Effect of antimicrobial Peptide, nisin, on the reproductive functions of rats.

Our previous studies have demonstrated that naturally occurring peptide, Nisin possess antibacterial activity and did not interfere with rabbit vaginal mucosa. In this study, the reproductive toxicity of the Nisin in male rats was evaluated. Rats were fed orally with Nisin (10, 25, and 50 mg/kg/day) for 13 weeks. No treatment related mortality was observed. The body weight gain, food consumption and serum biochemical parameters were at par with the control group. Histomorphology of the selected reproductive (testis, epididymis, ventral prostate, and seminal vesicle) and nonreproductive (liver and kidney) tissues was observed to be normal. There was no treatment-related increase or decrease in the expression of testis-specific genes (c-Kit, GATA-1, and HILS-1) and the activity levels of epididymal α-glucosidase, ventral prostate alkaline phosphatase (AlP), liver alanine aminotransferase (AlAT) and aspartate aminotransferase (AAT). Fructose and lactic acid levels in the seminal vesicles also remained unchanged. These studies suggest that Nisin did not affect the normal physiology of these organs. In addition, no adverse effects were observed on the reproductive performance of Nisin-treated male rats and their offspring. In conclusion, the current studies support our earlier studies, which demonstrated suitability of Nisin as a safe and effective microbicide.

Ageing down-modulates liver inflammatory immune responses to schistosome infection in mice.

Ageing is associated with several alterations in the immune system. Our aim in this study was to compare the development of immunity to Schistosoma mansoni infection in young versus aged C57Bl/6 mice using the liver as the main organ to evaluate pathological alterations and immune responses. In the acute phase, young mice had large liver granulomas with fibrosis and inflammatory cells. Chronic phase in young animals was associated with immunomodulation of granulomas that became reduced in size and cellular infiltrate. On the other hand, aged animals presented granulomas of smaller sizes already in the acute phase. Chronic infection in these mice was followed by no alteration in any of the inflammatory parameters in the liver. In concert with this finding, there was an increase in activated CD4+ T, CD19+ B and NK liver cells in young mice after infection whereas old mice had already higher frequencies of activated B, NK and CD4+ T liver cells and infection does not change these frequencies. After infection, liver production of inflammatory and regulatory cytokines such as IFN-gamma, IL-4 and IL-10 increased in young but not in old mice that had high levels of IL-4 and IL-10 regardless of their infection status. Our data suggest that the unspecific activation status of the immune system in aged mice impairs inflammatory as well as regulatory immune responses to S. mansoni infection in the liver, where major pathological alterations and immunity are at stage. This poor immune reactivity may have a beneficial impact on disease development.

Assessment of cervicovaginal cytokine levels following exposure to microbicide Nisin gel in rabbits.

Topical microbicides is an emerging female controlled strategy for preventing the acquisition and transmission of STIs/HIV infections. Since they are intended for repeated vaginal and/or rectal use it is essential to validate their safety. Nisin, a naturally occurring contraceptive antimicrobial peptide (AMP) is currently the focus of clinical trials. The present in vitro vaginal tissue explants culture studies revealed that Nisin did not effect vaginal cell viability analyzed at 15, 30, 45 and 60min following treatment with different concentrations of Nisin gel prepared in 1% polycarbophil gel (30.3, 60.6, 121.2, 242.4 and 484.8 microM/g tissue) and SDS (0.35, 0.70, 1.4, 2.8 and 5.6 microM/g tissue) gels compared to placebo gel treated groups. The levels of various pro-inflammatory (IL-6, IL-8 and TNF-alpha,) and immuno-regulatory cytokines (IL-10 and GM-CSF) in the explant culture supernatants of the Nisin treated cells were unaffected. Repeated intravaginal application of high dose of Nisin gel (15,150 microM/day/14 days) on cervicovaginal epithelium was evaluated in rabbits and the results were compared with SDS treated (56 microM) and 1% polycarbophil gel (placebo) groups. We examined vaginal cell morphology, structural integrity of vaginal epithelium and local production of cytokines (PICs) in the cervicovaginal lavage (CVL) of Nisin treated animals and compared with placebo and SDS treated groups. The results demonstrated no treatment related abnormalities either in the vaginal cell morphology or structural abnormalities in the mucosal epithelium. There was no change in the cytokine levels in cervicovaginal lavage (CVL) compared to SDS gel treated animals indicating Nisin gel did not induce irritation and/or inflammation in the vaginal epithelium. CVL cytokine levels were in accordance with immunohistochemical (IHC) localization of cytokines and flow cytometric evaluation of CD45 immune cell population in cervicovaginal epithelium. The levels of cytokines in the CVLs appear to be sensitive indicators in identifying and/or screening out suitable candidate microbicides before they enter phase-1 trials. In conclusion, the lack of vaginal toxicity of Nisin gel means that it has clinical potential as a safe, prophylactic contraceptive in addition to its antimicrobial activities to curb sexual transmission of HIV in human.

Inhibition of sperm-egg binding and fertilisation in mice by a monoclonal antibody reactive to 57-kDa human sperm surface antigen.

Monoclonal antibodies (mAbs) against spermatozoa are a popular approach to define sperm antigens involved in the process of fertilisation. The identification and characterisation of a 57-kDa fertility asssociated sperm antigen (FASA-57) from human spermatozoa was reported in an earlier paper by the authors. In the present report, studies to develop mAbs against partially purified FASA-57 are extended. From a panel of mAbs raised, one clone designated as 3H(4)B(9) was selected and characterised because it recognised native FASA-57. Indirect immunofluorescence studies revealed that FASA-57 localised on the acrosome of non-acrosome-reacted human spermatozoa and on the equatorial region after the acrosome reaction. Spermatozoa from several other mammalian species were also found to express this antigen, suggesting its evolutionary conservation across the species. The antigen localised specifically in spermatogonial cells and luminal spermatozoa of the testis and epididymis. Western blot studies showed the presence of a FASA-57-like protein in the mouse brain also, indicating that testis and brain share antigenic similarities. Further, the role of FASA-57 in sperm-egg interaction was investigated using a mouse model. The mAb 3H(4)B(9) inhibited sperm-egg binding and fusion in a dose-dependent manner with half-maximal inhibition at 2 microg mL(-1). In conclusion, FASA-57 appears to play an important role in sperm-egg recognition, fusion and fertilisation. Therefore, FASA-57 could be used as a diagnostic marker in the evaluation of male infertility.

Antimicrobial peptides: premises and promises.

Antimicrobial peptides (AMPs) are an important component of the natural defences of most living organisms against invading pathogens. These are relatively small (< 10kDa), cationic and amphipathic peptides of variable length, sequence and structure. During the past two decades several AMPs have been isolated from a wide variety of animals, both vertebrates and invertebrates, and plants as well as from bacteria and fungi. Most of these peptides are obtained from different sources like macrophages, neutrophils, epithelial cells, haemocytes, fat body, reproductive tract, etc. These peptides exhibit broad-spectrum activity against a wide range of microorganisms including Gram-positive and Gram-negative bacteria, protozoa, yeast, fungi and viruses. A few peptides have also been found to be cytotoxic to sperm and tumour cells. AMPs are classified based on the three dimensional structural studies carried out with the help of NMR. The peptides are broadly classified into five major groups namely (a) peptides that form alpha-helical structures, (b) peptides rich in cysteine residues, (c) peptides that form beta-sheet, (d) peptides rich in regular amino acids namely histatin, arginine and proline and (e) peptides composed of rare and modified amino acids. Most of these peptides are believed to act by disrupting the plasma membrane leading to the lysis of the cell. AMPs have been found to be excellent candidates for developing novel antimicrobial agents and a few of these peptides show antimicrobial activity against pathogens causing sexually transmitted infection (STI), including HIV/HSV. Peptides, namely magainin and nisin have been shown to demonstrate contraceptive properties in vitro and in vivo. A few peptides have already entered clinical trials for the treatment of impetigo, diabetic foot ulcers and gastric helicobacter infections. In this review, we discuss the source, structures and mode of action with special reference to therapeutic considerations of various AMPs.

Evaluation of antimicrobial peptide nisin as a safe vaginal contraceptive agent in rabbits: in vitro and in vivo studies.

In the midst of the global epidemics of both unwanted pregnancies and sexually transmitted infections (STIs), options that provide protection are ideal. In the present study, nisin, a known antimicrobial peptide, was evaluated for safety and contraceptive potential in vitro and in vivo in the rabbit. A concentration of 400 microg nisin per ml was found to be spermicidal in vitro, and the effect was dose and time dependent. In vivo studies indicated that intravaginal application of 1 mg nisin blocked conception in rabbits. Repeated application of nisin (50 mg/animal per day) in rabbits for 14 consecutive days did not cause local inflammation or damage to the vaginal epithelium. In addition, the rate of diffusion of nisin into the blood via the vaginal mucosal epithelium, and its clearance from the circulation was found to be rapid. No treatment-related changes were observed in the reproductive performance of rabbits after cessation of treatment. Furthermore, no changes were observed in the gestation period, subsequent growth and survival of neonates in these animals. When male rats were given nisin orally for 13 consecutive weeks, no effect was observed on reproductive performance. The number of pups born, survival and growth of pups were unaltered. The affinity studies of nisin revealed that spermatozoa are more susceptible to nisin than red blood cells and vaginal epithelial cells. We suggest that nisin with spermicidal and antimicrobial properties could serve as a safe vaginal contraceptive for future therapeutic interventions in STIs.

Studies on safety aspects of contraceptive Magainin-A in rabbits.

We have previously reported the contraceptive potential of Magainin-A in rats and rabbits under in vitro and in vivo condition. In this report we evaluated the effect of Magainin-A on the structural organisation of vaginal epithelium in rabbits. The effect of this compound on the erythrocytes and its rate of absorption and clearance from systemic circulation was also studied. The effective contraceptive dose of Magainin-A (1 mg) when administered intra-vaginally for five consecutive days did not induce any structural or morphological abnormalities in vaginal epithelial cells. No adverse effect was observed on the erythrocytes. The rate of Magainin-A absorption and clearance from the circulation was found to be rapid. These results suggest that Magainin-A may be used as a safe intra-vaginal contraceptive compound in future.

Isolation and purification of an early pregnancy factor-like molecule from culture supernatants obtained from lymphocytes of pregnant women: II. Identification of the molecule as a Fc-receptor-like molecule: a preliminary report.

PURPOSE: Early pregnancy factor (EPF)-like activity from culture supernatants obtained from stimulated lymphocytes of pregnant women was characterized and identified. METHODS: The enzyme-linked immunosorbent assay depending on the presence of "Fc" receptors on bovine spermatozoa was used to identify the EPF-like molecule purified by gel filtration and reverse-phase high-performance liquid chromatography. RESULTS: The results indicated that the crude lymphocyte culture supernatant, the EPF-positive G IV fraction obtained on gel filtration, and the EPF-positive reverse-phase high-performance liquid chromatography protein readily bound with the different concentrations of aggregated human gamma-globulin in a manner similar to that in which the standard control of aggregated human gamma-globulin binds to the bovine spermatozoa. CONCLUSIONS: EPF-like activity synthesized and secreted by lymphocytes during pregnancy may be a Fc-receptor-like molecule.

Isolation and purification of an early pregnancy factor-like molecule from culture supernatants obtained from lymphocytes of pregnant women.

PURPOSE: Our purpose was to determine whether lymphocytes synthesize proteins during pregnancy, to observe whether one of the proteins synthesized has early pregnancy factor (EPF)-like activity and to isolate and purify this molecule from culture supernatants obtained from stimulated lymphocytes of pregnant women. METHODS: Lymphocyte proliferation assay and 35S-methionine labeling were done to study de novo synthesis of proteins followed by autoradiography to confirm synthesis of proteins. The rosette inhibition assay was used for detection of the EPF-like molecule. Gel filtration on Sephadex G-100 and RPHPLC were used for purification of the EPF-like molecule. RESULTS: The rate of incorporation of 35S-methionine was significantly higher in the lymphocytes of pregnant women compared to those of the control, and autoradiography confirmed the synthesis of proteins during pregnancy. There is a total protein enhancement trend observed during the first trimester that declines toward term. The EPF-like molecule is observed to be synthesized during all the trimesters of pregnancy. This molecule, when purified, showed a single homogeneous biologically active peak. CONCLUSIONS: The results indicated that there is an enhancement of existing protein or synthesis of new proteins during pregnancy. The EPF-like molecule is one of the many proteins synthesized and secreted by lymphocytes during pregnancy that, when purified, is biologically active.

Evaluación de la calidad del cuidado infantil antes y después de la implantación de un programa de salud / Quality assessment of child healthcare before and after the establishment of a health program

El presente estudio ha evaluado la supervisión de la salud de niños menores de un año en una unidad de atención primaria de la salud en las afueras de Porto Alegre. La calidad de los cuidados de salud de los niños ha sido evaluada en dos períodos diferentes: antes y después de la implantación del programa del niño, respectivamente. Las visitas médicas realizadas durante ambos períodos -1986 y 1991- han sido estudiadas de acuerdo a: cobertura, frecuencia de las visitas a lo largo del primer año de vida, edad de los niños al tiempo de la primera visita y contenido de la primera visita. El cuidado médico adecuado ha sido establecido basándose en las referencias bibliográficas. Se recogieron los datos de historias existentes en cada visita desde la implantación de la unidad de atención primaria de la salud. La cobertura fue de del 35 por ciento y 90 por ciento respectivamente en 1986 y 1991. El número promedio de consultas para cada niño fue de 2 en 1986; 80 por ciento de los niños tuvieron hasta 6 consultas cada uno. En la primera visita la edad de los niños fue de 4 meses en 1986 y un mes en 1991. En 1991, el 56 por ciento de sus madres había tenido cuidados prenatales en esta unidad. Los resultados presentados muestran que la implantación de un programa dirigido a la infancia ha mejorado la calidad de los cuidados de salud de los niños

Evaluación de la calidad del cuidado infantil antes y después de la implantación de un programa de salud / Quality assessment of child healthcare before and after the establishment of a health program

El presente estudio ha evaluado la supervisión de la salud de niños menores de un año en una unidad de atención primaria de la salud en las afueras de Porto Alegre. La calidad de los cuidados de salud de los niños ha sido evaluada en dos períodos diferentes: antes y después de la implantación del programa del niño, respectivamente. Las visitas médicas realizadas durante ambos períodos -1986 y 1991- han sido estudiadas de acuerdo a: cobertura, frecuencia de las visitas a lo largo del primer año de vida, edad de los niños al tiempo de la primera visita y contenido de la primera visita. El cuidado médico adecuado ha sido establecido basándose en las referencias bibliográficas. Se recogieron los datos de historias existentes en cada visita desde la implantación de la unidad de atención primaria de la salud. La cobertura fue de del 35 por ciento y 90 por ciento respectivamente en 1986 y 1991. El número promedio de consultas para cada niño fue de 2 en 1986; 80 por ciento de los niños tuvieron hasta 6 consultas cada uno. En la primera visita la edad de los niños fue de 4 meses en 1986 y un mes en 1991. En 1991, el 56 por ciento de sus madres había tenido cuidados prenatales en esta unidad. Los resultados presentados muestran que la implantación de un programa dirigido a la infancia ha mejorado la calidad de los cuidados de salud de los niños

Ultrassonografia no diagnostico pre-natal das anomalias obstrutivas do trato gastrointestinal. / Ultrasonics in prenatal diagnosis of obstructive gastrointestinal anomalies

Os autores apresentam dois casos de obstucao intestinal fetal diagnosticados intra-utero, ressaltando a importancia clinica da avaliacao ultrassonografica no periodo pre-natal. Una anomalia obstrutiva do trato gastrointestinal deve ser suspeitada quando, associado a polihidramnio, se detectam estruturas cisticas no abdome feta