We would like to acknowledge the investigators, nurses, field wor

We would like to acknowledge the investigators, nurses, field workers and other personnel who contributed to the conduct of this trial; Mary Rusizoka, Beatrice Kamala, Wilbroad Shangwe, Francesca Lemme, Serafina Soteli, Clemens Masesa, and the HPV-021 trial team in Mwanza; Pius Magulyati, and the laboratory staff of the National Institute for Medical Research (NIMR) Mwanza Research Centre laboratory; the administrative staff of the Mwanza Intervention Trials Unit (MITU), NIMR Mwanza Research Centre, and Sekou Toure Hospital; Lucy Bradshaw, Gillian Devereux, Jayne Gould and Sue Napierala Mavedzenge and the research support staff at the London School of Hygiene and Tropical Medicine

(LSHTM). We thank Peter Hughes and the Clinical Diagnostic Laboratory of the MRC/UVRI Uganda Research Unit in Entebbe, selleck compound and David Warhurst and the Department of Pathogen Molecular Biology at LSHTM for their contributions to this work. We are grateful to the Ministry of Health and Social Welfare for granting permission to conduct this study. Conflict of interest statement Dr. Watson-Jones and Dr. Mayaud have received grant support through their institutions from GlaxoSmithKline Biologicals SA. During the trial, partial salary support for Drs. Watson-Jones,

Andreasen, Brown and Kavishe came from GSK Biologicals. There are no other conflicts of interest. Dr. Brown is supported by NIH-NIHM 1K01MH100994-01 and NIH-NCATS 8KL2TR000143-08. Richard Hayes, Saidi Kapiga, find more and Kathy Baisley receive support from the MRC and DFID (G0901756, MR/K012126/1). “
“Human papillomavirus (HPV) vaccines induce type-specific neutralizing antibodies which correlate with immunity to the corresponding HPV types [1], and World Health Organization guidelines recommend that assays which assess neutralization be used as the reference standard for measuring HPV vaccine responses [2]. Quadrivalent HPV (Q-HPV) Oxalosuccinic acid vaccine (Gardasil®, Merck Laboratories) consists of HPV 6, 11, 16 and 18 virus-like particles (VLP) and is licensed for a 3-dose

regimen. Post-Gardasil® antibody responses are typically measured by a proprietary multiplex competitive Luminex immunoassay (cLIA) [3], which is based on competitive binding of type-specific HPV antibodies in human sera with labelled monoclonal antibodies directed against neutralizing epitopes of the respective VLP types (HPV 6, 11, 16 and 18). It has been reported that HPV antibodies measured by the cLIA may decline to become undetectable over time, especially for HPV 18, despite continued vaccine efficacy in preventing infections [4] and [5]. The significance of the loss of detectable antibodies is unknown as protective levels of HPV antibodies remain undefined [1], [6] and [7] and vaccine efficacy remains near 100%. Recently, Merck Laboratories developed a total IgG Luminex immunoassay (TIgG) which measures antibodies against the entire VLP, i.e.

We observed a RIR (95% CI) of 1 09 (1 03, 1 15) for females versu

We observed a RIR (95% CI) of 1.09 (1.03, 1.15) for females versus males, which is similar to the result of our non-restricted analysis (Table 3). We then further restricted the event definition to include check details only specific types of adverse events

that would be expected following MMR vaccine. The four event types included, based on ICD-10 codes, were: fever, rash, febrile convulsions and viral enanthema [13] and [10]. The results of this restricted analysis showed a much larger RIR for females versus males of 1.23 (95% CI 0.99, 1.51) p = 0.06, which did not achieve nominal statistical significance due to the loss of events with the restricted event definition ( Table 4). Higher relative incidences in girls compared to see more boys were exhibited for each of the four event types, though none achieved nominal

statistical significance. We demonstrated that females had an increased risk of ER visits and/or hospitalizations during a specified ‘at risk’ period, immediately following the 12-month vaccination but not 2-, 4- and 6-month vaccinations. The increased risk associated with female sex translates to 192 excess events in females as compared to males, for every 100,000 infants vaccinated. As previously noted, the vaccine routinely administered at 12 months of age in Ontario during the entire period of study was MMR. A meningococcal disease (type C) vaccine was added to Ontario’s publicly-funded immunization schedule in September 2004. The time period

for increase in ER visits or hospitalizations following 12-month vaccination is consistent with the all known risk period following MMR vaccination [11], [13] and [18]. Our observations could either be explained by gender differences – the socially constructed distinction between the sexes, or by sex differences – the physiological differences between males and females. If gender differences accounted for our observation, one explanation would be that parents respond differently to similar adverse reactions between boys and girls, and are more likely to seek medical care for girls. Our analysis cannot find evidence to support or refute this hypothesis, although we may have expected lower acuity of presentation in girls if this were the case. In contrast, it is recognized in the medical literature that important physiological differences exist between males and females that govern their responses to infections and vaccines [19], [20], [21] and [22]. For example, estrogen can potentiate antibody responses to antigens, while both progesterone and androgens tend to have immunoregulatory or immunosuppressive actions [20], [22] and [23]. Sex differences in immune responses to measles vaccines have certainly been observed both in terms of immunogenicity [21] and [24] and short-term reactogenicity of both the live-attenuated rubella [1] and both high- and standard-titer measles vaccines [4], [25] and [26].

“Placenta percreta (PP) is a condition in which the placen

“Placenta percreta (PP) is a condition in which the placenta abnormally penetrates entirely through the myometrium and into the uterine serosa. This might be complicated by attachment CP-868596 nmr of the placenta to surrounding structures or organs, such as the urinary bladder or rectum. PP is a potentially fatal condition,

and mortality rate is correlated to the extent of involvement of surrounding structures. When PP is complicated by bladder invasion, mortality rates have been estimated as high as 9.5% and 24% for mother and child, respectively.1 Knowledge of this condition and expectant management are especially important, as the incidence is on the rise—an estimated 50-fold increase in the last 50 years—attributed to the increased frequency of Caesarean deliveries.2 A 38-year-old woman (G6P3023) at 24 weeks gestation presented with vaginal bleeding. She reported that 1 week before she awoke in a “puddle of fluid.” She denied gross hematuria. She had a history of 3 Caesarean sections.

Fetal ultrasound showed complete placenta previa with placental vessels invading the bladder confirming PP (Fig 1). She was admitted for expectant management. Maternal fetal medicine, anesthesia, neonatal intensive care, and urology were all consulted. Magnesium sulfate, antibiotics, and steroids were administered prophylactically. On hospital day #2, the patient had an increased oxygen requirement and tachycardia. A computed tomographic scan www.selleckchem.com/products/KU-55933.html of the chest revealed extensive bilateral pulmonary emboli. She underwent inferior vena cava filter placement, was transferred to the surgical intensive care unit, and continuous heparin infusion was initiated. On hospital day #6, the patient went into labor and was taken to the operating room for a multidisciplinary procedure. She underwent exploratory laparotomy and repeat Caesarean section through a fundal uterine incision by the obstetrics team. A viable female neonate was delivered with Apgar scores of 9 and 9. A total abdominal hysterectomy and lysis

of adhesions were then performed by the gynecologic oncology service. The anterior uterine wall was then recognized to be affixed to the bladder. Dissection of the anterior uterine wall from the posterior bladder was accompanied by large posterior cystotomy. On routine inspection, decreased efflux was noted from the mafosfamide right ureteral orifice, and the right ureter was markedly dilated. At this point, intraoperative urology consultation was requested. The right ureter was secured, and a suture was identified that appeared to be constricting it. This was released with immediate return of urine from the ureteral orifice. A double-J ureteral stent was placed, and cystorrhaphy was performed. No leak was identified on bladder irrigation, and an omental flap was placed between the bladder and the vaginal cuff. A Jackson-Pratt drain and a Foley catheter were placed.

Clearly, diagnosis tools that allow more rapid identification of

Clearly, diagnosis tools that allow more rapid identification of MTB

and characterization of drug susceptibility patterns will greatly benefit the management buy MK-2206 of TB. Due to the long generation time of MTB, traditional method using solid media for Mycobacterium identification required 6–7 weeks for growth, species identification, and susceptibility testing. In the last decades use of DNA hybridization technologies and liquid radiometric culture systems, such as BACTEC 460 TB (Becton Dickinson diagnostic Instrument Systems, Sparks, Md) has significantly reduced time of identification of Mycobacterium and determination of the drug susceptibility patterns. 6, 7 and 8 The direct detection of MTB in clinical samples has further been accredited for use only with acid test bacillus smears positive sputum. In the method of Mycobacteriophage-based assay that could be detect several Mycobacterial species, including MTB and characterize drug susceptibility patterns within 24–48 h of obtained positive culture. This novel approach utilizes genetically engineered reporter phage to defect viable Mycobacteria, which upon LRP infection produces quantifiable luminescence. In the presence of drug resistant of bacilli, retain their viability undergo phage infection Dactolisib order and also produce luminescence. In this way, quantification of photons with a luminometer could be used to reveal susceptibility

profile of each isolates. In this study revealed that host range of phAE 129 demonstrating its ability to identify primary clinical isolated of M. tuberculosis and to develop new modified method using chitin for homogenizing and decontaminating sputum sample ideal for using on LRP assay. 9 and 10 The chitin is a mild decontaminating agent and it was dissolved to concentrate sulfuric acid and further diluted to 5% H2S04. The hydrolysis of chitin by acid produces Calpain acetic acid and chitosamine

which as mucolytic action against sputum process. 11 In the present study revealed that modified chitin H2S04 method of sputum processed LRP assay allows rapid and reliable recognition of organism in M. tuberculosis complex with high degrees of specificity and sensitivity. This diagnostic technology is a step closer to clinical readiness. The suspected 292 sputum samples were collected from identified pulmonary tuberculosis patients at various district level of Tamil Nadu, India. The samples were analyzed by standard procedure. These samples were collected individual container (Metconey bottles) recommended by standard laboratory procedure. The most commonly recommended containers are a sterile wide mouth jar with tightly fitted screw cap lid. The diagnostic specimens were collected before the initiation therapy. All specimens were transported to the laboratory and ideally processed at the earliest of the collection. Note: delay in process leads to falls negative culture and increased bacterial contamination.

Self-reported incidences of clinically diagnosed genital warts co

Self-reported incidences of clinically diagnosed genital warts confirm that these are common in both women and men. Ever having had clinically diagnosed genital warts was reported by 10.6% of almost 70,000 Nordic women aged 18 to 45 years in 2005 and by 7.9% of almost 23,000 Danish check details men in the same age category in 2007 [9] and [10]. In 2000, in the UK, 4.1% of women and 3.6% of men aged 16–44 years reported ever being diagnosed with genital warts [11].

In the United States (1999–2004, age category 18–59) and Australia (2001–2002, age category 16–59), the cumulative incidence was 7.2% and 4.4% among women, respectively, and 4.0% among men [12] and [13]. Human papillomaviruses are small non-enveloped DNA selleck kinase inhibitor viruses that belong to the Papovaviridae family. The viral capsid is composed of two proteins: the major L1 and minor L2 proteins. There are 170 different HPV types identified, 40 of which infect the genital tract [14]. These mucosal HPV types

are classified as low-risk (LR) and high-risk (HR) types based on the prevalence ratio in cervical cancer and its precursors. LR-HPV types, such as 6 and 11, induce benign lesions with minimal risk of progression to malignancy, HR-HPV have higher oncogenic potential. Approximately 99% of cervical cancers contain HPV DNA of HR-HPV types, with type HPV16 being the most prevalent, followed by types 18, 31, 33, and 45 [15]. Most HPV infections are transient and are spontaneously cleared or suppressed by the host immune response. It is unclear whether these infections resolve by complete viral clearance or by maintenance of a latent phase in the basal cells of the epithelium, in which the virus replicates at extreme low levels without full viral expression [16]. Infections that are not cleared at an early Casein kinase 1 stage progress towards premalignant squamous intraepithelial lesions (SIL), histopathologically referred to as cervical intraepithelial neoplasia (CIN). Low-grade lesions, LSIL (cytological classification) or CIN1 (histological classification), represent a chronic HPV infection in which HPV DNA is episomal and intact virion production

and shedding occurs (both by high-risk HPV as well as low-risk HPV, e.g. HPV11). Lesions are frequently cleared by the immune system, however, some lesions do not spontaneously regress and can persist for a long period. Viral persistence within the host cells is an uncommon event that is necessary for progression to malignancy. Clonal progression of the persistently infected epithelium can lead to high-grade lesions (HSIL or CIN2-3), which in turn can progress towards invasive disease [16]. The progression towards high-grade disease (HSIL/CIN3) is often with a different strain of HPV and not necessarily a progression of low-grade disease. HIV infected women have a higher prevalence of HPV infection and are often infected with multiple HPV types.

Just a few viruses fell into subgroup 3B and group 6 (Fig 4, Fig

Just a few viruses fell into subgroup 3B and group 6 (Fig. 4, Fig. S4). Some isolates from North America, Europe and Asia belonged to groups 5 and 6, which have signature AA substitutions D53N, Y94H, I230V and E280A in HA1, with group 6 isolates carrying an additional AA substitution S199A. Viruses with low HI titres to post-infection ferret antisera raised against cell-propagated A/Victoria/361/2011 viruses were scattered throughout the HA tree and did not form monophyletic groups or share common AA substitutions. Genetic analysis OSI 906 of the HA sequences of several egg-propagated A/Victoria/361/2011-like

viruses were compared in order to see if low HI titres might be associated with amino acid substitutions linked to adaptation to growth in eggs. A number of such substitutions were noted in the HA of the initial egg-propagated A/Victoria/361/2011 wild-type virus, including a H156R substitution. A

subsequent R156Q change was acquired in the high growth reassortants IVR-165 and X221, although H156R was retained in the reassortant NIB-79. Changes in amino acid sequence in this area of the HA of A(H1N1)pdm09 viruses have been shown to alter their antigenic properties and, based on the ferret and human serology obtained for the egg-propagated A/Victoria/361/2011 virus, such substitutions may also have altered the antigenicity of this virus. Some egg-propagated viruses genetically similar to A/Victoria/361/2011, Ulixertinib such as A/Texas/50/2012, did not have similar adaptive substitutions in the 153–157 HA region. Egg-propagated A/Hawaii/22/2012 and the high growth reassortants made from this virus, X225 and X225A, had the substitution L157S and in HI assays antisera raised against A/Hawaii/22/2012 recognised the majority of test viruses with titres reduced more

than 4-fold compared to the homologous virus (Table 3). Vaccines containing influenza A/Victoria/361/2011 (H3N2)-like antigen stimulated anti-HA antibodies in all age groups that had reduced geometric mean HI titres to the majority of cell-propagated A(H3N2) viruses compared to the egg-propagated vaccine virus or other egg-propagated recent viruses (Fig. S5). The average reductions in HI GMT against cell-propagated A(H3N2) viruses compared to the egg-propagated vaccine virus were 66% Sodium butyrate for adults, 68% for the elderly and 64% for children. Based on surveillance data available in February 2013, it was concluded that the A(H3N2) component of the 2012–2013 Northern Hemisphere influenza vaccine should remain as a A/Victoria/361/2011-like virus. However it was stipulated that the like virus should be antigenically like the cell-propagated prototype virus. For this reason a new vaccine virus A/Texas/50/2012 and its reassortants, X-223 and X223A, were recommended for use in vaccines that are based on egg propagation. From September 2012 to February 2013, 832 influenza B viruses were analysed by WHO CCs.

24 Suitability of the methods towards the estimation of bulk drug

24 Suitability of the methods towards the estimation of bulk drug checked and found the mean recovery of 98.88 ± 0.45% this high percentage recovery proved that the method can adoptable for the estimation of TL in bulk. For the application of the proposed method to formulation the procured tablets were subjected to the analysis for their contents of TL by the proposed method and reported UV spectrophotometric method reported by Nanda et al.7 From test conducted about 99.91 and 99.67% assay was resulted with the proposed and existed method (Table 2). The results obtained are given in Table 3. The percentage relative standard deviation (% RSD) for inter, intra-day precision

was about 0.898 and 0.945 respectively which was very low and within the acceptance limits for precision experiments, PF-06463922 purchase evidencing repeatability

(precision) of the method. The resulted recovery at three levels was with the % RSD of 0.94–0.98% for TL (Table 3). The above % RSD were found within the acceptance limit for accuracy of <2% RSD this good accuracy of the purposed method. The effect of the MO was studied by measuring the absorbance of solutions containing TL (10 μg mL−1), and 0.5 mL of MO solution at various Selleckchem Doxorubicin concentration (0.025–0.15% wt/v). The results are portrayed in Fig. 5. As MO concentration of 0.05% wt/v gave a maximum absorbance. Results of quantity of MO to be added is given in Fig. 6. From the results it was established that Fossariinae 0.05 mL of 0.05% wt/v MO is sufficient to make complex with maximum absorbance. Volumes of above 0.05 mL reagent had no marked effect on the chromogen formation. The studied excipients

do not cause any interference in the estimation of the drug (Table 4). Likewise the placebo mixture of above excipients was prepared without the drug and studied at the wavelength of estimation for determining any absorbance for the chloroform extractable material in the placebo. Yellow color was not developed in the extract revealed the selectivity of the present method. Likewise the results of stability form the shown from Fig. 7 evidenced that the chromogen was stable more than 3.5 h. The results obtained were within the suggested limits for % RSD (<2%) (Table 5). Ruggedness was established by determining TL in the tablet formulation using two different spectrophotometer Shimadzu UV mini-1240 (system I) and SCINCO, Neosys-2000 DRS-UV provided with liquid sample analysis port (system II) and two different analysts (I and II). The results obtained were within the recommended % RSD limit (<2%) (Table 5). The proposed ion-pair extractive colorimetric estimation of tolterodine tartrate (TL) in bulk and in formulation is more sensitive, specific (selective), rapid and cost effective. The highest % recovery of the method proved that the present method was more accurate and comparable with that of reference method.

Final docking results were highlighted in the 3D models and minim

Final docking results were highlighted in the 3D models and minimum binding energies were calculated as per formula stated above. The three dimensional structure of B. megaterium tyrosinase with 4D87 was retrieved in .PDB format as in Fig. 1: In total 5 drugs were designed using the Chem Draw ultra 6.0 and further by using Chem3D, they were estimated for the structure minimum energy. The every drug details in IUPAC name and minimum energy in kcal/mol was shown in Fig. 2(A–E). In order to find out the potent binding energy among the drug and protein target, AutoDock 4.2 was set up to calculate the QSAR activity.

All five drugs have shown the minimum binding energy in the range of −6.00 kcal/mol. The details of each docking in the form of binding energy and docking location were highlighted in Fig. 3(A–E). Taken into consideration Proteasome activity VRT752271 supplier that in silico drug design and QSAR have been implicated extensively in recent time that ascertains probable success for the activity of bioactive agents. We have performed a QSAR analysis to determine tyrosinase inhibitor compounds those could regulate protein activity. The enzyme tyrosinase (EC is widely spread among species of different genera.1, 2, 3, 4, 5, 6 and 7 And also linked with melanogenesis disorders and hyper pigmentation therefore

tyrosinase is selected for the discovery of new tyrosinase inhibitors as it could be useful in therapy for pigmentation in Human. Unfortunately, three dimensional structure of human tyrosinase has not been elucidated yet.10 Hence we tried to dock the for five drugs designed for the tyrosinase of B. megaterium which was used

as a model protein in place of human tyrosinase. The QSAR data revealed that the all the drugs could bind with the target molecule with minimum binding energy in the range of −06.00 kcal/mol. It is also note worthy that the all five drugs bound to the same pocket of the target which suggest that the drugs are selecting particular pocket only for their binding as they have same drug backbone having the variable side groups. In this way, set of compounds was subjected to in silico screening and was detected for antityrosinase activity. Hence, via QSAR study the designed drugs could be tested in in vivo/cell line trials to determine their potential in therapy. All authors have none to declare. “
“Diuretics drugs increase the rate of urine flow and adjust the volume and composition of body fluids. Drug-induced diuresis is beneficial for the treatment of many maladies such as congestive heart failure (CHF), chronic renal failure, nephritis, cirrhosis, hypertension and pregnancy-induced toxemia.1 and 2 However, many of the diuretics currently used in clinical practice have been associated with a number of adverse effects, including electrolyte imbalance, metabolic alterations, the onset of diabetes, activation of the renin-angiotensin and neuroendocrine systems, and impairment of sexual function.

4 1) from Miltenyi Biotec CD3−

4.1) from Miltenyi Biotec. CD3− selleck products IAb+ CD11c+ PDCA-1+ cells were then sorted in a BD FACSAria III cell sorter. CD8+ cells were obtained from C57BL/6 mice (n = 2) s.c. infected with 104T. cruzi parasites.

Spleens were removed 15 days after infection. Following red blood cell lysis, a single cell suspension was stained with CD8 PE (53-6.7) from BD and positive cells were subjected to sorting in a BD FACSAria III cell sorter. As determined by FACS analysis, the purity of the CD8+ was 98%. Ex vivo ELISPOT (IFN-γ) or in vivo cytotoxicity assays were performed exactly as described previously [13] and [25]. Briefly, the in vivo cytotoxicity assays, C57BL/6 splenocytes were divided into two populations and labeled with the fluorogenic dye carboxyfluorescein diacetate succinimidyl diester (CFSE Molecular Probes, Eugene, Oregon, USA) at a final concentration of 10 μM (CFSEhigh) or 1 μM (CFSElow). CFSEhigh cells were pulsed for 40 min at 37 °C with 1 μM of the H-2 Kb ASP-2 peptide (VNHRFTLV) or TsKb-20. CFSElow cells remained unpulsed. Subsequently, CFSEhigh cells were washed and mixed with equal numbers of CFSElow cells before injecting intravenously (i.v.) 30 × 106

total cells per mouse. Recipient animals were mice that had been infected or not with T. AP24534 molecular weight cruzi. Spleen cells or lymph node cells of recipient mice were collected 20 h after transfer, fixed with 3.7% paraformaldehyde and analyzed by FACS as described above. The percentage of specific lysis was determined using the formula: 1−%CFSEhigh   infected/%CFSElow   infected%CFSEhigh   naive/%CFSElow   naive×100% The surface mobilization of CD107a and the intracellular expression of cytokines (IFN-γ

and TNF-α) were evaluated after in vitro culture of isothipendyl splenocytes in the presence or absence of an antigenic stimulus. Cells were washed 3 times in plain RPMI and re-suspended in cell culture medium containing RPMI 1640 medium (pH 7.4), supplemented with 10 mM Hepes, 0.2% sodium bicarbonate, 59 mg/L penicillin, 133 mg/L streptomycin, and 10% Hyclone fetal bovine sera (Hyclone, Logan, Utah). The viability of cells was evaluated using 0.2% Trypan Blue exclusion dye to discriminate between live and dead cells. The cell concentration was adjusted to 5 × 106 cells/mL in a cell culture medium containing anti-CD28 (2 μg/mL, BD Pharmingen), brefeldin A (10 μg/mL, BD Pharmingen), monensin (5 μg/mL, Sigma, St. Louis, MO), and FITC-labeled anti-CD107a (Clone 1D4B, 2 μg/mL, BD Pharmingen). In half of the cultures, VNHRFTLV peptide was added at a final concentration of 10 μM. Cells were cultivated in V-bottom 96-well plates (Corning) in a final volume of 200 μL in duplicates, at 37 °C in a humid environment containing 5% CO2.

Regulatory authorities have recognized the importance of stimulat

Regulatory authorities have recognized the importance of stimulating T cell responses to influenza

and have encouraged the exploration of T cell assays for evaluating vaccine efficacy in general [27] and [28] and, in particular, influenza vaccines including those aimed to protect against avian influenza [29] and [30]. However, standardized and reproducible assays of influenza-specific T cell responses that are too needed to make significant progress in the development of improved influenza vaccines have yet to be validated [29]. Herein, we report the validation of standardized assays of T cell responses that are likely to correlate with protection against influenza [13], [14] and [31]. The assays are based on the detection of effector molecules produced by peripheral see more blood mononuclear cells (PBMC) after ex vivo stimulation with live influenza virus. By using multiplex technology, multiple cytokines including IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, GM-CSF, IFN-γ, and TNF-α, could efficiently be detected in one sample of PBMC culture supernatant. In addition, a detection assay for granzyme B activity, an essential Selleckchem RG7420 effector molecule in the cytotoxic response of CD8+ T cells against virus-infected target cells [32], was validated in lysates of these virus-stimulated PBMC. The validation process was preceded by rigid standardization

of the assays and on-site training of the laboratory technicians following standard operating procedures (SOP) [33]. This work comprised determination of specificity, accuracy,

linearity, range, detection limit, intermediate precision, and robustness by three European and one Canadian laboratory. The validation results showed that these assays of the T cell response to influenza were reproducible and could measure the levels of granzyme B and cytokines in an accurate and specific manner. Human PBMC were isolated from buffy coats of healthy individuals by Lymphoprep (Axis Shield, Oslo, Norway) density centrifugation at 950 × g for 20 min. The PBMC were washed several times with PBS until the supernatant was clear. Subsequently, the PBMC were frozen in multiple aliquots in 90% FCS (Hyclone, Logan, Utah)/10% DMSO (Sigma–Aldrich, St. Louis, USA) and stored at −135 °C. Buffy coats were retrieved in accordance with the human experimental guidelines of Sanquin Blood Bank North West Region (project number S03.0015-X). Influenza Phosphoprotein phosphatase H3N2 A/Wisconsin/67/2005 was produced by infecting MDCK cells. As negative control (mock) medium of uninfected MDCK cells was used. The participating laboratories in alphabetical order, not in order in results, were: 1. National Centre for Epidemiology (NCE), Budapest, Hungary Frozen PBMC were thawed in AIM V medium and rested by incubation for 4 h at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Pilot experiments showed that this resting period is essential to obtain responses similar to responses with fresh cells (data not shown). Subsequently, PBMC (1.