These are easily measured by using a crystalline sample of a comp

These are easily measured by using a crystalline sample of a compound using standard DSC equipment. However, the PLS-DA modelling attempts resulted in non-significant models (data not shown). In the next step, we therefore also included Tg-related parameters, which are assumed to represent properties related to the molecular

mobility of the amorphous state. Interestingly, the most predictive ZD1839 mw model, shown in Fig. 1, did not include any parameter representing an absolute temperature parameter (Tm or Tg), as could be expected since the quality of the amorphous product formed often are related to difference between formation temperature and Tg ( Yamaguchi et al., 1992). Instead it was the balance between thermodynamic and

kinetic properties, i.e. the adjusted parameters involving both Tm and Tg, that carried most information. In this case, the predictivity was 81% for the test set ( Fig. 1A). The model was based on Tg,red, Tm − Tg, ΔSm, ΔGcr × Tg,red, ΔHm, ΔGcr/Tg,red and ΔGcr/Tg,red and hence, the analysis showed that the Tg-related properties indeed carry information of importance for the prediction of glass-forming ability. In a general context, larger molecules are commonly less prone to crystallize from a liquid state (Baird et al., 2010). Therefore, we wanted to evaluate the effect HDAC inhibitor of Mw on the predictions and hence, a new model was built including all former parameters, together with Mw-related properties. In this analysis, only the adjusted parameter Tg,red × Mw remained after model refinement and this property predicted 91% and 94% correctly of the training and test sets, respectively ( Fig. 1B). We also found that equal predictivity was obtained from Mw alone (accuracy of training and test sets of 88% and 94%, respectively, Fig. 1C). The results obtained herein, based on a large and structurally diverse drug-like dataset, strengthen previous findings of the importance of molecular size and Tg as predictors of glass-forming

ability ( Lin et al., 2009). In the scientific discussion, it is often ADP ribosylation factor referred to Kauzmann (1948) and Turnbull (1969) who suggested that compounds with a Tg,red higher than 2/3 are good glass-formers. The theoretical rationale for this effect is that compounds with smaller super-cooled liquid regime (i.e. high Tg,red) have a lower probability for nucleation when cooled below its melting temperature due to less time spent in that critical region. This has been confirmed in a study on a homologous series of cyclic stilbenes ( Ping et al., 2011), but in the same publication it was argued not to be true when looking at more diverse chemical structures. Recently it was shown by Baird et al., that for a set of drug compounds the Tg,red is not useful for predicting glass-forming ability ( Baird et al., 2010). This is partially in line with our observation that Mw is a good predictor by itself, and that the Tg,red contributes with minor information.

We are grateful to all patients who donated their blood samples f

We are grateful to all patients who donated their blood samples for this study and to Thongchai Hongsrimuang, Sunee Seethamchai, Pannadhat Areekul, Ratiporn Kosuwin, Urassaya Pattanawong, Teerayot Kobasa and the staff of the Bureau of Vector Borne Disease, Department of Disease Control, Ministry of Public Health, Thailand, for assistance in field work. This research was supported by grants from the National Research Council of Thailand and the Thai Government Research Budget

to S.J and C.P.; The Thailand Research Fund (RMU5080002) to C.P.; and from the National Institutes of Health (GM43940) to A.L.H. “
“Rotavirus is the most common cause of acute gastroenteritis in children under 5 years of age [1]. In developed countries, rotavirus gastroenteritis

remains a common AP24534 order cause of hospitalization at great cost to health services [2]. In England and Wales, the annual incidence Natural Product Library of rotavirus hospitalizations is estimated at 4.5 per 1000 children under the age of 5 years and the cost to the National Health Service estimated to be GBP 14.2 million per year [3]. The second generation of live oral rotavirus vaccines have demonstrated safety and efficacy [4] and [5] and are increasingly being used routinely as part of childhood immunization schedules in a number of middle and high income countries [6] and [7]. The Rotarix vaccine, made from the most common human serotype G1P1A[8], is recommended no by WHO as a two-dose schedule to be given at two and four months of age [8]. RotaTeq, a pentavalent vaccine developed from a bovine rotavirus strain and combined with reassorted strains of human serotypes G1, G2, G3, G4

and P1A[8], is WHO-recommended as a three-dose schedule to be given at two, four and six months of age [8]. In the United States, following the introduction of RotaTeq in 2006, there was a delay in the timing of peak incidence in the 2007–2008 season by two to four months and fewer cases overall compared to previous years [6]. This provides the first indication, post-licensure, that rotavirus vaccination reduces the burden of rotavirus disease in a large population and suggests that vaccination may also have an impact on transmission. Other high and middle income countries which have introduced rotavirus vaccination have shown similar effects [7] and [9]. In England and Wales, the introduction of rotavirus vaccination is currently under consideration. This study aims to develop a dynamic model of rotavirus transmission, and apply it to daily case reports of rotavirus disease from England and Wales. Using this model, we examine the potential epidemiological impact of a rotavirus mass vaccination programme. In temperate countries, most rotavirus disease occurs in late winter or early spring [10].

Forty-two community-dwelling people with stroke who were aged 70

Forty-two community-dwelling people with stroke who were aged 70 years old (SD 10) and 13 (31%) of whom were women participated. They were on average almost 3 years from the onset of stroke and approximately half of them were right hemiplegics. Twenty-one age-matched healthy controls who were aged 69 years old (SD 7) and 10 (48%) of whom were women also participated. The mean BMI of stroke survivors (26.4 kg/m2, SD 4.3) was slightly less thanthat of healthy controls (27.5 kg/m2, SD 3.9). Participants’ characteristics are presented in Table 1. People with stroke spent 79 min (95% CI 20 to 138) less time on their feet than healthy controls (Table 2). They spent significantly less

time in standing, MK-2206 chemical structure ascending and descending stairs, and transitions than healthy controls but not walking. On average, the observation period of the free-living physical activity of stroke survivors (10.8 hr) was significantly (p < 0.001)

less than that of the healthy controls (12.7 hr). After adjusting the observation period to 12 hr, there was no significant difference between groups in terms of time on feet (mean difference 36 min, 95% CI –27 to 99) ( Table 3). People with stroke spent 36 min (95% CI –17 to 89) less time not on their feet than healthy controls, which was not statistically significant (Table 2). They spent approximately the same time in sitting, reclining, or lying as healthy controls. After adjusting the observation Enzalutamide chemical structure period to 12 hr, the difference

remained statistically non-significant (Table 3). People with stroke carried out 5308 (95% CI 3171 to 7445) fewer activity counts than healthy controls. They carried out significantly fewer steps, transitions, and stair ascents and descents than healthy controls. After adjusting the observation period to 12 hr, they still carried out 4062 (95% CI 1787 to 6337) fewer activity counts than healthy controls (Table 3). This study found that ambulatory stroke survivors carry out less free-living physical activity both in terms of duration (time spent on feet) and frequency (activity counts) than age-matched healthy controls. No difference was found in terms of the time spent not on feet (sitting, reclining, or lying). However, the period of time that stroke much survivors were observed was shorter than for healthy controls. When data were adjusted to a standard observation period, the stroke survivors still carried out fewer activity counts but were on their feet for a similar amount of time, ie, although stroke survivors spent less absolute time on their feet than healthy controls, in relative terms it was much the same. The difference in the duration of the observation period between the stroke survivors and healthy controls therefore explains the difference in duration but not frequency of free-living physical activity. In terms of duration, the stroke survivors spent 10.8 hr (SD 3.

Each fetal head contained the appropriate number of appendages, a

Each fetal head contained the appropriate number of appendages, although the ears appeared disproportionately large for each head. Length and weight measurements were disproportionate for the fetus; the fetus weighed 690 g (26 weeks), the crown–rump length was 16 cm (20 weeks), the crown–heel length was GSK1120212 price 28 cm (22 weeks), and the heel–toe length was 5 cm (28 weeks). Both the hands and the feet appeared disproportionately large for the fetus, as demonstrated by the assigned gestational age by heel–toe length. Examination of the internal organs revealed abnormalities

predominantly within the thoracic cavity. Hypoplasia of the lungs was evident, with the right lung weighing 2.5 g and the left lung weighing 5.3 g (normal 24 week fetus would have a 17 gram combined lung weight). Furthermore, the right lung demonstrated a rudimentary fourth lobe. An adherent 0.4 cm diameter focus of selleck chemical ectopic pancreas was noted along the adventitia of the distal esophagus. The only abdominal duplication involved the formation of a bifid gallbladder. All other abdominal organs appeared appropriate in size and orientation. Of note, an additional focus of ectopic pancreas formation was evident as an adherent 0.2 cm diameter nodule along the

greater curvature. Microscopic analysis revealed extramedullary hematopoiesis in the liver, and congestion of the spleen. A single kidney was present on the right and left side and demonstrated vascular congestion. Mild abnormalities of the pelvic organs were noted, including a uterus with constriction along the superior aspect of the fundus. The remainder of the thoracic, abdominal, and pelvic organs appeared normal in orientation, although in size corresponded to a variable gestational age of 22–28 weeks. To our knowledge there are no published reports of the use of three-dimensional ultrasonography in clarifying this nonviable form of conjoined twins, although first trimester diagnosis

[3] and the use of MRI [4] to assist has been described. Recent reports have shown the value in both 2D and 3D ultrasound in the first trimester to classify conjoined twins and allow earlier reproductive choices [5], [6], [7] and [8]. Classification of conjoined twins is paramount for guiding obstetrical management. crotamiton Prenatal diagnosis can help guide decisions so that both fetal and maternal morbidity and mortality can be minimized. When considered as a whole, 75% of conjoined twins do not survive the first 24 h of life [9]. The fetal chance for survival has to be weighed against the potential surgical morbidity to the mother and feasibility of vaginal delivery [9]. In this case of non-viable conjoined twins, the use of 2D and 3D ultrasound correlated very closely with the postmortem autopsy report and measurement of the combined cephalic diameter allowed for a successful trial of vaginal delivery.

Whilst drugs may relieve symptoms, effect sizes are small to mode

Whilst drugs may relieve symptoms, effect sizes are small to modest at best and their toxicity/adverse event profile is unfavourable compared to conservative non-drug interventions (Zhang et al 2007). Indeed, all clinical guidelines advocate conservative non-drug strategies for hip osteoarthritis (Conaghan et al 2008, Hochberg et al 2012, Zhang et al 2008). In particular, guidelines recommend a focus ‘on self-help and patient-driven treatments rather than on passive

therapies delivered by health professionals’ (Zhang et al 2008). Treatment should be individualised check details and patient-centered, involving shared decision making between the patient and physiotherapist taking into account the patient’s preferences and wishes. Two recent systematic reviews have found that such patient-centred interaction enhances the therapeutic alliance (Pinto et al 2012a) and improves patient satisfaction with care (Oliveira et al 2012). Other aspects to consider Idelalisib in vivo in guiding treatment include: hip factors (adverse mechanical factors, impairments, obesity, physical activity, dysplasia); general factors (age, sex, co-morbidity); level of pain intensity and disability; and location and degree of structural damage (Zhang et al 2005). Given the broad impact of osteoarthritis and in accordance with a biopsychosocial approach to the management of chronic pain, it is logical that both biological

and psychosocial factors should be addressed in people with hip osteoarthritis. For hip osteoarthritis, core conservative treatments for all patients should include education and exercise. In addition, weight loss is also recommended for those with lower limb osteoarthritis who are overweight/obese (Conaghan et al 2008, Hochberg et al 2012, Zhang et al 2005, Zhang et al 2008). It is apparent that the treatments of exercise either and weight loss for osteoarthritis require behavioural changes and it is well known that these changes are difficult

to initiate and maintain. Therapists therefore need to assist the patient in formulating achievable shortand long-term goals and specific action plans. Patient education is a core component of hip osteoarthritis treatment as it is an indispensable element in promoting adequate self-management. Education delivery modes vary and can include informal discussion with the health care provider, provision of written materials, support groups, websites, and structured self-management programs. Self-management programs can also take various forms with differences in the content, mode of delivery (individual, group-based, telephone, internet), program length, and expertise of those delivering the material (lay leaders, health care professionals). Self-management programs typically include coping with behavioral change, educational information, and self-management techniques.

(2) The Grandi model does have a distinct fast Ito current, and

(2). The Grandi model does have a distinct fast Ito current, and so its conductance is altered directly. Models that have separate Ito components may be better for predictions based on screening Kv4.3 in future. We performed the simulation study three times in parallel, based on the following datasets: Quattro 5 channel (Q); Barracuda & Quattro 4 channel (B&Q2); and a third variant using the Quattro 5 channel screen but with hERG manual patch clamp IC50 values replacing the Quattro screening data. The manual data are taken from ICH-S7B Good Laboratory

Practice (GLP) studies featured in regulatory submission documents, and gathered by Gintant (2011). We refer to the third dataset as the Manual & Quattro (M&Q) dataset. Note that QTc Selleckchem SRT1720 is designed to be equal to QT at 1 Hz, so in the simulations we pace cells at 1 Hz (using the square wave stimulus current

with magnitude and duration as defined in the models’ CellML implementations, see below). We begin with a control simulation, pacing the model until it reaches a pseudo-steady state (see Supplementary Material S1.3 for details on steady state detection). Compound concentration is then increased from 1 nM to 100 μM, taking 20 increments equally spaced on a log10 scale. At each concentration, the data shown in Table 1 is used with Eqs. (1) and (2) to impose a new maximal conductance value for each of the screened ion currents. We then continue pacing until a new steady state is reached, and evaluate the action potential duration at 90% repolarisation

(APD90). The process is repeated with all permutations of mathematical model and dataset, giving a total of nine concentration–APD curves per compound. We use Adenylyl cyclase the method outlined in Elkins et al. (2013) to quantify the uncertainty on our APD90 predictions due to assay variability. In brief, we characterise variability associated with ion channel screens by examining the pIC50 distribution from the relevant control assays. A Bayesian inference scheme then produces a probability distribution for the mean of a large number of independent repeats. pIC50 values are then sampled from this distribution at random, and simulations are repeated with these values to build up a distribution of possible outcomes (as displayed in e.g. Fig. 3 and Fig. 4). The resulting intervals show where there is 95% probability that the simulation prediction lies, based on the variability we measured in the control screens for each channel. CellML is a machine-readable XML-based markup language used to describe models’ ordinary differential equations, initial conditions and parameters (Lloyd, Lawson, Hunter, & Nielsen, 2008). The ten Tusscher and Panfilov (2006), Grandi et al. (2010), and O’Hara et al. (2011) models were downloaded from the Physiome Project repository (

The latter approach has the advantage of being able to validate p

The latter approach has the advantage of being able to validate peptide selection by assessing in vitro recall responses using peripheral blood mononuclear cells (PBMC) from normal healthy donors. While a broad range of

potential universal epitopes have selleck chemicals been identified for both TT and DT [3], [4], [5], [6] and [7], a considerable amount of work has focused on TT830–844[8], [9] and [10]. Experimental evidence suggests that TT830–844 can be presented by up to ten different MHC class II alleles [3] and [6], although this has been disputed [11]. TT830–844 has been used as a helper peptide in various animal species including mice [12], [13] and [14], rats [15], rabbits [16] and rhesus macaques [17]. The predominant focus has been on using a helper peptide to improve a cytotoxic T lymphocyte (CTL) response for treating viruses and cancer [13], [14] and [15], rather than for enhancement of humoral immunity. Ku-0059436 concentration In one primate study, a CTL response was induced to simian immunodeficiency virus peptides from Nef and Gag proteins [17]. However, only two of eight primates demonstrated a proliferative response to the peptide. Helper peptides have been used in several clinical studies, again primarily focusing on inducing CTL responses for the treatment of human viruses or cancer. TT830–844

has been tested in vaccines to induce CTL responses for treatment of chronic hepatitis B virus [18] and human immunodeficiency virus infection PD184352 (CI-1040) [19], [20] and [21]. In addition, the helper peptide has been used to enhance CTL responses for the treatment of melanoma [22] and [23]. Those publications demonstrating recall response to TT830–844 report an average range of 60–75% of subjects responding [18] and [22]. However, in one report 91% of patients that received an immunization containing MHC class I restricted melanoma peptides plus TT830–844 demonstrated a recall response to the helper peptide, but 18% that did not receive the helper peptide also responded, presumably due to previous immunization with TT [23]. We have rationally designed a fully synthetic nanoparticle-based vaccine against nicotine for smoking cessation. However neither the B cell antigen, nicotine

nor the nanoparticle polymer contain T cell epitopes needed to provide help for B cell differentiation and antibody affinity maturation. Here we describe a ‘universal’ memory CD4 helper peptide that was designed and included in synthetic nanoparticle vaccines to provide promiscuous binding to a broad range of the most common MHC class II alleles in order to provide CD4 T cell help for B cell maturation and antibody production. We hypothesized that inclusion of a dimeric CD4 helper memory peptide (TpD) containing both TT and DT epitopes linked by a cathepsin linkage site, would result in improved antibody responses. We demonstrate that all 20 of tested normal human blood donors generated an in vitro memory recall response to the chimeric peptide.

, 2010) Reduced urinary levels of carnosine, glycine, serine, th

, 2010). Reduced urinary levels of carnosine, glycine, serine, threonine, alanine and histidine have also been observed in children with ASD, suggesting an imbalance of resident gut bacteria involved in both amino acid and carbohydrate selleck kinase inhibitor metabolism may be present ( Williams et al., 2011 and Ming et al., 2012). A reduced capacity for nutrient digestion and transport in children with ASD has been related to increased levels of Clostridium species, Bacteriodetes depletion, and loss of metabolites related to energy homeostastis (e.g disaccharidases, hexose transporters) ( Williams et al., 2011). Future efforts should focus on putative mechanisms by which microbe-dependent production of

neuromodulatory metabolites can result in neurodevelopmental dysregulation predictive of disease. The consequence of environmental stressors on gut microbiome composition in adults has been established for nearly four decades (Tannock and Savage, 1974). This association was first developed from observations that short-term environmental challenges – deprivation from food, water, and bedding – decreased the abundance of beneficial bacteria, such as Lactobacillus, and increased the susceptibility to opportunistic pathogens in mice ( Tannock and Savage, 1974). However, quantification of bacteria in these early studies

selleck chemical was limited to phyla that could be cultured in the lab, failing to account for >99% of microorganisms that could not be cultivated by standard techniques ( Hugenholtz et al., 1998). Recent advances in metagenomic analyses have identified microbial communities not previously cataloged, and captured a more complete representation

of the microbial composition in the intestine ( Leser et al., 2002, Dinan and Cryan, 2012, Lutgendorff et al., 2008 and Bendtsen et al., 2012). With these improved technologies, reduced Vasopressin Receptor microbial richness and opportunistic overgrowth of bacteria have been subsequently reported in animal models where adult chronic stress was examined, and where long-term programming changes in the HPA stress axis were found ( Bailey et al., 2010). Additionally, social stress-mediated depletion of Lactobacillus was associated with increased translocation of cutaneous-derived microflora to the inguinal and mesenteric lymph nodes ( Bailey et al., 2010, Bailey et al., 2006 and Bailey et al., 2011). Although the mechanistic significance of bacteria translocation in these lymphoid organs on HPA axis reprogramming is not clear, sympathetic and noradrenergic innervation of lymphoid organs plays a critical role in the neuroimmune modulation of the HPA axis ( Elenkov et al., 2000). Stress pathway dysregulation is the most common symptom in neuropsychiatric disorders, yet mechanisms involved in determining potential developmental windows of susceptibility are not fully understood.

Another approach, different from multivalent conjugate vaccines,

Another approach, different from multivalent conjugate vaccines, involves the use of highly conserved pneumococcal proteins. Pneumolysin toxoid (dPly) and histidine-triad protein D (PhtD) are potential candidates that have been shown to play a role in natural exposure [13] and induce disease protection in animal models [14], [15],

[16], [17] and [18]. We evaluated the safety, reactogenicity and immunogenicity of investigational vaccine formulations containing dPly and PhtD, either alone or in combination with the PS-conjugates of the 10-valent pneumococcal non-typeable Haemophilus influenzae INK 128 molecular weight protein D conjugate vaccine (PHiD-CV; Synflorix™, GlaxoSmithKline Vaccines), when administered to healthy toddlers. In healthy adults, these formulations were well-tolerated and appeared immunogenic [19]. The primary objective of this study focused on the incidence of grade 3 fever (rectal temperature >40 °C), as febrile reactions are common post-vaccination adverse reactions in children that have consequences for parents and healthcare providers, especially in terms of the resulting risk of febrile seizure. This phase II, randomized, observer-blind, controlled study (NCT00985751) was conducted in 10 centers in the Czech Republic between November 2009 and March 2011. The primary

objective was to assess the incidence of fever >40.0 °C (rectal temperature) within seven days following at least one primary

dose of the investigational vaccine compared to PHiD-CV. Secondary objectives included safety, reactogenicity and immunogenicity assessment of the investigational vaccines. The study protocol was reviewed and approved by the Ethics Committee for Multicentre Clinical Trials of Faculty Hospital first Hradec Kralove and local hospital ethics committees. The study was conducted in accordance with Good Clinical Practice and the Declaration of Helsinki. Written informed consent was obtained from the parents or legally acceptable representative of each child before enrolment. This study has been registered at (NCT00985751). A protocol summary is available at (study ID: 113171). Eligible participants were healthy toddlers (12–23 months at first vaccination), without history of any hypersensitivity reaction following previous vaccination, and who had not previously been vaccinated against S. pneumoniae. Toddlers were excluded if another vaccine had been administered, or planned, from 30 days before and up to 30 days after administration of a study vaccine dose. Participants were randomized (1:1:1:1:1) using a central internet randomization system (SBIR) to receive a 2-dose primary vaccination series followed by booster vaccination. The study comprised five visits at study months 0 (dose 1), 2 (dose 2), 3 (post-primary), 6 (pre-booster) and 7 (post-booster).

5 μg/ml TT in CM plus 5% PHS Because nearly 100% of the TT was a

5 μg/ml TT in CM plus 5% PHS. Because nearly 100% of the TT was adsorbed to the NP (see Section

3.1), an amount of 12.5 μg/ml was used for both NP-adsorbed and free Ag. Free CpGB and Poly (I:C) were used at a final concentration of 4.25 μg/ml, which was the same amount used for co-adsorption with Ag onto NP. Phytohaemaglutinin (PHA, 5 μg/ml, SIGMA) was used as a positive control of stimulation, and CM alone as a negative control. BSA-adsorbed NP, TT plus CpGB without NP, or chitosan alone were also used as controls. selleck kinase inhibitor Cell proliferation was assessed by incorporation into DNA of [3H]Td (GE Healthcare, Buckinghamshire, UK). The cells were pulsed with 0.5 μCi [3H]Td/well 18 h before harvesting, and counts per minute (c.p.m.) determined in a liquid scintillation β counter (1450 Microbeta Plus, Wallac Oy, Turku, Finland). Proliferation response was calculated see more as the mean ± SD of the c.p.m. from three replicates. Splenocytes from gp140-immune Balb/c mice were cultured for 3 days in the presence of 5 μg/ml gp140, either free or adsorbed to NP. Concanavalin-A (5 μg/ml, Sigma) was used as a positive control of stimulation. After 48 h, the cells were pulsed as for human cells, and 18 h later the cells were harvested

and the c.p.m. counted. Proliferation response was expressed as stimulation index (PI), calculated by dividing the mean of the c.p.m. from three replicates of the experimental by the mean c.p.m. of the not-stimulated cells. Determination of specific TT serum IgG, specific gp140 serum IgG, IgG1, IgG2a, and IgA, as well as specific gp140 IgG and IgA in vaginal and nasal lavages, and in feces was performed by ELISA. ELISA plates (MaxiSorp, Nalge-Nunc International, Rochester, NY) were coated overnight at room temperature with 4 μg/ml TT or 5 μg/ml gp140 in PBS. Blocking was performed for 1 h at 37 °C with PBS containing 1% BSA. Serially diluted samples were incubated for 1 h at 37 °C. Bound IgG, IgG1, and IgG2a were detected by incubation for 1 h at 37 °C with Vasopressin Receptor goat anti-mouse

Ig-HRP (AbD Serotec, Kidlington, Oxford, UK), or with biotinylated goat anti-mouse IgA Ab (SouthernBiotech, Birmingham, AL) to detect bound IgA. An amplification step was performed to detect IgA by incubating the plates with HRP-streptavidin conjugate (R&D Systems) for 1 h at 37 °C. Plates were developed by adding tetramethylbenzidine (TMB, Pierce-Endogen, Woburn, MA) and incubating the plates in the dark. The reaction was stopped using 1.0 N H2SO4, and optical densities (O.D.) read at 450 nm. A mix of pre-immune samples was run in 6-replicates per plate and the cut-off calculated (after subtracting the blank) as the mean of these 6 values plus 3 SD, except for that of feces where 5 SD were used. ELISA plates were coated with 1 μg/ml in PBS of affinity purified sheep anti-HIV-1-gp120 polyclonal antibody (AAlto Bio Reagents, Dublin, Ireland) and incubated overnight at room temperature.