DCs from GLA-SE but not SE-treated mice became active stimulators

DCs from GLA-SE but not SE-treated mice became active stimulators of the allogeneic mixed leukocyte reaction, inducing robust proliferation of both CD4+ and CD8+ T cells (Fig. 5C). To further evaluate the capacity of DCs to become immunogenic following antigen capture in vivo, mice were injected with anti-DEC-HIV gag and either GLA-SE or SE. After 4 h, splenic DCs were purified by cell sorting and injected into naïve mice i.v. In addition, to check that antigen presentation was performed by the transferred and not recipient DCs, MHCII−/− DCs were used as negative controls. Only WT DCs, after targeting with anti-DEC-gag and stimulated with GLA-SE in vivo, were capable

of inducing gag-specific T-cell immunity (Fig. 5D). These data indicate that GLA induces the full maturation of spleen and lymph node NVP-LDE225 DCs in vivo. The discovery of receptors

responsible for stimulating innate immunity, such as the TLR and RIG-like receptor pattern recognition receptors, makes it possible to test chemically defined agonists as new adjuvants to trigger the DC link between innate and adaptive immunity. To understand adjuvant action, these agonists need to be characterized in vivo at the level of antigen presenting DCs. Our experiments at this direct level indicate that a synthetic TLR4 agonist, GLA-SE, serves as an effective adjuvant and enhances MLN0128 mw the capacity of DCs in vivo to immunize against protein antigens. The adjuvant role of GLA-SE was dependent on TLR4. Similar results have been reported by Baldwin et al. where GLA induced production of IL-6 by click here monocyte-derived DCs in culture, and this was blocked with anti-TLR4 but not TLR2 antibodies 27. Our results extend prior research by showing a complete dependency of TLR4 stimulation for the induction of adaptive responses in vivo by GLA-SE. DCs are the major link between the innate and the adaptive immune system, and its appropriate activation and maturation by agonists for innate signaling receptors should allow for the induction of

an adaptive response 41, 42. However, much of the evidence involves studies of DCs stimulated in cell culture with adjuvants 43. In the current study, we demonstrated that GLA-SE injection together with a protein antigen allows the antigen-capturing DCs to quickly become immunogenic in vivo. Enhanced T-cell responses were detected when antigen was targeted to DCs. We did not detect qualitative difference in adaptive responses between untargeted or targeted protein. However, lower doses of antigen were required using anti-DEC-HIV gag p24 to achieve detectable responses. This finding highlights the importance of DCs for initiating adaptive T-cell immunity. After showing that DCs were essential for the generation of T-cell responses in lymph nodes to an s.c.

This study aims to elucidate the role of radiation induces Akt ex

This study aims to elucidate the role of radiation induces Akt expression in regulatory T cells (Tregs). The surgically removed BCa tissue was collected from 26 patients treated with or without radiotherapy. The frequency of Tregs and apoptotic Tregs in BCa tissue was assessed by

flow cytometry. A cell culture model was employed to investigate the mechanism by which the tumour-infiltrating Tregs survive from radiation. After radiotherapy, the frequency of Treg was increased in the BCa tissue; the apoptotic Tregs were decreased; the expression of Akt was increased in remained Tregs. The results were reproduced in vitro with a cell culture model. The addition of Akt inhibitor blocked the radiation-induced Treg survival in Adriamycin culture. Akt plays an important

MI-503 manufacturer role in the radiation-induced tumour-infiltrating Treg survival in BCa. The bladder carcinoma (BCa) is the fifth most common cancer, which accounts for 85-90% of the primary carcinomas with increasing incidence worldwide [1, 2]. Although the research on BCa was advanced rapidly in the last decade, the pathogenesis of BCa remains unknown; the prognosis of patients with BCa is unsatisfactory [3]. Regulatory T cells (Tregs) are a subtype of T cells. A majority of Tregs is CD4+ CD25+ Foxp3+ Tregs [4]. Tregs express a set of immune suppressive molecules, such as transforming growth factor (TGF)-β and interleukin (IL)-10, to suppress other effector T cells’ activities [5]. Thus, Tregs are an important cell population in the maintenance of homoeostasis in the body. On the other hand, Tregs also suppress the activities of the antitumour immune cells, such as cytotoxic CD8+ T cells [6]; cancer cells thus get the chance to grow. Some investigators propose to get rid of Tregs from the body, using monoclonal anti-CD25

antibodies to promote the therapeutic effect of cancer Ribonuclease T1 [7]. How the increase in tumour-infiltrating Tregs occurs is unclear. Protein kinase B is also known as Akt. Akt is a serine/threonine protein kinase that plays an important role in a number of cellular processes such as glucose metabolism, cell proliferation, apoptosis, transcription and cell migration. Cumulative reports indicate that Akt plays an important role in cancer cell survival [8]. Direct inhibition of the serine/threonine kinase Akt provides another avenue to pharmacologically suppress tumour cells’ activity [9]. Yet, whether the expression of Akt in cancer tissue has any association with Treg survival is unclear. Thus, we collected surgically removed BCa tissue and found an increase in Akt expression in the tumour-infiltrating Tregs, which greatly promoted the Treg’s survival. Reagents.  The fluorescently labelled antibodies were purchased from BD Bioscience (Shanghai, China). Monoclonal antibodies of Foxp3, CD4, CD25, Akt, CD3 and CD28 were purchased from Santa Cruz Biotech (Santz Cruz, CA, USA).

ELISA experiments showed that TNF-α was not secreted by mock-infe

ELISA experiments showed that TNF-α was not secreted by mock-infected cells or HB101-treated cells (Fig. 7E). These results were expected, because tnf-α mRNA was not detected by RT-PCR. However, E2348/69 infection activated TNF-α secretion at a high value (252 ± 8 ng/ml) at 2 h of infection, which decreased at 4 h post-infection (151 ± 13 ng/ml). In E22-infected cells, there was no decrease and TNF-α secretion was similar at 2 h (252 ± 8 ng/ml) and 4 h (247 ± 13 ng/ml) post-infection (Fig. 7E).

Therefore, as with E2348/69, E22 infection activates TNF-α synthesis and secretion. E22Δeae infection caused IWR-1 molecular weight contrary effects on TNF-α secretion depending on the infection time. At 2 h of E22Δeae infection, TNF-α secretion was of 282 ± 8 ng/ml, while at 4 h of infection, cells secreted 50% less TNF-α (126 ± 13 ng/ml) than cells infected with E22 WT (247 ± 13 ng/ml). TNF-α secretion in cells infected

with E22ΔescN ABT263 was not reduced (236 ± 8 ng/ml) at 2 h and decreased at 4 h (192 ± 13 ng/ml), whereas the secretion at 2 h in cells infected with E22ΔespA was 191 ± 8 ng/ml and at 4 h of 116 ± 13 ng/ml. Thus, T3SS is involved in the activation of TNF-α release. E22ΔfliC infection caused a reduced secretion of TNF-α at 2 h (201 ± 8 ng/ml), and at 4 h TNF-α was completely absent from the supernatants (Fig. 7F). Evidently, flagellin is a factor which is necessary to activate TNF-α secretion, and it is essential to maintain this cytokine in the supernatants of infected cells (strikingly similar is the effect of flagellin in IL-8 release). Inflammation induced by EPEC results from

the balance of positive and negative factors [39]. Here, we analysed the role of the EPEC virulence factors T3SS, EspA, intimin and flagellin, on the epithelial inflammatory response. Sirolimus chemical structure The evaluation comprised TLR5 signalling activated by EPEC flagellin [25], activation of ERK1/2 [28] and NF-κB [27] pathways and transcription of proinflammatory cytokine genes [33, 39]. EPEC-induced cell signalling was reproduced and unified in an in vitro epithelial cell infection model, which consisted in using HT-29 cells infected with the prototype strain E2348/69 and the strain E22, which is a strain pathogenic for rabbits, which contains LEE but no BFP [40], and can be considered an atypical EPEC. The role of the virulence factors was studied using isogenic E22 mutants, to be able to corroborate the results in vivo through the experimental rabbit infection model [33]. The significance of EPEC flagellin in the activation of proinflammatory response is well established [25]. However, TLR5 expression, localization and functionality in intestinal epithelial cells have all been unclear [38], and previous studies have been focused on TLR5 distribution in polarized cells [41, 42].

The membrane was incubated with primary antibody and an appropria

The membrane was incubated with primary antibody and an appropriate secondary horseradish peroxidase-conjugated antibody. Signals were detected by enhanced chemiluminescence (GE Healthcare Bio-sciences, Little Chalfont, UK). The immunoreactive https://www.selleckchem.com/products/Fulvestrant.html bands were scanned to produce digital images that were quantified employing SCION Image software, and fold phosphorylation was calculated from the amount of phospho-protein relative to the corresponding non-phospho loading control. The IgE-sensitized cells (1×106) were loaded with 4 μM Fluo3-AM (Dojindo, Kumamoto, Japan) for 30 min at 37°C. The cells were resuspended in 1×Tyrode’s

buffer, and then changes in dye fluorescence upon the addition of stimulants were monitored employing flow cytometry. [Ca2+]i mobilization was expressed as the relative fluorescence intensity. Data shown are the mean±SD. Statistical analysis was performed using Student’s t-test. Probability values <0.05 were considered to indicate statistically significant differences. This work was supported by the grants-in-Aid for private universities from the Ministry of Education, Culture, Sports, Science (C. Ra), and Technology of Japan, the

Grants-in-Aid for Scientific Research from the Nihon University (C. Ra). Conflict of interest: The authors declare no financial or commercial conflict of interest. “
“Although data show check details the importance of type I interferons (IFNs) in the regulation of the innate and adaptive immunity elicited in response to viral, bacterial and parasitic infections, the functional activities of these cytokines during fungal infections are poorly

understood. We examined here the impact of IFN-β on the response of human monocyte-derived dendritic cells (DCs) infected in vitro with Aspergillus fumigatus. Having found that A. fumigatus-infected DCs do not express IFN-β, we evaluated the effect of the exogenous addition of IFN-β on the maturation of human DCs induced by the infection with A. fumigatus conidia. Although the phagocytosis of the fungus was not affected by IFN-β treatment, the expression of CD86 and CD83 induced upon A. fumigatus challenge was enhanced in IFN-β-conditioned DCs, which also showed an increased expression of C-X-C chemokine receptor type 7 (CXCR-7) IL-27 and IL-12p70, members of IL-12 family. Through these modifications, IFN-β improved the capacity of DCs to promote an anti-Aspergillus T helper type 1 response, as evaluated by mixed leucocyte reaction, which plays a crucial role in the control of invasive aspergillosis. Our results identified a novel effect of IFN-β on anti-Aspergillus immune responses which, in turn, might open new perspectives on the use of IFN-β in immunotherapy for fungal infections aimed at enhancing the immunological functions of DCs. Aspergillus fumigatus ( A. fumigatus) conidia are ubiquitous in the environment.

To our knowledge, this test was replicated by another research gr

To our knowledge, this test was replicated by another research group in a Norwegian cohort of adult CD patients [7,8]. In the present study we validated this method in a cohort of 14 young CD patients recruited in the south of Italy, and estimated the level of its reproducibility by exposing the same individual twice to gluten consumption. After the first

in-vivo challenge we observed a significant increase of IFN-γ-secreting cells in response to gliadin 6 days after the wheat intake, confirming the data reported in both Australian and Norwegian adult coeliac patients [4,7,8,23]. Similarly, the magnitude of the IFN-γ responses was comparable to the values Selleck NVP-LDE225 found in previous studies [4–7]. When we looked at individual responses we found that, upon wheat consumption, the frequency of IFN-γ-releasing cells to whole gliadin increased at least three times in eight of 14 (57%) subjects, barely within the average obtained in previous studies, that ranged from 40% [23] to 90% [5] of exposed coeliac patients. In agreement with these studies, the specific response to gluten elicited by the in-vivo challenge was mediated Roxadustat chemical structure by CD4+ T cells and was DQ2-restricted. Furthermore, the IFN-γ-producing cells expressed

beta-7 integrin, indicating a phenotype of gut-homing cells. Short-term gluten consumption also induced a significant increase of T cells reacting to the immunodominant 33-mer peptide, although contrasting findings were reported on the

frequency of responder patients [2,3]. Anderson and co-workers reported that the great majority of coeliacs reacted to 33-mer (or to truncated peptide, α-gliadin (57–73) ZD1839 research buy [5,6], while in a more recent study reactivity was observed in only six of 10 patients [23]. Our results are in agreement with this latter finding, as we found an evident increase of IFN-γ responses induced by immunodominant gliadin peptide in 8 of 14 patients at first challenge. Unexpectedly, upon the second challenge the number of reacting subjects was far fewer (three of 13 subjects challenged). In this regard, we found that approximately 50% of intestinal T cell lines generated from south Italian CD patients who were assayed in vitro reacted to 33-mer, suggesting that only a subgroup of our coeliac donors seems to display a response to this epitope [2]. These data are not surprising because, despite its strong immunogenicity, 33-mer is one of several gliadin-derived T cell epitopes active in coeliac patients [2,6], and this could explain the increased magnitude of IFN-γ-positive cells found in response to whole gliadin digest. In contrast to previous studies, in which the immune reactivity to gluten was very low, or totally absent, before wheat consumption at day 0, we also found substantial IFN-γ production instead.

The Vβ8 2+ cells that were present in the skin of HEL and CT immu

The Vβ8.2+ cells that were present in the skin of HEL and CT immunized mice expressed the transcription factors Tbet and RORγt, and also both IFN-γ and IL-17, which is indicative of Th1 and Th17 differentiation (Fig. 4B and C). As shown in Fig. 4D, the DTH response

was dependent on IL-17 and partially dependent on IFN-γ activity, as blocking these cytokines during the challenge clearly affected the induction of the DTH response. These results indicate that immunization in the ear with both CT and with CTB induces a signature DTH response www.selleckchem.com/products/bay-57-1293.html that is characterized by IL-IFN-γ. Considering the robust IFN-γ and IL-17 production by CD4+ T cells that is induced by ear immunization with low doses of antigen in combination with CT or CTB (which translates in the induction of a DTH response), we evaluated the role of migrating skin DCs in CD4+ T-cell differentiation DNA Damage inhibitor by elimination of the immunization site. The antigen presentation that was induced by CT or CTB was not notably affected by the absence of migrating cells from the ear (Fig. 5A). Remarkably, cytokine production following immunization with 0.3 μg HEL and 1 μg CT or CTB was dependent on the presence of migrating cells, as

we observed virtually no cytokine expression by HEL–re-stimulated CD4+ T cells when the immunization site was removed after 90 min (Fig. 5B). The intracellular expression of IFN-γ was also considerably reduced in mice in which the inoculation site was removed, even when a saturating dose of antigen was used (Fig. 5C and D). When the site of inoculation was removed 24 h after immunization, the percentage of IFN-γ+ cells were similar to those obtained from animals in which

the ear was not removed (Fig. 5D). These results indicate that after ear immunization with HEL in combination with either CT or CTB, CD4+ T-cell differentiation is dependent on the presence of cells migrating from the ear to the dCLNs. Several strategies for skin immunization Reverse transcriptase have been developed 10, 12, 14, 24. However, the nature of the CD4+ T-cell response that is dominant in the skin and the role of migrating DCs in the presence of different adjuvants in shaping the immune response are important issues that need to be investigated. Here, mice of varying genetic backgrounds were immunized in the ear with model antigens in combination with CT or CTB as an adjuvant. We present evidence that, following ear immunization, both CT and CTB preferentially induced IFN-γ– and IL-17-producing CD4+ T cells over IL-4- or IL-5-producing cells. This response was dependent on migrating cutaneous DCs. Immunization with CT, as well as with the non-toxic CTB subunit, resulted in the induction of a DTH response that was dependent on IL-17 and to a lesser extent on IFN-γ.

EE was actually found to exacerbate symptoms in female transgenic

EE was actually found to exacerbate symptoms in female transgenic SOD1(G93A) ALS mice, in a sexually

dimorphic manner [28]. It is possible that in these ALS mice the increased synaptic drive induced by EE could have accelerated specific excitotoxic mechanisms in motor neurones, a possibility which remains to be tested. Furthermore, the limitations of such transgenic animal models which involve overexpression of a specific familial human gene mutation [29], with respect to ‘genetic construct validity’, means that the direct relevance of such EE effects to the majority of ALS cases (which are sporadic and genetically heterogeneous) requires further investigation. The present article will review the effects of EE on brain disorders, with a focus on animal models of neurodegenerative diseases. I will address specific molecular, cellular and behavioural effects of EE in these models, MG-132 potential mechanisms selleck and implications for future therapeutic interventions for neuroprotection and brain repair. Huntington’s disease (HD) is a fatal, autosomal dominant neurodegenerative disorder which presents as a triad of cognitive, psychiatric and motor symptoms. HD is caused by a tandem repeat (CAG trinucleotide)

expansion encoding an extended tract of glutamines in the huntingtin protein. Understanding the pathogenesis of HD has been greatly accelerated by the development

of transgenic and knock-in animal models, the first of which were the R6 transgenic mouse lines [30]. These and other genetically targeted animal models have demonstrated that the cognitive, psychiatric and motor symptoms are associated with specific effects of the HD mutation in selective neural circuitries and tissues [31,32]. Furthermore, knock-in and transgenic animal models of HD have provided new insights Y-27632 2HCl into mechanisms of pathogenesis, including molecular deficits, synaptic dysfunction and progressive abnormalities in neurones and other cell types [33–35]. The effects of EE were first investigated in the R6/1 HD transgenic mouse model [8]. HD and wild-type mice were randomized post-weaning into either EE and standard housed (SH) conditions. EE was shown to dramatically delay onset of motor deficits in R6/1 HD mice and ameliorate neurodegenerative loss of peristriatal cerebral volume [8]. Subsequently, it has been demonstrated that EE can also ameliorate cognitive deficits [36] and depressive-like abnormalities [10] in R6/1 HD mice. Furthermore, evidence has been provided, in R6/2 transgenic mice, that EE initiated around the time of motor onset can also slow progression of the movement disorder [37]. These studies in HD mice have been followed up in clinical cohorts with epidemiological studies.

Supernatants were harvested and counted on an automated gamma cou

Supernatants were harvested and counted on an automated gamma counter. Percent specific lysis was calculated as [(sample 51Cr releasespontaneous

51Cr release)/(maximum 51Cr releasespontaneous 51Cr release)]×100. In vivo cytotoxicity experiments were performed as described with modifications 35. Naïve splenocytes (target cells) were pulsed with 1, 0.1 or 0.01 μg/mL of JEV NS4b S9, WNV NS4b S9 peptide or control influenza NP 366–374 peptide (1 μg/mL) for 45 min at 37°C. Cells were stained with 1 μM Cell Trace Far Red 7-hydroxy-9H- (1,3-dichloro-9,9-dimethylacridin-2-one)-SE (DDAO-SE; Invitrogen, Carlsbad, CA, USA) and serial dilutions of CFSE (5 μM, 1.5 μM, 0.4 μM, 0.1 μM; Invitrogen). Target https://www.selleckchem.com/products/PLX-4032.html cells in PBS (2×107 cell/mL) were injected i.v. into JEV-immunized or naïve mice 8 days post immunization. Splenocytes were harvested 2 h later

and analyzed using a FACSAria. Percent specific lysis was calculated by the formula 1(Ratio immune/Ratio naïve)×100, where Ratio=(♯ events of JEV or WNV peptide/♯ events of control influenza peptide). Recombinant H-2Db:Ig fusion protein (4 μg; BD Biosciences) was loaded with variant peptides (>90% purity) at 640 molar excess peptide in PBS (pH=7.2) at 37°C overnight according to manufacturers guidelines. Peptide-loaded dimer was incubated with 2.4 μg APC-anti-mouse IgG (BD Biosciences, mAb A85-1) followed by incubation with purified mouse IgG isotype control (4 μg; BD Biosciences; mAb A111-3). Splenocytes were resuspended in PBS, stained with Live/Dead Aqua and incubated with anti-CD16/32 (2.4G2; BD Bioscience), followed by staining with 4 μg of peptide-loaded dimer. Cells selleck products were surface stained with anti-CD44, anti-CD62L, anti-KLRG1 and anti-CD127 conjugated with FITC, PE-Cy7 and PerCP-Cy5.5, washed and resuspended in BD stabilizing buffer. Peptide-loaded dimer staining levels in naïve mice were subtracted from experimental Tideglusib values in infected mice. The gating strategy is shown in Supporting Information Fig. 3A and B. On days 3 and 7 post JEV or WNV infection, spleen, brain and serum were obtained

and flash frozen at −70°C. Tissues were homogenized to give a 10% (spleen) or 20% (brain) homogenate based on tissue weight using a Qiagen mixer mill. Serial dilutions were made in MEM and titers were determined on Vero cells as described 36. Plates were incubated for 2 (WNV) or 4 days (JEV Beijing and SA14-14-2) prior to second agar overlay. The limit of detection was 50 pfu/mL for serum, 250 pfu/g for brain and 500 pfu/g for spleen. Means, medians and standard errors were calculated using GraphPad Prism (GraphPad Software, LaJolla, CA, USA). Comparisons of variables between JEV and WNV infection groups were performed with log transformed data using the Mann–Whitney U test on STATA software (StataCorp, College Station, TX, USA). p<0.05 was considered significant. This work was supported by contract N01-AI25490 and grants U19 AI057319, P30 DK032520 and T32 AI007349 (D.W.

Interestingly, the CD11c LuciDTR

mice exhibit increased b

Interestingly, the CD11c.LuciDTR

mice exhibit increased bacterial burden after DT injection in the same pyelonephritis model. Thus, in the absence of the confounding effects of the early neutrophilia, a role for CD11c+ cells in reducing rather than increasing pathogen burden can be revealed. The findings of Tittel et al. [30] raise the question of whether the conclusions from other studies using CD11c.DTR or CD11c.DOG mice need to be revised. For example, in a recent study, Autenrieth et  al. [33] found that animal survival was significantly increased upon DC depletion in CD11c.DOG mice in a model of Yersinia enterocolitica infection see more and that the enhanced survival was mediated by increased neutrophil and monocyte activity. The authors concluded that DCs could regulate neutrophil and monocyte

function in the steady state as well as during bacterial infection. However, when considering the results of Tittel et  al. [30], it is also possible that enhanced survival was due to increased bacterial killing by recruited neutrophils. Thus, DCs could have a smaller role in the regulation of phagocyte activity than might be apparent at first glance [33]. Similarly, in a model of peripheral vesicular stomatitis virus (VSV) infection, DC depletion in CD11c.DTR mice did not affect viral clearance in the first 48 h, even though type I interferon production, which is critical for early VSV clearance, was markedly impaired [34]. These unexpected results could again be explained by the induction Sirtuin activator of neutrophilia and monocytosis in CD11c.DTR mice, as neutrophils and monocytes can mount an early innate immune response that limits viral replication. If this were the case, the authors’ conclusion Vitamin B12 that DCs are of limited importance to the early response to peripheral VSV infection would need to be revised [34]. Of note, some of the DC-depleted mice failed to control virus replication in the brain and developed fatal VSV encephalitis, suggesting

that the brain might be excluded from any protective neutrophilia and monocytosis induced by DT treatment of CD11c.DTR mice [34]. Interestingly, the same study showed that after DC depletion VSV-specific CD4+ T-cell responses were not affected, while the expansion of CD8+ T cells was severely impaired [34]. As DCs have been ascribed a crucial role in both CD4+ and CD8+ T-cell activation, the unaltered CD4+ T-cell response is surprising. The authors suggest that there might be another antigen-presenting cell, such as a macrophage, that supports CD4+ T-cell priming. While this may certainly be the case, it is important to determine to what extent such antigen-presenting macrophages/DCs are a result of the monocytosis induced by DC depletion. In summary, although the CD11c.DTR and CD11c.

This could be attributable to the foreign

antigen express

This could be attributable to the foreign

antigen expression. FK506 chemical structure Another important difference in the current study is the utilization of a frozen inoculum, mandated by the NIH; our prior study utilized freshly grown organisms centrifuged from stationary phase broth cultures. Virulence factors are regulated by temperature in a complex fashion in L. monocytogenes, and its ability to adapt to and grow at low temperatures is of importance for food safety, as reviewed recently (39). Some strains have a greater “growth lag phase” after cold storage (40). Cryotolerance (freeze/thaw tolerance) appears to be strain dependent, and growth temperatures may affect this (41). It is beyond the scope of this paper to further examine reasons for the poor immune responses observed. Live attenuated bacterial vectors for oral delivery of vaccine antigens have unfortunately not been highly successful in this or other human studies. Perhaps

Depsipeptide price these highly-attenuated, safe strains could be used in other applications requiring transient delivery of other molecules or pharmacologic “payloads” to the gut lumen. This work was supported in part by NIH/NIAID-NERCE/BEID Career Development Fellowship 5 U54 A1057159–03 (BMB), NIH/NIAID R01 AI51206 (ELH) and grants M01-RR-01066 (Massachusetts General Hospital GCRC) and UL1 RR025758–01 (Harvard Clinical and Translational Science Center) from the National Center for Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health. We acknowledge the generosity of the Cerus Corporation, Concord, CA, USA, for providing us with the L. monocytogenesΔactA/inlB strain as well as the LLO peptide pool. We especially thank our volunteers, and the inpatient clinical research center nursing staff and clinical microbiology laboratory at Massachusetts General Hospital. “
“Considerable interest has emerged towards phagocytosis of apoptotic cells, due to its intricate

molecular mechanisms and important regulatory functions in development, homoeostasis, and immune tolerance. Impaired clearance of apoptotic cells leads to immune-mediated Non-specific serine/threonine protein kinase disorders. Current quantification methods of the engulfment of apoptotic cells by macrophages are potentially flawed by several limitations. Adherent macrophage populations are overlaid with apoptotic targets in suspension and then co-cultured for a definite period, which may give rise to two different features: (1) engulfed and (2) non-engulfed macrophages that are surface-bound cell populations. Rigorous washing to dislodge surface-bound apoptotic cells before assessment of phagocytosis may lead to loss of phagocytes, thereby skewing the apparent magnitude of the overall phagocytic response.