A partner monoclonal antibody to Moab 730 kills 100% of DU145 and PC3 androgen-independent cancer cells.

A number of therapeutic options are available for patients with prostate carcinoma till the time that the tumour is hormone dependent. However, no fully effective therapy is available for the treatment of androgen-independent prostate carcinomas. Antibodies directed at epitopes unique to or overexpressed on the cancer cells could be of therapeutic utility. A monoclonal antibody (Moab) 2C4 has been generated, which binds with cells of two androgenindependent prostate cancers, DU145 and PC3, and does not bind to peripheral blood leukocytes (PBLs) of healthy donors. This antibody, along with the previously developed Moab 730, kills 100% of both DU145 and PC3 cells in the presence of complement and does not have a deleterious effect on PBLs of healthy males. The anti-tumour action of the two antibodies prevents the establishment of DU145 cell tumour in nude mice in vivo. Moab 2C4 in combination with 730 has potential for use as therapy for androgen-independent cancers.

Spectrum of Lactobacillus species present in healthy vagina of Indian women.

Background & objectives: Lactobacilli are depleted in vagina of women suffering from recurring episodes of bacterial vaginosis with vaginal pH >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 specifi c primers were further characterized by employing species specifi c primers followed by sequencing of 16S rDNA. Isolates of the same species were differentiated by random amplifi ed polymorphic DNA (RAPD) profi les. 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 identifi cation. None of the isolates had similar RAPD profi le. Interpretation & conclusions: Our fi ndings 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.

Spermicidal & contraceptive properties of Praneem polyherbal pessary.

BACKGROUND & OBJECTIVES: Though a number of barrier methods and potent spermicides are available, most of these have nonoxynol-9 (N-9) as the active ingredient which is observed to cause inflammation and genital ulceration on repeated use. The present study was undertaken to develop a safe spermicide with conjoint microbicidal properties. METHODS: A polyherbal pessary was formulated with purified ingredients from neem (Azadirachta indica) leaves, Sapindus mukerossi (pericarp of fruit) and Mentha citrata oil. Spermicidal action on human sperm was tested by Sander-Cramer slide test in vitro and by post coital tests in vivo. Contraceptive action was tested in rabbits. RESULTS: The combination of the three herbal ingredients resulted in the potentiation of the spermicidal action by 8-folds. The post coital tests confirmed the spermicidal properties of the Praneem polyherbal pessary (PPP) in women with high cervical mucous score around mid estrus. It also prevented in most women the migration of sperm into the cervical mucous. In 15 rabbits studied pregnancy was prevented by the intravaginal administration of PPP, whereas 13 of the 15 animals in the control group became pregnant. INTERPRETATION & CONCLUSION: The Praneem polyherbal pessary has potent spermicidal action on human sperm in vitro and in vivo. When applied in the vagina before mating, it prevented rabbits from becoming pregnant.

The impact of new technologies on vaccines.

Vast changes are taking place in vaccinology consequent to the introduction of new technologies. Amongst the vaccines included in the Expanded Programme of Immunization (EPI), the pertussis vaccine has been replaced by acellular purified fractions devoid of side-effects. Non-pathogenic but immunogenic mutants of tetanus and diptheria toxins are likely to replace the toxoids. An effective vaccine against hepatitis B prepared by recombinant technology is in large-scale use. Conjugated vaccines against Haemophilus influenzae b, S. pneumococcus and meningococcus are now available, as also vaccines against mumps, rubella and measles. Combination vaccines have been devised to limit the number of injections. Vaccine delivery systems have been developed to deliver multiple doses of the vaccine at a single contact point. A genetically-engineered oral vaccine for typhoid imparts better and longer duration of immunity. Oral vaccines for cholera and other enteric infections are under clinical trials. The nose as a route for immunization is showing promise for mucosal immunity and for anti-inflammatory experimental vaccines against multiple sclerosis and insulin-dependent diabetes mellitus. The range of vaccines has expanded to include pathogens resident in the body such as Helicobacter pylori (duodenal ulcer), S. mutans (dental caries), and human papilloma virus (carcinoma of the cervix). An important progress is the recognition that DNA alone can constitute the vaccines, inducing both humoral and cell-mediated immune responses. A large number of DNA vaccines have been made and shown interesting results in experimental animals. Live recombinant vaccines against rabies and rinderpest have proven to be highly effective for controlling these infections in the field, and those for AIDS are under clinical trial. Potent adjuvants have added to the efficacy of the vaccines. New technologies have emerged to 'humanize' mouse monoclonals by genetic engineering and express these efficiently in plants. These recombinant antibodies are opening out an era of highly specific and safe therapeutic interventions. Human recombinant antibodies would be invaluable for treating patients with terminal tetanus and rabies. Antibodies are already in use for treatment of cancer, rheumatoid arthritis and allergies. An advantage of preformed antibodies directed at a defined target and given in adequate amounts is the certainty of efficacy in every recipient, in contrast to vaccines, where the quality and quantum of immune response varies from individual to individual.

Safety of intrauterine administration of purified neem seed oil (Praneem Vilci) in women & effect of its co-administration with the heterospecies dimer birth control vaccine on antibody response to human chorionic gonadotropin.

Praneem Vilci (PV), purified neem oil was reported to exercise a reversible antifertility effect after a single intrauterine instillation in rodents and primates without any adverse effects. After toxicology, drug regulatory and ethical clearances, a phase I clinical trial was conducted on PV. Eighteen healthy tubectomised women were enrolled to evaluate the safety of a single intrauterine instillation of PV and to determine the effect of its co-administration on anti-hCG response to the heterospecies dimer (HSD) hCG vaccine. Eight women received PV alone and ten women were given the HSD-hCG vaccine in addition. Base-line and post-treatment haematological and biochemical profiles were determined as also the mid-luteal serum progesterone. Endometrial biopsies were examined to assess ovulatory status and the effect of intrauterine treatment with PV on the endometrium. Anti-hCG antibody titres were estimated in women who were concurrently immunized with the HSD vaccine. No untoward reaction was observed in any woman. Menstrual pattern and ovulatory status remained unaltered. Endometrial biopsy after PV instillation in one woman showed non-specific endometritis but she remained asymptomatic. Mild eosinophilia was seen in two women and this reverted to normal on its own. All women receiving PV and the HSD vaccine generated antibodies against hCG. Our data show that intrauterine administration of PV is safe and does not prevent the antibody response to HSD-hCG vaccine.

Monoclonal antibodies against antigenic epitopes common between Setaria cervi and Brugia malayi.

Several common antigens between the bovine (Setaria cervi) and human (Brugia malayi) filarial parasites have been demonstrated [Immunol Investig, 16 (1987) 139]. Hybridoma cell lines producing monoclonal antibodies against such common antigenic epitopes were obtained by immunizing the BALB/c mice with S. cervi antigen, fusing the spleen cells with Sp2/0 myeloma cells and screening the culture supernatants for antibody against both S. cervi and B. malayi antigens by ELISA. Nine monoclonal antibodies directed against antigenic epitopes common between the bovine and human filarial parasites were identified. Two monoclonal antibodies (I3B4 and I5D6) showed reactivity with the antigen(s) present in filariasis patients serum and thus may have potential for detecting circulating antigen in filaria infected individuals.

Immunodiagnosis of group-A streptococci by latex agglutination assays with monoclonal or monospecific polyvalent antibodies.

Seven clones of murine monoclonal antibodies specific for group-A streptococci were generated. All of them were of IgM isotypes and recognized trypsinized as well as nontrypsinized group-A streptococci and polysaccharide. These were devoid of reactivity with streptococci-B, -C, -G and Staphylococcus aureus. Polyclonal antibodies against group-A polysaccharide (APS) were also raised in rabbits by linking APS to bovine serum albumin, and rendered monospecific by adsorption. Latex agglutination assays were developed employing both types of antibodies. The assay employing monospecific polyvalent antibodies had a sensitivity of 12.5 ng APS/ml as compared to 1 microgram APS/ml for latex sensitized with monoclonal antibodies. Both assays were specific, as no agglutination was observed with polysaccharides obtained from streptococci-B, -C, -G, Staph. aureus, Cornybacterium diptheriae, Candida albicans, Candida spp., Morexella catarrhalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Streptococcus agalactiae, Strep. pneumoniae. Salmonella typhi and S. paratyphi A and B. Throat swabs from children obtained in duplicate, when tested for the presence of streptococci-A, showed a good correlation of the results obtained by latex agglutination assay with the microbial culture test and serogrouping.

Characteristics of monoclonal antibodies against porcine zona pellucida-3 and their functional relevance.

Seven monoclonal antibodies (MAs) against 55 kDa glycoprotein family of porcine zona pellucida (ZP3) reacting with either ZP3 alpha (MA-7, MA-27, MA-28) or ZP3 beta (MA-1, MA-2, MA-11, MA-30) have been described. MA-1, -2, -27, -28 and -30 do not recognize carbohydrate determinants as shown by their reactivity to the deglycosylated (DG) ZP3 alpha and ZP3 beta. Indirect immunoperoxidase studies showed that all MAs reacted with zona pellucida from porcine and monkey ovaries. Only MA-1 and -27 reacted with ZP from rabbit ovary as well, while none of the MAs recognised mouse ZP, MA-7, -11, -27, -28 and -30 inhibited in vitro, the zona lysis by trypsin as well as the binding of ZP3 to sperm membrane vesicle as investigated by ELISA.

Amphiphilic and hydrophilic domains of human sperm membrane antigens recognized by immunoinfertile sera.

Selected sera from married couples with immunological infertility were used to identify antigens on hydrophilic and amphiphilic domains of human sperm membrane. Out of eight sera, six recognized proteins from the hydrophilic as well as amphiphilic regions of the sperm membrane. Sera were either reactive to acrosome or to equator and tail of human sperms in indirect immunofluorescence assay.

Induction of lepromin positivity by a candidate anti-leprosy vaccine Mycobacterium w in lepromin negative healthy contacts of multibacillary leprosy patients.

In a hospital based study, 362 household contacts of multibacillary leprosy patients were screened for evidence of leprosy and 54 (14.9%) were found to be having leprosy. The remaining 308 apparently healthy contacts were lepromin tested and 109 (35.4%) were observed to be negative to Mitsuda lepromin. M.w vaccine was administered intradermally to 95 of these 109 lepromin negative contacts. Sixty eight of them could be retested for lepromin A reactivity. Fifty six (82.35%) manifested lepromin conversion. The twelve subjects who did not show lepromin conversion, received a second dose of the vaccine, and eleven subsequently became lepromin positive. The overall lepromin conversion rate was thus 98.5% (67 out of 68). Follow-up of these contacts upto a period of 30 months did not demonstrate reversion of lepromin positivity back to negativity status. No untoward effects of vaccination were observed except for local ulceration at the site of vaccine administration.

Over-expression and characterization of recombinant beta subunit of the human chorionic gonadotropin hormone synthesized in insect cells infected with a genetically engineered baculovirus.

A recombinant baculovirus, vAc beta hCG, having a replacement of the viral polyhedrin gene with the cDNA encoding the beta subunit of hCG was used to express beta hCG, an extensively glycosylated hormone, in insect cells. Virus-infected cells, 72 hr pi, secreted approximately 8.02 micrograms beta hCG/2 x 10(6) cells/ml. The recombinant beta hCG purified from insect cells exhibited increased mobility on SDS-PAGE as compared to authentic urinary beta hCG, a reflection on differences in glycosylation between insect and mammalian systems. The insect derived beta hCG, however, was identical to the native hormonal peptide in terms of immunoreactivity and bioactivity on association with alpha-subunit, as evident by its binding to rat testicular receptors and induction of steroidogenesis in a mouse Leydig cell bioassay system. The implications of using the baculovirus system to study the importance of carbohydrates for biological activity are also discussed.

Cloning and nucleotide sequencing of sheep growth hormone cDNA.

cDNA was prepared from the mRNA isolated from sheep anterior pituitary glands. On cloning cDNA in E. coli, a clone coding full sequence of sheep pre-growth hormone was determined. The sequence for the sheep growth hormone (GH) is in agreement with the amino acid sequence of the protein determined previously except for the asparagine residue at position 99 rather than aspartic acid and the arginine residue at position 146 in place of threonine. The cDNA sequence presented is also in accordance with the genomic sequence for the sheep GH gene that has been reported.

Cloned single copy DNA sequences of Mycobacterium tuberculosis as DNA probes.

A recombinant genomic clone was isolated from a lambda gt 11 library of M. tuberculosis on the basis of lack of hybridization with M. avium and M. kansasi. The specificity and sensitivity of M. tb DNA probes, 2.5 and 2.3 kb in size, were assessed by Southern blot and dot blot hybridization. These did not cross hybridize to DNA of mycobacteria other than members of M. tb complex, nor with DNA of non mycobacterial origin. Sensitivity was determined to be 200 pg which is equivalent to 10(4) bacilli. Genomic Southern hybridization indicated single copy nature of the probes.

Construction, purification and characterization of a recombinant baculovirus containing the gene for alpha subunit of human chorionic gonadotropin.

A cDNA encoding the alpha subunit of human chorionic gonadotropin, a placental glycoprotein hormone, was cloned downstream to the viral polyhedrin gene promoter of Autographa california nuclear polyhedrosis virus and the recombinant transfer vector was used to co-transfect Spodoptera frugiperda cells growing in culture. Recombinant baculovirus carrying the alpha hCG gene was detected and isolated after dot hybridization using supernatant from co-transfected cells. Recombinant vAc alpha hCG having a replacement of the viral polyhedrin gene, which is hyper-transcribed very late in the infection cycle, with the alpha hCG cDNA was purified after a single round of plaque purification. Insect cell culture infected with vAc alpha hCG, secreted high levels of hCG which was biologically active.

Molecular cloning of human satellite DNA sequences and their use in detecting DNA polymorphism.

A approximately 400 bp HaeIII human genomic satellite DNA band was cloned into pUC18 to construct a partial library. A fragment of bacteriophage M13 containing a sequence homologous to the human minisatellite core was cloned in pUC18 and was used as a probe to isolate a approximately 350 bp human satellite clone (pTRF5.6) from the partial library. Other clones from this library showed a wide variation in terms of size and hybridization to the pTRF5.6 clone. Human DNA from different individuals was digested with restriction enzymes, Southern transferred and probed with TRF5.6. Individual-specific complex pattern of DNA bands was produced. TRF5.6, therefore, could be useful as a probe for detecting genetic polymorphism.

Comparative evaluation of enzyme immunoassays based on synthetic glycoconjugates and phenolic glycolipid-I for immunodiagnosis of leprosy.

Enzyme immunoassays (EIAs) based on synthetic glycoconjugates containing the terminal monosaccharide (M-BGG) or disaccharide (ND-BSA) residue of the trisaccharide component of phenolic glycolipid-I (PGL-I), for immunodiagnosis of leprosy are described. The results of the assays were compared with that of the EIA using PGL-I. All the three assays were highly specific for leprosy. The per cent positivity of active lepromatous leprosy (LL) patients with M-BGG was 78.05 in comparison to 85.36 with ND-BSA and 82.11 with PGL-I. Similarly, the positivity of tuberculoid (TT) leprosy patients in M-BGG assay was lower than that in EIAs using ND-BSA or PGL-I. However, the difference in the positivity of individual category of leprosy patients in the three EIAs was not statistically significant. The correlation between absorbance values of leprosy sera in EIAs based on M-BGG and PGL-I, as well as that in assays using ND-BSA and PGL-I was statistically significant.

Generation and characterization of a human monoclonal antibody against phenolic glycolipid-I of M. leprae.

The development of an Epstein-Barr virus transformed human B-cell line secreting a monoclonal antibody (MoAb), KR2/B5 is described. KR2/B5 is an IgM type of antibody and is highly specific for phenolic glycolipid-I (PGL-I) a component unique to M. leprae. The MoAb appears to be directed against the terminal sugar residue of the immunodominant trisaccharide component of PGL-I.