Of all the available materials, calcium phosphate was selected as core of choice as it is ceramic (structurally most regular materials) and crystalline in nature (high degree of order). The surface exhibits high level of surface energy which favors the binding

of carbohydrate on surface film. Cisplatin In the second step, extent of sugar loading was quantified by using anthrone method. The method is based on hydrolysis of carbohydrates to simple sugars in presence of acid followed by dehydration of sugars to furfural derivatives, e.g. hydroxyl methyl furfural. Furfural derivatives react with anthrone to form a deep green color with an absorption maximum at 625 nm. The sugar adsorption on core was confirmed using FTIR spectroscopy. Further drug is adsorbed over sugar loaded core particles through non-covalent and ionic interactions. The pimozide loaded aquasomes exhibited

smaller particle size than that of pimozide pure drug. Hence it can be concluded that, the aquasomal formulation had lead to reduction of particle size to nanometer range. Improved dissolution was observed with aquasome formulation of pimozide than that of pure drug, which can be accounted for nanosize and aqueous environment of the aquasomes. The release followed the first order kinetics which supported the mechanism of immediate release of pimozide. Ceramic nanoparticles were developed as a technological innovation for the pimozide delivery via the peroral route. Co-precipitation by sonication technique Vorinostat was found to give more yield

than other methods. Size analysis indicated spherical particles in the size range of aquasomes. Release studies of aquasomes showed greater dissolution than that of pure drug. Thus aquasomes can be used for enhancing the solubility of poorly soluble drugs. All authors have none to declare. Authors would like to express thanks to Vasudha Pharma Chemical Ltd, Hyderabad for providing the ADAMTS5 pimozide gift sample. Authors would also like to express their thanks to Dr Sathesh, HOD, Pharmaceutics for his guidance and support. “
“The physiological environment within a living organism is mostly chiral. Therefore, chiral discrimination has been an issue in the development and use of pharmaceutical drugs. Enantiomers of racemic drugs often differ in pharmacokinetic behavior or pharmacological action.1 In recent years, research has been intensified to understand the aspects of the molecular mechanism for stereoselective biological activities of the chiral molecules. The development of analytical methods for the assessment of enantiomeric purity is challenging due to the fact that enantiomers possess virtually identical properties.2 In the pharmaceutical industry, much emphasis is put on chiral analysis. The reason is the potentially different behavior of the enantiomers of a chiral drug molecule after administration.

Compliance

to Alisertib cell line vaccine intake was high for subsequent doses; only 3.5% of infants did not receive all the three intended doses. Vaccine efficacy in children up to 2 years of age was 55.1% (95% CI 39.9 to 66.4; p < 0.0001); the vaccine efficacy in the second year of life of 48.9 (95% CI 17.4 to 68.4; p = 0.0056) was only marginally less than that in the first year of life [56.3% (95% CI 36.7 to 69.9; p < 0.0001)]. The results were similar in the intent-to-treat (ITT) population where up to 2 years efficacy of 55.8% (95% CI 41.3 to 66.7; p < 0.0001) did not differ substantially from that in the first [57.2% (95% CI 38.9 to 70.1; p < 0.0001)] or the second year of life at 49% (95% CI 17.5 to 58.4; p = 0.0055). There was no significant interaction of treatment group and site with vaccine efficacy (p = 0.4802). The secondary endpoint analyses strongly supported the primary analysis (Table 1). In the second year of life, the vaccine efficacy

against RVGE of any severity requiring hospitalization or supervised rehydration therapy, RVGE requiring hospitalization ≥6 h and severe GE of any etiology were 34.3% (95% CI 17.2 to 47.8), 35.9% (95% CI −9.1 to 62) and 10.9 (95% CI −17 to 31.8) respectively. For the genotype specific analysis, there were a total of 199 episodes of severe RVGE that occurred in 195 subjects up to 2 years of age. For this particular analysis, a subject could contribute more than one primary event if associated with a different genotype. Four subjects had more than one episode of severe RVGE with different genotypes; three in the vaccine group and one in the placebo. The most prevalent selleck products (85%) rotavirus genotypes identified in the 199 episodes were G1P[8]

(37%; n = 74), G2P[4] (31%; n = 61), G12P[6] (11%; n = 21) and G12P[8] (7%; n = 13). A post hoc analyses on ADAMTS5 the genotype specific efficacy is consistent with the overall protective efficacy. The G9P[4] genotype had an imbalance of cases with nine in the vaccine group and one in the placebo group ( Table 2). Survival curves in the vaccine group compared with the placebo group showed a significantly increased cumulative proportion of infants without severe RVGE (Fig. 2). We calculated that 40 infants would need to be immunized to prevent one episode of severe RVGE in the first 2 years of life (95% CI 28.0 to 63.0) and 21 had to be immunized to prevent RVGE of any severity in the same period (95% CI 16.0 to 32.0). Fig. 3 displays the incidence rate ratios for the primary outcome and several secondary outcomes as a forest plot. In children up to 2 years of age, the incidence of severe RVGE per 100 person years was 1.3 in the vaccine group and 2.9 in the placebo group for an incidence rate ratio of 0.45 (95% CI 0.34 to 0.60) and an absolute rate reduction of 1.6 (95% CI 0.9 to 2.2). In the first year of life, the incidence of severe RVGE per 100 years was 2.0 in the vaccine group and 4.

The Lys residues contained in this probe are capped and therefore have no charge. Owing to the presence of 8 CAARs, the renal uptake of the probe would increase substantially. The positively charged Lys was found to reduce the renal uptake of the radiolabeled somatostatin analogs pentetreotide, octreotide, and octreotide (containing a single Lys residue each) through a putative competitive mechanism [12], [13], [14] and [28]. In the present study, co-injection with Lys did not reduce the renal uptake of 64Cu-cyclam-RAFT-c(-RGDfK-)4, possibly

because of the lack of charged Lys residues. In addition to the number and type of CAARs, factors such as their structure LY2109761 cost and distribution inside a molecule may also contribute to renal reabsorption mechanisms. Unlike Lys, GF reduced the renal uptake of all the radiolabeled peptides examined [19], [26] and [28], including 64Cu-cyclam-RAFT-c(-RGDfK-)4 investigated in this study. This could be because GF is a polypeptide-based succinylated gelatin composed of several molecules of varying sizes and structures, with both negative and positive CAARs; it may therefore possess the ability to interact with several binding domains of megalin simultaneously, thereby Verteporfin solubility dmso efficiently blocking the renal

reabsorption of various molecules. Aside from co-injection with Lys and GF, other strategies have been reported to reduce the renal uptake and retention of radiolabeled peptides, especially somatostatin analogs [13], [29], [30], [31] and [32]. In addition to these, modification of the peptide by coupling it with another molecule (such as polyethylene glycol) can

alter the pharmacokinetics by increasing the size and hydrophilicity of the molecule and masking its charges [11], which may also be considered in future studies for reducing the renal below accumulation of 64Cu-cyclam-RAFT-c(-RGDfK-)4. In addition, our subsequent studies on the development of 64Cu-cyclam-RAFT-c(-RGDfK-)4 internal radiotherapy will also focus in estimating and determining the therapeutic but non-nephrotoxic doses of this radioactive compound. Co-injection with GF effectively reduced uptake of 64Cu-cyclam-RAFT-c(-RGDfK-)4 in mouse kidney. l-lysine alone had no effect on the probe biodistribution, but the combined use of Lys and GF tended to enhance the effect of GF. Dynamic PET imaging enabled visualization and quantification of the spatiotemporal change in renal radioactivity caused by GF and strongly suggested that the mechanism of action of GF at least partially occurs via inhibition of renal tubular reabsorption of 64Cu-cyclam-RAFT-c(-RGDfK-)4. The use of GF should be included in future studies exploring the therapeutic potential of 64Cu-cyclam-RAFT-c(-RGDfK-)4. We would like to thank the Molecular Probe Program (MPP) for supplying the 64Cu produced for this study; the Cyclotron Operation Section for cyclotron operation; and Mr.

One Russian government respondent noted: “seroprevalence data for some regions show high antibodies; however, we do not have exact Onalespib chemical structure data for most regions in different age groups.” Overall, the published epidemiological data in Russia were quite variable, suggesting variations in measurement, reporting, or interpretation [27], [28] and [29]. In Russia, the literature reported several outbreaks in cities [30] and following natural disasters [31], [32] and [33], some of which

were mentioned by respondents. In India and Mexico, respondents and the literature agreed that the hepatitis A epidemiological evidence is weak, but some respondents did not find this alarming. In India, two respondents said there were no epidemiologic data available: “[We have] no mortality, no morbidity, no estimates of economic loss for the poor. But the technical advisory groups need to have these

data to review to make decisions.” A few respondents noted recent studies not yet completed and published. The literature review confirmed the lack of recent seroprevalence data in most areas of India [34], [35], [36], [37], [38] and [39]. Meanwhile, several respondents believed hepatitis A disease is not in India and that seroprevalence in India has not changed: “We don’t have [data] and we really don’t need it.” Policy articles from 1995 through 2011, however, indicate a growing recognition of the epidemiological transition in India and the growing threat of outbreaks [40], [41], GDC-0199 in vitro [42], [43], [44], [45], [46] and [47]: “The epidemiological transition needs to be documented as well as the potential for outbreak; Kerala was one state with a recent outbreak.” A 2005 outbreak in Hyderabad suggested a change in adult seroprevalence, warranting further assessment for vaccination [48]. Currently, there

is no national PD184352 (CI-1040) surveillance system to track outbreaks and the burden of hepatitis A in India. In Mexico, respondents noted there is no data by age group, geography, or socioeconomic status, or data capturing private immunizations, disease severity and the extent of fulminant disease. The overall body of Mexican literature on hepatitis A epidemiology was relatively small, with old (1996) seroprevalence data for Mexico City [49] and more recent data through 2006 for other areas [50], [51] and [52]. Older data suggest the initiation of the epidemiological transition in Mexico [53]. The majority of stakeholders in 5 out of 6 countries reported that economic and financial data were very important in the decision making process (Table 3). A government implementer in Mexico noted the Ministry of Health is “quite willing to have a discussion on hepatitis A; that is why we need cost-effectiveness [data].” However, the literature and internet search identified only 4 economic analyses on hepatitis A in the six countries.

AMA1 also contains a transmembrane domain, which spans the plasma membrane and anchors the protein to the cell surface. Two glycosylation mutants (GM) of AMA1 were constructed by mutation of putative N-glycosylation sites (Fig. 1a). Alignment of all known P. falciparum AMA1 genes revealed that most of the glycosylation sites were conserved. For AMA-GM1, the glycosylation sites that were not conserved between isolates were modified to be similar to the rare non-glycosylated isolates and glycosylation sites that were conserved were modified such that the asparagine (N) residue

was replaced with a glutamine (Q). In AMA1-GM2, all of the potential glycosylation sites were removed by substitutions with amino acids present in other selleck chemicals AMA1 alleles among different species of Plasmodium [34] and [39]. Both GM forms retained the native signal sequence. In the intracellular form of AMA1, AMA1-IC, the signal sequence was deleted to retain the protein within the cytoplasm after translation in transduced cells. All forms of AMA1 were engineered for expression from E1/E3/E4-deleted Ad5 vectors with expression cassettes driven by the murine cytomegalovirus (mCMV) immediate early gene promoter inserted at the site of the E4 deletion ( Fig. 1b). The glycosylation status of the four AMA1 variants was monitored by gel migration following digestion with

enzymes that cleave the carbohydrate moieties of glycosylated proteins. We observed a shift in mobility of

the native, but not the modified (GM1, GM2, and IC) AMA1 antigens following treatment of infected Bumetanide cell lysates with Afatinib PNGase F (Fig. 1c). These results indicate that the native AMA1 antigen is N-glycosylated when expressed in mammalian cells following adenovector delivery and that the mutants with altered glycosylation sites or a deleted signal sequence are not N-glycosylated. To determine the cellular localization of the various adenovectors expressing AMA1, we transduced A549 cells with the adenovectors and then assayed for cell location by immunofluorescence in the presence or in the absence of saponin, using the conformational specific anti-AMA1 monoclonal antibody 4G2. Comparison of the staining pattern in the presence or in the absence of saponin showed that the native as well as the GM1 and GM2 versions of AMA1 are located at the cell surface and that most AMA1-IC is located intracellularly (Fig. 2). To evaluate the immunogenicity of adenovectors expressing the different forms of AMA1, mice were immunized with one or two doses of vector. AMA1-specific T cell responses were evaluated by interferon-γ ELIspot with freshly isolated splenocytes as effectors and transfected A20 target cells as target APCs. Following a single dose of adenovector, all cell surface associated forms of AMA1 induced better T cell responses compared to the intracellular form; there was little difference between the glycosylated or non-glycosylated forms (Fig. 3a).

Thus, the ORF of NS1 was used for inserting Brucella sequences in this study. The A/Puerto Rico/8/34 (H1N1) strain was used as the backbone for obtaining influenza A virus vectors expressing Brucella L7/L12 or Omp16 sequences

in the form of fusion proteins with the N-terminal 124 amino acid residues of NS1. Our previous studies have shown that a bivalent vaccine formulation VE-822 order comprising a mixture of recombinant influenza A virus subtype H5N1 or H1N1 expressing the ribosomal L7/L12 or Omp16 proteins in prime and booster immunization mode (via conjunctival injection) generated antigen specific humoral and Th1-cellular immune responses in laboratory animals, and most importantly provided a high level of protection equivalent to the commercial B. abortus vaccine S19 (unpublished data). On this basis, a logical continuation of our research is to evaluate the immunogenicity and protectiveness

of the proposed new live vector vaccine in cattle, which is the purpose of the present study. All viruses were generated by a standard reverse genetics method using eight bidirectional plasmids pHW2000 [26]. Briefly, Vero cells were co-transfected by the LonzaNucleofector™ (Cologne, Germany) technique with 0.5 μg/μl of plasmids encoding the PB1, PB2, PA, NP, M gens and NS (chimeric) genes of the A/Puerto Rico/8/34 (H1N1) virus; and the HA and NA genes of the A/chicken/Astana/6/05 (H5N1) or A/New Caledonia/20/99 (H1N1) strains. The HA protein selleck compound sequence of the H5 virus was attenuated by means of exchanging its polybasic cleavage site to one containing a trypsin-dependent sequence. The NS genes were modified to express NS1 fusion proteins containing the sequence encoding the 124 N-terminal amino acids of the NS1 protein coupled with the sequences of B. abortus-derived proteins: L7/L12 (GenBank: AAA19863.1) or Omp16 (GenBank: AAA59360.1), followed by a double stop codon. Brucella sequences were obtained synthetically. very The supernatants of the transfected cell cultures were used to inoculate 10-day-old embryonated

chicken eggs (CE; Lohmann Tierzucht GmbH, Cuxhaven, Germany) which were incubated at 34 °C for 48 h. Vaccine batches were produced in CE after three egg passages of the viral constructs (Flu-NS1-124-L7/L12-H5N1, Flu-NS1-124-Omp16-H5N1, Flu-NS1-124-L7/L12-H1N1 и Flu-NS1-124-Omp16-H1N1). Vaccine samples were prepared from the viral constructs Flu-NS1-124-L7/L12-H5N1, Flu-NS1-124-Omp16-H5N1, Flu-NS1-124-L7/L12-H1N1 and Flu-NS1-124-Omp16-H1N1, which accumulated in 10-day-old CE (Lohmann Tierzucht GmbH) at 34 °C for 48 h. The obtained allantoic suspensions of viral constructs with the same antigenic structure (H5N1 or H1N1) were combined in a single pool in a 1:1 ratio to obtain the bivalent vaccine formulation.

A

summary of recommendations including grade of recommendation is presented in colour-coded organisation Duvelisib nmr on pages 4–29. These cover evidence for organisation of services, stroke recognition and pre-hospital care, early assessment and diagnosis, acute medical and surgical management, secondary prevention, rehabilitation, managing complications, community participation and long term recovery, and cost and socioeconomic implications. This is followed by detailed chapters that discuss the specific evidence that underpins each recommendation. Many sections are relevant to physiotherapy, such as the organisation of services, the amount, timing, and intensity of rehabilitation, management of sensorimotor impairment, rehabilitation of physical activity, managing complications such as contracture, pain, cardiorespiratory fitness, UMI-77 and falls, and long term recovery. All references (990) are provided at the end of the document. Appendices include information on the National Stroke Audit,

and priorities for research. This is a comprehensive, multidisciplinary document that provides detailed, latest evidence for the management of individuals presenting with stroke or TIA. “
“The evidence-based practice (EBP) movement has gained ground steadily in physiotherapy over the past decade. Influential researchers and clinicians have argued that physiotherapists have a moral and professional obligation to move away from assessment and treatment methods based on anecdotal testimonies or opinion (Grimmer-Somers

2007). However, the growing volume isothipendyl of high-quality clinical research makes it difficult for clinicians to keep pace with the latest evidence. Simultaneously, the practice of physiotherapy has become increasingly complex due to changes in health care systems that entail higher demands on physiotherapists to provide effective and efficient management of patients amidst high patient turnover. Research on implementation of EBP in physiotherapy has established many barriers to developing a more evidence-based physiotherapy practice. Most frequently identified barriers include factors such as time restrictions, limited access to research, poor confidence in skills to identify and critically appraise research, and inadequate support from colleagues, managers and other health professionals (Jette et al 2003, Iles & Davidson 2006, Grimmer-Somers et al 2007). Limited research in some areas of physiotherapy also constitutes an obstacle to practising evidence-based physiotherapy (Fruth et al 2010). Some authors express the influences on EBP in physiotherapy as facilitators rather than barriers.

Disagreements on eligibility were first resolved by discussion and decided by a third reviewer (CL) if disagreement persisted. Design • Repeated measures between raters Participants • Symptomatic and

asymptomatic individuals Measurement procedure • Performed passive (ie, manual) physiological or accessory movements in any of the joints of the shoulder, elbow, or wrist-hand-fingers Outcomes • Estimates of inter-rater reliability Description: We extracted data on participants (number, age, clinical characteristics), raters (number, profession, training), measurements (joints and movement direction, position, movement performed, method, outcomes click here reported), and inter-rater reliability (point estimates, estimates of precision). Two reviewers (RJvdP and EvT) extracted data independently and were not blind to journal, authors, or results. When disagreement between reviewers could not be resolved by discussion, a third reviewer (CL) made the final decision. Quality: No validated instrument is available for assessing Hormones antagonist methodological quality of inter-rater reliability studies. Therefore, a list of criteria for quality was compiled derived from the QUADAS tool, the STARD Statement, and criteria used for assessing studies on reliability of measuring

passive spinal movements ( Bossuyt et al 2003a, Bossuyt et al 2003b, Van Trijffel et al 2005, Whiting et al 2003). Criteria were rated ‘yes’, ‘no’, or ‘unknown’ where insufficient information was provided ( Box 2). Criteria 1 many to 4 assess external validity, Criteria 5 to 9 assess internal validity, and Criterion 10 assesses statistical methods. External validity was considered sufficient if Criteria 1 to 4 were rated ‘yes’. With respect to internal validity, Criteria 5, 6, and 7 were assumed to be decisive in determining risk of bias. A study was considered to have a low risk of bias if Criteria 5, 6, and 7 were all rated ‘yes’, a moderate risk if two of these criteria were rated ‘yes’, and a high risk if none or only one of these criteria were rated ‘yes’. After training, two reviewers (RJvdP, EvT) independently assessed methodological quality

of all included studies and were not blind to journal, authors, and results. If discrepancy between reviewers persisted after discussion, a decisive judgement was passed by the third reviewer (CL). 1. Was a representative sample of participants used? Data were analysed by examining ICC and Kappa (95% CI). ICC > 0.75 indicated an acceptable level of reliability (Burdock et al 1963, cited by Kramer and Feinstein 1981). Corresponding Kappa levels were used as assigned by Landis and Koch (1977) where <0.00 = poor, 0.00–0.20 = slight, 0.21–0.40 = fair, 0.41–0.60 = moderate, 0.61–0.80 = substantial, and 0.81–1.00 = almost perfect reliability. In addition, reliability was analysed relating it to methodological quality and risk of bias.

Overall, when examining all trials together over all affective episodes, the mean change (weighted mean difference;

baseline to endpoint) in Hamilton Depression Rating Scale (HDRS) scores indicated a significant difference in favour of treatment. A number of small-scale clinical trials have examined the role of cortisol biosynthesis inhibitors such as ketoconazole, aminoglutethimide and metyrapone in the Inhibitors,research,lifescience,medical treatment of depression [Murphy, 1997; Murphy et al. 1998; O’Dwyer et al. 1995; Thakore and Dinan, 1995; Wolkowitz et al. 1993] and these are discussed in the section on ‘Metyrapone and treatment-resistant depression’ below. Another strategy which has been used to target the HPA axis for the treatment of depression is the use of GR antagonists [e.g. RU486 (mifepristone), Org34517]. The mechanism of action of these drugs has not been fully elucidated, but Inhibitors,research,lifescience,medical it

is speculated that they may act by enhancing MR function or by a rebound increase in GR function [Thomson and Craighead, 2008] suggesting the possibility that short-term treatment may exert persistent effects. Inhibitors,research,lifescience,medical Studies with RU486 suggest that it can reduce the psychiatric symptoms associated with Cushing’s disease [van der Lely et al. 1991]. There have also been a number of studies conducted using mifepristone in (non-Cushing’s) patients with pMDD. Positive findings in the initial open studies [Belanoff et al. 2001, 2002; Simpson et al. 2005] and randomized controlled trials [DeBattista et al. 2006; Flores et al. 2006] have been followed by a selleck larger negative trial [Blasey et al. 2011], which

used reduction in psychotic symptoms as the outcome measure. The authors argue that higher Inhibitors,research,lifescience,medical mifepristone doses may have led to a more robust response. An organon compound which acts as an antagonist at GRs has also been reported to have Inhibitors,research,lifescience,medical antidepressant properties in a poster and abstract but not in a full paper [Hoyberg et al. 2002]. CRH receptor antagonists have been developed and tested extensively in preclinical models below to investigate their anxiolytic and antidepressant properties [Jones et al. 1998; van Gaalen et al. 2002; Zorrilla et al. 2002]. Most of the clinical trials done in this area are small scale and a definite role for these drugs can only be established after large-scale trials. A CRH receptor antagonist, R121919, was able to significantly reduce depression and anxiety scores in a cohort of 20 patients in an open-label clinical trial [Zobel et al. 2000]. Further studies showed that R121919 was effective in improving sleep and showed a good tolerability profile in patients with depression [Held et al. 2004; Kunzel et al. 2003, 2005; Zobel et al. 2000]. The increased liver enzymes seen in some patients after treatment [Zobel et al. 2000] has led to the discontinuation of development of this product.

Normal AZD6244 concentration control monkey serum was used as a negative control. Standard curves were derived using serum from a macaque immunised with HIV-1W61D gp120 [28].

Antibody titres and concentrations of immunoglobulin were corrected for dilution factor derived from weight of sample/weight of sample + 600 assuming a density of 1 mg μl−1[19]. Neutralising antibody responses were measured against tier 1 and tier 2 HIV-1 envelope-pseudotyped viruses, prepared by transfection of 293T/17 cells, using a standardised luciferase-based assay in TZM.bl cells [29] and [30]. The 50% inhibitory concentration (IC50) titre was calculated as the dilution of serum that gave a 50% reduction in relative luminescence units (RLU) compared to the virus control wells after subtraction of cell control RLUs. Murine leukaemia virus (MuLV) negative controls were included in all assays. Dissected spleen tissue and lymph nodes or marrow washed from the bone were dissociated in RPMI by sieving through a 100 μm mesh and then centrifuged

at 4 °C for 10 min at 400 × g. Supernatant was removed and the pellet resuspended in residual media and washed once more with 10 ml RPMI. Cells were resuspended in 25 ml RPMI and were then filtered through a 50 μm filcon (BD Biosciences, Oxford, UK) before being layered onto Histopaque-1077 (Sigma, UK) and centrifuged at room temperature for 30 min at 1500 × g. Interface cells were collected and viable mononuclear cells counted. Ex vivo amplified Gefitinib datasheet Histone demethylase ELISpot assays were based on the method described by Bergmeier et al. [31]. PVDF membrane plates (Muliscreen HTSIP, Millipore) were treated with 35% ethanol for 1 min, washed three times with sterile PBS and coated with either recombinant CN54 gp140 or KLH (Calbiochem) at 10 μg ml−1 overnight at 4 °C. Following a further 6 washes with PBS-T, reactive sites were Modulators blocked by incubation with RPMI 1640 medium containing 10% FCS and pen/strep for 1 h at room temperature. Freshly recovered tissue MNCs were added to triplicate wells at 1 × 105

and 5 × 105 cells/well and incubated for 24 h at 37 °C in an atmosphere of 5% CO2. After further washing in PBS-T, bound secreted antibody was detected with either goat anti-monkey IgG-HRP (Serotec) diluted 1/2000 or with goat anti-monkey IgA-biotin (Acris) at 1/1000 followed by avidin–HRP (Sigma) diluted 1/2000. Spots were detected by addition of TMB substrate (Sureblue TMB 1-component peroxidise substrate, KPL) and enumerated with a reader. Total IgG and IgA ASC were assayed by the same method using plates coated with goat anti-monkey IgG (γ-chain-specific) (KPL) or goat anti-monkey IgA (α-chain-specific) (KPL) as capture antibodies. Specified analyses were performed using SigmaPlot version 11 software.