Acute expression of constitutively active CREB in hippocampal neurons increased the protein levels of KIF17, NR2B, and NR2A (Figures

8C and 8D). The kif17 and nr2b mRNA levels were also increased, but the nr2a level was not ( Figures 8E and 8F). Inhibition of CREB activity by expression of a dominant-negative form of CREB caused significant decreases in the mRNA and protein levels for NR2B and in the mRNA level for KIF17 ( Figures 8E and 8F). These data suggest that activation of CREB upregulates KIF17 and NR2B by increasing the transcription of each coding gene, but that other mechanisms underlie the increase HDAC inhibitor in the level of NR2A. KIF17 has been implicated in the transport of NR2B subunits (Guillaud et al., 2003 and Setou et al., 2000). Consistently, our results revealed a decreased level of motility of NR2B-EGFP clusters (Figures 2G–2J; Movie S1), but normal motility of NR2A-EGFP clusters (Figures 2K–2N; Movie S2) in kif17−/− mouse neurons. These data suggest that KIF17 transports the NR2B subunit, but not the NR2A

subunit. The NR2A subunit is likely to be transported by a different molecular motor. Recent papers have shown data suggesting that Kv4.2 and other neurotransmitter receptors, namely GluR5 and KA2, might also be cargoes for KIF17 (Chu et al., 2006 and Kayadjanian et al., 2007). We studied the expression of Kv4.2, GluR5, and KA2 in the kif17−/− mouse hippocampus.

No significant differences in the levels of these proteins were observed Selleckchem GSK126 between kif17+/+ and kif17−/− mice ( Figure 1B). One possibility is that some molecular motors other than KIF17 support the transport of these proteins, compensating for the loss of function of KIF17 in kif17−/− neurons. Interestingly, significant reductions in the levels of both NR2B and NR2A in kif17−/− mouse synapses were observed (Figures 1G, 2A–2F, and S2B). Further analysis revealed that the level of NR2A in kif17−/− mouse neurons is downregulated at a posttranslational level, but that of NR2B is downregulated at a transcriptional level (Figures 1B–1F and 3A–3C). The NR2A level is decreased in kif17−/− mouse Ribonucleotide reductase neurons due to its accelerated degradation in dendrites ( Figures 3I and 3L). Consistent with this finding, other investigators have reported that NR2A-containing NMDA receptors are easily degraded ( Yashiro and Philpot, 2008), whereas the NR2B subunit is preferentially driven to a recycling pathway ( Lavezzari et al., 2004 and Scott et al., 2004). We also showed that the NR2A degradation is dependent on the ubiquitin-proteasome system ( Figures 3D–3F). Considering these data together with studies reporting KIF17-mediated NR2B transport (Guillaud et al., 2003 and Setou et al., 2000), it is plausible that the accelerated NR2A degradation is caused by the reduction in NR2B transport in kif17−/− mouse neurons.

This finding suggests that NgR1 requires a coreceptor to inhibit synapse development. Genome-wide RNA sequencing revealed that of the known NgR1 coreceptors,

only Lingo-1 and TROY are expressed at appreciable levels in 7 DIV neuronal neurons (data not shown). Since Lingo-1 is largely expressed on axons (Lee et al., 2008), we focused on TROY as a potential NgR1 coreceptor that might function in dendrites to inhibit synapse development. Immunostaining with protein-specific antisera revealed that TROY is expressed along the dendrites of cultured neurons and overlaps significantly with all NgR family members (Figure S4E). In addition, TROY knockdown (Figures S4E, S4I, S4J, and S8B) caused a significant increase in synapse density in cultured hippocampal neurons (Figure 4H). Together, DAPT datasheet these findings are consistent

with TROY being the coreceptor that mediates the inhibitory effects of NgR1 on synapse development. To determine whether TROY is required for NgR1-dependent suppression of synapse development, WTNgR1 was overexpressed with or without TROY knockdown (shTROY) and synapse density was quantified. TROY knockdown reversed selleck chemicals llc the reduction in synapse number observed with NgR1 overexpression (Figure 4I). An increase in synapse density was observed, similar to that seen upon TROY knockdown alone. Similar epistasis studies with WTNgR2 and WTNgR3 overexpression revealed that TROY is required for the suppression of synapse development by NgR2 and NgR3 (Figure S4K). Moreover, binding experiments using recombinant TROY protein incubated with heterologous cells expressing different NgR family members show that TROY is capable of binding NgR1 and NgR2, but not NgR3 (Figure S4F), suggesting that NgR1 and NgR2 may signal through TROY directly. It remains unclear whether the affinity of the NgR3-TROY interaction falls below the detection limit of this assay or whether NgR3 acts through an alternative coreceptor. Taken together, these findings identify TROY as a potential

coreceptor for the NgR family that mediates their ability to restrict excitatory synapse number. To address whether the NgR family contributes to synaptic development in vivo, we crossed NgR mutant mice with the GFPM line (Feng et al., 2000), in which a small subset of neurons are genetically labeled with the Thy1-GFP Bay 11-7085 allele, thus enabling visualization of dendritic spines from hippocampal pyramidal neurons. Knockout of any one NgR family member alone was not sufficient to affect the density of dendritic spines in vivo (Figure 5B). Given our previous finding that all three NgR family members play a similar role in limiting synapse development in vitro (Figures 2G and S2I), we hypothesized that these family members might functionally compensate for one another in vivo. To address this possibility, we generated triple knockout mice (NgRTKO−/−).

Nevertheless, the engagement of a large fraction of the brain by olfactory afferents is consistent with the widespread ramification of fibers revealed by labeling of individual OB glomeruli with nontransneuronal anterograde tracers (Sosulski et al., 2011) and probably reflects ERK inhibitor solubility dmso the importance of olfaction for both learned and innate behaviors in mice. One obvious application of

this technology is the transneuronal labeling of pathways engaged by neurons expressing a specific, behaviorally relevant olfactory receptor, e.g., those involved in pheromone detection (Mombaerts et al., 1996). However, the efficiency of labeling in the MOE and VNO was low, presumably due to the inhibition of viral spread by mucus, and in pilot experiments we were unable to detect labeling in mice expressing Cre recombinase under the control of a specific olfactory receptor, MOR28, that is expressed in ∼1% of ORNs (Mombaerts, 2006). Nevertheless, our preliminary data suggest that this problem may be overcome by injection of the virus into the olfactory bulb, where infection of ORN axons can occur followed by retrograde transport to the cell body and recombination in ORNs (Figures S1R and S1S). The methodology described here, while powerful, has certain limitations.

First, like other replicating transneuronal tracers (Callaway, 2008 and Ekstrand selleck chemicals llc et al., 2008), HSV-based tracers are toxic and kill infected neurons, as well as eventually the whole animal (see Table S2). This limits the number of days that an injected animal can be maintained before analysis. Furthermore, there is unpredictable variability in survival times, reflecting variability in the initial level of infection. This makes it currently difficult to perform prospective time course studies of the progression of labeling in a given pathway, except in a retrospective manner (Figures S5C and S5D). In addition, due to viral cytotoxicity, in animals sacrificed after longer incubation times the initial sites of infection have often been cleared

from the brain by macrophages, obscuring until the identification of initial relays in a pathway. This limitation may be overcome by analyzing animals exhibiting mild symptoms and/or after shorter survival times, in order to identify the pattern of labeling in early structures. Second, although virus released endogenously from infected neurons appears to be taken up exclusively by dendrites and transported in the anterograde direction (Zemanick et al., 1991), exogenously injected virus can clearly infect nerve terminals and undergo retrograde transport to the cell body, as reported previously (Barnett et al., 1995, Rinaman and Schwartz, 2004 and Song et al., 2009) and confirmed here in the olfactory and cerebellar systems (Figures S1P–S1S).

, 2012), and thus may be less sensitive to fluctuations in R∗ lifetime than the peak amplitude (see also the discussion in Hamer et al., 2003). It has been claimed that the diffusion

Apoptosis Compound Library screening of cGMP and/or of calcium plays a central role in SPR reproducibility, acting as a “variability suppressor” (Bisegna et al., 2008; Caruso et al., 2010; Shen et al., 2010; Caruso et al., 2011). In WT rods, the experimentally determined longitudinal diffusion coefficient for cGMP (Gross et al., 2012) is large enough that the maximal decrease in cGMP concentration is small (∼15%) even when R∗ deactivation is slowed ∼2-fold (Figure 2). Thus, the limited diffusion of cGMP does not contribute to reduction of SPR amplitude variability through saturating local channel closure. Furthermore, the spatial profile of calcium is not determined by the diffusion coefficient of calcium, but rather by the spatial profile of cGMP, which

governs calcium influx (Gross et al., 2012). However, the fall in cGMP can reduce the rate of cGMP hydrolysis in the absence of GCAPs-mediated PLX-4720 in vivo feedback (compare gray and colored traces in Figure 5A), producing compression of PDE activity relative to R∗ lifetime, as noted above. In this sense, the local fall in cGMP tends to self-limit the PDE activity, a phenomenon that can contribute to SPR reproducibility (“cGMP hydrolysis saturation effects”; Caruso et al., all 2011). With the lifetimes of R∗ and G∗-E∗ measured from the ΔTsat data ( Figures 1 and 3; Table 1), a remarkably accurate account can be given of the SPRs of rods with genetic manipulations of R∗ deactivation, both with and without calcium feedback to cGMP synthesis ( Figures 4A and 4B). The diffusion of cGMP is sufficiently rapid to insure maximal amplification ( Gross et al., 2012), while the delayed decline in calcium drives cGMP synthesis more strongly for longer R∗ lifetimes ( Figure 5) in rods with normal

GCAPs expression. As a consequence, the amplitude of the mean SPR is stabilized against genetic perturbations to R∗ lifetime ( Figure 4), and the trial-to-trial SPR amplitude is more reproducible in rods with functional calcium feedback ( Figure 6). In general, then, these results reveal how a fast feedback mechanism, operating at a downstream stage in a GPCR cascade, can sharpen the timing of a signal and reduce its variability while maintaining high signal amplification. Mice were cared for and handled following an approved protocol from the Institutional Animal Care and Use Committee of the University of California, Davis and in compliance with the National Institutes of Health guidelines for the care and use of experimental animals. Mice were reared in 12 hr cyclic lighting conditions and euthanized by CO2 narcosis followed by decapitation. All mice were between 1 and 6 months of age when used for experiments.

We have also identified an intimate link between PHF6 and the PAF1 transcription elongation complex that plays an essential role

in neuronal migration in the cerebral cortex. Finally, we have identified Neuroglycan C/Chondroitin sulfate proteoglycan 5 (NGC/CSPG5) as a downstream target of PHF6 and the PAF1 complex in the control of neuronal migration. Our findings define a pathophysiologically relevant cell-intrinsic transcriptional pathway that orchestrates neuronal migration in the cerebral cortex. To interrogate PHF6 function in the mammalian brain, we first characterized the expression of PHF6 in the developing 17-AAG cerebral cortex. We found that PHF6 was highly expressed during early phases of development in primary cortical neurons and in the developing mouse brain (see Figures S1A and S1B available online). PHF6 AZD9291 concentration was broadly expressed in the mouse cerebral cortex at embryonic day 17 (E17) (Figure S1C). The temporal profile of PHF6 expression raised the possibility that PHF6 might play a role in cortical development. To determine PHF6 function in cortical development, we used a plasmid-based method of RNA interference (RNAi) to acutely knockdown PHF6 in the developing cerebral cortex (Gaudilliere et al., 2002). Expression of three short hairpin

RNAs (shRNAs) targeting distinct regions of PHF6 mRNA induced knockdown of exogenous PHF6 protein in 293T cells and endogenous PHF6 in primary mouse cortical neurons (Figures 1A, 1B, S1D, and S1E). We next employed an in utero electroporation method to induce knockdown of PHF6 in the developing mouse cerebral cortex in vivo. The PHF6 RNAi plasmids were electroporated together with a plasmid encoding GFP in the developing cortex in mice at Mephenoxalone E14, when superficial layer neurons are generated. Embryos were allowed to

develop in utero until E19, and brains were harvested and subjected to immunohistochemical analyses. We first confirmed that PHF6 RNAi triggered the downregulation of endogenous PHF6 in the cerebral cortex in vivo (Figures 1C and S1F). Upon characterizing the consequences of PHF6 knockdown on cortical development, we found a striking migration phenotype. Neurons in control animals differentiated and migrated properly to the superficial layers of the cortical plate. By contrast, cortical neurons in PHF6 knockdown animals failed to migrate to the proper location in the upper cortical plate (Figures 1D and 1E). PHF6 RNAi reduced the percentage of neurons reaching the upper cortical plate by 2- to 3-fold and increased the percentage of neurons in the intermediate zone by 3- to 5-fold. The extent of the migration defect correlated with the degree of PHF6 knockdown (Figure 1A).

Children are less time conscious than adults and to recall details of specific events from the past places considerable demands on their cognitive abilities. Proxy reports by parents and/or teachers have been employed in some studies but accurate recall of young children’s

PA by adults is difficult and confounded by children’s intermittent PA patterns. Young people’s moderate intensity PA tends to be non-planned PD0332991 datasheet and less memorable than more intense PA and is therefore often underestimated by self- or parental-recall. Vigorous PA is generally overestimated, particularly when carried out in a sporting context where, say, football may be reported as 90 min of vigorous PA whereas it actually consisted of rest and moderate intensity PA interspersed with short intermittent bouts of vigorous PA. Comparisons of data from a range of

self-report instruments have indicated wide discrepancies in estimates of HPA at an individual level.3 Some researchers have estimated energy expenditure from self-report instruments but as there are no comprehensive reference values for children and adolescents energy costs of activities are usually derived from adult values. This methodology introduces additional errors as with young people energy costs may be underestimated by more than 20% using adult values as a proxy.20 Although errors may be substantial in estimating an individual’s HPA several large, well-designed, national and multinational surveys have provided valuable descriptions of

young people’s HPA at a population level.21, 22 and 23 As PA involves movement of the whole or parts of small molecule library screening the body Org 27569 motion sensors provide an objective means of measuring HPA. The most common motion sensor used in early studies of HPA was the pedometer which was first used in this context by Lauter24 in 1926, although Leonardo da Vinci designed a pedometer to measure distance by counting steps somewhat earlier.25 More sophisticated mechanical, electronic and magnetic counters have gradually replaced pedometers26 and 27 and in recent years accelerometers have become the motion sensor of choice in the study of young people’s HPA.5 Pedometers are simple motion sensors which are normally used to detect and record the number of steps taken over a period of time. Advantages include the low cost and non-reactivity whereas disadvantages include the inability to record intensity, duration or frequency of PA and susceptibility to noise during activities such as cycling. Interpretation of step counts per day during youth must take into account factors such as stature and stride length as pedometer data are not directly comparable across ages during growth and maturation. Despite problems with assigning the appropriate number of steps recorded to specific PA guidelines pedometers do give an overall indication of HPA and are useful tools for large scale studies.

For AHSV serotypes 1, 3, 7, 8 and 9, open reading frames based on amino acid sequences of VP2 proteins (GenBank accession number: CAP04841; U01832; AAN74570; ABI96883, respectively), were designed for optimized expression in insect cells

(Gene Art, Regensburg, Germany). VP2 genes were amplified by PCR with specific primers containing BamHI or SmaI site for cloning purposes into the transfer vector pAcYM1 [27]. Recombinant vectors pAcYM1 with VP2 genes were purified and co-transfected into Sf9 cells with linearized baculovirus DNA (strain BAC10:KO1629), using Cellfectin® II Reagent (Invitrogen) according to the manufacturer’s instruction. On day six after transfection, 200 μl of the supernatants were transferred to fresh Sf9 cells in 12-wells plates. After click here the first passage,

supernatants were transferred to fresh Sf9 cells every 3–5 days until virus infection was confirmed by light microscopy. The virus titer was measured by standard plaque assay using Sf21 cells. Recombinant R428 purchase baculoviruses expressing AHSV VP2 were used to infect Sf9 cells with a multiplicity of infection (moi) of 5. Infected cells were incubated at 28 °C for 72 h. Then, infected cells were harvested by centrifugation, washed with phosphate buffered saline (PBS) and pelleted by centrifugation. Cell pellets were suspended in 25 mM sodium bicarbonate (NaHCO3, pH 8.39) at 1.0 × 107 cells/ml. Cells were disrupted by dounce homogenization and after centrifugation at 6000 rpm for 3 min, supernatants containing soluble VP2 protein were collected. To examine the amount of VP2 proteins, soluble VP2 were mixed with equal volumes of SDS-PAGE sample buffer (10 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 2% β-mercaptoethanol,

20% glycerol, 0.05% bromophenol blue). After heating at 95 °C for 1 min, the samples were analyzed by SDS-PAGE with BSA as concentration standard and protein molecular weight standard (Page Ruler, SM0671, Fermentas). Concentrations of all samples were adjusted to 100 μg of VP2 per ml by 25 mM sodium bicarbonate and stored at −80 ° C until use. All experiments with live animals were performed under the guidelines of the European GBA3 Community (86/609) and were approved by the Committee on the Ethics of Animal Experiments of the Central Veterinary Institute (Permit numbers: 2011-042 and 2011-170). Adult female guinea pigs were purchased from a registered breeding farm for guinea pigs and were randomly divided into groups of six animals. Nine groups were immunized with VP2 protein from each AHSV serotype, two groups were immunized with cocktails of different combinations of VP2 proteins (one consisting of serotypes 1, 3, 7, 8 and other, serotypes of 2, 4, 5, 6, 9, respectively) and one group was immunized with phosphate buffered saline (PBS). Shortly before immunization, recombinant VP2 proteins or PBS in 1.5 ml were warmed to 37 °C and mixed with an equal volume of Montanide 206VG (Seppic) by vortexing.

1). In comparison, protein bands were observed at ∼150 kDa for all Calu-3 cell lysates and were the strongest

for cells at a high passage number cultured at the ALI (Fig. 1). The mouse anti-human MDR1 antibodies UIC2 and MRK16 were subsequently used for immunohistochemistry and flow cytometry. A positive immunohistochemical signal was obtained with both antibodies on the apical membranes of all but HEK293 cell layers investigated (Fig. 2). This was however discontinuous on NHBE and low passage Calu-3 layers (Fig. 2). Both MDCKII-WT and MDCKII-MDR1 cell layers stained positively, possibly due to the cross-reactivity of the antibodies with the canine mdr1 expressed in the cells [29]. Staining ABT-263 mouse GSK3 inhibitor appeared nevertheless more intense for the transfected cells. Flow cytometry using the UIC2 antibody produced a low MFI value of 1.3 with the negative control MDCKII-WT cells, whereas the MDCKII-MDR1 positive cell control generated a MFI value of 7.5, demonstrating the UIC2 antibody reacts specifically with MDR1. At low passage, 36% of Calu-3 cells were shown to express the MDR1 transporter in comparison with 70% at a high passage, resulting in a MFI of 5.2 and 15.0, respectively (Fig. 3). In contrast, only 6% of NHBE cells expressed MDR1 (MFI = 1.3). Similar trends in MDR1 expression levels were

obtained with the MRK16 antibody with, however, lower fluorescence values recorded, likely due to a weaker affinity of this antibody for MDR1 (Fig. S1; Supplementary information). The well-established MDR1 substrate digoxin is often used to probe MDR1 in biological systems, both in vitro and in vivo [13] and [17]. However, the drug has also been reported to be a substrate for other transporters detected at the gene level in our broncho-epithelial cell layers (e.g. some of the OATP) [20] and [21]. Hence, in order to verify the functionality of MDR1 in bronchial

epithelial cells, we performed an UIC2 antibody shift assay in presence of the much potent MDR1 inhibitor PSC833 as an alternative to measuring digoxin efflux ratios. This assay is based on the observation that binding of MDR1 ligands alters the conformation of the transporter, which increases the affinity of the UIC2 antibody for the MDR1 protein and causes a shift in fluorescence intensity [30] and [31]. Relative MFI values of 1.8 and 1.06 were obtained when MDCKII-MDR1or MDCKII-WT cells, respectively, were pre-incubated with PSC833, in line with their role as positive and negative controls ( Fig. S2; Supplementary information). Values of 1.27 and 1.26 were calculated for Calu-3 cells at a low or high passage, respectively, while NHBE cells produced a relative MFI of 1.16 ( Fig. S2; Supplementary information), indicating the presence of a MDR1 activity in bronchial epithelial cells.

We computed figure-ground

measure for population response (FG-m, Equation 1; see Figure 3Ai). FG-m was defined by subtracting the population response (average over pixels) in the background from the circle for the contour and noncontour conditions and then taking the difference between the two conditions. This index indicated how well the “figure” (circle area) is differentiated from the “ground” (background area). FG-m was calculated for each recording session separately. equation(Equation 1) FG-m=(Pc−Pb)cont−(Pc−Pb)non−cont,FG-m=(Pc−Pb)cont−(Pc−Pb)non−cont,where Pc and Pb are the population responses in the circle and background areas, respectively, cont and non-cont are the contour and noncontour conditions, respectively. The subtraction of the noncontour from the contour condition also enabled us to eliminate any response differences Capmatinib in space due to uneven staining. We also computed the differential Baf-A1 (contour minus noncontour) circle or background response ( Equations 2 and 3). Figure 3Bi depicts the circle differential response (Pcdiff) and background differential response (Pbdiff) as function of time. equation(Equation 2) Pcdiff=Pccont−Pcnon−contPcdiff=Pccont−Pcnon−cont equation(Equation 3) Pbdiff=Pbcont−Pbnon−contPbdiff=Pbcont−Pbnon−cont To study

the behavioral performance in the contour saliency experiments, we computed the probability of contour detection. This was normalized to the contour and noncontour conditions by setting the probability of contour detection to 1 in the contour condition, 0 in the noncontour condition and varying accordingly the probability for the jittering orientation conditions (Figure 5B). The purpose of this

normalization was to overcome the slight variation in behavioral performance due to the animal’s motivation. We verified that the nonnormalized and normalized psychometric curves showed similar results (Figure S4A). To study the effects of contour saliency on the population response, the neurometric curve was computed by calculating the FG-m as a function of orientation jitter (FG-mjitt; Equation 4). equation(Equation 4) FG−mjitt=(Pc−Pb)jitt−(Pc−Pb)non−cont,FG−mjitt=(Pc−Pb)jitt−(Pc−Pb)non−cont,where Pc and Pb are the population responses in the circle and background why areas, respectively, and jitt and non-cont are the different jitter conditions and noncontour condition, respectively (the contour condition is defined by jitter = 0). The neurometric curve values were normalized to maximal and minimal values in each recording session (to overcome the variable staining quality across recording sessions; Figure 5C). We verified that the nonnormalized and normalized curves showed similar results (Figure S4B). The population response for each pixel (VSDI amplitude, normalized as in the previous section) was computed as function of the orientation jitter condition. This yielded the neurometric curve for each pixel, which was then computed for each time frame.

28 in this study. The Guinea-Bissau cohort [14] reported a proportion of 0.40 and it was one in three infections for the Mexican cohort [13]. The measure of pathogenicity is very sensitive to the accuracy of detection of asymptomatic infections which usually have low viral excretion and thus the estimate of Guinea-Bissau where neither serology nor molecular techniques were used could possibly be overestimated. Though rotavirus infects children throughout the first three years of life, in some developing country settings it displays an affinity toward neonates.

In this study, 18% of the children were infected find more in the first month. This phenomenon has been reported earlier in various studies [19], [20], [21] and [22] and in hospitalized settings [23] and [24]. One explanation could be that a newborn, exposed to an environment saturated with the virus, is more likely to get infected or that neonates might be infected with specific strains that could bind to receptors not expressed in the post-neonatal period [25]. While rotavirus infections occurred throughout follow up, disease was seen mainly between the ages of 4–12 months. During early infancy, the child seemed to be protected from developing diarrhea due

to rotavirus, as evident from the proportionately higher asymptomatic infections in the first three months. Beyond three months, rotavirus produced symptoms more often. As the child crossed the age Parvulin of one year, the proportion selleck chemical of rotavirus infections developing into disease decreased and stayed low until the end of the follow-up. This was also demonstrated by Velazquez et al. [26] where rotavirus associated diarrhea was found to peak between 4 and 6 months and asymptomatic infections were more frequent in the first three months and beyond 10 months. Description of the natural history of rotavirus, especially of asymptomatic infections is limited. The Kaplan Meier estimates from the Mexican cohort [13] showed that 34% of the children were infected

by six months, 67% by one year and 96% by the age of two years. The West African cohort found that 26% infected by six months, 46% by one year and 74% by the age of two years [14]. While the survival curves of these two cohorts were gradual and uniform, the Vellore cohort displayed a steeper curve initially with a high incidence rate and 43% infected by six months. The late infancy window of a high rate of symptomatic rotavirus infection has been reported previously in many studies [27], [28] and [29]. This may occur following the waning of the maternal antibodies known to be protective against disease and preceding the steady build-up of child’s immune system, or corresponding to weaning, and increased levels of contamination.