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Problem: To manage population of dogs (Canis familiaris), the efficacy of recombinant proteins‐based contraceptive vaccines to inhibit fertility has been evaluated in female beagle dogs. Method of study: Female beagle dogs (n = 4) were immunized with physical mixture of Escherichia coli‐expressed recombinant porcine ZP3 with promiscuous T cell epitope of tetanus toxoid (TT‐KK‐pZP3) and porcine ZP4 with promiscuous T cell epitope of bovine RNase (bRNase‐KK‐pZP4), or with a fusion protein encompassing dog ZP3 fragment and two copies of GnRH with appropriate promiscuous T cell epitopes (dZP3‐GnRH2); control animals received only alum, the adjuvant. The immunized animals were followed‐up for antibody titres by ELISA as well as for fertility status subsequent to mating with male dogs. Results: Active immunization of female dogs following a three injections schedule at 4‐week intervals with a physical mixture of TT‐KK‐pZP3 + bRNase‐KK‐pZP4 as well as dZP3‐GnRH2, led to generation of significant antibody titres against respective recombinant proteins. Active immunization with dZP3‐GnRH2 also led to generation of antibodies reactive with both dZP3 and GnRH. A booster dose on day 383 led to an increase in antibody titres and circulating antibodies against respective recombinant proteins could be observed on day 528. Antibodies in immune serum samples from dogs immunized with TT‐KK‐pZP3 + bRNase‐KK‐pZP4 or dZP3‐GnRH2 reacted with native canine ZP as assessed by an indirect immunofluorescence assay. Mating studies revealed a reduced number of pregnancies as well as a significant reduction in the number of pups born in the female dogs immunized with dZP3‐GnRH2 as compared to the adjuvanted control. Curtailment of pregnancy in dZP3‐GnRH2 immunized group was associated with antibody titres against dZP3‐GnRH2. However, immunization with recombinant TT‐KK‐pZP3 + bRNase‐KK‐pZP4 did not significantly decrease the number of pups born as compared to the adjuvanted control. Conclusion: These studies revealed the potential of recombinant dZP3‐GnRH2‐based contraceptive vaccine to curtail fertility in female dogs. Large scale studies to establish the efficacy and safety of this recombinant protein for the management of community dog population are thus warranted. [ABSTRACT FROM AUTHOR]

Problem: The miRNAs show placenta‐specific expression patterns, which alter during pregnancy‐related complications. In present study, the role of miR‐27b‐5p during forskolin‐mediated BeWo cells fusion has been investigated. Method of Study: The fusion of BeWo cells in response to forskolin treatment (25 µM) was studied by desmoplakin I+II staining. Expression profile of miR‐27b‐5p by qRT‐PCR and its targets HSD3β1 and WNT2B by qRT‐PCR and in Western blot were studied. The effect of overexpression of miR‐27b‐5p and silencing of HSD3β1 & WNT2B by siRNA on forskolin‐mediated BeWo cells fusion and secretion of hCG and progesterone by ELISA was investigated. Results: Time‐dependent down‐regulation in the expression of miR‐27b‐5p in forskolin‐treated BeWo cells has been confirmed by qRT‐PCR. Overexpression of miR‐27b‐5p significantly inhibits forskolin‐mediated BeWo cells fusion as well as hCG & progesterone secretion. HSD3β1 and WNT2B were identified as targets of miR‐27b‐5p and are up‐regulated in forskolin‐treated BeWo cells. Overexpression of miR‐27b‐5p in BeWo cells downregulates their expression. Further, luciferase reporter assay revealed that miR‐27b‐5p directly target expression of both HSD3β1 and WNT2B. Silencing of both HSD3β1 and WNT2B leads to a significant reduction in forskolin‐mediated BeWo cells fusion with concomitant decrease in the secretion of progesterone or/and hCG. Decrease in forskolin‐mediated cells fusion observed in miR‐27b‐5p mimic transfected BeWo cells could be rescued by the overexpression of both HSD3β1 and WNT2B. Conclusion: These observations suggest that reduced miR‐27b‐5p in forskolin‐treated BeWo cells leads to increased secretion of progesterone and hCG due to loss of repressional control on HSD3β1 and WNT2B. [ABSTRACT FROM AUTHOR]

Problem: Requirement of multiple injections of contraceptive vaccines to achieve infertility is one of the important impediments for their application. In the present study, attempts have been made to reduce the number of injections of contraceptive vaccine. Method of study: Fusion protein encompassing C‐terminus fragment of sperm protein Sp17 (aa residues 76‐126) and two copies of gonadotropin‐releasing hormone along with T‐cell epitopes and dilysine linkers (abbreviated as Sp17C‐GnRH2) was expressed in Escherichia coli. Its immunogenicity and contraceptive efficacy have been evaluated in female FVB/J mice using different adjuvants and delivery platforms. Results: Immunization of female mice with recombinant Sp17C‐GnRH2 (25 μg/injection/mouse) emulsified with squalene‐arlacel A following two injections schedule led to failure of 88.8% immunized animals to conceive, which was not significantly different from mice immunized with same protein along with alum following three injections schedule. To make single‐dose vaccine, poly d,l‐lactic acid–based microparticles (PLA‐MPs) entrapping Sp17C‐GnRH2 were prepared. Immunization of female mice with a combination of soluble Sp17C‐GnRH2 (12.5 μg/injection/mouse) along with Sp17C‐GnRH2 entrapped in PLA‐MPs (12.5 μg/injection/mouse) in alum showed higher antibody titres and contraceptive efficacy as compared to mice immunized with Sp17C‐GnRH2 entrapped in PLA‐MPs alone in alum. Immunization with recombinant Sp17C‐GnRH2 led to long‐term infertility as second mating (150 days after immunization) of various groups of immunized mice showed similar infertility as observed during first mating. Conclusion: Single‐dose immunization with PLA‐MPs entrapping Sp17C‐GnRH2 along with soluble recombinant protein in alum generated long‐lasting infertility in female mice. [ABSTRACT FROM AUTHOR]

Abstract: Betulinic acid (BA), a naturally occurring plant‐derived pentacyclic triterpenoid, has gained attention in recent years owing to its broad‐spectrum biological and medicinal properties. Despite the pharmacological activity of BA, it has been associated with some drawbacks, such as poor aqueous solubility and short half‐life in vivo, which limit therapeutic application. To solve these problems, much work in recent years has focused on enhancing BA's aqueous solubility, half‐life, and efficacy by using nanoscale drug delivery systems. Several different kinds of nanoscale delivery systems—including polymeric nanoparticles, magnetic nanoparticles, liposomes, polymeric conjugates, nanoemulsions, cyclodextrin complexes, and carbon nanotubes—have been developed for the delivery of BA. Here, we focus on the recent developments of novel nanoformulations used to deliver BA in order to improve its efficacy. [ABSTRACT FROM AUTHOR]

In an attempt to find bio-active small molecules, a series of novel 3-(5-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)pyridine 5a-i and 5-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbaldehyde 6a-i were designed, synthesized, and evaluated for their in vitro cytotoxic activity against a panel of human cancer cell lines namely; HeLa (human cervix), NCI-H460 (human lung), PC-3 (human prostate), and NIH-3T3 (mouse embryo fibroblasts) normal cell line. Most of these compounds exhibited moderate to good cytotoxic activity against the tested cancer cell lines and weak toxicity against normal cell line. Analogues 5b, 5f, 5g, 6b, and 6g showed significant cytotoxicity as compared to standard drug etoposide. Among all the synthesized compounds, compound 6g displayed superior cytotoxicity with an IC50 value of 7.98 ± 1.08 μM for Hela cancer cell line. [ABSTRACT FROM AUTHOR]

Problem Necessity to elicit antibody response above the protective threshold titres by sexually active women immunized to prevent pregnancy. Method of study Recombinant hCGβ- LTB vaccine expressed as both DNA and protein. Balb C mice employed for testing immunogenicity. Results Necessity to give three primary injections of the vaccine to elicit proper antibody response. Immunization twice with DNA form of the vaccine at fortnightly interval followed by the protein elicits a distinctly higher antibody response than proteinic vaccine alone. Antibodies generated are bio-effective against hCG. Conclusion Immunization with the DNA form of the recombinant hCGβ- LTB vaccine twice at fortnightly interval followed by the proteinic form of the vaccine induces distinctly higher antibody response. [ABSTRACT FROM AUTHOR]

Gupta SK, Srinivasan VA, Suman P, Rajan S, Nagendrakumar SB, Gupta N, Shrestha A, Joshi P, Panda AK. Contraceptive vaccines based on the zona pellucida glycoproteins for dogs and other wildlife population management. Am J Reprod Immunol 2011; 66: 51-62 Zona pellucida (ZP) glycoproteins, by virtue of their critical role in fertilization, have been proposed as candidate antigens for the development of contraceptive vaccines. In this review, the potential of a ZP-based contraceptive vaccine for the management of wildlife population, with special reference to street dogs, is discussed. Immunization of various animal species, including female dogs, with native porcine ZP led to inhibition of fertility, which was associated with the ovarian dysfunction. Immunization of female dogs with Escherichia coli-expressed recombinant dog ZP glycoprotein-3 (ZP3) either coupled to diphtheria toxoid or expressed as fusion protein with 'promiscuous' T non-B-cell epitope of tetanus toxoid also led to inhibition of fertility. To improve the contraceptive efficacy of ZP-based contraceptive vaccine, various groups are working on improving the immunogen, use of DNA vaccine as prime-boost strategy, and delivering the zona proteins/peptides presented on either virus-like particles or entrapped in microsphere. Host-specific live vectors such as ectromelia virus and cytomegalovirus have also been used to deliver mouse ZP3 in mice. Various studies show the enormous potential of the ZP-based vaccine for the management of wildlife population, where permanent sterilization may be desirable. [ABSTRACT FROM AUTHOR]

An internal cDNA fragment (978 bp) corresponding to bonnet monkey (Macaca radiata ) zona pellucida glycoprotein-C (bmZPC), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was cloned in pQE-30 vector and the protein expressed as inclusion bodies in Escherichia coli . Recombinant bmZPC (r-bmZPC) was solubilized from purified inclusion bodies in the absence of a high concentration of chaotropic agents and was subsequently refolded. Use of a low concentration of urea (2 m) during solubilization of r-bmZPC helped to minimize the extent of protein aggregation during refolding of the recombinant protein, and retain the existing native-like secondary structure that was essential for proper folding. Purified r-bmZPC appeared as a dominant band of 43 kDa on SDS/PAGE and Western blot. Although it lacked carbohydrate moieties, the purified and refolded r-bmZPC bound to the head region of bonnet monkey spermatozoa, confirming the existence of a native-like conformation. CD revealed a maximum at 200 nm and a single broad minimum extending from 209 to 216 nm, indicating the presence of both α-helical and β-sheet conformations in the refolded r-bmZPC. Two different phases of transition were observed by urea-gradient electrophoresis, suggesting the existence of multiple intermediate stages during the unfolding of r-bmZPC. The availability of refolded r-bmZPC will help in elucidating its role during the complex cascade of events during fertilization. [ABSTRACT FROM AUTHOR]