii (MIP) in Balb/c mice. Unimmunized or control group mice were administered with phosphate buffer saline. Each group was then challenged with syngeneic 4T-1 cells to induce the growth of breast tumor. Tumor growth was monitored to evaluate the efficacy of immune-response in preventing the growth of cancer cells. RESULTS: Preventive immunization with 20 µg recombinant Survivin and MIP was effective in suppressing growth of 4T-1 mouse model of breast cancer (P = 0.04) but 50 µg dose was ineffective in suppressing tumor growth. However, combination of Survivin and LHRH fusion protein was more effective in suppressing tumor growth (P = 0.02) as well as metastasis in vivo in comparison to LHRH fusion protein as vaccine antigen alone. CONCLUSION: Recombinant Survivin and MIP suppress tumor growth significantly. Combining LHRH fusion protein with Survivin and MIP enhances tumor suppressive effects marginally which provides evidence for recombinant Survivin and LHRH fusion protein as candidates for translating the combination cancer immunotherapy approaches.
RESUMEN
The first evidence for the efficacy of a birth control vaccine in humans is now available from the Phase II trials on the human chorionic gonadotrophin vaccine in India. Several sperm antigens have been identified as potential contraceptive immunogens and zona pellucida antigens have been reported that reversibly control fertility.
PIP: Birth control vaccines, once proven to be safe, effective, and reversible, may be superior to available methods in terms of the reduced number of doses required, the lack of administration of pharmacological agents, and the absence of risk due to improper use. The reproductive process can be interrupted by immune effectors at several points, making several potential birth control vaccines possible. For example, pregnancy can be blocked by immune effectors which can either inactivate an hormone which is indispensable to reproduction or counteract a gamete antigen crucial for gamete development and/or fertilization. First evidence of a birth control vaccine in humans has emerged from Phase II trials on the human chorionic gonadotrophin vaccine in India. Several sperm antigens have been identified as potential contraceptive immunogens and zona pellucida antigens have been reported which reversibly control fertility. Several problems still have to be resolved before fertility control vaccines become available for routine use, but research is nonetheless yielding encouraging results. Sections discuss human chorionic gonadotrophin vaccines, gonadotropin releasing hormone vaccine, carrier-induced suppression, sperm antigen vaccines, egg antigens, other candidate immunogens, and future perspectives.
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Anticoncepción/métodos , Vacunas/inmunología , Gonadotropina Coriónica/inmunología , Hormona Liberadora de Gonadotropina/inmunología , Humanos , Masculino , Óvulo/inmunología , Espermatozoides/inmunologíaRESUMEN
A polyherbal vaginal pessary (Praneem) has been formulated that has antimicrobial properties against genital pathogens in addition to spermicidal action. Thus, it has dual potential as a barrier method for contraception and for providing protection against some sexually transmitted infections. The present study reports the findings of a multicentre trial that was conducted to evaluate the safety of this product. Trials were carried out in 23 women in three centres in India: the Postgraduate Institute of Medical Education and Research, Chandigarh; Safdarjang Hospital, New Delhi; and Kamla Nehru Memorial Hospital, Allahabad. Thorough clinical and pelvic examinations were carried out as well as cervical cytology, blood biochemistry and haematology before and after use of the polyherbal pessary intravaginally once daily for 7 consecutive days. No toxicity was observed on clinical examination or by laboratory investigations. Daily intravaginal use of this pessary for 7 days had no adverse effects on cervical cytology or on metabolic and organ functions.
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Antiinfecciosos/efectos adversos , Fitoterapia/efectos adversos , Extractos Vegetales/administración & dosificación , Quinina/administración & dosificación , Enfermedades de Transmisión Sexual/prevención & control , Espermicidas/efectos adversos , Administración Intravaginal , Adulto , Antiinfecciosos/administración & dosificación , Combinación de Medicamentos , Femenino , Humanos , Pesarios , Espermicidas/administración & dosificación , Frotis VaginalRESUMEN
Human lung tumor cells (ChaGo) established in culture from a bronchogenic squamous cell carcinoma synthesize and secrete large amounts of human chorionic gonadotropin (HCG), predominantly the alpha subunit of the glycoprotein hormone. ChaGo cells lose their transformation phenotypes following treatment with anti-alpha-HCG antibody or following inhibition of intracellular synthesis of alpha-HCG by the anti-sense RNA technique. We report that tumors induced by ChaGo cells in female athymic mice undergo necrotic degeneration following local or intraperitoneal administration of alpha-HCG-specific-antibody. The alpha-HCG antibody did not affect the growth of tumor induced by alpha-HCG nonproducing human tumor cells. Histopathological examinations of the anti-alpha-HCG antibody-treated tumor tissues showed active necrosis. When the antibody treatment was discontinued, the tumorigenesis process resumed. When anti-alpha-HCG antibody was administered simultaneously with ChaGo cells, a concentration-dependent inhibition of tumor growth in athymic mice was completely prevented at higher concentrations of the specific antibody.
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Carcinoma de Células Escamosas/patología , Gonadotropina Coriónica/inmunología , Hormonas Glicoproteicas de Subunidad alfa/fisiología , Neoplasias Pulmonares/patología , Animales , Anticuerpos , División Celular/fisiología , Gonadotropina Coriónica/farmacología , Femenino , Humanos , Ratones , Ratones Desnudos , Necrosis , Trasplante de Neoplasias , Células Tumorales CultivadasRESUMEN
We report here a large scale, double blind immunoprophylactic trial of a leprosy vaccine based on Mycobacterium w (Mw) in an endemic area of Kanpur Dehat, Uttar Pradesh, India. A population of 420,823 spread over 272 villages was screened where 1226 multibacillary (MB) and 3757 paucibacillary (PB) cases of leprosy were detected. A total of 29,420 household contacts (HHC) of these patients were screened for evidence of active or inactive leprosy. After exclusion of 1622 contacts for any of the different exclusion criteria, a total of 24,060 HHC could be vaccinated for vaccine or placebo under coding (20,194 administered two doses and 3866 received single dose). The vaccine consisted of 1 x 10(9) heat killed bacilli (Mw) in normal saline for the first dose and half of the first dose, i.e. 5 x 10(8) bacilli for the second dose, given 6 months after the first dose. The placebo consisted of 1/8th dose of the normal dose of tetanous toxoid. Both placebo and vaccine were given under double-blind coding, The contacts were followed up during three surveys at 3, 6 and 9 years after the initial vaccination, for detection of post-vaccination cases (PVCs) and observing any side-effects caused as a result of vaccination. The codes were opened on 24th January 2001, after the analysis of the data following completion of the third and final follow-up survey. When only contacts received the vaccine, Mw vaccine showed a protective efficacy (PE) of 68-6% at the end of first, 59% at the end of the second and 39.3% at the end of the third follow-up survey. When both patients and contacts received the vaccine, the protective efficacy observed was 68%, 60% and 28% at the end of the first, second and third surveys, respectively. When patients, and not the contacts, received the vaccine, a PE of 42.9% in the first, 31% in the second and 3% in the third survey was shown. These results suggest that the vaccination of the contacts is more valuable in achieving the objective of immunoprophylaxis than that of patients, and the vaccine effects are noted maximally in children (as compared to adolescents and adults) who constitute the most responsive group The effect of vaccine is sustained for a period of about 7-8 years, following which there is a need to provide a booster vaccination for the sustained protection.
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Vacunas Bacterianas/administración & dosificación , Transmisión de Enfermedad Infecciosa/prevención & control , Lepra/prevención & control , Lepra/transmisión , Vacunación/métodos , Adolescente , Adulto , Anciano , Niño , Preescolar , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Composición Familiar , Femenino , Estudios de Seguimiento , Humanos , India , Lepra/inmunología , Masculino , Persona de Mediana Edad , Probabilidad , Valores de Referencia , Medición de Riesgo , Factores de TiempoRESUMEN
Vaccinia virus (VV) expression vector was used to clone the genes for coding alpha and beta subunits of human chorionic gonadotropin (hCG). Recombinant viruses VSL3 and VSS1 containing these genes were selected as blue coloured plaques on the basis of co-expression of Escherichia coli beta-galactosidase in the infected cells. CV-1 cells when infected with VSL3 or VSS1 secreted 2.4 and 1.8 micrograms of alpha and beta hCG subunits, respectively, per 3 x 10(6) cells after 24 h of infection. The subunit proteins expressed individually had immunoreactivity with monoclonal and polyclonal antibodies specific to hCG. The subunit hormonal peptides associated with each other during co-infection to form the complete hCG dimer, which was biologically active as evident from the induction of steroidogenesis in a mouse Leydig cell system.
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Gonadotropina Coriónica/biosíntesis , Clonación Molecular , Genes Virales , Virus Vaccinia/genética , Animales , Anticuerpos Monoclonales , Línea Celular , Gonadotropina Coriónica/genética , Gonadotropina Coriónica/inmunología , Gonadotropina Coriónica Humana de Subunidad beta , Hormonas Glicoproteicas de Subunidad alfa/biosíntesis , Hormonas Glicoproteicas de Subunidad alfa/genética , Humanos , Ratones , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Plásmidos , Conformación Proteica , Radioinmunoensayo , Transfección , Ensayo de Placa Viral , beta-Galactosidasa/genéticaRESUMEN
Trichoplusia ni and Spodoptera littoralis larvae were infected with a recombinant AcNPV, having the viral polyhedrin gene replaced with the cDNA encoding firefly luciferase. Both S. littoralis and T. ni synthesized very high levels of luciferase representing greater than or equal to 25% and greater than or equal to 15%, respectively of the total Coomassie blue stainable protein. Luciferase was apparently not secreted into the hemolymph but was contained within the body tissue. Expression in S. littoralis larvae suggests that luciferase can be an excellent reporter enzyme to study virus infection, dissemination and expression in different tissues, host range determination, insect physiology and also to monitor the release of recombinant virus in the environment when used as a biocide.
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Escarabajos/genética , Virus de Insectos/genética , Luciferasas/genética , Mariposas Nocturnas/enzimología , Animales , Escarabajos/enzimología , Técnicas Genéticas , Vectores Genéticos , Larva , Luciferasas/biosíntesis , Recombinación Genética , Mapeo RestrictivoRESUMEN
A recombinant baculovirus, vAc alpha hCG, having a replacement of the viral polyhedrin gene with the cDNA encoding the alpha subunit of hCG was used to express alpha hCG, an extensively glycosylated hormone, in insect cells. Virus-infected cells, 72 h pi, secreted approximately 11.3 micrograms alpha hCG/2 x 10(6) cells/ml which was identical to the native hormonal peptide in terms of electrophoretic mobility, immunoreactivity and bioactivity on association with beta subunit, as evident by its binding to rat testicular cells and induction of steroidogenesis in a mouse Leydig cell bioassay system. The alpha hCG secreted into the medium represented approximately 20-30% of the total hCG synthesized by vAc alpha CG infected insect cells. The implications of using a very late promoter, in a baculovirus expression system, for directing the transcription of a gene whose gene product requires extensive post-translational modifications are discussed.
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Baculoviridae/genética , Gonadotropina Coriónica/biosíntesis , Animales , Western Blotting , ADN/genética , Electroforesis en Gel de Poliacrilamida , Regulación Viral de la Expresión Génica , Genes Virales , Vectores Genéticos , Humanos , Insectos , Mapeo RestrictivoRESUMEN
We demonstrate that the expression of extensively modified and secreted heterologous proteins synthesized in the baculovirus expression vector system (BEVS) depends on the temporal nature of the promoter transcribing the foreign gene. The beta subunit of the human chorionic gonadotropin, an extensively modified secretory glycoprotein hormone was expressed under the transcriptional control of the AcNPV basic protein gene promoter (MP) and the polyhedrin gene promoter (POL), respectively. MP, activated late in the infection cycle, is a weaker promoter when compared to the stronger very late POL promoter. Levels of secretion, immunoreactivity and bioactivity of recombinant proteins, beta hCGMP and beta hCGPOL synthesized under control of the MP and POL promoter were compared. Secretion of beta h CGMP was relatively higher. Enzymatic analysis revealed that the synthesized protein was sialylated. Receptor binding assays and testosterone stimulation assays in a mouse Leydig cell system demonstrated that on a unit protein basis, beta hCGMP was biologically more active than beta hCGPOL.
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Baculoviridae/genética , Gonadotropina Coriónica/genética , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional , Proteínas Virales/genética , Animales , Células Cultivadas , Gonadotropina Coriónica/metabolismo , Electroforesis en Gel de Poliacrilamida , Insectos , Proteínas de la Matriz de Cuerpos de Oclusión , Plásmidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Proteínas Estructurales ViralesRESUMEN
This paper describes the adaptation of a cellular enzyme-linked immunosorbent assay (CELISA) for the detection of antibodies to cell-surface antigens. This CELISA has the advantages of convenience and rapidity and is therefore ideally suited for the screening of a large number of hybridoma culture supernatants. The basic procedure involves the direct drying of cell suspensions onto the wells of enzyme immunoassay (EIA) plates and a subsequent EIA with appropriate blocking reagents. In order to overcome high background binding of primary antibodies to Fc receptors and of secondary antibodies to surface Ig (sIg), this method involves a blocking step consisting of unlabelled secondary antibodies. Once CELISA plates are prepared, they can be stored for a period of at least 6 months and hence this assay does not rely on the availability of fresh, viable cells for each assay. This assay is simple, reproducible and sensitive. The results can be assessed in an objective manner and can also be adapted for the detection of cellular antigens. This paper describes a CELISA for the detection of antibodies to blood group antigens and human leukocyte (HLA) antigens.