In anesthetic-free, chronic experiments (n = 5), for the surgery

In anesthetic-free, chronic experiments (n = 5), for the surgery the animals were anesthetized Nutlin-3a in vitro with a mixture (4 ml/kg) of ketamine (25 mg/ml), xylazine (1.3 mg/ml). Silicon probes were implanted above the thalamus attached to a custom-manufactured microdrive. After 1 week of recovery, the probes were moved

gradually, and recordings were made at several depth locations. Tungsten wires (50 μm) were implanted to both primary somatosensory of motor cortices, also in hippocampus in three cases. Three of the five chronic animals yielded narrow spike units of clusterable quality. For juxtacellular recording and labeling, glass micropipettes (20–70 MΩ) filled with 1.5% Neurobiotin (Vector Laboratories) were used. After perfusion, 60-μm-thick coronal brain sections were cut on a Vibratome

and incubated with avidin-biotin-peroxidase complex (Vector Laboratories). The labeled cells were visualized using nickel-intensified diaminobenzidine (DAB) reaction. Labeled neurons and axonal trees were reconstructed using Camera Lucida. In case of dual nRT-VB recording experiments first a silicon probe was lowered into VB, and the receptive field of the multiunit was determined. Next, nRT units with a matching receptive field were then sought with several penetrations of a juxtacellular recording pipette. Lesion experiments were performed by recording a baseline session from VB, followed by iotophoresis of 1% kainic acid (−2 μA, 7 s on/off cycle) Volasertib cost for 20 min without moving the electrodes. Several (3–4) hours later postlesion session was recorded from the same electrode. Parvalbumin-channelrhodopsin and vesicular GABA-transporter-channelrhodopsin mouse strains were generated by crossing PV-cre or vGAT-cre (The Jackson Laboratory) mice with -129S-Gt(ROSA)26Sortm32(CAG-COP4∗H134R/EYFP-Hze (The Jackson Laboratory) reporter strains. For optical stimulation a 473 nm DPSS laser (LaserGlow) was used via a fiberport (Thorlabs) and a patch cord (Thorlabs) to the brain-implanted optic fiber. The optic fiber was either attached to the silicon probe in

also close proximity (<200 μm) of the recording site (axonal stimulation), or inserted directly into the nRT (soma-dendritic stimulation). Light intensity was modulated through the DPSS power supply, with a MATLAB-controlled DAQ-board (National Instruments). Stimulus strength was adjusted to span a range from near-threshold (∼0.1 mW) to maximal effect (∼10 mW). Extracellular signals were high-passed filtered (0.3 Hz), amplified (2,000 times) by a 64-channel amplifier, and digitized at 20 kHz with two National Instruments PCI-6259 cards. After detection, units were grouped by the semiautomatic “cluster cutting” algorithm (“KlustaKwik”; available at http://github.com/klusta-team) followed by manual clustering (Csicsvari et al., 2003). Auto- and cross-correlograms were inspected to verify the clustering procedure.

We designed an expression construct encoding a short hairpin RNA

We designed an expression construct encoding a short hairpin RNA that efficiently and specifically

knocked down Rnd3 expression (Rnd3 shRNA #2; Figures S2A, S2B, and S6A) and introduced this Rnd3 shRNA together with EGFP expressed from the same construct by in utero electroporation in the cerebral cortex at E14.5. Examination of the electroporated brains at E17.5 ( Figure 2A) revealed a marked defect in the migration of Rnd3 shRNA-treated cells compared with control shRNA-treated cells. Rnd3 silencing resulted after 3 days in a significant increase in the fraction of electroporated cells remaining in the VZ/SVZ (23.8 ± 1.8% of Rnd3 shRNA-electroporated cells compared with 12.3 ± 2.0% of control shRNA-electroporated cells) www.selleckchem.com/products/lee011.html and the IZ (39.1 ± 3.5% versus 23.3 ± 1.8%) and a significant decrease in the fraction of cells reaching the CP (37.1 ± 3.4% versus 64.4 ± 3.3%) and particularly the median (11.9 ± 1.6% versus 23.3 ± 1.9%) and upper parts of the CP (9.5 ± 2.7% versus 23.3 ± 4.3%; Figure 2A). To rule Akt inhibitor out a mere

delay in migration, which has been observed when silencing some migration-promoting genes ( Creppe et al., 2009), we electroporated the Rnd3 shRNA at E14.5 and harvested the treated brains at postnatal day (P) 2. A significant migration defect was still observed in Rnd3-silenced neurons at this stage ( Figure S3A), indicating that Rnd3 is absolutely required for cortical neuron migration to proceed. Electroporation of Rnd3 shRNA in the cortex did not induce the death of migrating neurons or defects in radial glia processes ( Figures S2C and S2E). However, it altered neural progenitor proliferation as shown by an increase in the fraction of BrdU-incorporating cells in the VZ and SVZ ( Figure S2D). This suggests that Rnd3 Montelukast Sodium inhibits cell-cycle progression of cortical progenitors, a result consistent with previous

studies demonstrating a role for Rnd3 in fibroblast and tumor cell proliferation ( Bektic et al., 2005, Poch et al., 2007 and Villalonga et al., 2004). This finding raised the possibility that the reduced migration of Rnd3-silenced cells that we observed was a secondary consequence of the failure of progenitor cells to exit the cell cycle. To address this idea, we electroporated the Rnd3 shRNA at E14.5 and we maintained electroporated brains in organotypic slice cultures for 4 days in the continuous presence of BrdU. When analyzing the migration of electroporated cells, we identified BrdU-negative cells as being already postmitotic when Rnd3 was knocked down at the beginning of the experiment. These Rnd3 shRNA-treated postmitotic cells presented a similar block in their migration as observed in previous experiments ( Figures S2B and S2F).

(Figure 3D, open circles versus filled circles, and Figure S4) <

(Figure 3D, open circles versus filled circles, and Figure S4). Onalespib The failure to observe an increase in performance

accuracy with longer go signals was surprising, given that Rinberg et al. (2006) did find such an increase using apparently similar conditions. Therefore, we next turned to examine whether overlooked differences in task structure might account for this discrepancy. We first noted that while we had tested subjects on a given go-signal delay for hundreds of trials in a row, Rinberg et al. randomly interleaved go signals of different delays in a single session. Previous studies have shown that the ability to anticipate the time at which a brief stimulus will be presented can affect reaction time and accuracy of performance (Griffin et al., 2001; Nobre, 2001; Correa et al., 2006; Katzner et al., 2012). We therefore hypothesized that expectation of (or

readiness to respond to) the timing of the go signal would also affect performance in this task. Specifically, we reasoned that when go-signal delays vary randomly from trial-to-trial, the subject may not respond as accurately as when responses are self-paced or instructed by a go signal delivered at a constant delay. The predictability of random go-signal times has been Selleckchem Sunitinib formalized by the notion of “hazard rate,” defined as the probability that a signal will occur given that it has not already occurred (Luce, 1986). The “subjective hazard rate” (Janssen and Shadlen, 2005) is an extension of this concept that takes into account the finding that the variance of subjective time estimation increases proportionally to the interval duration (Gibbon, 1977; Gallistel and Gibbon, 2000). By calculating the subjective hazard rate for the experimental distribution of go-signal times, a quantitative prediction of performance as a function of not go-signal delay can be obtained. To test the idea that hazard rate impacts go signal performance, we compared performance of subjects on two different distributions of go signals, formed using uniform and exponential probability

densities, which have very different hazard rates. These distributions, their hazard rates and subjective hazard rates are depicted in Figure S4. The subjective hazard rate for go signals in the uniform condition rises with time toward the end of the distribution interval; therefore performance in this condition is expected to increase relatively slowly over the distribution interval. In contrast, the exponential distribution has a much flatter subjective hazard rate; therefore, performance in this condition is expected to rise relatively more quickly resulting in relatively better performance at short delays. Rats were tested first on the uniform distribution for several consecutive sessions (phase I), then on the exponential distribution (phase II) and then again on the uniform distribution (phase III) (Figure 4A).

We also measured TAM

We also measured TAM check details receptor and ligand mRNAs in the retina and eye using quantitative

RT-PCR with mRNA prepared from multiple isolated tissues: RPE, choroid, eye cup (tissue remaining after removal of neural retina and RPE), ciliary body (CB), and neural retina (minus RPE; see Experimental Procedures) (Figure 6). Although the apical microvilli of RPE cells express both Mer and Tyro3 (Prasad et al., 2006), Mertk−/− single gene mutants yield a strong PR degeneration phenotype ( Figures 2 and 3), whereas Tyro3−/− mutants have a normally configured ONL ( Prasad et al., 2006). To assess the relative expression level of the two TAM receptor genes in these cells, we measured the relative expression of Mertk and Tyro3

mRNAs in isolated RPE. As before ( Prasad et al., 2006), we used a collagenase/hyaluronidase dissociation protocol to cleanly peel off the RPE layer from adult 129/C57Bl/6 retinae, and prepared mRNA from this purified RPE layer. Quantitative RT-PCR revealed that RPE cells express slightly more than four times as much Mertk mRNA as Tyro3 mRNA, and no detectable Axl mRNA ( Figure 6A). In these same qRT-PCR experiments, we measured Gas6 and Pros1 mRNA levels. Gas6 ( Figure 6B) and Pros1 ( Figure 6C) mRNAs were detected in all ocular tissues, with measured Gas6 mRNA levels being approximately an order of magnitude higher than those for Pros1

mRNA throughout the eye. We observed a modest (∼2.5-fold) upregulation of Pros1 mRNA expression in the CB of Gas6−/− mutants, with no substantial signaling pathway change in this mRNA in other Gas6−/− ocular tissues ( Figure 6C). Similarly, there was no substantial change in Gas6 mRNA levels measured in the retina of either Pros1fl/-/Nes-Cre or Pros1fl/-/Trp1-Cre mice ( Figure 6B). The complete degeneration phenotype evident in the central retina of the Pros1fl/fl/Nes-Cre/Gas6−/− mice suggests that blood-derived Protein S is unlikely to be a major reservoir of ligand for the Mer receptor of the RPE. The TAM RTKs play key roles in immune regulation, the phagocytosis of apoptotic cells (ACs) and membranes, the facilitation of viral infection, and the progression of cancer (Lemke and Burstyn-Cohen, 2010; Lemke and STK38 Rothlin, 2008; Meertens et al., 2012; Morizono et al., 2011; Verma et al., 2011). However, the relative importance and contribution of the ligands that activate these receptors has yet to be assessed genetically in any setting in vivo. Although there have been previous biochemical studies that document Protein S and/or Gas6 binding to or activation of Tyro3, Axl, or Mer (Lemke and Rothlin, 2008), most of these in vitro studies were performed with cultured cells or membranes in which endogenous TAM receptor and ligand expression were unknown.

When first introduced, the broad-spectrum anthelmintics, includin

When first introduced, the broad-spectrum anthelmintics, including benzimidazoles

(BZ), nicotinic acetylcholine receptor (nAChR) agonists (e.g., levamisole, LEV) and the macrocyclic lactones (ML), were highly efficacious. selleck screening library However, intensive use has selected drug-resistant parasite populations globally in many animal species within a decade of the introduction of every anthelmintic class, and AR is now a major global problem in small ruminants (Kaplan, 2004, Jabbar et al., 2006, Waghorn et al., 2006 and Kaplan and Vidyashankar, 2012) and horses (Molento et al., 2012 and Reinemeyer, 2012), and is emerging in cattle (El-Abdellati et al., 2010, Sutherland and Leathwick, 2011 and Kaplan and Vidyashankar, 2012). For the purposes of this guideline, AR may be simply defined as a heritable change in susceptibility to an anthelmintic in a population of parasitic nematodes such that a dose which normally provides ≥95% clearance of adult worms provides ≤80% clearance. Anthelmintic resistance is arguably the greatest threat to the sustainable control of helminthoses in the short- to medium-term and the problem is compounded by the fact that many of these parasite populations are resistant to more than one class of anthelmintic (van Wyk et al., 1997, Love et al., 2003, Anziani et al., EPZ5676 cell line 2004, Wrigley et al.,

2006, Sargison et al., 2007, Sutherland

et al., 2008, Gasbarre et al., 2009a, Gasbarre et al., 2009b, Cezar et al., 2010, Baker et al., 2012, Molento et al., 2012 and Reinemeyer, 2012). Despite the scarcity of well-structured surveys, it is generally accepted that the prevalence of AR is increasing globally in the ruminant livestock industries and in horses. Drug combinations are commonly used for chemotherapeutic indications in human medicine, including cancer as well as viral, bacterial and protozoan parasitic infections (White, 1999, Miles et al., 2002, Anonymous, 2004, Harrigan et al., 2005, Lane, 2006, Airley, 2009, World Health Organization, 2009, Bang, 2010 and Hastings, 2011). The principle that combinations of chemotherapeutic agents benefit from patients by maintaining drug efficacy in the presence of resistance has been repeatedly demonstrated in this context for diverse pathogens and builds on knowledge gained from insecticide, pesticide and herbicide use (Wood and Mani, 1981, Curtis, 1985, Mani, 1985, Comins, 1986 and Diggle et al., 2003). Currently, combinations of two or more anthelmintics are primarily being used to manage AR in ruminants (i.e., by delaying the emergence and spread of resistance, and/or controlling parasite populations with existing resistance), and to enlarge/expand the spectrum of efficacy.

Only English language publications which included children and ad

Only English language publications which included children and adolescents aged 4–18 years old were included. How active are obese children? Are they less active than their non-obese counterparts? It would seem that regardless of the measurement device, (accelerometry, heart rate measurement, or doubly-labeled water), the obese children generally exhibit lower levels of activity. For example, Maffeis and colleagues14 used heart rate monitoring to estimate PA in a small group of 8–10-year-old obese (body Osimertinib cost mass index (BMI) >97th percentile) and non-obese children. Non-obese children spent about 100 min a day more being physically active (all activity above sedentary behavior) than the obese children. Moderate

Torin 1 research buy to vigorous PA was similar in the two groups, so the difference in total daily activity was accounted for by light intensity activity. The authors also found that the obese children spent more time (approximately 100 min a day) engaged in sedentary pursuits compared to the non-obese children. These findings are similar to Yu et al.15 who also used heart rate derived estimates of energy expenditure to compare total activity (activity above sedentary

behavior) and sedentary behavior between 18 obese (BMI ≥95th percentile) and 18 non-obese 6–18-year-olds. The obese youngsters in Yu et al.’s15 study spent 30% less of the monitored time engaged in physically active pursuits (no data are provided for the breakdown of light, moderate or vigorous activity), but 51% more time engaged in sedentary activities during the waking hours. Accelerometry studies also found lower PA levels in obese youngsters. In a group of 53 obese (BMI ≥98th percentile) and 53 non-obese boys and girls (mean age 8.6 years), Hughes and colleagues16

found total activity time (mean accelerometer counts/min) second was lower in the obese (648 counts/min) compared to non-obese children (729 counts/min). Interestingly there was no difference in time spent being sedentary between the two groups of children in this study. When the proportion of the time spent engaged in activities of moderate to vigorous intensity was compared this was marginally less (2.4%) in the obese (average of about 16 min a day) compared to the non-obese (average of about 23 min a day). Similarly, Page et al.17 found that time spent being moderately to vigorously active was slightly less in 14 obese (BMI >99th percentile; average of about 10 min a day) compared with 54 non-obese (average of about 13 min a day) 10-year-olds. Although total activity and PA are generally less in the obese children and adolescent compared to the non-obese, total activity energy expenditure is not always reduced. Ekelund et al.18 assessed total activity energy expenditure and PA using a combined doubly-labeled water and accelerometer approach in 18 obese (BMI >30 kg/m2) and 18 non-obese adolescents.

Taken together, these circuit-activity mapping experiments reveal

Taken together, these circuit-activity mapping experiments reveal the functional significance of

the inhibitory THVTA-LHb pathway in regulating midbrain activity. In vivo, pharmacological inhibition of the LHb increases dopamine in forebrain regions such as the striatum (Lecourtier et al., Selumetinib cost 2008). Likewise, we observed that in vivo activation of the THVTA-LHb pathway increased the firing rate of midbrain dopaminergic neurons ( Figure 6). Therefore, we hypothesized that in vivo activation of the THVTA-LHb::ChR2 pathway would result in a reward-related phenotype. To test this hypothesis, we implanted bilateral optical fibers ( Sparta et al., 2012) aimed directly above the LHb in THVTA-LHb::ChR2 mice ( Figure S6) and determined the behavioral ramifications of selectively activating the THVTA-LHb::ChR2 pathway. Using a real-time place preference assay, as previously described ( Stamatakis and Stuber, 2012), THVTA-LHb::ChR2 mice exhibited a significant preference for the side of the chamber that was paired with optical stimulation. In contrast, littermate controls (THVTA-LHb::Control) displayed no preference, demonstrating that activation ABT-199 purchase of the THVTA-LHb::ChR2 pathway produces reward-related behaviors ( Figures 7A–7C). This preference was dependent on GABAA signaling within the LHb, Ketanserin as intra-LHb microinjections of a GABAA

receptor antagonist (gabazine) through guide cannulas

interfaced with the optical fibers ( Jennings et al., 2013) blocked the preference for the stimulation-paired side ( Figures 7D and S7). In contrast, intra-LHb microinjection of a dopamine receptor antagonist (D1 and D2) cocktail did not block the rewarding phenotype ( Figures 7D and S7), whereas a systemic injection of the dopamine antagonist cocktail did disrupt the preference ( Figures 7E and S7). These data suggest that the observed reward-related phenotype induced by optical stimulation of the THVTA-LHb::ChR2 pathway does not depend on dopamine signaling within the LHb, but rather on downstream dopamine signaling in brain regions such as the NAc. Finally, to determine if activation of the THVTA-LHb::ChR2 pathway is reinforcing, we trained mice to nose-poke for optical stimulation of the THVTA-LHb::ChR2 pathway ( Figures 7F–7H). THVTA-LHb::ChR2 mice made significantly more nose-pokes to receive optical stimulation than THVTA-LHb::control mice ( Figure 7F). Taken together, these data demonstrate that although activation of THVTA-LHb::ChR2 terminals does not result in detectable dopamine release in the LHb, selective activation of this pathway promotes reward-related behavior by suppressing LHb activity through the release of GABA, leading to disinhibition of VTA dopaminergic neurons.

As previous research has focused on the direct influence of the p

As previous research has focused on the direct influence of the physical and social environmental factors (e.g., accessibility, social support) on PA participation,21 the plausible mediation effect between those factors and PA participation were neglected. The current study allowed for a more comprehensive view of this situation in an attempt to better understand these relationships and to offer guidance for application

of the findings. From this we have clarified that interventions aimed at the enabling and reinforcing factors should focus on increasing the predisposing factors (e.g., perceived competence, self-efficacy) in order to ultimately promote PA Selleck Pexidartinib participation. In accordance with previous studies, our study provides additional evidence of the importance of environmental factors on PA participation, in particular its indirect effect.19 and 20

However, the influence of environmental factors on PA participation remains unclear overall. For example, in a previous study objectively measured environmental variables were significantly related to PA, whereas self-reported environmental variables were not.36 That said, there is clearly growing interest in the relationship between PA and the built environment with much still to be learned.37 and 38 Finally, although previous research has suggested a relationship between language fluency and PA participation,35 this was not supported in our study. This may be because the majority of participants in our study were graduate students and their admission into graduate school in the U.S. was at least partially contingent upon their English language

buy Buparlisib fluency. Compared to previous studies, the proposed final model Sodium butyrate highlights the direct influences of the predisposing factors and the indirect effects of the enabling and reinforcing factors. In future studies it would be interesting to compare how the YPAP model could be different among different groups (e.g., between American and Chinese college students). Likewise, continued refinement of the model will help maximize its utility and clarify it generalizability across different subgroup populations. Our findings are limited by convenience sampling, the retrospective study design, and the self-reported nature of the data obtained. Specifically, the sample was not randomly selected and may not be fully representative. Those who participated seemed relatively active compared to previous reports of this subgroup population. This may represent a social desirability bias too. However, those meeting vs. those not meeting the PA guidelines did report higher activity levels on a separate measure (i.e., LTEQ), which offers some evidence of construct validity. Future studies should continue to test and modify the YPAP model for the Chinese international student population. Where feasible studies should use objective measures to measure PA and the environmental factors.

, 2009) The hippocampal and prefrontal cortex (PFC) appear to pl

, 2009). The hippocampal and prefrontal cortex (PFC) appear to play special roles as “hubs” of interstructure communication. As such, both of these structures are capable of orchestrating the activity of many cortical and subcortical areas subserving cognitive

functions such as working memory, memory acquisition and consolidation, and decision making (see Benchenane et al., 2011 and Schwindel and McNaughton, 2011, for reviews). Both structures receive converging input from the higher sensory/associative areas. Furthermore, the PFC is one of the few neocortical areas that receives direct input from the hippocampus itself. Consistent with this link, activity oscillations in PFC and the hippocampus are coherent (see e.g., Sirota et al., 2008), and the degree of coherence covaries with working memory (Jones and Wilson, 2005) and decision making (Benchenane et al., 2010) Ibrutinib research buy demands. In this issue of Neuron, Fujisawa and Buzsáki (2011) aim to extend our understanding of oscillatory coherence in the context of working memory by studying the simultaneous activity of the rat PFC, the hippocampal CA1 subfield and the VTA, a basal ganglia nucleus containing dopaminergic (DA) cells, which sends neuromodulatory signals to much of the brain. The authors analyzed the activity of ensembles of single neurons and local field potentials (which reflect local

averages of membrane currents) in these areas while rats performed a working memory task on a T-maze. The animals were HSP inhibitor trained to choose either the left or right target arm, based either on association STK38 with an odor sampled at the departure point, or to alternate between arms. In both cases, while the rat was in transit to the choice-point, information about the arm to be chosen (or which arm was most recently chosen) has to be maintained in working memory, a function that, in rats, has been shown to require the integrity of the hippocampal-PFC network ( Floresco et al.,

1997). In analyzing the spectral coherence between PFC and VTA, the authors indentify a novel slow rhythm centered at 4 Hz. In this frequency range, both regions engage in coherent oscillations that are modulated by behavior, where the strongest and most coherent oscillations are observed on the central arm, or “choice-point,” of the T-maze (i.e., where working memory is necessary for correct decision-making). Those oscillations were not present during performance of a forced-choice control task that did not require working memory (Figure 1). Concurrently, oscillatory coherence at theta frequencies (∼8 Hz) was observed between the PFC and the hippocampus, as previously shown during working memory maintenance (Jones and Wilson, 2005), and surprisingly between the hippocampus and VTA as well.

Following the first HPV vaccination, pain was

Following the first HPV vaccination, pain was reported by 49% of subjects when administered concomitantly with MenACWY-CRM and Tdap, by 36% when given 1 month after Tdap, and by 42% when given 1 month after MenACWY-CRM (Table 5). The second and third HPV vaccinations were administered alone in all three vaccine groups, and had similar percentages of subjects reporting pain across all vaccine groups, at a slightly higher rate following the third HPV vaccination

Ribociclib cell line (40–43% and 45–47% after the second and third HPV vaccinations, respectively). Severe pain was reported by <5% of subjects across all vaccine groups and for all HPV vaccinations. Although lower, the percentages of subjects reporting erythema and induration showed a similar trend to those observed for pain: following the first HPV vaccination, the percentages were higher when HPV was administered concomitantly with MenACWY-CRM and Tdap than when it was administered alone (erythema: 14% versus 7% and 9%, respectively; induration: 10% versus 5% and 5%, respectively) (Table 5). Following the second and third HPV vaccinations, the reporting rates were similar across

vaccine groups and slightly higher after the third HPV vaccination Selleckchem Capmatinib (erythema: 10–12% and 12%, respectively; induration: 8–11% and 10–12%, respectively) (Table 5). The percentages of subjects reporting any solicited systemic reactions

after MenACWY-CRM alone were 51% before Tdap and 43% after Tdap (Table 6). The frequency was slightly higher when all three vaccines were administered concomitantly (58%) (Table 6). Across the vaccine groups, the most commonly reported systemic inhibitors reactions were headache, myalgia, and malaise. In the concomitant group these were reported by 40%, 27%, and 25%, respectively, compared with 36%, 19%, and 20%, respectively, when MenACWY-CRM was administered alone before the other vaccines, and 27%, 16%, and 18%, respectively, when MenACWY-CRM was given alone after previous Tdap vaccination. When Tdap was administered alone the respective rates were Adenylyl cyclase 37%, 26%, and 21%, respectively, when given before MenACWY-CRM, and 25%, 16%, and 18% when given 1 month after MenACWY-CRM vaccination. Rates with HPV were lower and similar for all doses (Table 6). The percentages of subjects experiencing any unsolicited AEs were similar between vaccine groups (28–29%). Serious AEs were also similar between vaccine groups (<1–1%). No SAEs were considered to be possibly or probably related to the study vaccines, and no deaths occurred. Nine subjects reported pregnancies during the study. No further vaccinations were administered to these subjects and they were followed up until delivery or termination.