S3). This suggests that modulation of DCs by B10 cells observed in other tissue compartments [17] does not occur in the liver. Having demonstrated that hepatic B cells comprise fewer regulatory subsets than splenic B cells, a question not addressed in this study is why Bregs appear not to contribute to the overall tolerogenic liver environment. One possibility may be to prevent overinhibition of immune responses in the liver. As shown in this report and by others [15-17], the TLR-4 ligand LPS, a normal constituent of portal venous blood, is a potent stimulator of B10 cells. The absence of B10 cells and the presence of B cells with proinflammatory potential in an overall tolerogenic liver environment

could help to balance the hepatic capacities of immune tolerance and immune stimulation. Our data presented here show that the absence of hepatic B cells compromises further the capacity of mDCs to respond to LPS (Fig. 3). To obtain sufficient numbers of liver Fulvestrant purchase mDCs for

analysis, Flt3L-treated mice were used in Fig. 3 and Supplementary Figs S2 and S3. We are aware of the caveat that Flt3L might modify the composition of mDC subsets as well as other cells. Extended experiments using animal models are BEZ235 clinical trial needed to confirm the positive regulation of liver mDCs and liver immune responses by hepatic B cells. Future research to understand more clearly the mechanisms underlying hepatic B cell activation and function is merited, and may lead to improved understanding and therapy of different liver-related pathological conditions. The authors thank Dr David Rothstein for the gift of IL-10 reporter mice and Thomson laboratory members for helpful discussion. The work was supported by NIH grant P01AI81678 (A.W.T.), Anidulafungin (LY303366) grant (874279717) from the Roche Organ Transplantation Research Foundation (A.W.T.) and by an American Society of Transplantation Basic Science Fellowship awarded to Hong Zhang. Hong Zhang did most of the experiments and wrote the manuscript, Donna

Beer Stoltz performed immunofluorescence, Geetha Chalasani provided direction for B cell subset analysis and Angus W. Thomson provided intellectual input and guided the preparation of the manuscript. The authors declare no financial or commercial conflicts of interest. Fig. S1. Expression of cell surface activation markers on murine B cells following in-vivo poly I:C administration. C57BL/6 (B6) mice were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS). On days 0 and 1 post-injection, the mice were examined for the expression of the indicated surface molecules on spleen versus hepatic B cells; n = 4 mice per group. On day 1, both liver and splenic B cells up-regulated expression of CD39, CD40, CD80 and CD86; *P < 0·05. No significant difference was observed between the liver and spleen. Data are representative of two independent experiments. Fig. S2. Close proximity of B cells (CD19+) and dendritic cells (DCs) (CD11c+) in liver parenchyma.

G. Dranoff, Dana-Farber Cancer Institute, Boston, MA, USA), replaced every other day. On day 6, BMDC were detached with enzyme-free digestion buffer (Sigma-Aldrich, St. Louis, MO, USA). BMDC pulsed with α-GalCer (200 ng/mL, Kirin) or vehicle (Tween-20) in medium for 3 h at 37°C. BMDC were subsequently washed with PBS and

fixed with 0.02% glutaraldehyde (Sigma-Aldrich) for 1 min click here before being used in experiments. Single cell suspensions from spleens were prepared by standard techniques. Liver MNC were isolated as previously described 17 without prior Collagenase digestion. Briefly, livers were perfused with PBS, minced and iNKT cells were enriched by centrifugation in a two-step Percoll gradient. Enriched populations typically contained 20–30% iNKT cells. Human iNKT cell lines were

established by sorting PBMC with iNKT-mAb 6B11 and expanding with mitogen as described 26. Lines were maintained by periodic re-stimulations and purity checked with Vα24 mAb 26. iNKT cells from livers were stimulated in the presence of either plate-bound PBS57-loaded CD1d monomers or α-GalCer-pulsed and Glutaraldehyde-fixed BMDC. PBS57-loaded CD1d monomers were plate-bound overnight in PBS at 4°C, blocked and washed with complete culture medium before cells were added. Cytokine-specific ELISA assays (eBioscience, San Diego, CA, USA) were performed following the manufacturers instructions. Sera were diluted 1:10 in PBS/1% BSA. RNA isolations using TRIzol (Invitrogen, Carlsbad, CA, USA) and RT reactions were performed as described 27. Real-time

NADPH-cytochrome-c2 reductase PCR using 1/20 volume of reverse BIBW2992 price transcription reactions and primers specific for adenosine receptors A1R (F, 5′-CATTGGGCCACAGACCTACT-3′, R, 5′- CAAGGGAGAGAATCCAGCAG-3′), A2aR (F, 5′- CACGCAGAGTTCCATCTTCA-3′, R, 5′-ATGGGTACCACGTCCTCAAA-3′), A2b (F, 5′- TGCTCACACAGAGCTCCATC-3′ R, 5′- AGTCAATCCAATGCCAAAGG-3′), A3R (F 5′-GCTGATCTTCACCCATGCTT-3′, R, 5′- ATCCAAACTGACCACGGAAC-3′), and GAPDH (F, 5′-aactttggcattgt-3′, 5′-acacatttgggggta-3′) were performed using Quantitect SYBR Green in a Corbett (Qiagen, Valencia, CA, USA). Target gene expression was normalized against levels of GAPDH and normalized against standards with known copy numbers (102–105/reaction) of adenosine receptors. Subsequent to blocking with anti-CD16/32 mAb cells were stained with CD3-FITC, NK1.1-PE and CD1d tetramer-APC. NKT cells were gated as CD3+NK1.1+CD1d-tetramer+ and sorted to purities >95% using a FACSAria (all BD Biosciences, San Jose, CA, USA). Intracellular stainings for IL-4 and IFN-γ were performed using Cytofix/cytoperm (BD Biosciences) according to manufacturer’s instructions. Results are expressed mean±SD. For statistical analyses, the one-way-ANOVA with Newman-Keuls post-test was used. Values of p<0.05 were considered as significant.

After centrifugation at 5000 g 10 min, supernatants were frozen at −80°C until used. Extracts (50 µg protein/lane) subjected to 10% SDS-PAGE were immunoblotted with antibodies to total Bad, phosphorylated Bad (Santa Cruz Biotechnology) and revealed by enhanced chemiluminescence (ECL) detection system (Pierce). Densitometric analysis of protein levels was performed with ImageQuant software. The frequency of

apoptotic acini cells was also assessed by flow cytometry analysis with Annexin V/IP double staining following the manufacturer’s recommendations (BD). Flow cytometry data were acquired in a FACSAria cytometer® and results analysed using WinMDI software®. For bax expression assays, acinar cells were homogenized either freshly or after induction with TNF-α and RT–PCR experiments were carried out as indicated FG-4592 manufacturer above and previously [16]. Statistical significance of differences was determined by the two-tailed t-test check details for independent populations. When multiple comparisons were necessary, the Student–Newman–Keuls test was used after analysis of variance. Differences between groups were considered significant at P < 0·05. Figure 1a shows the expression kinetics of VIP and their receptors in submandibular

glands isolated from NOD mice of different ages from postnatal day 2 to 20 weeks of age. Compared to normal mice, NOD mice showed the highest level of VIP expression at 4 weeks of age and decreased thereafter. The progressive decrease in VIP expression from the fourth week takes place with no changes in VPAC1 and VPAC2 receptors. A clear reduction

of VIP levels was evident in NOD submandibular glands at 16 weeks ever of age (Fig. 1a), which was confirmed by qRT–PCR (Fig. 1b). The decline in VIP/VPAC1 relative expression over time is similar to the kinetics of neural nitric oxide synthase (nNOS) activity and salivary secretion loss shown previously [12]. NF-κB appears as an intracellular pivotal determining factor that conditions the apoptotic or survival fate of TNF-α-stimulated cells [28]. Thus, we analysed NF-κB activation and apoptosis in response to TNF-α in NOD acinar cells. As shown in Fig. 2a, acinar cells from NOD glands present a basal translocation of p65 of NF-κB to the nucleus (merge image with PI-stained nuclei) that is not seen in normal BALB/c mice. Consistent with this, WB analysis of I-κB in the cytosolic fraction or p65 in the nuclear fraction revealed that p65 appeared located to the nucleus, while I-κB expression was increased in cytosol of acini in basal conditions (Fig. 2b). Moreover, when treated in vitro with TNF-α, NOD mice acinar cells showed an abnormal NF-κB activation kinetics compared with BALB/c acinar cells (Fig. 2a,b).

e. able to induce full T-cell differentiation 27, 38, 39. BALB/c ByJ and OT-I TCR-transgenic (Charles Rivers), C57BL/6J (Janvier), and ubiquitin–GFP-expressing mice 23 (Jackson) were housed and bred BIBW2992 in our SPF animal facility. Unless otherwise specified in the legend of the figures, wt C57BL/6 mice were used in the experiments. This study was carried out in strict accordance with the recommendations in the Guide

for the Care and Use of Laboratory Animals of the Commitee of Animal Care and Use of the Regional Cote d’Azur. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Institut de Pharmacologie Moléculaire et Cellulaire (Permit Number: B-06-152-5, delivered by the Veterinary Services of the Alpes-Maritimes Prefecture) and by the animal use committees at the Albert Einstein

College of Medicine. All efforts were made to minimize suffering and provide humane treatment to the animals included in the study. We used the L. monocytogenes 10403s background strain in all experiments, either wt or deleted in the secA2 gene, expressing or not GFP 16. Wt Lm-OVA was a kind gift from Hao Shen (University of Pennsylvania, PA, USA). For infections, Lm were grown to log phase (OD600∼0.05–0.15) in broth heart infusion (BHI) medium (Sigma-Aldrich), diluted in PBS and injected in the lateral tail vein. For Lm titers, organs were Ensartinib mw dissociated on metal screens (water 0.1% Triton X-100), and serial dilutions plated onto broth heart infusion plates. Spleens were digested 20 min at 37°C in HBSS (Invitrogen) containing 4000 U/mL collagenase I (Invitrogen) and 0.1 mg/mL

DNase I (Roche). Red blood cells were lysed for 5 min in 170 mM NH4Cl, 17 M Tris-HCl and pH 7.4. All fluorochrome-labeled mAbs are listed in the Supporting Information Table S1. PE-conjugated LLO91-99/H2-Kd Fludarabine tetramers were obtained from the NIH tetramer core facility. Splenocytes were stained with the specified antibodies in PBS containing 0.5% BSA (FACS buffer). For surface staining, cells were incubated for 20 min on ice. For intracellular staining, splenocytes were incubated for 4 h at 37°C, 5% CO2 in RPMI1640 (Invitrogen) 5% FBS, 2 μg/mL Golgi Plug (BD) with or without 100 nM LLO91–99 peptide (Mimotopes), fixed in 1% paraformaldehyde/FACS buffer 10 min, incubated 20 min in 1× Perm/Wash (BD). Cells were analyzed on a FACSCalibur cytofluorometer (BD). When indicated, cells were sorted on a FACSVantage SE cell sorter (BD). Organs were homogenized in PBS containing a complete protease inhibitor cocktail (Roche), centrifuged 10 min 12 000×g. The supernatants were incubated with the BD Cytometric Bead Assay Flex Sets and analyzed using a FACS Array (BD).

The effect of OPN on osteoclasts suggests that the bone loss secondary to endodontic infection that we observed in OPN-deficient mice might be restricted by the osteoclast defect, and could be more severe in the absence of this defect. Alternatively, factors may be produced during the course of the response to infection that can override the osteoclast defect, as has been suggested in bone loss associated with metastatic tumour growth in the bone.20,58 Mice infected with M. bovis develop granulomas, and the number and size of these granulomas are higher in mice deficient for OPN expression.31 This effect was shown to be unrelated to the adaptive immune response; rather there was a defect in bacterial killing

by OPN-deficient macrophages. Hence, the effect of OPN in our model of endodontic infection seems to resemble the host response to M. bovis. It is not clear if the mechanism RG7420 price of host response is the same in both these models, but this similarity illustrates the generality of the OPN dependency of aspects of the innate immune response. In conclusion, our results suggest that OPN has a protective effect in endodontic infections at least partially through an effect

on neutrophil persistence. A possible mechanism for these observations is that OPN deficiency may affect macrophage recruitment or function, such that macrophage-dependent neutrophil BI 2536 cell line clearance is impaired. Understanding the mechanism of action of OPN in these infections may lead to new therapeutic approaches to treat polymicrobial infections. The authors thank Martha O’Hara for help with immunohistochemistry, and Justine Dobeck for expert tissue sectioning. This work was supported by grant DK067685 from the NIDDK/NIH (SRR) and by the High-Tech Research Center Program

at Private Universities from the Japanese Ministry of Education, Culture, Sports, Science, and Technology. The authors report no conflicts of interest. Megestrol Acetate “
“Specific pro-inflammatory cytokine profiles in plasma may characterize women with recurrent miscarriage (RM) but the dynamics of the cytokine profiles with progressing pregnancy is largely unknown. Plasma was repeatedly sampled in the first trimester from 47 RM patients. The concentrations of five cytokines including tumour necrosis factor alpha (TNF-α) were measured. TNF-α levels were correlated to carriage of five TNFA promoter polymorphisms. TNF-α levels increased (P = 0.014) with progressing pregnancy, with higher levels in secondary than primary RM (P = 0.042) but with no significant impact on outcome. Carriage of TNFA -863C and TNFA -1031T was associated with higher TNF-α levels, and the former was found more often in secondary than primary RM (P < 0.02). Plasma TNF-α levels increase during early pregnancy in RM women regardless of outcome, but are higher in secondary than primary RM, which may be partly genetically determined. "
“Tuberculosis (TB) constitutes the major cause of death due to infectious diseases.

1 GN,[62] murine diabetic nephropathy,[63, 64] and the non-immune-mediated renal disease models UUO[65, 66] and IR injury.[67, 68] CCR2 and CX3CR1 KO mice displayed significant renoprotection from IR injury, whereas CCL2 KO mice do not show attenuation of disease possible because of compensatory actions from other ligands.[67] It is unclear whether CCR2 and CX3CR1 are acting in synergy or independently of each

other within this model, but CCR2 Ly6Chi monocyte infiltration within atherosclerotic plaques is CX3CR1 dependent.[69] Cytokines also play a major role in monocyte recruitment to the kidney following injury and the production of CSF-1 protein Afatinib nmr is pivotal to the macrophage response. Both the glomerular and tubulointerstitial compartments produce CSF-1 during chronic injury,[70] renal cell carcinoma[71] and in in vitro cell culture[72, 73] with the tubular epithelium

being the major site for CSF-1 production during chronic experimental kidney disease.[70] In the autoimmune lupus nephritis model in MRL-Faslpr mice, CSF-1 production fuels the intrarenal accumulation, proliferation and activation of macrophages that leads to disease progression.[74, 75] The therapeutic potential of targeting CSF-1 signalling in renal buy Metformin macrophages has recently been investigated using small-molecule inhibitors of tyrosine kinase activity of the CSF-1 receptor (CSF-1R).[76] The inhibitor effectively prevented complete monocyte/macrophage accumulation in the obstructed rat kidney together with reduced tubular apoptosis.[76] However, in experimental models of acute renal disease, CSF-1 exerts M2-reparative effects on macrophages Cyclooxygenase (COX) resulting in improved renal structural and functional recovery.[28] CSF-1 also induces growth-promoting effects in the embryonic kidney with a clear expansion of macrophages that leads to an increased number of ureteric branch tips and developing nephrons.[77]

The pro-inflammatory cytokines TNF-α, IL-1, and IFN-γ also promote monocyte and macrophage infiltration to sites of renal injury. These pro-inflammatory cytokines induce the expression of adhesion molecules on the endothelium to mediate monocyte migration into tissue and stimulate further production of cytokines.[57] Following monocyte infiltration into the kidney, conditions within the local microenvironment govern the selective differentiation into macrophages or DCs. The precise mechanism by which monocytes differentiate into these cells is highly controversial and unclear because of their phenotypic and functional similarities.[78] Like macrophages, DCs also represent a heterogeneous population of cells that are subdivided based on phenotype, function and tissue distribution.[79] There are two major classes of DCs, these include classical DCs and plasmacytoid DCs. Classical DCs are professional antigen-presenting cells that activate and regulate the adaptive immune response.

Moreover, in the subgroup of check details patients without previous immunosuppressant treatment, there was no disability progression during the treatment period. Hence, mycophenolate mofetil might serve as an alternative therapy for RRMS [41]. Moreover, recent studies examined the safety and efficacy of combinations of ‘classic’ immunosuppressive

drugs with recombinant IFN-β and showed equivocal results [42]. Moreover, some novel oral immunomodulatory drugs have recently been tested alone or in combination with IFN-β or GA in Phase III trials in patients with CIS or RRMS (see below). A parallel approach, however, is lacking in CIDP. Mitoxantrone is an anthracenedione derivative related to the anthracyclines doxorubicin and daunorubicin. It interacts with topoisomerase-2, stabilizes its cleavable complex with DNA, and thus prevents the ligation of DNA strands and consecutively delays cell-cycle progression. Preparations and administration: mitoxantrone is approved in Europe for the disease-modifying monotherapy of patients with highly active RRMS and SPMS

Temsirolimus mouse (‘escalation therapy’) [43]. Its use, however, is limited by cardiotoxicity (the standard cumulative lifetime dose of mitoxantrone is 96 mg/m2, which can be extended up to a maximum lifetime dose of 140 mg/m2 under careful risk–benefit weighting and monitoring) and the risk of therapy-associated leukaemia (especially acute myelogenous leukaemia, AML). Given these limitations and the broadening spectrum of drugs available for patients with highly active RRMS, the use of mitoxantrone is limited in clinical practice to patients with SPMS. Mitoxantrone is administered intravenously at a dosage of 12 mg/m2 every 3 months for a total of 2 years, according to the mitoxantrone

in MS study (MIMS) [44]. To extend the total administration period, the dosage can be reduced to 5 mg/m2 upon clinical stabilization. PIK3C2G Clinical trials: there are no recent clinical trials with mitoxantrone in MS. Moreover, due to a lack of evidence from randomized, controlled clinical trials the use of mitoxantrone in CIDP is not established. Adverse effects, frequent: secondary amenorrhoea/azoospermia, nausea and vomiting, myelosuppression; infrequent: alopecia, cardiotoxicity, secondary leukaemia (especially AML) [45, 46]. Contraindications: severe active infections, chronic or relapsing infections, cardiomyopathy, treatment with other cardiotoxic drugs, severe liver or kidney dysfunction, pregnancy and lactation. Due to a lack of evidence from randomized, controlled clinical trials, the use of cyclophosphamide in MS and CIDP is not properly established [25, 47]. Teriflunomide is the biologically active metabolite of leflunomide, which is approved for the treatment of rheumatoid arthritis.

Transfer experiments of iNKT cell subsets reveal the pathogenic role of CD4− iNKT cells containing the iNKT17 cell population in the development of diabetes. Reconstitution of immunodeficient

NOD mice with CD4− iNKT cells enhanced the incidence of diabetes after injection of a low dose of BDC2.5 T cells. Similar exacerbation of diabetes incidence was observed Trichostatin A in vivo after reconstitution with the NK1.1− CD4− iNKT cell population, which exhibits a high frequency of iNKT17 cells. However, due to cell number limitations most of our experiments were performed with the whole CD4− iNKT cell population. Treatment with anti-IL-17 antibodies abolished the pathogenic role of CD4− iNKT cells suggesting that iNKT17 cells are the critical players in the exacerbation Rucaparib order of diabetes, however, we cannot rule out that other cell types producing IL-17 are also participating.

Unfortunately, we could not directly demonstrate that only iNKT17 cells were involved in the deleterious effect of CD4− iNKT cells since there is presently no specific surface marker to purify this cell population. IFN-γ is also produced by CD4− iNKT cells and this cytokine could also participate in the exacerbation of diabetes; however, no exacerbation was observed after reconstitution with NK1.1+ CD4− iNKT cells producing high amounts of IFN-γ but low levels of IL-17. Of note, CD4− iNKT cells alone do not induce diabetes after transfer into immunodeficient NOD mice (data not shown). Therefore, we can propose that iNKT17 cells enhanced diabetes Venetoclax purchase incidence through different mechanisms. In vitro data have shown that IL-17 synergizes with other cytokines

such as IFN-γ and IL-1α/β to induce iNOS expression and subsequent NO production in insulinoma cells or in pancreatic islets of NOD mice 42. Similarly in the pancreas, IL-17 produced by iNKT cells could synergize with IFN-γ secreted by BDC2.5 T cells to induce high expression of NO in β-cells resulting in their destruction. A deleterious loop could take place since β-cell death induced by NO would promote self-antigen presentation by DCs to BDC2.5 T cells. This mechanism could explain the higher frequency of BDC2.5 T cells observed in the PLNs and the pancreas of mice transferred with CD4− iNKT cells as compared with mice devoid of iNKT cells. Furthermore, it has been shown that IL-17A and IL-17F can induce CXCL10 chemokine expression in lung epithelial cells 43, 44. Production of CXCL10 by pancreatic β-cells could contribute to the recruitment of auto reactive T cells expressing the CXCR3 chemokine receptor as previously shown in several mouse models of type 1 diabetes (T10) 45, 46. Thus, iNKT17 cells might not be involved in the initiation of the insulitis but rather could participate in the exacerbation of -β-cell death and diabetes onset. Our data reveal a functional dichotomy between CD4+ and CD4− iNKT cell subsets in the control of diabetes development.

6). We found no significant changes in the expression of activation or apoptosis markers on CD4+ or CD8+ T cells or in the fractions of the DC subsets. Because of a low number of subjects BMS-777607 clinical trial converting to QFT negative after treatment (4/20), we could not perform statistical analyses of possible differences between converters and subjects

who remained QFT positive (13/20). However, there seems to be a trend towards increased expression of HLA-DR and CD38 on CD8+ T cells in subjects who remained QFT positive indicating persistent immune activation. The subjects converting to QFT negative contributed predominantly to the increase in foxp3+ Treg seen after therapy (data not shown). The role of the various T cell and DC subsets in TB infection and their contribution to immunopathogenesis in disease progression has not been clarified. We found that the level of blood Treg,

identified as CD4+CD25+CD127− T cells, was higher in both the active TB and the LTBI groups compared to QFT-negative controls. In contrast, increased T cell activation was predominately found in the active TB group. The proportions of mDC and pDC subsets were comparable between the study groups. After 3 months of preventive anti-tuberculous therapy, there was an increase in the fraction of AZD1208 molecular weight foxp3+ Treg in patients with LTBI , but we observed no differences in the expression of activation or apoptosis markers on T cells. Increased levels of T cell activation have been described in patients with active pulmonary TB and are even more pronounced in HIV/TB co-infected patients [2, 3]. Consistent with these studies, we found an increased expression of the activation markers CD38 and HLA-DR and a corresponding lower expression of the co-stimulatory molecule CD28 on CD8+ T cells from patients with active TB. The level of CD4+ T cell activation was also increased in patients with active TB. Although large variations among the subjects in the LTBI group were seen, our data indicate that immune activation Liothyronine Sodium gradually increases throughout the various stages of TB infection corresponding to the level of bacterial burden. There have been few

studies of Treg in patients with LTBI [21]. High levels of circulating Treg have previously been found in patients with active TB [10–12], but our data demonstrate that CD127-negative Treg are elevated already from the latent stage of infection. Studies have shown that CD4+CD25high+foxp3+ Treg cells are elevated in active TB compared with both uninfected controls [10] and subjects with LTBI [11, 12]. In another study, the level of Treg in patients with active TB decreased after 1 month of anti-tuberculous therapy [13]. In a TB case contact study, the level of foxp3 mRNA was lower in the TB ELISPOT-positive contacts compared to the TB ELISPOT-negative contacts and both groups had lower levels than that found in patients with active TB [22].

C57BL/6 mice (2 months old) were i.n. infected with 5 HAU of influenza virus. After 3 days, lung mononuclear cells were isolated from infected mice or uninfected mice, and then the cell suspensions were layered on a Histopaque-1083 gradient (Sigma-Aldrich), and centrifuge at 400 × g for 30 min at room temperature. Subsequently NK cells were purified using a negative selection mouse NK cell enrichment kit (StemCell Technologies), and labeled by CellTrace™

Violet (Invitrogen Corporation). As described previously [52, 53], 2 × 106 NK cells in 0.25 mL PBS were injected i.v. into recipient mice via the tail vein. On the same selleck chemicals day, the mice were i.n. infected with 5 HAU of influenza A/PR8 virus. After infection, NK cells from lung and spleen were analyzed by flow cytometry 15 h later. The survival rate and body weight of

infected mice were monitored daily. Two months Midostaurin chemical structure old C57BL/6 mice were i.n. infected with 5 HAU of influenza virus or normal egg allantoic fluid on day 0. At days 2, 4, and 6 after infection, mice were euthanized and lungs were isolated and fixed in 10% buffered formalin, then embedded in paraffin and sectioned. Specimens were stained with H&E and examined using a Zeiss Axio Imager M1 microscope equipped with an AxioCam HRc camera under control of AxioVision 4 software (Carl Zeiss Canada Ltd.). GraphPad Prism 4.00 (GraphPad Software, Inc., San Diego, CA, USA) was used for all analyses. Differences among experimental groups were assessed by one-way ANOVA followed by Tukey multiple comparison test. Unpaired t-test (two-tailed) was used to compare pairs of groups. Survival curves were assessed by survival analysis in Prism. Values were reported as the mean ± SEM. This work was supported by operating grants from the Canadian Institutes for Health Research (to K.P.K.). We thank Suellen Lamb, Dr. L. Tyrrell Laboratory, University of Alberta for making histologic sections

and performing hematoxylin and eosin staining. We thank Donger Gong for her technical support. The authors declare no financial or commercial conflict of interest. “
“The description of highly many exposed individuals who remain seronegative (HESN) despite repeated exposure to human immunodeficiency virus (HIV)-1 has heightened interest in identifying potential mechanisms of HIV-1 resistance. HIV-specific humoral and T cell-mediated responses have been identified routinely in HESN subjects, although it remains unknown if these responses are a definitive cause of protection or merely a marker for exposure. Approximately half of HESN lack any detectible HIV-specific adaptive immune responses, suggesting that other mechanisms of protection from HIV-1 infection also probably exist.