Therefore, it is unsurprising that evidence of microvascular dysfunction often predates evidence of clinically recognized target organ damage. The retinal microcirculation is a site where this important predictive role is recognized and, at least in those with diabetes, clinically exploited. The epidemiology of retinopathy is reviewed in detail within this edition [52] and therefore will not be covered in great detail here, except its key position

in establishing the importance of microcirculation as an early predictor of disease. The retina is a unique site where the microcirculation can be imaged directly, Selleck Pifithrin�� providing an opportunity to study in vivo the structure and pathology of the human circulation, and the possibility of detecting changes in microvasculature relating to the development of cardiovascular

disease. Diabetic retinopathy is the biggest cause of premature blindness in Western society as well as being a strong risk marker for cardiovascular mortality [33,61], hence the establishment of the annual screening program for individuals with diabetes [3,30]. The presence of retinopathy, however, may predate the occurrence of type 2 diabetes, suggesting that the diabetic phenotype may have a microvascular etiology [70]. This is consistent with reports Selleck R788 that skin microcirculatory abnormalities also predate new future diabetes [28]. In the nondiabetic population, retinopathy also carries an important prognostic role. The microvasculature of the eye is often regarded as an extension of the cerebral circulation. Therefore, its predictive role of future stroke is unsurprising, although the almost fivefold increased risk is greater than many commentators would expect. The Atherosclerosis Risk In Communities study looked prospectively at a population-based cohort for risk factors associated with future cardiovascular events [69]. The two

measures of microvascular damage assessed, retinopathy and cerebral 3-oxoacyl-(acyl-carrier-protein) reductase white matter lesions detected on MRI, were commonly associated with each other. Volunteers with evidence of retinopathy had a 4.9-fold (95% CI: 2.0–11.9) increased risk of future strokes after adjustment for age, gender, ethnicity, and vascular risk factors. Cerebral white matter lesions carried an adjusted hazard ratio of 3.4 (1.5–7.7); however, if both were present, the adjusted hazard ratio for future strokes was 18.9 (5.9–55.4), suggesting a compound effect of microvascular damage on the cerebral circulation. A similar predictive role of retinopathy in the risk of future congestive heart failure has been described [71]. Over seven years, retinopathy is associated with a twofold increased risk of congestive heart failure (HR: 1.96; 95% CI: 1.

We found that the surface protein A (SasA) of S. aureus could protect mice from lethal challenge of the bacteria. Staphylococcus aureus, a conditional pathogenic Gram-positive bacterium, is the leading cause of bloodstream, lower respiratory tract and skin/soft-tissue infections, accounting for 20–25% of all nosocomial infections (1,2,3). Bacteremia is the most prevalent type of S. aureus infections in hospitalized patients, followed by lower respiratory tract infections and skin/soft tissue infections (4,5). S. aureus is able

to adapt to new antibiotics and acquire antibiotic resistance (6). The extensive use of antibiotics has resulted in increased resistance among S. aureus clinical isolates. In patients with large area burn, it was found that more than 90% of S. aureus isolates were resistant to 11 types of antibiotics, including ampicillin, cefazolin, ciprofloxacin, gentamicin, levofloxacin, clidamycin, erythromycin, oxacillin, penicillin(16). MK2206 Due to multi-drug resistance and the ability to selleck kinase inhibitor acquire resistance to new antibiotics quickly, it is more and more difficult to treat S. aureus infection, especially with the emergence of vancomycin resistant S. aureus strains (7,8). As a result, many investigators resort to immunological approaches to contain S. aureus infection (9). Many components of S. aureus, such as capsular polysaccharide (9), poly-N-acetylglucosamine

(10), clumping factor A (11), clumping factor B (12), iron-regulated surface determinant (IsdB) (13) and fibronectin-binding protein (FnBP) (14), can generate immune responses that afford partial protection against S. aureus challenge in experiment animals. It is difficult to develop S. aureus vaccines because there are many pathogenic determinants in S. aureus and different clinical isolates may have different pathogenic determinants. Ideal vaccine candidates for S. aureus should be expressed broadly in different S. aureus

clinical isolates and be consistent among different strains. Vaccines consisting of several components may induce better protective immunity against infective 4��8C S. aureus (15). In this study, to screen good vaccine candidates against S. aureus, a panel of pathogenic proteins of S. aureus was expressed and dot blotted with sera from mice infected with S. aureus USA300, 546 and 1884, respectively. The proteins that interact with the sera were selected to immunize BALB/c mice. The immunized mice were then challenged with S. aureus USA300. A protein named SasA was found to be able to induce protective immunity against lethal challenge of S. aureus USA300. Staphylococci were cultured on tryptic soy agar or in broth at 37 °C. S. aureus USA300 were obtained from ATCC. This strain does not produce toxic shock syndrome toxin. The lethal dosage of S. aureus USA300 or S. aureus 546 was determined before as in respectively. S. aureus 546 and S. aureus 1884 were obtained from China Veterinary Culture Collection Center (CVCC). E.

To reduce background phosphorylation, NK92

were incubated overnight in fresh media lacking IL-2 prior to incubation with fixed K562 targets. Western blotting.  Cell lysates transferred to PVDF membranes were evaluated by western blot. Primary antibody was diluted in 3% BSA/TBST and incubated with membranes overnight at 4 °C with shaking. After Ivacaftor ic50 washing, membranes were probed with appropriate HRP-linked secondary antibody for 1 h in 3% BSA/TBST and then developed with Millipore Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica MA, USA) and imaged using a UVP Bioimaging Systems EpiChemi3 Darkroom operating LabWorks Ver 4.6 (UVP Inc., Upland, CA, USA). Antibodies used from Cell Signaling Technology (Danvers, MA, USA) were rabbit anti-phospho-p38 MAP kinase, rabbit anti-total-ERK and HRP-linked anti-rabbit IgG secondary antibody.

Santa Cruz Biotechnology mouse anti-phospho-ERK and HRP-linked goat anti-mouse IgG secondary antibody (KPL, Gaithersburg, MD, USA) were also employed. Idasanutlin price For re-probing membranes were stripped for 10–20 min using glycine stripping buffer (200 mm Glycine, 0.1% SDS, 1% Tween-20, pH2.2) and re-subjected to the same western protocol using a different primary antibody. Antibodies specific for phosphorylated protein were always used prior to stripping as stripping may de-phosphorylate proteins. Mouse anti-GAPDH (Abcam, Cambridge, MA, USA) was used to ensure an equal amount of protein was loaded in each lane. Chromium release killing assay.  Target cells were labelled with chromium-51 by incubating one million cells with 2 MBq of Na251CrO4 (NEN Research Products, Boston, MA, USA) for 90 min in standard tissue culture conditions. Labelled target cells were incubated with an equal volume of effector cells under various conditions on a 96-well plate. After incubation for 4 h in standard tissue culture conditions, the cells were pelleted

at 250 G for 5 min. 100 μl of supernatant was collected and radioactivity measured. Percentage of specific lysis was calculated by the following equation: (a−b/c−b) × 100, where a is the radioactivity of the supernatant of target cells mixed with effector cells, b is that in the supernatant of target cells incubated alone, and c is that in the supernatant after lysis Montelukast Sodium of target cells with 1% Nonidet P-40. Statistical analysis.  Statistical analysis was conducted using One-way anova with Tukey’s post-hoc test using graphpad prism statistical software. A p-value of 0.05 or less was considered significant, 95% confidence interval. RT-PCR analysis on the cDNA of NK92 cells using LLT1 FP 5′- GAA TTG CCT GCA AAC CCA GGT TGT CTG –3′ and LLT1 RP 5′- TTG GAA CAA ATC CAC TTC CTC TCT GTG – 3′ revealed an approximately 430 bp that corresponded to the correct size of LLT1 (Fig. 1A). Flow cytometric analysis of NK92 cells using the anti-human OCIL/LLT1 monoclonal antibody (Fig. 1C) and 4C7 anti-LLT1 monoclonal antibody (Fig. 1D) indicates that LLT1 is expressed on the surface of these cells.

Clotting in the dialysis circuit is triggered by both the extrinsic and the intrinsic pathways at the same time but to different degrees depending on the composition of the dialysis membrane and design and composition of the lines. Once the blood flow is initiated, plasma proteins deposit on the dialyser surface, and factor XII and high-molecular-weight kallikrein accumulate and act as initiating factors for contact coagulation

Depsipeptide molecular weight – the Intrinsic Pathway. Peripheral blood leucocytes and monocytes, which contact the dialyser membrane, become adherent or activated and release blebs of surface membrane rich in tissue factor – activating the Extrinsic pathway. Platelets become activated by contact and in response to turbulent flow and high shear stress. The surface of platelets provides an enhancing environment promoting the interaction of coagulation cascade components. These triggers activate the clotting cascade, platelet aggregation, activation and degranulation, cytokine release and activation of circulating white cells, all of which can contribute in differing degrees to the triggering of or progressive activation buy LEE011 of the clotting cascade leading to thrombosis in the dialysis circuit. Anticoagulation is routinely required to prevent clotting of the dialysis lines

and dialyser membranes, in both PI3K inhibitor acute intermittent haemodialysis and

continuous renal replacement therapies.5 As the field of anticoagulation is constantly evolving it is important to regularly review advances in knowledge and changing practices in this area.6 The responsibility for prescribing and delivering anticoagulant for haemodialysis is shared between the dialysis doctors and nurses. Dialysis is a medical therapy, which must be prescribed by an appropriately trained doctor. The prescribing doctor usually determines which anticoagulant agent will be used and the dosage range. The doctor’s prescription may include broad instructions such as ‘no heparin’, ‘low heparin’ or ‘normal heparin’. In a mature dialysis unit the dose and delivery of anticoagulant is, however, the responsibility of professional and experienced dialysis nurses, who have latitude within parameters determined by detailed written policies or standing orders. Dosing regimens, while generally safe and effective, are somewhat unscientific. In terms of monitoring, most units do not practise routine monitoring, although the anticoagulant effect of unfractionated heparin (UF heparin) can be monitored with some accuracy by the APTT or the activated clotting time tests where indicated. The dialysis nurses know there is too much anticoagulation if the needle sites continue to ooze excessively for a prolonged period (e.g. more than 15 min) after dialysis.

An effective way of visualizing the receptor-binding motif is by using sequence logos. Sequence logos were introduced by Schneider and Stephens14 to graphically represent the sequence motif contained in a set of aligned sequences, where at each position, the frequency of all amino acids is displayed as a stack of letters. The height of a column in the logo is given as the information content in bits of the alignment at that particular position, and the relative height of individual letters is proportional to the frequency of the corresponding amino

acid at that position. MG-132 purchase In this paper, we use such sequence logos to display the HLA-DP and HLA-DQ binding specificities identified by NNAlign. The five HLA-DP allelic variants were chosen by Wang et al.7 to cover a high percentage of the human population. Only considering the β-chain, more polymorphic than the α-chain and the main determinant for HLA-DP binding,15,16 the allele choice provides coverage of about 92% of the average population at the DPB1 locus.9 The sequence motifs identified by NNAlign for the five HLA-DP molecules are shown in Fig. 1. In general, all variants share a common pattern characterized by anchors at positions P1 and P6, with strong preferences for phenylalanine (F) and

other aromatic or hydrophobic amino acids. Additionally, some molecules appear to buy RAD001 have a hydrophobic Sunitinib cell line preference at P9 especially for leucine (L). This P9 anchor was previously described for DPB1*04:02,17 but here we observe it also

for other variants such as DPA1*02:01-DPB1*01:01 and DPA1*02:01-DPB1*05:01. In some instances, and notably for DPA1*03:01-DPB1*04:02, the residues at position P7 appear to have influence on the binding specificity of the molecule. This has not been described in previous reports. Another small exception to the P1–P6 hydrophobic/aromatic pattern is observed in the allelic variant DPA1*02:01-DPB1*05:01, where the positively charged amino acids R and K are moderately preferred at P1 together with hydrophobic ones, as was also previously noted.9 Taken as a whole, there appears to be a large overlap in the peptide-binding specificities of the five DP molecules, characterized by strong hydrophobic/aromatic anchors at P1 and P6, with the few exceptions noted above. Consistent with these observations, previous studies have found considerable overlaps in the peptide repertoires that can bind different DP alleles, and suggested the existence of a DP supertype encompassing the most common variants.9,17 Greenbaum et al.,18 on the basis of shared binding repertoires, suggested the presence of two DP supertypes: a ‘main DP’ supertype (composed of DPB1*01:01, 05:01 and 04:02) and a DP2 supertype (DPB1*02:01 and 04:01).

Such studies have important implications for the design of future clinical studies. The search for further surface markers to aid the isolation of purer or more potent Treg populations led to studies investigating markers such as CD121a/CD121b, TGF-β/ latency associated peptide (LAP) [59] and CD39 [60]. However, all these proteins are expressed only on activated Tregs and

would be of use only to re-isolate Tregs after expansion. This may not be feasible, in view of the costs of re-isolating billions of Tregs on a per-patient basis. Other studies complicate the story even further. Ito et al. [61] showed that FoxP3+ Tregs could be grouped into two subsets based on the expression of the inducible T cell co-stimulator (ICOS). They showed that while ICOS–FoxP3+ Tregs mediate their suppressive function via TGF-β, NVP-BGJ398 molecular weight AZD8055 nmr ICOS+FoxP3+ Tregs additionally secrete IL-10. Therefore, depending on the type of immune response to be suppressed, it may be useful to isolate subsets of Tregs which have specific

mechanisms of action. Moreover, a recent study by Ukena et al. [62] compared different Treg isolation strategies in order to define the most promising Treg target cell population for cellular therapy. They compared CD4+CD25hi enrichment, CD4+CD25hi enrichment and depletion of CD127+, enrichment of CD4+CD25hiCD45RA T cells, depletion of CD49d+ (a marker of proinflammatory cytokine-producing effector T cells) and CD127+ T cells and enrichment of CD4+CD25hi ICOS+ and ICOS– Tregs. They concluded that while CD4+CD25hiCD127– and CD4+CD25hiICOS+

Tregs are the most promising Tregs for fresh cell infusions in clinical trials with respect to cell yield, phenotype, function and stability, the CD4+CD25+ Tregs qualify as the best candidate for in-vitro expansion. Such studies, therefore, paint a complicated picture that when choosing the Treg marker for cell isolation we should also bear in mind Metalloexopeptidase other factors other than simply purity, i.e. isolating potent cells with a mechanism of action to suppress the immune response of interest and cells with the desired expansion profiles. Despite this, however, what limits choice when devising a clinically applicable protocol is that isolation techniques need to be good manufacturing practice (GMP)-compliant, and GMP purification reagents for all the various markers outlined above are not yet available. The clinical Treg selection protocols used to date in the United Kingdom have used a combination of depletion and positive selection steps, with the isolation tools involving mainly the automated CliniMACS plus system (Miltenyi Biotec, Bisley, UK). This enables GMP-compliant cell selection by magnetic bead activated cell sorting [63].

Traditionally, naive and memory cells are characterized ex vivo by their mutually exclusive expression of CD45RA and CD45RO molecules, respectively. However, it is known that upon activation and proliferation in vitro naive T lymphocytes first acquire the expression of CD45RO and subsequently lose the expression of CD45RA, making the timing of the analysis of CD45 molecule expression in vitro critically important [23]. Nevertheless, it can be assumed that the percentage of CD4+CFSElow cells with the CD45RA-CD45RO+ phenotype also reflects

more or less accurately the frequency of memory cell-derived antigen-specific precursor T cells in our experimental system. Therefore, based on our data it this website seems that in children with CD CD4+ T cells specific to gTG have mainly a memory phenotype, whereas Pifithrin-�� in vitro in healthy children these cells are more predominantly of naive origin. Consequently, in children with CD the stronger proliferative responses observed to gTG also presumably reflect the higher

frequency of memory CD4+ T cells specific to gTG in vivo. We also examined the expression of the gut-homing β7 integrin and observed that gTG-specific T cells expressed high levels of the molecule. This finding suggests that the CD4+ T cells specific to gTG are generated in the gut mucosa and are capable of trafficking back to the intestine. The specificity of the increased β7 integrin expression by gTG-specific T cells was demonstrated by a considerably lower expression of the molecule by TT-specific T cells that are primed by subcutaneous injections and thus lack the capacity to traffic to the gut. Our current results corroborate earlier reports demonstrating the

expression of β7 integrin by CD4+ T cells specific to gTG [12,14]. In conclusion, we have shown that CD4+ T cells specific to gTG are detectable in the peripheral blood of more than half of children with newly diagnosed CD, whereas this was significantly less common among Clomifene healthy control children. In contrast, the responses to native gliadin did not differ between children with CD and healthy controls. We also demonstrate that in children with CD the CD4+ T cells specific to gTG have a memory cell phenotype and express β7 integrin as a marker of gut homing. Taken together, our results support the widely accepted model for the importance of T cell responses to gTG epitopes in the pathogenesis of CD. Moreover, further development of assays to detect specific CD4+ T cell responses to gTG in the peripheral blood may also have practical applications for the diagnostics of the disease, as demonstrated recently by HLA-tetramer staining in patients with uncertain CD [24]. We thank Virpi Fisk for the skilful technical assistance. The study was supported financially by the Finnish Cultural Foundation, the Finnish Coeliac Society and the Finnish Medical Foundation. The authors confirm that there are no conflicts of interest.

We first show that kidney recipients selected for clinical stability (good graft function at least 5 years post-transplantation) displayed heterogeneous TCR patterns from Gaussian to highly selected profiles. Given the large size of the analyzed cohort, we looked for correlation of the TcL topology with the biological and clinical variables

collected in the GenHomme database. The factor with the strongest correlation (ρ=0.58, p<0.01) was the CD8+/CD4+ T-cell ratio. Stable recipients displaying TSA HDAC mouse class 1 TcL patterns have low to moderate CD8+/CD4+ T-cell ratios, whereas those with classes 3 and 4 patterns have a higher CD8+/CD4+ T-cell ratios. This observation and the fact that altered TCR patterns were positively correlated with the CD8+/CD4+ T-cell ratio are not surprising since CD8+ T cells have been shown to be the main contributor of the alterations of T-cell repertoire in different situations including healthy individuals 18, 19, HIV-infected patients 20, EBV-infected patients 21, 22 and kidney graft recipients 10. We thus identified a sub-group of highly clinically stable patients that accumulated antigen-experienced

CD8+ T cells. This observation was strengthen by the fact that inflammation related genes (i.e. GZMB and T-bet) were increased and regulatory associate gene (i.e. FOXP3) was decreased in patients with a skewed Vβ repertoire. We also found that TCR repertoire usage was significantly different Sorafenib price between operationally tolerant recipients and patients with chronic rejection. Patients with chronic rejection displayed SSR128129E peaked Vβ transcript CDR3-LD associated with higher quantity of transcripts, indicating accumulation of oligo

or monoclonal Vβ expansions. This skewed TCR usage was not found in patients with chronic renal failure (RFA), suggesting that T-cell alterations reflected rejection process and not kidney dysfunction (Supporting Information Fig. 3). Such results are in agreement with those of Matsutani et al., who reported that the level of alterations of TCR usage was significantly greater in recipients with graft failure 23. Both persistent and non-persistent viruses have been shown to induce a highly biased T-cell repertoire 21, 24, 25. Among the virus-specific T cells, the T-cell response to CMV has been shown to be large, comprising up to 10% of all CD8 T cells 26–29. In this study, only a low correlation was found between CMV seropositivity status and peripheral TCR repertoire usage of the patients with stable graft function. Briefly, 18% of the patients within TcL class 1 have anti-CMV IgG, whereas 36% of the patients with a stable graft function, whose TcL belong to classes 3 and 4, have anti-CMV IgG. Based on this observation, CMV reactivation was also found to be more frequent in patients with the TcL classes 3 and 4 than in patients with a TcL class 1.

Real time (RT) PCR was performed in triplicate using FAM-labeled Assay-on-Demand reagent sets for IL-10 (Hs00174086_m1) and Foxp3 (Hs00203958_m1). RT-PCR reactions were multiplexed using VIC-labeled 18S primers and probes (Hs99999901_s1) as an endogenous control and analyzed using SDS software version 2.1

(Applied Biosystems), according to the 2-(∆∆Ct) method. Results are presented as mean ± SEM, unless indicated. Data were assessed for normality and equal variation after which the appropriate parametric or nonparametric test was performed (see individual NVP-LDE225 figure legends). Differences were considered significant at the 95% confidence level. Correlations were verified with the Pearson’s correlation test or the Spearman’s rank correlation coefficient, as indicated in the figure legend. Z. U. was initially funded by an MRC CASE PhD studentship, held in association with Novartis Institute for Biomedical Research, Horsham, UK. D. R. and Z. U were also supported through funding

by EURO-Thymaide. E. S. C. is funded through an MRC British Thoracic Society/Morriston https://www.selleckchem.com/products/ink128.html Davies Trust Capacity Building PhD studentship. E. X. by a British Lung Foundation Fellowship. C. H. gratefully acknowledges financial support from the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College click here Hospital NHS Foundation Trust. A. G. is the recipient of a BMA James Trust Fellowship. L. G., J. C., and A. O. G. are funded by MRC, UK. At KCL, we thank C Reinholtz and K Jones, our research nurses. At MRC National Institute for Medical Research we thank: A. Rae, G. Preece, and N. Biboum for assistance in flow cytometry cell sorting; Biological Services Unit

and Xumei Wu for animal husbandry and breeding. We thank Bernard Malissen INSERM-CNRS Universite de la Mediterranee, France and Adrien Kissenpfennig, Queen’s University, UK for their generosity in providing the Foxp3GFP C57BL/6 mice. The authors declare no financial or commercial conflict of interest. Disclaimer: Supplementary materials have been peer-reviewed but not copyedited. Table 1: Characteristics of pediatric asthma patients. For more information see Bush & Saglani, 2010 [47]. Figure 1: Blocking TGF-β signaling diminishes the frequency of Foxp3+ T cells in 1α25VitD3 treated cultures. Figure 2: 1α25VitD3 maintains Foxp3 expression of murine regulatory T cells. Figure 3: Blocking IL-10 signaling promotes the proliferation of Foxp3+ T cells in 1α25VitD3 treated cultures. Figure 4: Purity of peripheral blood Treg and effector T cells isolated by cell sorting. Figure 5: Treg gating strategy in bronchoaveolar lavage fluid. “
“Despite the high prevalence of highly pathogenic H5N1 influenza A viruses in Indonesia, epidemiology information on seasonal human influenza is lacking.

PBMC, 1 × 106, were stained using a total of 5 µg of AHG- AlexaFluor® 488; 5 µl of anti-CD4-Pacific Blue™ or allophycocyanin (APC) and 5 µl of either phycoerythrin (PE) or PE-cyanin 7 (Cy7™) anti-human CD25 for 20 min at room temperature (RT). The

fluorescence was acquired using a fluorescence activate cell sorter (FACS)Caliber (BD Bioscience). The CH5424802 concentration data were analysed using FlowJo software from Treestar (Ashland, OR, USA). First the gates were drawn using forward- and side-scatter. The lymphocyte population was then gated for the CD4+ lymphocytes. Thereafter, the CD4+ gated population was analysed further for CD25+ and AlexaFluor® 488–AHG binding population. TCC was isolated from pooled normal sera, as described previously [25]. An additional step

to purify TCC further was performed by subjecting the TCC to chromatographic separation on Superose™ 6 YK (GE Healthcare, Los Angeles, CA, USA). The TCC-containing fractions were examined for C9 polymerization Vadimezan price by monitoring the generation of the neo-epitope (using clone aE11) using an enzyme-linked immunosorbent assay (ELISA) system. The TCC-containing fractions were pooled, concentrated and then stored at −70°C. The non-lytic dose of TCC was determined by incubating 1 × 106 Jurkat cells with varying concentrations from 0·25 to 5 µg of protein for 4 h and monitoring of apoptosis and necrosis using Vybrant® Apoptosis Assay #3 from Invitrogen, as per the manufacturer’s suggested protocol. From these experiments a dose of 2·5 µg was considered optimal, as more than 95% cells remained viable with trypan blue dye and propidium iodide staining. The expanded human naive CD4+ T cells purified from normal donors were used; 1 × 106 cells were activated by placing in serum-free medium for 4 h and treated with ICs (2·0 µg) or ICs (2·0 µg) in the presence of non-lytic TCC (2·5 µg).

The cells were collected post-2 h and used directly for experiments. The 2 h time interval was selected based on our previous observation of the T cell activation in response to treatment with ICs and TCC [26]. Lysates from cells treated with various stimuli were prepared from 1 × 106 cells using 0·5 ml of radioimmunoprecipitation assay (RIPA) buffer (50 mm Tris-HCL; 1% NP40; 0·25% Na-deoxycholate; 1 mm tetraacetic Urease acid (EDTA); 1 mm phenylmethylsulphonyl fluoride (PMSF); 1 mm Na3VO4; 1 mm sodium fluoride (NaF); and 1 µg/ml each aprotinin, leupeptin, pepstatin). Thereafter, 2 µg of monoclonal anti-FcγRIIIA/B antibody was added to the lysates and this mixture was then incubated at RT for 1 h. A 50-µl suspension of Protein G sepharose beads in saline (PBS) was then added and the mixture was incubated further at 4°C overnight. Subsequently, the Protein G beads were washed with excessive RIPA buffer and suspended into 1X reducing loading buffer from Invitrogen. These samples were heated and beads were separated by centrifugation. Protein content of samples was measured with micro bicinchoninic acid method (Sigma).