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Studies have long sought specific cytokines that could characterize endometriosis. Either due to variations between study designs regarding the assessment criteria for the cytokine or to low power resulting from small sample size, no factor proved to be sufficiently specific to endometriosis. In other clinical fields, a combination of several markers proved to be more powerful than a single-molecule approach. As well, in the context of endometriosis, simultaneous assessment of several cytokines present in the peritoneal fluid might help in unveiling patho-physiological processes, thus contributing to a better understanding of the condition. Therefore, the objective of this study was to investigate peritoneal fluid cytokines-derived of endometriotic women. For this retrospective case-control study, peritoneal fluid samples were obtained at laparoscopy and assessed by multiplex. Our data showed distinct patterns of peritoneal fluid cytokine concentrations in endometriotic women most notably a marked increase in EGF, FGF-2, IL-1α, MIP-1β, TGFα, PDGF-AA, PDGF-BB, MCP-3, sCD40L, Gro Pan, IL-17α, MDC and Rantes. The overall effect of fertility status revealed a significant difference for only one cytokine, namely MDC. Furthermore, FLT-3L and IP-10 levels were decreased in endometriosis patients, the former in both menstrual cycle phases and the latter in the secretory phase. A significant inverse Pearson correlation (p<0.05) was noted between pro-angiogenic cytokines EGF and FGF and the anti-angiogenic cytokine IP-10 in endometriosis patients at stages III-IV and in the secretory phase. These changes may exacerbate the local peritoneal angiogenic and proliferative reaction observed in women with endometriosis, and contributes to its pathophysiology., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Large numbers of melanoma lesions develop resistance to targeted inhibition of mutant BRAF or fail to respond to checkpoint blockade. We explored whether modulation of intratumoral antigen-presenting cells (APCs) could increase responses to these therapies. Using mouse melanoma models, we found that CD103(+) dendritic cells (DCs) were the only APCs transporting intact antigens to the lymph nodes and priming tumor-specific CD8(+) T cells. CD103(+) DCs were required to promote anti-tumoral effects upon blockade of the checkpoint ligand PD-L1; however, PD-L1 inhibition only led to partial responses. Systemic administration of the growth factor FLT3L followed by intratumoral poly I:C injections expanded and activated CD103(+) DC progenitors in the tumor, enhancing responses to BRAF and PD-L1 blockade and protecting mice from tumor rechallenge. Thus, the paucity of activated CD103(+) DCs in tumors limits checkpoint-blockade efficacy and combined FLT3L and poly I:C therapy can enhance tumor responses to checkpoint and BRAF blockade., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Visceral obesity is independently related to numerous cardiometabolic alterations, with adipose tissue dysfunction as a central feature., Objective: To examine whether omental (OM) and subcutaneous (SC) adipocyte size populations in women relate to visceral obesity, cardiometabolic risk factors and adipocyte lipolysis independent of total adiposity., Design and Methods: OM and SC fat samples were obtained during gynecological surgery in 60 women (mean age, 46.1±5.9 years; mean BMI, 27.1±4.5 kg/m² (range, 20.3-41.  kg/m²)). Fresh samples were treated with osmium tetroxide and were analyzed with a Multisizer Coulter. Cell size distributions were computed for each sample with exponential and Gaussian function fits., Results: Computed tomography-measured visceral fat accumulation was the best predictor of larger cell populations as well as the percentage of small cells in both OM and SC fat (P<0.0001 for all). Accordingly, women with visceral obesity had larger cells in the main population and higher proportion of small adipocytes independent of total adiposity (P≤0.05). Using linear regression analysis, we found that women characterized by larger-than-predicted adipocytes in either OM or SC adipose tissue presented higher visceral adipose tissue area, increased percentage of small cells and homeostasis model assessment insulin resistance index as well as higher OM adipocyte isoproterenol-, forskolin- and dbcAMP-stimulated lipolysis compared to women with smaller-than-predicted adipocytes, independent of total adiposity (P≤0.05)., Conclusion: Excess visceral adipose tissue accumulation is a strong marker of both adipocyte hypertrophy and increased number of small cells in either fat compartment, which relates to higher insulin resistance index and lipolytic response, independent of total adiposity., (© 2016 European Society of Endocrinology.)

Objective: To assess the ability of CT-derived measurements including adipose tissue attenuation and area to predict fat cell hypertrophy and related cardiometabolic risk., Methods: Abdominal adipose tissue areas and radiologic attenuation were assessed using 4 CT images in 241 women (age: 47 years, BMI: 26.5 kg/m(2)). Fat cell weight was measured in paired VAT and SAT samples. Fasting plasma lipids, glucose and insulin levels were measured., Results: Adipose tissue attenuation was negatively correlated with SAT (r=-0.46) and VAT (r=-0.67) fat cell weight in the corresponding depot (p<0.0001 for both). Women with visceral adipocyte hypertrophy had higher total-, VLDL-, LDL- and HDL-triglyceride and apoB levels as well as a higher cholesterol/HDL-cholesterol ratio, fasting glucose and insulin levels compared to women with smaller visceral adipocytes. Adjustment for VAT area minimized these differences while subsequent adjustment for attenuation eliminated all differences, with the exception of fasting glycaemia. In SAT, adjustment for VAT area and attenuation eliminated all adipocyte hypertrophy-related alterations except for fasting hyperglycaemia., Conclusion: CT-derived adipose tissue attenuation and area both contribute to explain variation in the cardiometabolic risk profile associated with the same biological parameter: visceral fat cell hypertrophy.

Importance: Cutaneous verruca vulgaris lesions (warts) and oral squamous cell papillomas are common lesions caused by human papillomavirus (HPV). Multiple reports have described cases of wart resolution following quadrivalent HPV vaccination. We report the case of a patient with chronic oral papillomas with resolution after quadrivalent HPV vaccination and perform a review of the literature., Observations: An immunocompetent man in his 60s presented with chronic verrucous papules on the lips, tongue, and buccal mucosa refractory to multiple excisions. Biopsy showed squamous cell papilloma, and DNA sequencing revealed HPV-32. He received the quadrivalent HPV vaccine resulting in clearance of all lesions after 3 months. We found 8 reported cases of disseminated, recurrent warts with resolution after quadrivalent HPV vaccination. Improvement was seen within 4 weeks of vaccination, and resolution after 3 to 8 months., Conclusions and Relevance: We report the case of recurrent oral papillomas caused by HPV-32 with complete resolution after quadrivalent HPV vaccination and reviewed reports of resolution of recalcitrant and disseminated warts after vaccination. Production of cross-protective immunoglobulins and cytotoxic T cells is a possible mechanism. There remains a critical need for randomized clinical trials to assess efficacy of quadrivalent HPV vaccination for treatment of oral squamous papillomas and cutaneous verruca vulgaris.

Treatment with ionizing radiation (IR) can lead to the accumulation of tumor-infiltrating regulatory T cells (Treg cells) and subsequent resistance of tumors to radiotherapy. Here we focused on the contribution of the epidermal mononuclear phagocytes Langerhans cells (LCs) to this phenomenon because of their ability to resist depletion by high-dose IR. We found that LCs resisted apoptosis and rapidly repaired DNA damage after exposure to IR. In particular, we found that the cyclin-dependent kinase inhibitor CDKN1A (p21) was overexpressed in LCs and that Cdkn1a(-/-) LCs underwent apoptosis and accumulated DNA damage following IR treatment. Wild-type LCs upregulated major histocompatibility complex class II molecules, migrated to the draining lymph nodes and induced an increase in Treg cell numbers upon exposure to IR, but Cdkn1a(-/-) LCs did not. Our findings suggest a means for manipulating the resistance of LCs to IR to enhance the response of cutaneous tumors to radiotherapy.

We sought to define cellular immune mechanisms of synergy between tumor-antigen-targeted monoclonal antibodies and chemotherapy. Established B16 melanoma in mice was treated with cytotoxic doses of cyclophosphamide in combination with an antibody targeting tyrosinase-related protein 1 (αTRP1), a native melanoma differentiation antigen. We find that Fcγ receptors are required for efficacy, showing that antitumor activity of combination therapy is immune mediated. Rag1(-/-) mice deficient in adaptive immunity are able to clear tumors, and thus innate immunity is sufficient for efficacy. Furthermore, previously treated wild-type mice are not significantly protected against tumor reinduction, as compared with mice inoculated with irradiated B16 alone, consistent with a primarily innate immune mechanism of action of chemo-immunotherapy. In contrast, mice deficient in both classical natural killer (NK) lymphocytes and nonclassical innate lymphocytes (ILC) due to deletion of the IL2 receptor common gamma chain IL2γc(-/-)) are refractory to chemo-immunotherapy. Classical NK lymphocytes are not critical for treatment, as depletion of NK1.1⁺ cells does not impair antitumor effect. Depletion of CD90⁺NK1.1⁻ lymphocytes, however, both diminishes therapeutic benefit and decreases accumulation of macrophages within the tumor. Tumor clearance during combination chemo-immunotherapy with monoclonal antibodies against native antigen is mediated by the innate immune system. We highlight a novel potential role for CD90⁺NK1.1⁻ ILCs in chemo-immunotherapy., (©2015 American Association for Cancer Research.)

Depression and anxiety disorders are associated with increased release of peripheral cytokines; however, their functional relevance remains unknown. Using a social stress model in mice, we find preexisting individual differences in the sensitivity of the peripheral immune system that predict and promote vulnerability to social stress. Cytokine profiles were obtained 20 min after the first social stress exposure. Of the cytokines regulated by stress, IL-6 was most highly up-regulated only in mice that ultimately developed a susceptible behavioral phenotype following a subsequent chronic stress, and levels remained elevated for at least 1 mo. We confirmed a similar elevation of serum IL-6 in two separate cohorts of patients with treatment-resistant major depressive disorder. Before any physical contact in mice, we observed individual differences in IL-6 levels from ex vivo stimulated leukocytes that predict susceptibility versus resilience to a subsequent stressor. To shift the sensitivity of the peripheral immune system to a pro- or antidepressant state, bone marrow (BM) chimeras were generated by transplanting hematopoietic progenitor cells from stress-susceptible mice releasing high IL-6 or from IL-6 knockout (IL-6(-/-)) mice. Stress-susceptible BM chimeras exhibited increased social avoidance behavior after exposure to either subthreshold repeated social defeat stress (RSDS) or a purely emotional stressor termed witness defeat. IL-6(-/-) BM chimeric and IL-6(-/-) mice, as well as those treated with a systemic IL-6 monoclonal antibody, were resilient to social stress. These data establish that preexisting differences in stress-responsive IL-6 release from BM-derived leukocytes functionally contribute to social stress-induced behavioral abnormalities.

Pelvic inflammation is a hallmark of endometriosis pathogenesis and a major cause of the disease's symptoms. Abnormal immune and inflammatory changes may not only contribute to endometriosis-major symptoms, but also contribute to ectopic endometrial tissue growth and endometriosis development. A major pro-inflammatory factors found elevated in peritoneal fluid of women with endometriosis and to be overexpressed in peritoneal fluid macrophages and active, highly vascularized and early stage endometriotic lesions, macrophage migration inhibitory factor (MIF) appeared to induce angiogenic and inflammatory and estrogen producing phenotypes in endometriotic cells in vitro and to be a possible therapeutic target in vivo. Using a mouse model where MIF-knock out (KO) mice received intra-peritoneal injection of endometrial tissue from MIF-KO or syngeneic wild type (WT) mice and vice versa, our current study revealed that MIF genetic depletion resulted in a marked reduction ectopic endometrial tissue growth, a disrupted tissue structure and a significant down regulation of the expression of major inflammatory (cyclooxygenease-2), cell adhesion (αv and β3 integrins), survival (B-cell lymphoma-2) and angiogenic (vascular endothelial cell growth) factors relevant to endometriosis pathogenesis, whereas MIF add-back to MIF-KO mice significantly restored endometriosis-like lesions number and size. Interestingly, cross-experiments revealed that MIF presence in both endometrial and peritoneal host tissues is required for ectopic endometrial tissue growth and pointed to its involvement in endometrial-peritoneal interactions. This study provides compelling evidence for the role of MIF in endometriosis development and its possible interest for a targeted treatment of endometriosis.

Background: Langerhans cells (LCs) are self-renewing epidermal myeloid cells that can migrate and mature into dendritic cells. Recipient LCs that survive cytotoxic therapy given in preparation for allogeneic hematopoietic cell transplantation may prime donor T cells to mediate cutaneous graft-versus-host disease (GVHD). This possible association, however, has not been investigated in the setting of nonmyeloablative allografting., Methods: We prospectively studied the kinetics of LC-chimerism after sex-mismatched allogeneic hematopoietic cell transplantation with nonmyeloablative (n=23) or myeloablative (n=25) conditioning. Combined XY-FISH and Langerin-staining was used to assess donor LC-chimerism in skin biopsies obtained on days 28, 56, and 84 after transplant. The degree of donor LC-chimerism was correlated with the development of skin GVHD., Results: We observed significantly delayed donor LC-engraftment after nonmyeloablative transplantation compared with other hematopoietic compartments and compared with LC-engraftment after myeloablative conditioning. In most recipients of nonmyeloablative transplants, recipient LCs proliferated in situ, recruitment of donor-LCs was delayed by two months, and full donor LC-chimerism was only reached by day 84 after transplant. Although persistence of host LCs on day-28 after transplant was not predictive for acute or chronic skin GVHD, the recruitment of donor-derived LCs was associated with nonspecific inflammatory infiltrates (P=0.009)., Conclusions: These results show that LCs can self-renew locally but are replaced by circulating precursors even after minimally toxic nonmyeloablative transplant conditioning. Cutaneous inflammation accompanies donor LC-engraftment, but differences in LC conversion-kinetics do not predict clinical or histopathological GVHD.

Subcutaneous adipose tissue expansion through adipogenesis is increasingly recognized as a major determinant of body fat distribution and obesity-related cardiometabolic alterations. Our objective was to assess whether adipogenic rates of cultured human primary preadipocytes from the visceral and subcutaneous compartments relate to visceral obesity and cardiometabolic alterations. We recruited 35 women undergoing gynecological surgery and assessed body fat distribution by CT as well as fasting plasma lipids and glycemia. Fat samples from the greater omentum and abdominal subcutaneous (SC) compartments were used to assess mature adipocyte cell size and establish primary preadipocyte cultures. Differentiation was induced using adipogenic media and adipogenic rates were assessed using Oil Red O (ORO) absorbance/DNA content ratio and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) activity/DNA of differentiated cells. We found a lower adipogenic capacity of omental (OM) preadipocytes than SC preadipocytes originating from the same women (P < 0.05). Whereas only OM cell size was different among groups of low vs high OM adipogenic rate, SC adipogenic rates were clearly related to increased OM cell size and dyslipidemia when women were separated on median value of either ORO/DNA or G3PDH activity/DNA ratios. When matched for BMI, women with low SC preadipocyte adipogenic rates had a higher visceral adipose tissue area (P < 0.01), omental adipocyte hypertrophy (P < 0.05), higher VLDL-lipid content (P < 0.01) and higher fasting glycemia (P < 0.05) than those with low SC adipogenic rates. In conclusion, low abdominal subcutaneous preadipocyte differentiation capacity in vitro is associated with visceral obesity, visceral adipocyte hypertrophy, and a dysmetabolic state.

Langerhans cell histiocytosis (LCH) is a clonal disorder with elusive etiology, characterized by the accumulation of CD207(+) dendritic cells (DCs) in inflammatory lesions. Recurrent BRAF-V600E mutations have been reported in LCH. In this study, lesions from 100 patients were genotyped, and 64% carried the BRAF-V600E mutation within infiltrating CD207(+) DCs. BRAF-V600E expression in tissue DCs did not define specific clinical risk groups but was associated with increased risk of recurrence. Strikingly, we found that patients with active, high-risk LCH also carried BRAF-V600E in circulating CD11c(+) and CD14(+) fractions and in bone marrow (BM) CD34(+) hematopoietic cell progenitors, whereas the mutation was restricted to lesional CD207(+) DC in low-risk LCH patients. Importantly, BRAF-V600E expression in DCs was sufficient to drive LCH-like disease in mice. Consistent with our findings in humans, expression of BRAF-V600E in BM DC progenitors recapitulated many features of the human high-risk LCH, whereas BRAF-V600E expression in differentiated DCs more closely resembled low-risk LCH. We therefore propose classification of LCH as a myeloid neoplasia and hypothesize that high-risk LCH arises from somatic mutation of a hematopoietic progenitor, whereas low-risk disease arises from somatic mutation of tissue-restricted precursor DCs.

Neutrophils are the most abundant cell type in the immune system and play an important role in the innate immune response. Using a diverse range of mouse models with either defective dendritic cell (DC) development or conditional DC depletion, we provide in vivo evidence indicating that conventional DCs play an important role in the regulation of neutrophil homeostasis. Flk2, Flt3L, and Batf3 knockout mice, which have defects in DC development, have increased numbers of liver neutrophils in the steady state. Conversely, neutrophil frequency is reduced in DC-specific PTEN knockout mice, which have an expansion of CD8(+) and CD103(+) DCs. In chimeric CD11c-DTR mice, conventional DC depletion results in a systemic increase of neutrophils in peripheral organs in the absence of histological inflammation or an increase in proinflammatory cytokines. This effect is also present in splenectomized chimeric CD11c-DTR mice and is absent in chimeric mice with 50% normal bone marrow. In chimeric CD11c-DTR mice, diphtheria toxin treatment results in enhanced neutrophil trafficking from the bone marrow into circulation and increased neutrophil recruitment. Moreover, there is an increased expression of chemokines/cytokines involved in neutrophil homeostasis and reduced neutrophil apoptosis. These data underscore the role of the DC pool in regulating the neutrophil compartment in nonlymphoid organs.

Unlabelled: Previous studies have suggested altered triglyceride (TG) storage in patients with abdominal obesity and blood lipid disorders., Objective: We hypothesized that women with abdominal obesity and a dysmetabolic profile have low DGAT activity in their abdominal fat compartments., Methods: Paired omental (OM) and subcutaneous (SC) adipose tissue samples were obtained surgically from 39 women undergoing abdominal hysterectomies. Body composition and fat distribution were measured by dual energy x-ray absorptiometry and computed tomography. DGAT activity was measured by acylation of sn-l,2-diacylglycerol with [(14)C] oleoyl-CoA in microsomal fractions isolated from whole adipose tissue homogenates. DGAT activity was calculated on the basis of picomoles (pmol) TG synthesized in the assay per min per mg lipid, per μg protein or per 1000 cells., Results: No depot differences were found when DGAT activity was reported per μg microsomal protein or per 1000 cells. DGAT activity in either depot was not associated with adipocyte diameters and blood lipid profile variables. DGAT activity per mg lipid was higher in OM than in abdominal SC adipose tissue (0.43 ± 0.20 vs. 0.34 ± 0.18 pmol/min/mg lipid, p < 0.05). OM DGAT activity was negatively correlated with OM adipocyte diameter and visceral adipose tissue area (r = -0.43, p < 0.01 and r = -0.38, p < 0.05 respectively). Plasma total, LDL and HDL TG levels were negatively associated with OM DGAT activity independent of total body fat mass (r = -0.39, p < 0.05, r = -0.46, p < 0.001 and r = -0.40, p < 0.05 respectively)., Conclusion: A defect in adipose tissue DGAT activity is predictive of adiposity and blood lipoprotein TG enrichment only when considering activity per tissue lipid mass., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

miR-126 is a microRNA expressed predominately by endothelial cells and controls angiogenesis. We found miR-126 was required for the innate response to pathogen-associated nucleic acids and that miR-126-deficient mice had greater susceptibility to infection with pseudotyped HIV. Profiling of miRNA indicated that miR-126 had high and specific expression by plasmacytoid dendritic cells (pDCs). Moreover, miR-126 controlled the survival and function of pDCs and regulated the expression of genes encoding molecules involved in the innate response, including Tlr7, Tlr9 and Nfkb1, as well as Kdr, which encodes the growth factor receptor VEGFR2. Deletion of Kdr in DCs resulted in reduced production of type I interferon, which supports the proposal of a role for VEGFR2 in miR-126 regulation of pDCs. Our studies identify the miR-126-VEGFR2 axis as an important regulator of the innate response that operates through multiscale control of pDCs.

Objective: To investigate prostaglandin (PG) biosynthesis and catabolism pathways in eutopic and ectopic endometrium of women with endometriosis., Design: Retrospective study., Setting: Human reproduction research laboratory., Patient(s): Forty-five women with endometriosis and 29 normal controls., Intervention(s): Endometrial and endometriotic tissue samples were obtained during laparoscopic surgery., Main Outcome Measure(s): Cyclo-oxygenases (Coxs 1 and 2), PGE2 synthases (microsomal [m] PGES 1 and 2 and cytosolic [c] PGES), PGF2α synthases (aldoketoreductase [AKR]-1C3 and AKR-1B1), and the PG catabolic enzyme 15-hydroxyprostaglandin dehydrogenase messenger RNA expression by quantitative real-time polymerase chain reaction and protein localization by immunohistochemistry., Result(s): This study showed a marked increase in the key PG biosynthesis enzymes Cox-2, mPGES-1, mPGES-2, cPGES, and AKR-1C3 in ectopic endometrial tissue of women with endometriosis, particularly in the earliest and most active stages of the disease, without a noticeable change in the expression of the PG catabolic enzyme 15-hydroxyprostaglandin dehydrogenase. Meanwhile, the significant increase in rate-limiting Cox-2 expression upstream was correlated downstream by a significant stage- and cycle phase-dependent decrease in the terminal specific synthase mPGES-2, thereby revealing the presence of counter-regulatory mechanisms, which operate in the eutopic endometrium of women with endometrium but seem to be lacking in the ectopic implantation sites., Conclusion(s): This study reveals for the first time multiple defects in PG biosynthesis pathways, which differ between eutopic intrauterine and ectopic endometrial tissues and may, owing to the wide spectrum of PG properties, contribute to the initial steps of endometrial tissue growth and development and have an important role to play in the pathogenesis and symptoms of this disease., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Despite accumulating evidence suggesting local self-maintenance of tissue macrophages in the steady state, the dogma remains that tissue macrophages derive from monocytes. Using parabiosis and fate-mapping approaches, we confirmed that monocytes do not show significant contribution to tissue macrophages in the steady state. Similarly, we found that after depletion of lung macrophages, the majority of repopulation occurred by stochastic cellular proliferation in situ in a macrophage colony-stimulating factor (M-Csf)- and granulocyte macrophage (GM)-CSF-dependent manner but independently of interleukin-4. We also found that after bone marrow transplantation, host macrophages retained the capacity to expand when the development of donor macrophages was compromised. Expansion of host macrophages was functional and prevented the development of alveolar proteinosis in mice transplanted with GM-Csf-receptor-deficient progenitors. Collectively, these results indicate that tissue-resident macrophages and circulating monocytes should be classified as mononuclear phagocyte lineages that are independently maintained in the steady state., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In comparison to murine dendritic cells (DCs), less is known about the function of human DCs in tissues. Here, we analyzed, by using lung tissues from humans and humanized mice, the role of human CD1c(+) and CD141(+) DCs in determining the type of CD8(+) T cell immunity generated to live-attenuated influenza virus (LAIV) vaccine. We found that both lung DC subsets acquired influenza antigens in vivo and expanded specific cytotoxic CD8(+) T cells in vitro. However, lung-tissue-resident CD1c(+) DCs, but not CD141(+) DCs, were able to drive CD103 expression on CD8(+) T cells and promoted CD8(+) T cell accumulation in lung epithelia in vitro and in vivo. CD1c(+) DCs induction of CD103 expression was dependent on membrane-bound cytokine TGF-β1. Thus, CD1c(+) and CD141(+) DCs generate CD8(+) T cells with different properties, and CD1c(+) DCs specialize in the regulation of mucosal CD8(+) T cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A role for macrophages in erythropoiesis was suggested several decades ago when erythroblastic islands in the bone marrow, composed of a central macrophage surrounded by developing erythroblasts, were described. However, the in vivo role of macrophages in erythropoiesis under homeostatic conditions or in disease remains unclear. We found that specific depletion of CD169(+) macrophages markedly reduced the number of erythroblasts in the bone marrow but did not result in overt anemia under homeostatic conditions, probably because of concomitant alterations in red blood cell clearance. However, CD169(+) macrophage depletion significantly impaired erythropoietic recovery from hemolytic anemia, acute blood loss and myeloablation. Furthermore, macrophage depletion normalized the erythroid compartment in a JAK2(V617F)-driven mouse model of polycythemia vera, suggesting that erythropoiesis in polycythemia vera remains under the control of macrophages in the bone marrow and splenic microenvironments. These results indicate that CD169(+) macrophages promote late erythroid maturation and that modulation of the macrophage compartment may be a new strategy to treat erythropoietic disorders.

Colony stimulating factor-1 (Csf-1) receptor and its ligand Csf-1 control macrophage development, maintenance, and function. The development of both Langerhans cells (LCs) and microglia is highly dependent on Csf-1 receptor signaling but independent of Csf-1. Here we show that in both mice and humans, interleukin-34 (IL-34), an alternative ligand for Csf-1 receptor, is produced by keratinocytes in the epidermis and by neurons in the brain. Mice lacking IL-34 displayed a marked reduction of LCs and a decrease of microglia, whereas monocytes, dermal, and lymphoid tissue macrophages and DCs were unaffected. We identified IL-34 as a nonredundant cytokine for the development of LCs during embryogenesis as well as for their homeostasis in the adult skin. Whereas inflammation-induced repopulation of LCs appears to be dependent on Csf-1, once inflammation is resolved, LC survival is again IL-34-dependent. In contrast, microglia and their yolk sac precursors develop independently of IL-34 but rely on it for their maintenance in the adult brain., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Endometriosis is often associated with a chronic pelvic immuno-inflammatory process, which is closely related to disease pathogenesis and major symptoms. Our studies led to the detection of a marked imbalance between IL-1 and its natural inhibitor IL-1 receptor type 2 (IL1R2) in women with endometriosis. This points to a deficiency in the local control of IL-1 that, in view of the cytokine's elevated levels and potent proinflammatory, angiogenic, and growth-promoting effects, may contribute to endometriosis development. Using an in vivo model in which human endometrial tissue was inoculated into nude mice and left to establish before any further treatment, our data showed that sIL1R2 interferes with the capability of endometrial tissue to invade, grow, disseminate, and stimulate angiogenesis into the host tissue. sIL1R2 significantly down-regulated the expression of major cell adhesion receptors (αv and β3 integrins), matrix metalloproteinases (MMP-2 and -9), and vascular endothelial cell growth factor. Interestingly, treatment with sILR2 (5 μg/kg) led to a concomitant upregulation of matrix metalloproteinases natural inhibitors (TIMP1 and TIMP2) and down-regulation of BclII, a potent anti-apoptotic protein. This creates an imbalance between pro- and anti-proteolytic and apoptotic factors and may further contribute to IL1R2 growth-inhibitory effects. This study provides evidence that sIL1R2 alters ectopic endometrial tissue growth, remodeling, and survival in vivo and may represent an interesting potential therapeutic tool., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Although peroxisome proliferator-activated receptor γ (PPARγ) has anti-inflammatory actions in macrophages, which macrophage populations express PPARγ in vivo and how it regulates tissue homeostasis in the steady state and during inflammation remains unclear. We now show that lung and spleen macrophages selectively expressed PPARγ among resting tissue macrophages. In addition, Ly-6C(hi) monocytes recruited to an inflammatory site induced PPARγ as they differentiated to macrophages. When PPARγ was absent in Ly-6C(hi)-derived inflammatory macrophages, initiation of the inflammatory response was unaffected, but full resolution of inflammation failed, leading to chronic leukocyte recruitment. Conversely, PPARγ activation favored resolution of inflammation in a macrophage PPARγ-dependent manner. In the steady state, PPARγ deficiency in red pulp macrophages did not induce overt inflammation in the spleen. By contrast, PPARγ deletion in lung macrophages induced mild pulmonary inflammation at the steady state and surprisingly precipitated mortality upon infection with Streptococcus pneumoniae. This accelerated mortality was associated with impaired bacterial clearance and inability to sustain macrophages locally. Overall, we uncovered critical roles for macrophage PPARγ in promoting resolution of inflammation and maintaining functionality in lung macrophages where it plays a pivotal role in supporting pulmonary host defense. In addition, this work identifies specific macrophage populations as potential targets for the anti-inflammatory actions of PPARγ agonists.

Immune-endocrine interplay may play a major role in the pathogenesis of endometriosis. In the present study, we have investigated the interaction between macrophage migration inhibitory factor (MIF), a major pro-inflammatory and growth-promoting factor markedly expressed in active endometriotic lesions, and estradiol (E(2)) in ectopic endometrial cells. Our data showed a significant increase of MIF protein secretion and mRNA expression in endometriotic cells in response to E(2). MIF production was blocked by Fulvestrant, an estrogen receptor (ER) antagonist, and induced by ERα and ERβ selective agonists propyl-pyrazole-triol (PPT) and diarylpropionrile (DPN), respectively, thus demonstrating a specific receptor-mediated effect. Cell transfection with MIF promoter construct showed that E(2) significantly stimulates MIF promoter activity. Interestingly, our data further revealed that MIF reciprocally stimulates aromatase protein and mRNA expression via a posttranscriptional mRNA stabilization mechanism, that E(2) itself can upregulate aromatase expression, and that inhibition of endogenous MIF, using MIF specific siRNA, significantly inhibits E(2)-induced aromatase. Thus, the present study revealed the existence of a local positive feedback loop by which estrogen acts directly on ectopic endometrial cells to upregulate the expression of MIF, which, in turn, displays the capability of inducing the expression of aromatase, the key and rate-limiting enzyme for estrogen synthesis. Such interplay may have a considerable impact on the development of endometriosis., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Although much progress has been made in the understanding of the ontogeny and function of dendritic cells (DCs), the transcriptional regulation of the lineage commitment and functional specialization of DCs in vivo remains poorly understood. We made a comprehensive comparative analysis of CD8(+), CD103(+), CD11b(+) and plasmacytoid DC subsets, as well as macrophage DC precursors and common DC precursors, across the entire immune system. Here we characterized candidate transcriptional activators involved in the commitment of myeloid progenitor cells to the DC lineage and predicted regulators of DC functional diversity in tissues. We identified a molecular signature that distinguished tissue DCs from macrophages. We also identified a transcriptional program expressed specifically during the steady-state migration of tissue DCs to the draining lymph nodes that may control tolerance to self tissue antigens.

GM-CSF (Csf-2) is a critical cytokine for the in vitro generation of dendritic cells (DCs) and is thought to control the development of inflammatory DCs and resident CD103(+) DCs in some tissues. Here we showed that in contrast to the current understanding, Csf-2 receptor acts in the steady state to promote the survival and homeostasis of nonlymphoid tissue-resident CD103(+) and CD11b(+) DCs. Absence of Csf-2 receptor on lung DCs abrogated the induction of CD8(+) T cell immunity after immunization with particulate antigens. In contrast, Csf-2 receptor was dispensable for the differentiation and innate function of inflammatory DCs during acute injuries. Instead, inflammatory DCs required Csf-1 receptor for their development. Thus, Csf-2 is important in vaccine-induced CD8(+) T cell immunity through the regulation of nonlymphoid tissue DC homeostasis rather than control of inflammatory DCs in vivo., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Langerhans cells (LCs) are the dendritic cells (DCs) of the epidermis, forming one of the first hematopoietic lines of defense against skin pathogens. In contrast to other DCs, LCs arise from hematopoietic precursors that seed the skin before birth. However, the origin of these embryonic precursors remains unclear. Using in vivo lineage tracing, we identify a first wave of yolk sac (YS)-derived primitive myeloid progenitors that seed the skin before the onset of fetal liver hematopoiesis. YS progenitors migrate to the embryo proper, including the prospective skin, where they give rise to LC precursors, and the brain rudiment, where they give rise to microglial cells. However, in contrast to microglia, which remain of YS origin throughout life, YS-derived LC precursors are largely replaced by fetal liver monocytes during late embryogenesis. Consequently, adult LCs derive predominantly from fetal liver monocyte-derived cells with a minor contribution of YS-derived cells. Altogether, we establish that adult LCs have a dual origin, bridging early embryonic and late fetal myeloid development.

Delta-like ligand 4 (Dll4)-Notch signaling is essential for T cell development and alternative thymic lineage decisions. How Dll4-Notch signaling affects pro-T cell fate and thymic dendritic cell (tDC) development is unknown. We found that Dll4 pharmacological blockade induces accumulation of tDCs and CD4(+)CD25(+)FoxP3(+) regulatory T cells (T(reg) cells) in the thymic cortex. Both genetic inactivation models and anti-Dll4 antibody (Ab) treatment promote de novo natural T(reg) cell expansion by a DC-dependent mechanism that requires major histocompatibility complex II expression on DCs. Anti-Dll4 treatment converts CD4(-)CD8(-)c-kit(+)CD44(+)CD25(-) (DN1) T cell progenitors to immature DCs that induce ex vivo differentiation of naive CD4(+) T cells into T(reg) cells. Induction of these tolerogenic DN1-derived tDCs and the ensuing expansion of T(reg) cells are Fms-like tyrosine kinase 3 (Flt3) independent, occur in the context of transcriptional up-regulation of PU.1, Irf-4, Irf-8, and CSF-1, genes critical for DC differentiation, and are abrogated in thymectomized mice. Anti-Dll4 treatment fully prevents type 1 diabetes (T1D) via a T(reg) cell-mediated mechanism and inhibits CD8(+) T cell pancreatic islet infiltration. Furthermore, a single injection of anti-Dll4 Ab reverses established T1D. Disease remission and recurrence are correlated with increased T(reg) cell numbers in the pancreas-draining lymph nodes. These results identify Dll4-Notch as a novel Flt3-alternative pathway important for regulating tDC-mediated T(reg) cell homeostasis and autoimmunity.

Acute graft-versus-host disease (GVHD) results from the attack of host tissues by donor allogeneic T cells and is the most serious limitation of allogeneic hematopoietic cell transplantation (allo-HCT). Host antigen-presenting cells are thought to control the priming of alloreactive T cells and the induction of acute GVHD after allo-HCT. However, whereas the role of host DC in GVHD has been established, the contribution of host macrophages to GVHD has not been clearly addressed. We show that, in contrast to DC, reducing of the host macrophage pool in recipient mice increased donor T cell expansion and aggravated GVHD mortality after allo-HCT. We also show that host macrophages that persist after allo-HCT engulf donor allogeneic T cells and inhibit their proliferation. Conversely, administration of the cytokine CSF-1 before transplant expanded the host macrophage pool, reduced donor T cell expansion, and improved GVHD morbidity and mortality after allo-HCT. This study establishes the unexpected key role of host macrophages in inhibiting GVHD and identifies CSF-1 as a potential prophylactic therapy to limit acute GVHD after allo-HCT in the clinic.

Objective: The objective of this study was to assess adolescents' attitudes towards the contraceptive vaginal ring (CVR). We also aimed to identify the factors associated with their willingness to use it., Methods: We conducted a descriptive cross-sectional study. Fifty-nine female volunteers, aged 14 to 18 years, were recruited in a clinical setting. The participants completed a self-administered questionnaire collecting information on sexual and contraceptive history as well as personal and demographic data. After reading information describing the CVR, they were asked about their perceptions of and willingness to adopt this contraceptive method. We used a logistic regression model to assess factors associated with their willingness to use the CVR., Results: Our population was mostly composed of coitally experienced (86%), high school level (71%) adolescents living with their parents (97%). Only a minority (17%) were willing to use the CVR, while 39% remained undecided. Participants were concerned about experiencing vaginal discomfort and difficulty in inserting or removing the ring. They also feared that it would interfere with their sexual activities or cause urinary tract infections, leukorrhea, or vaginitis. Willingness to adopt the CVR increased with the number of contraceptive methods already used (OR = 1.92; P = 0.015) and was not associated with a lack of contraceptive compliance., Conclusions: A minority of adolescents are willing to use the CVR, but many are ambivalent. Health care professionals should focus on addressing concerns about vaginal insertion. Adolescents who tend to be the most interested in the CVR are those who have experience with a greater number of contraceptives.

Objective: To investigate interleukin (IL) 1 receptor accessory protein (IL1RAcP) expression in the eutopic endometrium of women with endometriosis., Design: Retrospective study., Setting: Human reproduction research laboratory., Patient(s): Sixty-six women with endometriosis and 60 healthy women with no laparoscopic evidence of endometriosis., Intervention(s): Endometrial tissue samples were obtained during laparoscopic surgery., Main Outcome Measure(s): IL1RAcP protein expression was analyzed by immunohistochemistry, Western blot, and ELISA, and IL1RAcP mRNA expression was analyzed by quantitative real-time polymerase chain reaction., Result(s): This study showed a significant downregulation of soluble (s)IL1RAcP expression in the eutopic endometrium of women with endometriosis compared to normal women, occurring at the protein or mRNA level. This finding appeared to vary according to endometriosis stage, being more obvious in the earliest (I and II) than the latest (III and IV) stages of the disease. However, the membrane-bound IL1RAcP did not show any noticeable endometriosis-related change, neither at the protein or mRNA level., Conclusion(s): In view of the wide spectrum of IL-1 inflammatory and growth-promoting effects, downregulation of sIL1RAcP, which is known for inhibiting IL1, indicates a profound defect in the capability of endometrial cells of women with endometriosis to counterregulate IL-1 effects and may represent an important mechanism underlying the ability of these cells to implant and develop into host tissues., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Hematopoietic stem cells (HSCs) reside in specialized bone marrow (BM) niches regulated by the sympathetic nervous system (SNS). Here, we have examined whether mononuclear phagocytes modulate the HSC niche. We defined three populations of BM mononuclear phagocytes that include Gr-1(hi) monocytes (MOs), Gr-1(lo) MOs, and macrophages (MΦ) based on differential expression of Gr-1, CD115, F4/80, and CD169. Using MO and MΦ conditional depletion models, we found that reductions in BM mononuclear phagocytes led to reduced BM CXCL12 levels, the selective down-regulation of HSC retention genes in Nestin(+) niche cells, and egress of HSCs/progenitors to the bloodstream. Furthermore, specific depletion of CD169(+) MΦ, which spares BM MOs, was sufficient to induce HSC/progenitor egress. MΦ depletion also enhanced mobilization induced by a CXCR4 antagonist or granulocyte colony-stimulating factor. These results highlight two antagonistic, tightly balanced pathways that regulate maintenance of HSCs/progenitors in the niche during homeostasis, in which MΦ cross talk with the Nestin(+) niche cell promotes retention, and in contrast, SNS signals enhance egress. Thus, strategies that target BM MΦ hold the potential to augment stem cell yields in patients that mobilize HSCs/progenitors poorly.

Context: Prostaglandins (PGs) E2 and PGF2α are produced in the endometrium and are important for menstruation and fertility. Dysmenorrhea is associated with increased production of PGF2α relative to PGE2, and the opposite is true for menorrhagia. The pathways leading to PGE2 biosynthesis are well described, but little is known for PGF2α. Aldoketoreductase (AKR)-1C3, the only PGF synthase identified in the human, cannot explain the production of PGF2α by endometrial cells. AKR1B1 appears to be an alternate candidate with promising therapeutic value., Objective: The objective of the study was to address whether AKR1B1 (gene ID 231) is a functional PGF2α synthase in the human endometrium and a valid therapeutic target for menstrual pain., Design: The design of the study was basic laboratory analyses to identify gene expression and protein levels associated with PGF2α production in endometrial tissues and endometrial cells from cycling women aged between 23 and 52 yr undergoing biopsies or hysterectomy for diverse gynecological disorders., Results: AKR1B1 is expressed at a high level during the menstrual cycle during the secretory phase and in both epithelial and stromal cells, whereas AKR1C3 was found only in epithelial cells. Purified recombinant AKR1B1 protein, gene silencing, and transient transfection experiments all concur to demonstrate that this enzyme is a functional PGF synthase. Ponalrestat, a specific inhibitor developed to block AKR1B1 activity, reduced PGF2α production in response to IL-1β in both cultured endometrial cells and endometrial explants., Conclusions: The human aldose reductase AKR1B1 currently associated with diabetes complications is also a highly functional PGF synthase responsible for PGF2α production in the human endometrium and a potential target for treatment of menstrual disorders.

Epidermal and hair follicle development from surface ectodermal progenitor cells requires coordinated changes in gene expression. Histone deacetylases alter gene expression programs through modification of chromatin and transcription factors. We find that deletion of ectodermal Hdac1 and Hdac2 results in dramatic failure of hair follicle specification and epidermal proliferation and stratification, phenocopying loss of the key ectodermal transcription factor p63. Although expression of p63 and its positively regulated basal cell targets is maintained in Hdac1/2-deficient ectoderm, targets of p63-mediated repression, including p21, 14-3-3σ, and p16/INK4a, are ectopically expressed, and HDACs bind and are active at their promoter regions in normal undifferentiated keratinocytes. Mutant embryos display increased levels of acetylated p53, which opposes p63 functions, and p53 is required for HDAC inhibitor-mediated p21 expression in keratinocytes. Our data identify critical requirements for HDAC1/2 in epidermal development and indicate that HDAC1/2 directly mediate repressive functions of p63 and suppress p53 activity., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Microglia are the resident macrophages of the central nervous system and are associated with the pathogenesis of many neurodegenerative and brain inflammatory diseases; however, the origin of adult microglia remains controversial. We show that postnatal hematopoietic progenitors do not significantly contribute to microglia homeostasis in the adult brain. In contrast to many macrophage populations, we show that microglia develop in mice that lack colony stimulating factor-1 (CSF-1) but are absent in CSF-1 receptor-deficient mice. In vivo lineage tracing studies established that adult microglia derive from primitive myeloid progenitors that arise before embryonic day 8. These results identify microglia as an ontogenically distinct population in the mononuclear phagocyte system and have implications for the use of embryonically derived microglial progenitors for the treatment of various brain disorders.

Ex vivo generated dendritic cells are currently used to induce therapeutic immunity in solid tumors. Effective immune response requires dendritic cells to home and remain in lymphoid organs to allow for adequate interaction with T lymphocytes. The aim of the current study was to detect and track Feridex labeled human dendritic cells in murine models using magnetic resonance imaging. Human dendritic cells were incubated with Feridex and the effect of labeling on dendritic cells immune function was evaluated. Ex vivo dendritic cell phantoms were used to estimate sensitivity of the magnetic resonance methods and in vivo homing was evaluated after intravenous or subcutaneous injection. R2*-maps of liver, spleen, and draining lymph nodes were obtained and inductively coupled plasma mass spectrometry or relaxometry methods were used to quantify the Feridex tissue concentrations. Correlations between in vivo R2* values and iron content were then determined. Feridex labeling did not affect dendritic cell maturation or function. Phantom results indicated that it was possible to detect 125 dendritic cells within a given slice. Strong correlation between in vivo R2* values and iron deposition was observed. Importantly, Feridex-labeled dendritic cells were detected in the spleen for up to 2 weeks postintravenous injection. This study suggests that magnetic resonance imaging may be used to longitudinally track Feridex-labeled human dendritic cells for up to 2 weeks after injection., (Copyright © 2010 Wiley-Liss, Inc.)

Foxp3(+) Treg are crucial for the maintenance of self-tolerance and have been shown to control CD8(+) T-cell effector functions. In addition, Treg are thought to control the priming of CD8(+) T cells, which recognize the same antigens as Treg. Taking advantage of our model of peripheral tolerance induction to influenza hemagglutinin (HA) after HA gene transfer, we found that HA-specific Treg suppress antigen-linked CTL responses through early blockade of CD8(+) T-cell expansion. Confronted with their cognate antigen, Treg expand more rapidly than CD8(+) T cells and are highly suppressive only during the initial stages of immune priming. They nullify HA-specific CD8(+) T-cell responses, local inflammatory responses and rejection of HA transduced cells. When HA gene transfer is performed with extensive tissue inflammation, HA-specific Treg are less effective but still reduce the frequency of newly primed HA-specific CD8(+) T cells and the ensuing frequency of memory CD8(+) T cells. Our results demonstrate that Treg control CTL priming in an antigen-specific manner at the level of T-cell expansion, highlighting how self-reactive Treg could prevent the induction of autoimmune responses through selective blockade of autoreactive T-cell proliferation.

CX(3)CR1(+) and CD103(+) dendritic cells (DCs) in intestinal lamina propria play a key role in mucosal immunity. However, the origin and the developmental pathways that regulate their differentiation in the lamina propria remain unclear. We showed that monocytes gave rise exclusively to CD103(-)CX(3)CR1(+) lamina propria DCs under the control of macrophage-colony-stimulating factor receptor (M-CSFR) and Fms-like thyrosine kinase 3 (Flt3) ligands. In contrast, common DC progenitors (CDP) and pre-DCs, which give rise to lymphoid organ DCs but not to monocytes, differentiated exclusively into CD103(+)CX(3)CR1(-) lamina propria DCs under the control of Flt3 and granulocyte-macrophage-colony-stimulating factor receptor (GM-CSFR) ligands. CD103(+)CX(3)CR1(-) DCs but not CD103(-)CX(3)CR1(+) DCs in the lamina propria constitutively expressed CCR7 and were the first DCs to transport pathogenic Salmonella from the intestinal tract to the mesenteric lymph nodes. Altogether, these results underline the diverse origin of the lamina propria DC network and identify mucosal DCs that arise from pre-DCs as key sentinels of the gut immune system.

Objective: To evaluate the levels of interleukin-1beta (IL1beta) and its inhibitory soluble interleukin-1 receptor type II (IL1R2) in the peritoneal fluid (PF) of normal women and patients with endometriosis suffering from pelvic pain and infertility., Design: Retrospective study using ELISA to measure peritoneal fluid IL1beta and soluble IL1R2., Setting: Gynecology clinic and human reproduction research laboratory., Patient(s): Sixty-eight normal women and 154 women with endometriosis., Intervention(s): Peritoneal fluid samples were obtained at laparoscopy., Main Outcome Measure(s): IL1beta and soluble IL1R2 concentrations in the PF samples., Result(s): This study showed a marked decrease in peritoneal soluble IL1R2 levels in women with endometriosis compared to normal women and a concomitant increase in the levels of IL1beta. Both fertile and infertile women with endometriosis had lower soluble IL1R2 and higher IL1beta concentrations than fertile women having a normal gynecological status, but the difference was more significant in infertile endometriosis patients. Compared with normal controls, the decrease in soluble IL1R2 levels was less significant in women with endometriosis than without pelvic pain, whereas the increase in IL1beta concentrations was statistically significant only in women with endometriosis reporting pelvic pain., Conclusion(s): This study revealed an imbalance between IL1beta and its decoy inhibitory receptor type 2 in women with endometriosis, which was particularly obvious in those who were infertile, and suggests that a defect in the local control of IL1 may be involved in the pathophysiology of endometriosis and related infertility.

Purpose: The purpose of the current study is to explore third- year medical students' interest in learning about family planning, exposure to family planning (contraception and abortion) and perceived barriers and benefits to family planning education in their obstetrics and gynecology rotation., Method: We conducted four focus groups with 27 third-year medical students near the end of their rotation in obstetrics and gynecology., Results: Students desired education in family planning but perceived limited exposure during their rotation. Most students were aware of abortion but lacked factual information and abortion procedural skills. They felt systemic and faculty-related barriers contributed to limited exposure. Students discussed issues such as lack of time for coverage of contraception and abortion in the curricula and rotation itself. Perceived benefits of clinical instruction in family planning included increased knowledge of contraceptive management and abortion the ability to care for and relate to patients, opportunity for values clarification, and positive changes in attitudes towards family planning., Conclusions: Medical students who desire full education in family planning during their obstetrics and gynecology rotation may face barriers to obtaining that education. Given that many medical students will eventually care for reproductive-age women, greater promotion of opportunities for exposure to family planning within obstetrics and gynecology rotations is warranted.

Foxp3+ regulatory T cells (Tregs) play a pivotal role in the maintenance of peripheral T cell tolerance and are thought to interact with dendritic cells (DC) in secondary lymphoid organs. We analyzed here the in vivo requirements for selective expansion of Ag-specific Treg vs CD4+CD25- effector T cells and engagement of Ag-specific Treg-DC interactions in secondary lymphoid organs. Using i.v. Ag delivery in the absence of inflammation, we found that CD4+CD25+Foxp3+ Tregs undergo vigorous expansion and accumulate whereas naive CD4+CD25-Foxp3- T cells undergo abortive activation. Quantifying directly the interactions between Tregs and CD11c+ DC, we found that Tregs establish cognate contacts with endogenous CD11c+ DC in spleen and lymph nodes at an early time point preceding their expansion. Importantly, we observed that as few as 10(3) Tregs selectively expanded by i.v. Ag injection are able to suppress B and T cell immune responses in mouse recipients challenged with the Ag. Our results demonstrate that Tregs are selectively mobilized by Ag recognition in the absence of inflammatory signals, and can induce thereafter potent tolerance to defined Ag targets.

Langerin is a C-type lectin receptor that recognizes glycosylated patterns on pathogens. Langerin is used to identify human and mouse epidermal Langerhans cells (LCs), as well as migratory LCs in the dermis and the skin draining lymph nodes (DLNs). Using a mouse model that allows conditional ablation of langerin(+) cells in vivo, together with congenic bone marrow chimeras and parabiotic mice as tools to differentiate LC- and blood-derived dendritic cells (DCs), we have revisited the origin of langerin(+) DCs in the skin DLNs. Our results show that in contrast to the current view, langerin(+)CD8(-) DCs in the skin DLNs do not derive exclusively from migratory LCs, but also include blood-borne langerin(+) DCs that transit through the dermis before reaching the DLN. The recruitment of circulating langerin(+) DCs to the skin is dependent on endothelial selectins and CCR2, whereas their recruitment to the skin DLNs requires CCR7 and is independent of CD62L. We also show that circulating langerin(+) DCs patrol the dermis in the steady state and migrate to the skin DLNs charged with skin antigens. We propose that this is an important and previously unappreciated element of immunosurveillance that needs to be taken into account in the design of novel vaccine strategies.

We describe the Phase II HapMap, which characterizes over 3.1 million human single nucleotide polymorphisms (SNPs) genotyped in 270 individuals from four geographically diverse populations and includes 25-35% of common SNP variation in the populations surveyed. The map is estimated to capture untyped common variation with an average maximum r2 of between 0.9 and 0.96 depending on population. We demonstrate that the current generation of commercial genome-wide genotyping products captures common Phase II SNPs with an average maximum r2 of up to 0.8 in African and up to 0.95 in non-African populations, and that potential gains in power in association studies can be obtained through imputation. These data also reveal novel aspects of the structure of linkage disequilibrium. We show that 10-30% of pairs of individuals within a population share at least one region of extended genetic identity arising from recent ancestry and that up to 1% of all common variants are untaggable, primarily because they lie within recombination hotspots. We show that recombination rates vary systematically around genes and between genes of different function. Finally, we demonstrate increased differentiation at non-synonymous, compared to synonymous, SNPs, resulting from systematic differences in the strength or efficacy of natural selection between populations.

With the advent of dense maps of human genetic variation, it is now possible to detect positive natural selection across the human genome. Here we report an analysis of over 3 million polymorphisms from the International HapMap Project Phase 2 (HapMap2). We used 'long-range haplotype' methods, which were developed to identify alleles segregating in a population that have undergone recent selection, and we also developed new methods that are based on cross-population comparisons to discover alleles that have swept to near-fixation within a population. The analysis reveals more than 300 strong candidate regions. Focusing on the strongest 22 regions, we develop a heuristic for scrutinizing these regions to identify candidate targets of selection. In a complementary analysis, we identify 26 non-synonymous, coding, single nucleotide polymorphisms showing regional evidence of positive selection. Examination of these candidates highlights three cases in which two genes in a common biological process have apparently undergone positive selection in the same population:LARGE and DMD, both related to infection by the Lassa virus, in West Africa;SLC24A5 and SLC45A2, both involved in skin pigmentation, in Europe; and EDAR and EDA2R, both involved in development of hair follicles, in Asia.

A major impediment to gene replacement therapy is immune elimination of genetically modified cells. In principle, this can be dealt with by inducing a strong, specific, and enduring tolerance through engraftment of transgene-modified autologous bone marrow (BM). Because usual myeloablation and/or immunosuppression are risk factors in most pathologies, we assessed the potential of monospecific CD4(+)CD25(+) regulatory T cells (Tregs) to engraft minor-mismatched BM without preconditioning. We found that as few as 5 x 10(4) Tregs directed to the male DBY protein promote the engraftment of foreign male BM into sex-mismatched female hosts, establishing sustained chimerism in all hematopoeitic compartments. We achieved concomitantly strong tolerance to all foreign antigens expressed in the BM, likely occurring through induction of anergy and/or deletion of antidonor T cells. Chimerism was obtained in thymectomized mice too, underlining the major role of peripheral tolerance mechanisms in our system. This allowed us to engraft gene-modified tissues while preserving full immunocompetence to third-party antigens. Our results demonstrate that very few donor-specific Tregs are effective as the sole conditioning to induce mixed molecular chimerism and long-term tolerance to multiple foreign antigens.

This work addresses the problem of efficient control of gene expression in the context of viral vectors, which still represents a difficult challenge. A number of lentiviral vectors incorporating the different elements of regulatable transcriptional systems have been described, but they fail to perform satisfactorily either because of a poor dynamic range of transcription levels or because they display high background activities in the uninduced state and mediocre inducer response. We report here on the systematic comparison of vector designs containing the elements of the doxycycline-inducible Tet-on system in their most advanced versions (rtTA2S-M2 transactivator and tTS(Kid) repressor). We show that a simple "all-in-one" vector can be obtained and used for efficient control of transgene expression in long-term tissue culture and in the hematopoietic system of mice following bone marrow transplantation. Using this vector, the uninduced state can be kept at background levels and induction factors of 100-fold are repeatedly obtained over months both in tissue culture and in vivo. Interestingly, the low background activity of the all-in-one vector renders the use of the tTS repressor dispensable, avoiding the problem of progressive loss of inducibility over time associated with irreversible modifications of the chromatin surrounding proviral sequences.

Haemophilia A is an attractive target for gene therapy. We designed a haemophilia A gene therapy strategy involving the genetic modification of haematopoietic stem cells to achieve tissue-specific expression of a factor VIII (FVIII) transgene in the megakaryocytic lineage. Platelets would then serve as vehicles to store the expressed FVIII and deliver the coagulation factor at the site of vascular injury. A local correction of the haemostasis defect could, therefore, be expected following platelet activation and secretion. In this study, we demonstrated that a model of haematopoietic cell lines (Dami cells) could produce a correctly processed FVIII. FVIII transgenes were placed under the control of the human platelet glycoprotein IIb (GPIIb) promoter and used for stable transfection of the Dami megakaryocytic cell line. The highest FVIII production was obtained when the FVIII transgene contained a factor IX intron 1 gene sequence inserted in the FVIII intron 1 and 13 sites. Reverse transcription polymerase chain reaction demonstrated that the splicing of these introns was complete. Recombinant FVIII (rFVIII) produced in Dami cells was a biologically active molecule (specific activity: 5664 IU/mg) that was correctly glycosylated and sulphated. This recombinant FVIII protein exhibited biochemical characteristics after deglycosylation or thrombin activation that were comparable to a commercially available B-domainless rFVIII. These results demonstrate the advantages of a modified FVIII transgene and represent the first biochemical characterization of megakaryocyte-produced FVIII.