Direct quantification of cytosolic delivery of drug nanocarriers using FlAsH-EDT2.

The delivery of therapeutics across the cell membrane and into the cytoplasm is a major challenge that limits the development of new therapies. This challenge is compounded by the lack of a general assay for cytosolic delivery. Here we develop this assay based on the pro-fluorophore CrAsH-EDT2, and provide cytosolic penetration results for a variety of drug delivery agents (polyethyleneimine, poly-arginine, Ferritin, poly [maleic anhydride-alt-isobutene] grafted with dodecylamine, and cationic liposomes) into HeLa and T98G cells. Our results show that this method can be widely applicable to different cells and drug delivery agents, and yield statistically robust results. We later use this method to optimize and improve a model drug delivery agent's (Ferritin) cytosolic penetration.

Covid-19 vaccination during the third trimester of pregnancy: rate of vaccination and maternal and neonatal outcomes, a multicentre retrospective cohort study.

OBJECTIVE: To evaluate the impact of Covid-19 vaccination (Pfizer-BioNTech BNT162b2) during the third trimester of pregnancy on maternal and neonatal outcomes. DESIGN: A multicentre, retrospective computerised database. POPULATION: Women who gave birth at >24 weeks of gestation in Israel, between January and April 2021, with full records of Covid-19 disease and vaccination status. METHODS: Women who received two doses of the vaccine were compared with unvaccinated women. Women who were recorded as having disease or a positive Covid-19 polymerase chain reaction (PCR) swab during pregnancy or delivery were excluded from both study groups. Univariate analysis was followed by multivariate logistic regression. MAIN OUTCOME MEASURES: Composite adverse maternal outcomes. Secondary outcomes were vaccination rate and composite adverse neonatal outcomes. RESULTS: The overall uptake of one or both vaccines was 40.2%; 712 women who received two doses of the Covid-19 vaccine were compared with 1063 unvaccinated women. Maternal composite outcomes were comparable between the groups; however, women who received the vaccine had higher rates of elective caesarean deliveries (CDs) and lower rates of vacuum deliveries. An adjusted multivariable logistic regression analysis demonstrated that Covid-19 vaccination was not associated with maternal composite adverse outcome (aOR 0.8, 95% CI 0.61-1.03); a significant reduction in the risk for neonatal composite adverse outcomes was observed (aOR 0.5, 95% CI 0.36-0.74). CONCLUSIONS: In a motivated population covered by a National Health Insurance Plan, we found a 40.2% rate of vaccination for the Covid-19 vaccine during the third trimester of pregnancy, which was not associated with adverse maternal outcomes and, moreover, decreased the risk for neonatal adverse outcomes. TWEETABLE ABSTRACT: Covid-19 vaccine during pregnancy is safe for both mother and fetus.

Risk of major congenital malformations following first-trimester exposure to vaginal azoles used for treating vulvovaginal candidiasis: a population-based retrospective cohort study.

OBJECTIVE: To evaluate the risk for major malformations following first-trimester exposure to vaginal azoles. DESIGN: A population-based retrospective cohort study of women exposed to vaginal azoles from the first day of the last menstrual period until the 90th gestational day. SETTING: A combination of four computerised databases: medications, birth, infant hospitalizations, and pregnancy terminations. POPULATION: All women who gave birth or underwent a pregnancy termination at Soroka Medical Center, Beer-Sheva, Israel, between 1999 and 2009. METHODS: Crude and adjusted relative risks for major congenital malformations and for specific malformations according to organ systems were calculated using a multivariate negative binomial regression. Potential confounders were assessed and controlled for included parity, maternal age, ethnicity, maternal diabetes, smoking, and year of birth or pregnancy termination. Additional analysis using propensity score matching was performed for selected malformations. MAIN OUTCOME MEASURES: Major malformations as well as specific malformations according to organ systems. RESULTS: Of 101 615 pregnancies, 1993 (1.96%) were exposed to clotrimazole vaginal tablets and 313 (0.31%) to miconazole vaginal tablets during the first trimester of pregnancy. No association was found between first-trimester exposure to clotrimazole and major or specific malformations. An association was found between miconazole exposure and musculoskeletal malformation in general and other congenital musculoskeletal anomalies in particular. However, no association was detected when propensity score matching was used. CONCLUSIONS: Intrauterine exposure to vaginal azoles during the first trimester of pregnancy was not associated with either major or specific malformations according to organ systems. TWEETABLE ABSTRACT: First-trimester exposure to vaginal azoles is not associated with either major or specific malformations.

Contrasting effects of aspirin on prostate cancer cells: suppression of proliferation and induction of drug resistance .

BACKGROUND: Aspirin is widely used as a preventive measure against occlusive vascular diseases. Since the age group in which aspirin use has become prevalent is similar to the one presenting with prostate cancer, we decided to examine the potential effects of aspirin on prostate cancer. METHODS: We studied the effects of plasma-attainable concentrations of aspirin (0.5-2 mM) on the human prostate cancer cell lines LNCaP, PC-3, and DU 145, employing cytotoxicity assays and flow cytometric analyses. RESULTS: Incubation with aspirin for 3 days reduced cellular proliferation by up to 35-55% in each cell line studied, but induced a tripling of the percentage of cells expressing P-glycoprotein (an efflux pump conferring multidrug resistance) only in the LNCaP cells. Both effects were dose-dependent. The effect on P-glycoprotein expression was reflected in the induction of resistance against adriamycin cytotoxicity. Furthermore, this protective effect of aspirin was reversed by a specific P-glycoprotein inhibitor, PSC833. The cellular expression of P-glycoprotein returned to normal within 3 days following the removal of aspirin. Aspirin did not affect the cell cycle distribution of LNCaP cells. CONCLUSIONS: This study suggests that aspirin enhances the ability of androgen-responsive prostate cancer cells to resist chemotherapeutic drugs. These findings could potentially have significant clinical ramifications for prostate cancer patients taking aspirin shortly before or during chemotherapeutic sessions.

Aspirin enhances multidrug resistance gene 1 expression in human Molt-4 T lymphoma cells.

BACKGROUND: We recently found that aspirin induces the expression of P-glycoprotein (P-gp), a protein mediating drug resistance, in human prostate cancer cells. The purpose of this study was to evaluate the effect of aspirin on the expression of P-gp in a different human cancer type, i.e., T lymphoma. Furthermore, we analyzed this effect at the level of the gene encoding P-gp, MDR1, and of the transcription factor (NF-IL6), regulating this gene. MATERIALS AND METHODS: NF-IL6 was assayed by the electrophoretic mobility shift assay, MDR1 mRNA was assayed by the reverse transcriptase polymerase chain reaction (RT-PCR), and P-gp was assayed by Western blotting. RESULTS: aspirin, at plasma attainable levels, induced NF-IL6 DNA-binding activity, and increased MDR1 mRNA expression (by up to 140%), as well as the expression of P-gp, in Molt-4 cells. CONCLUSIONS: This study suggests that treatment with aspirin induces a cellular signal culminating in the enhancement of P-gp expression in T lymphoma Molt-4 cells.

[The clinical pharmacist in internal medicine--impact on the quality of therapeutic care].

Several studies have documented the impact of clinical pharmacy services on patient care and drug costs in hospital wards. However most hospitals in Israel do not provide such services and until recently their benefits in local health care have not been demonstrated. We therefore determined whether the activity of a pharmacist in the medical department of a medical center leads to improved quality of drug utilization and reduced costs. During the first 3 months of the clinical pharmacist's work all interventions and consultation were documented. The effect of these interventions on drug costs was calculated by the change in drug acquisition costs during the study period compared with those of preceding months, as well as in the other 5 medical departments of the hospital without clinical pharmacy services. During the study period the pharmacist joined 44 clinical rounds in which he documented 40 consultations in response to physicians' requests for drug information and 42 interventions on his own initiative. The pharmacist's recommendations were accepted in 38 of the 42 cases (90%). In 10 cases the pharmacist's initiative in improving the quality of drug therapy led to an increase in drug acquisition costs. However, the overall drug costs during the study period decreased 12.6%. During the same period drug costs in the other medical departments decreased only 2.2%. The results of this study conform with those of many other studies that show a beneficial impact of the clinical pharmacist on the quality of drug therapy and on drug costs. They indicate that the clinical pharmacist can play a crucial role in the medical department.

Atrial natriuretic peptide induces acrosomal exocytosis of human spermatozoa.

Acrosomal exocytosis in mammalian spermatozoa is a process essential for fertilization. We report here that atrial natriuretic peptide (ANP) markedly stimulates acrosomal exocytosis of capacitated human spermatozoa. Typically, ANP exerts some of its actions via activation of the ANP receptor (ANPR-A), a particulate guanylyl cyclase-linked receptor, and subsequent formation of guanosine 3',5'-cyclic monophosphate (cGMP). We found that ANP-stimulated acrosome reaction was inhibited by the competitive ANPR-A antagonist anantin, indicating a receptor-mediated process. A linear fragment of ANP, ANP-(13-28), and another ANP-like compound, brain natriuretic peptide, were inactive. The stimulatory effect of ANP on acrosome reaction was mimicked by the permeable cGMP analog, 8-bromo-cGMP (8-BrcGMP). Addition of the protein kinase C (PKC) inhibitors, staurosporine and GF-109203X, resulted in a dose-related inhibition of ANP-induced acrosome reaction. Also, downregulation of endogeneous PKC activity resulted in inhibition of ANP- but not 8-BrcGMP-induced acrosome reaction. Removal of extracellular Ca2+ abolished ANP-induced acrosome reaction. Thus ANP via Ca2+ influx, PKC activation, and stimulation of particulate guanylyl cyclase may play a role in the induction of acrosome reaction of human spermatozoa.

Ultrastructural localization of protein kinase C in human sperm.

We localized protein kinase C (PKC) in human sperm cells at the ultrastructural level by the immunogold technique. The sperm head PKC was localized in the acrosome, equatorial segment, and post-acrosomal region. In the flagellum, PKC was associated with the segmented column of the neck and was distributed along the mid, principal, and end pieces. Immunoreactive sites were observed in patches along the axoneme and outer dense fibers and were evenly distributed between these regions. Pre-absorption of the antibody used with rat brain PKC (alpha and beta) eliminated gold labeling of the sperm head but only reduced labeling of the sperm tail. The co-localization of PKC with various cytoskeletal and other structural elements suggests that the proteins involved are potential substrates for sperm PKC subspecies. The localization of PKC in distinct structures of the human sperm (head, neck, and tail) strongly suggests a role for this enzyme in various aspects of sperm physiology.

Ca(2+)-independent induction of acrosome reaction by protein kinase C in human sperm.

We report that activated protein kinase C (PKC) can induce acrosome reaction independently of elevated Ca2+. Addition of 12-O-tetradecanoyl phorbol-13-acetate or the membrane-permeable diacylglycerol analog 1-oleoyl-2-acetylglycerol to ejaculated human sperm resulted in stimulation of acrosomal reaction (2- to 3-fold), provided the sperm underwent capacitation. Induction of acrosome reaction by 12-O-tetradecanoyl phorbol-13-acetate was blocked by the PKC inhibitor staurosporine or by down-regulation of endogenous PKC, but not by removal of extracellular Ca2+. Acrosome reaction was also enhanced by the Ca2+ ionophore ionomycin in a Ca(2+)-dependent, PKC-independent fashion. Immunohistochemical analysis with type-specific PKC antibodies revealed the presence of PKC alpha and PKC beta II in the equatorial segment, whereas PKC beta I and PKC epsilon staining was found in the principal piece of the tail. Acrosome reaction, thus far believed to be induced only by elevated Ca2+, can therefore be triggered by activated PKC in a Ca(2+)-independent fashion. The PKC subtypes potentially involved in acrosome reaction are most likely alpha and beta II, whereas the beta I- and epsilon-subspecies might be involved in regulation of flagellar motility of human sperm.

Role of protein kinase C in the acrosome reaction of mammalian spermatozoa.

Mammalian spermatozoa undergo a Ca(2+)-dependent exocytotic event before fertilization which is known as the acrosome reaction. The process of exocytosis in several cell systems is mediated by a protein kinase C (PKC)-catalysed phosphorylation. Addition of phorbol 12-myristate-13-acetate or the membrane-permeant diacylglycerol analogue 1-oleoyl-2-acetylglycerol, which are potent activators of PKC, to bovine spermatozoa resulted in stimulation of the acrosome reaction. This stimulation was inhibited by low concentrations (50% inhibition at 0.7 nM) of the PKC inhibitor staurosporine. PKC specific activity in bovine spermatozoa is extremely low in comparison with other cells; however, it is comparable with the activity found in human spermatozoa. Immunohistochemical analysis using anti-PKC antibodies revealed staining in the equatorial segment, the post-acrosomal region and the upper region of the head. We propose that PKC is involved in the mammalian sperm acrosome reaction.

The use of an electric freezer in human semen banking.

The use of an electric freezer for cryostorage of human semen is described. A simple method for semen freezing in liquid nitrogen after dilution with 7.5% glycerol was used. Thawing for quality analysis revealed only a small decrease (10%) in post-thaw sperm motility (59 +/- 1.4 vs. 53 +/- 1.8%; mean +/- SE, n = 12, P less than 10(-5)) and 14% in post-thaw sperm vitality (85.0 +/- 1.3 vs. 72.9 +/- 1.8%). After the freezing process, the samples, three of each donor, were cryopreserved in a regular electric freezer which maintained temperatures in the range of -85 degrees C (+/- 2 degrees C). The samples were stored for 1 week, 2 and 6 months, thawed and then assayed for motility and vitality. No effect of storage was found for a period up to 2 months. An additional decrease of 17.2% in sperm motility and 18% in sperm vitality were noted only after 6 months of preservation. The final motility and vitality rates of these sperm samples were 44 +/- 2.4 and 60 +/- 3.0%, respectively. According to these results, in cases of sperm storage for limited periods, it is recommended to cryopreserve human semen by the use of a combination of freezing in liquid nitrogen and storage of the samples in an electric freezer at -85 degrees C.

Further studies on the involvement of protein kinase C in human sperm flagellar motility.

Addition of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) or the membrane-permeable diacylglycerol analog, 1-oleoyl-2-acetylglycerol to human sperm resulted in increased motility. The biologically inactive 4 alpha-phorbol 12,13 didecanoate had no effect on flagellar motility. Basal motility was markedly reduced in the absence of Ca2+ in the incubation medium, but TPA-induced sperm motility persisted even in the absence of Ca2+. Sperm motility was also enhanced by the Ca2+ ionophore ionomycin in a Ca2(+)-dependent, protein kinase c (PKC)-independent fashion. Although all stimulants examined here reached maximal response at about 15 min of incubation, nevertheless whereas the effect of TPA and 1-oleoyl-2-acetylglycerol declined at 60 min of incubation, that of ionomycin still persisted. Human sperm PKC activity is extremely low and represents only about 20% and 25% of the specific activity recovered from PC-12 and rat pituitary cells, respectively. Immunohistochemical analysis using various type-specific PKC antibodies revealed staining only in the equatorial segment and the principal piece of the tail. Thus, PKC is present in human ejaculated sperm and is involved in flagellar motility.

Protein kinase C is present in human sperm: possible role in flagellar motility.

We report the presence of protein kinase C (PKC) in ejaculated human sperm as revealed by enzymatic activity assay and indirect immunohistochemistry. PKC is localized in the equatorial segment and in the principal piece of the tail. Addition of phorbol 12-myristate 13-acetate resulted in increased flagellar motility that was blocked by known PKC inhibitors such as sphingosine, staurosporine, and 1-(5-isoquinoylinylsulfonyl)-2-methylpiperazine. A very good correlation (r = 0.9, P less than 0.001) was found between the percentage of PKC-stained sperm cells and motility. We propose that PKC is involved in the regulation of flagellar motility in human sperm.

Determination of urinary luteinizing hormone for prediction of ovulation.

Detection of the approximate time of ovulation is important in the in vitro fertilization-embryo transfer programs in order to avoid a possible early surge of luteinizing hormone (LH) before human chorionic gonadotropin administration in the induction of ovulation and for timing of artificial insemination. In the present study correlation between the detection of LH in urine and serum levels (mean +/- SEM) of estradiol greater than 557 +/- 118 pg/ml, progesterone less than 1.9 +/- 0.37 ng/ml and leading follicle diameter greater than 26.2 +/- 1.3 mm (mean +/- SEM) was found. Thus, the prediction of ovulation via detection of urinary LH in urine by a dipstick procedure is a good semiquantitative method, accurate enough, reliable, cheap and acceptable by the women.

Clomiphene citrate treatment in oligozoospermia: comparison between two regimens of low-dose treatment.

Clomiphene citrate (CC) is a well-known drug in fertility clinics that is used for increasing gonadotropin secretion. The present study was planned in order to evaluate the efficiency of a new regimen of treatment by 25 mg on alternate days (group A), compared to a daily dose of 25 mg (25 days on, 5 days off, group B), for 4 months. Semen quality was assessed in two matched groups, which consisted of 45 and 44 normogonadotropic oligoterato-asthenozoospermic (OTA) men, respectively. Nine men in group A and 22 in group B did not respond to therapy by improvement in semen quality. The statistical evaluation of the results revealed group A to yield the highest improvement in sperm concentration (P less than 0.0008) and total sperm count (P less than 0.004). Sperm motility was improved only in group A. No changes were recorded in the morphology of the sperm cells or in semen volume. Pregnancy rate after 6 months of follow-up was 26.7% and 20.5%, in couples of groups A and B, respectively. This study implicates the use of CC (25 mg on alternate days) in andrologic clinics as one of the recommended drugs for normogonadotropic OTA subfertile men in order to achieve a significant increase in sperm concentration and total sperm count.