Production Of Immune Cells Research Articles

Clonal analysis of lineage fate in native haematopoiesis.

SUMMARYHematopoiesis, the process of mature blood and immune cell production, is functionally organized as a hierarchy, with self-renewing hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells sitting at the very top1,2. Multiple models have been proposed as to what the earliest lineage choices are in these primitive hematopoietic compartments, the cellular intermediates, and the resulting lineage trees that emerge from them3–10. Given that the bulk of studies addressing lineage outcomes have been performed in the context of hematopoietic transplantation, current lineage branching models are more likely to represent roadmaps of lineage potential rather than native fate. Here, we utilize transposon (Tn) tagging to clonally trace the fates of progenitors and stem cells in unperturbed hematopoiesis. Our results describe a distinct clonal roadmap in which the megakaryocyte (Mk) lineage arises largely independently of other hematopoietic fates. Our data, combined with single cell RNAseq, identify a functional hierarchy of uni- and oligolineage producing clones within the MPP population. Finally, our results demonstrate that traditionally defined long-term HSCs (LT-HSCs) are a significant source of Mk-restricted progenitors, suggesting that the Mk-lineage is the predominant native fate of LT-HSCs. Our study provides evidence for a substantially revised roadmap for unperturbed hematopoiesis, and highlights unique properties of MPPs and HSCs in situ.

Immune-pineal axis - acute inflammatory responses coordinate melatonin synthesis by pinealocytes and phagocytes.

Melatonin is well known for its circadian production by the pineal gland, and there is a growing body of data showing that it is also produced by many other cells and organs, including immune cells. The chronobiotic role of pineal melatonin, as well as its protective effects in vitro and in vivo, have been extensively explored. However, the interaction between the chronobiotic and defence functions of endogenous melatonin has been little investigated. This review details the current knowledge regarding the coordinated shift in melatonin synthesis from the pineal gland (circadian and monitoring roles) to the regulation of acute immune responses via immune cell production and autocrine effects, producing systemic interactions termed the immune-pineal axis. An acute inflammatory response drives the transcription factor, NFκB, to switch melatonin synthesis from pinealocytes to macrophages/microglia and, upon acute inflammatory resolution, back to pinealocytes. The potential pathophysiological relevance of immune-pineal axis dysregulation is highlighted, with both research and clinical implications, across several medical conditions, including host/parasite interaction, neurodegenerative diseases and cancer. LINKED ARTICLES: This article is part of a themed section on Recent Developments in Research of Melatonin and its Potential Therapeutic Applications. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.16/issuetoc.

Effects of energy supply and nicotinic acid supplementation on phagocytosis and ROS production of blood immune cells of periparturient primi- and pluriparous dairy cows

Effects of energy supply and nicotinic acid (NA) supplementation on the phagocytic activity of polymorphonuclear leukocytes (PMN) and peripheral blood mononuclear cells (PBMC), and on ROS production in PMN of periparturient cows differing in parity were examined. 29 pluriparous and 18 primiparous cows were allocated to four different feeding groups from 42days prepartum until 100days postpartum. They were fed either a ration with a low concentrate proportion of 30% (LC) or a high concentrate proportion of 60% (HC). After parturition all animals received 30% concentrate which was increased to 50% either within 16 (LC) or within 24days (HC). The different concentrate feeding strategies aimed at triggering differences in postpartum lipolysis. Half of the animals per group were supplemented with 24g per day of NA from 42days prepartum until 24days postpartum. All investigated parameters varied significantly over time compared to parturition (p<0.05). Numbers of phagocytosing PMN and PBMC increased in the course of the experiment, whereas the amount of engulfed bacteria per cell decreased between 42 and 11days prepartum. Percentage of basal ROS producing PMN decreased strongly before parturition and reached initial values only at 28days in milk again. Mean fluorescence intensity (MFI) in these ROS producing cells, however, increased before parturition. Oxidative burst stimulation in PMN was reduced around parturition but the amount of ROS produced in the stimulated cells was increased. Pluriparous cows exhibited higher numbers of basal ROS producing PMN and phagocytic PBMC. NA supplementation influenced phagocytosis in blood leukocytes.

627 A single sunburn influences the immune component of human skin for up to 14 days

Sunburn is prevalent and while the clinical effects disappear quickly, repeated episodes have long term consequences such as skin cancer. Pro-inflammatory eicosanoid expression and leukocyte infiltration characterise the peak of the sunburn response, however, little is known of these processes during the resolution phase. We examined changes in eicosanoid expression and leukocyte infiltration up to 14 days post acute sunburn. Photoprotected buttock skin was exposed to 3xMED of broadband UVR and skin erythema measured over 14 days (n=13; 20-58yrs, 8F, phototype I-III). Biopsies and suction blister fluid were sampled from unexposed and UVR-exposed skin over the 14 day time-course and analysed by immunohistochemistry (neutrophil elastase, CD4, CD8, CD68, CD1a, COX-2, EP4) and LC-MS/MS (eicosanoids). Erythema, COX-2, eicosanoid production and infiltration of innate immune cells (neutrophils and macrophage), peaked at D1 post UVR. By D7 erythema showed 60% resolution, while expression of the prostaglandin E2 receptor EP4 remained elevated at D14. Infiltration of adaptive immune cells (CD4+, CD8+ T cells) peaked at D4-D7 and remained above baseline at D14 (p<0.01, p<0.001 respectively). Moreover, the CD4+:CD8+ ratio rose post-UVR, peaking at 4.3:1 at D1, compared with 2:1 at D0. In contrast, epidermal Langerhans cell numbers showed maximum reduction (∼50%) at D4-D7 (p<0.01), only fully returning to baseline at D14. Acute UVR exposure has profoundly impacts epidermal antigen presenting cell number persisting for up to 2 weeks following the initial insult. Thus a single sunburn may trigger a prolonged immunosuppressive environment compromising ability to respond to neoplastic change and exogeneous challenge.

Modulation of occluding junctions alters the hematopoietic niche to trigger immune activation.

Stem cells are regulated by signals from their microenvironment, or niche. During Drosophila hematopoiesis, a niche regulates prohemocytes to control hemocyte production. Immune challenges activate cell-signalling to initiate the cellular and innate immune response. Specifically, certain immune challenges stimulate the niche to produce signals that induce prohemocyte differentiation. However, the mechanisms that promote prohemocyte differentiation subsequent to immune challenges are poorly understood. Here we show that bacterial infection induces the cellular immune response by modulating occluding-junctions at the hematopoietic niche. Occluding-junctions form a permeability barrier that regulates the accessibility of prohemocytes to niche derived signals. The immune response triggered by infection causes barrier breakdown, altering the prohemocyte microenvironment to induce immune cell production. Moreover, genetically induced barrier ablation provides protection against infection by activating the immune response. Our results reveal a novel role for occluding-junctions in regulating niche-hematopoietic progenitor signalling and link this mechanism to immune cell production following infection.

Ebola Virus Glycoprotein Induces an Innate Immune Response In vivo via TLR4.

Ebola virus (EBOV), a member of the Filoviridae family, causes the most severe form of viral hemorrhagic fever. Although no FDA licensed vaccine or treatment against Ebola virus disease (EVD) is currently available, Ebola virus glycoprotein (GP) is the major antigen used in all candidate Ebola vaccines. Recent reports of protection as quickly as within 6 days of administration of the rVSV-based vaccine expressing EBOV GP before robust humoral responses were generated suggests that the innate immune responses elicited early after vaccination may contribute to the protection. However, the innate immune responses induced by EBOV GP in the absence of viral vectors or adjuvants have not been fully characterized in vivo. Our recent studies demonstrated that immunization with highly purified recombinant GP in the absence of adjuvants induced a robust IgG response and partial protection against EBOV infection suggesting that GP alone can induce protective immunity. In this study we investigated the early immune response to purified EBOV GP alone in vitro and in vivo. We show that GP was efficiently internalized by antigen presenting cells and subsequently induced production of key inflammatory cytokines. In vivo, immunization of mice with EBOV GP triggered the production of key Th1 and Th2 innate immune cytokines and chemokines, which directly governed the recruitment of CD11b+ macrophages and CD11c+ dendritic cells to the draining lymph nodes (DLNs). Pre-treatment of mice with a TLR4 antagonist inhibited GP-induced cytokine production and recruitment of immune cells to the DLN. EBOV GP also upregulated the expression of costimulatory molecules in bone marrow derived macrophages suggesting its ability to enhance APC stimulatory capacity, which is critical for the induction of effective antigen-specific adaptive immunity. Collectively, these results provide the first in vivo evidence that early innate immune responses to EBOV GP are mediated via the TLR4 pathway and are able to modulate the innate-adaptive interface. These mechanistic insights into the adjuvant-like property of EBOV GP may help to develop a better understanding of how optimal prophylactic efficacy of EBOV vaccines can be achieved as well as further explore the potential post-exposure use of vaccines to prevent filoviral disease.

Abstract 3689: Jak3 mutations in colorectal neoplasia- Preliminary data on a not so silent minority

Abstract As a tumorigenesis model, colorectal cancer is associated with multiple gene mutations accumulating progressively but also has mutations that may alter disease course and provide a therapeutic target. Good examples of this are EGFR and VEGF. In melanoma, PD-1 interactions involving the immunobiome are therapeutically important. We conducted a study to detect potential mutations that might enhance therapy in colorectal cancer guided by expression of p87, a product of innate immune system Paneth cells. Methods: Adnab-9 immunohistochemistry or ELISA was used to define significant p87 Adenoma-associated antigen field effects (FE) in 10 patients with &amp;gt;1cm large high grade dysplastic adenomas (LHiGDA) and 3 with smaller high grade dysplastic adenomas (SHiGDA). We postulated that SHiGDA are not immunologically recognized by host defenses leading to negative outcomes. We used Ion Torrent™ sequencing (ITS) to find mutations in DNA (QiaAmp kit) extracted from 4 normal-appearing colonic segments taken from 1 patient in each group. Novel mutations found on ITS would then be sought using PCR with appropriate primers and subsequent sequencing of the PCR product circulating DNA extracted from available serum samples. These samples were taken from the Large- and SHiGDA groups described above and from 17 patients undergoing colonoscopy for diverse indications. Results: p87 FE were found in 40% of 10 LHiGDA and 0% of 3 SHiGDA patients. The ITS in the 2 representative patients showed unique mutational fields in: KRAS, APC, p53 in the LHiGDA and Jak3, PIK3Ca, p53, APC in the SHiGDA patient, both of whom lacked p87 FE. PCR using the Jak3 primers used in the ITS and subsequent sequencing revealed the same non-synonymous mutation in the serum of a FAP patient after colonic resection and an additional colonic segment of the selected ShiGDA patient but not in his serum. Other Jak3 mutations were found in 1 of 8 LHiGDA, 1 (the selected patient) of 3 SHiGDA, 1 of 7 patients with FAP and 1 of 10 colonoscopy patients with a family history of colorectal cancer with a FE and an untoward outcome. The selected SHiGDA patient subsequently contracted and died of NSCLC adenocarcinoma. The positive LHiGDA and FAP patients had a severely dysplastic anal condyloma and severe pancreatitis, respectively. Overall, the non-synonymous mutation occurring in the non-FAP SHiGDA patient occurred in the absence of p87 FE. Conclusions: In this pilot study we demonstrate the presence of Jak3 mutations likely associated with the lack of p87 expression in patients with high grade dysplastic adenomas and 1 FAP patient. Most of these patients had a clinical course which may have differed from their group members suggesting an altered immune system milieu. If confirmed in SHiGDA and FAP, Jak 3 mutations, associated with the SCID and late onset combined immunodeficiency, may allow for intervention with currently available medications to potentially avert a deleterious clinical outcome. Citation Format: Martin Tobi, Xiaoping Zhao, Darshana Jhala, Rebecca Rodriguez, Fadi Antaki, Edi Levi, Paula Sochacki, John Lieb, MaryAnn Rambus, Tapan Ganguly, Martin Bluth, Michael J. Lawson. Jak3 mutations in colorectal neoplasia- Preliminary data on a not so silent minority [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3689. doi:10.1158/1538-7445.AM2017-3689

Treatment with anti-FcεRIα antibody exacerbates EAE and T-cell immunity against myelin

Objective:To investigate the effects of targeting the high-affinity receptor for immunoglobulin E (FcεRI), that plays a central role in allergic responses and is constitutively expressed on mast cells and basophils, in clinical disease and autoimmune T-cell response in experimental MS.Methods:Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein 35–55. Anti-FcεRI α-chain antibody was administered intraperitoneally. CNS immunohistochemistry, flow cytometry analysis of immune cell populations, IgE and histamine serum concentration, immune cell proliferation, and cytokine measurement were performed. In BALB/c mice, EAE was induced by immunization with myelin proteolipid protein 185–206.Results:Treatment with anti-FcεRIα antibody resulted in exacerbation of EAE and increased CNS inflammation in C57BL/6 mice. Treated mice displayed long-lasting complete depletion of basophils in the blood stream and peripheral lymphoid organs and increased antigen-induced immune cell proliferation and production of interferon-γ, interleukin (IL)-17, IL-6, and granulocyte-macrophage colony-stimulating factor. In BALB/c mice, which are T-helper (Th) 2 prone and resistant to EAE, treatment with anti-FcεRIα antibody restored susceptibility to EAE.Conclusion:Our observations that anti-FcεRIα antibody increases Th1 and Th17 responses against myelin antigen and exacerbates EAE suggest that FcεRI, basophils, and possibly other FcεRI-bearing cells that might be affected by this antibody play important roles in influencing the severity of CNS autoimmunity.

DOCK8 regulates signal transduction events to control immunity.

Genetic mutations in the gene encoding DOCK8 cause an autosomal recessive form of hyper immunoglobulin E syndrome (AR-HIES), referred to as DOCK8 deficiency. DOCK8 deficiency in humans results in the onset of combined immunodeficiency disease (CID), clinically associated with chronic infections with diverse microbial pathogens, and a predisposition to malignancy. It is now becoming clear that DOCK8 regulates the function of diverse immune cell sub-types, particularly lymphocytes, to drive both innate and adaptive immune responses. Early studies demonstrated that DOCK8 is required for lymphocyte survival, migration and immune synapse formation, which translates to poor pathogen control in the absence of DOCK8. However, more recent advances have pointed to a crucial role for DOCK8 in regulating the signal transduction events that control transcriptional activity, cytokine production and functional polarization of immune cells. Here, we summarize recent advances in our understanding of DOCK8 function, paying particular attention to an emerging role as a signaling intermediate to promote immune responses to diverse external stimuli.

Immune-Neuro-Endocrine Reflexes, Circuits, and Networks: Physiologic and Evolutionary Implications.

The existence of a network of interactions between the immune and nervous systems that influences host defenses and brain functions is now well-established. Here we discuss how immune and classical neuro/sensorial signals are processed in the brain and how neuro-endocrine immunoregulatory and behavioral responses are integrated. Considering the ability of brain cells to produce cytokines, originally described as immune cell products, we propose that the tripartite synapse plays a central role in the integration of neuro-endocrine-immune interactions. We also propose that the immune-neuro-endocrine responses that influence the course of transmissible and other diseases predisposing to infections can be relevant for evolution, either by restoring health or by mediating an active process of negative selection.

Bone Marrow (BM) Hematopoietic Dysfunction in Chronic Lymphocytic Leukemia (CLL) - Association with Leukemic Burden and Reversibility with Therapeutic Responses.

Introduction: CLL is the most prevalent leukemia in the United States (≥120,000 cases) with approximately 5000 deaths per year. The dominant cause of mortality in CLL patients is serious infection. Indeed, about 80% of CLL patients will sustain infectious complications at some point during their disease course and infection risk increases with length of the disease. Steady-state hematopoiesis in BM is required to sustain a peripheral repertoire of blood cells and maintain immune competence. There have been few studies evaluating the hematopoietic capacity of CLL BM and how it may relate to immune competence. Thus, the goal of this study was to determine the status of BM hematopoiesis in untreated and treated CLL patients.Methods and findings: The hematopoietic stem (HSC) and progenitor cell pool (HSPC) in human BM is defined by expression of CD34 and lack of surface markers (Lin-) expressed on their mature progeny. Frequencies of Lin- CD34+ progenitors in BM between age matched healthy subjects (HS) (n=22) and untreated CLL patients (n=10) were compared. We note that the Lin+ cocktail used included an antibody to CD19, eliminating dilution of CD34+ HSPC frequency due to variation in leukemic B cell infiltration. CD34+ HSPCs were significantly reduced in untreated CLL patients (6.33±3.01% vs. 2.04±2.33% in HS vs. CLL, p=0.0004). Next we evaluated total CFU responding cells within the residual CD34+ cells to determine if they were functionally compromised. Numbers of total CFU-responsive CD34+ progenitors (CFU-GM, CFU-E, and CFU-GEMM) were reduced when assayed from purified CLL patient CD34+ cells compared to HS. Thus, while HSPC are present in CLL patient BM, they are functionally compromised even after removal from the tumor microenvironment. To determine if reductions in CFUs were accompanied by decreased myeloid and erythroid lineage differentiated progeny, we evaluated frequencies and absolute numbers of CD14+ monocytes and CD71+ erythroid progenitors by flow cytometry and found both were severely reduced in CLL patient BM. To determine if the reductions in myeloid/erythroid lineage cells was due to deficiencies in their precursor HSPCs, we evaluated frequencies and absolute numbers of HSC, multipotent progenitors (MPP), and common myeloid progenitors (CMP). Proportions, but not absolute numbers, of HSC, MPP, and CMP were significantly reduced in CLL patient BM. Frequencies of CD14+ monocytes and CD71+ erythroid precursors were also significantly reduced in patients with low BM tumor burden (designated as 20-40% BM involvement), suggesting that the hematopoietic dysfunction is not solely due to tumor burden. Comparative analysis of frequencies of CD14+ monocytes in BM to frequencies of monocytes in peripheral blood revealed a strong association, suggesting that for some immune cell types the blood immune repertoire may be reflective of BM dysfunction. In addition to BM evaluation of untreated CLL patients, we also studied a cohort of post-therapy patients (n=23). Not surprisingly, we found considerable heterogeneity in the extent of BM hematopoietic reconstitution. Nevertheless, we observed robust BM hematopoiesis post therapy in a subset of treated patients (n=9 of 23).Summary: Our findings reveal BM hematopoietic dysfunction (HD) in CLL not solely due to the degree of tumor burden. Importantly, the BM HD is reversible by effective therapies. The HD is manifested by reduced numbers and function of HSPCs and production of their mature progeny. Of significance is the severe reduction in monocytic and granulocytic cells found in CLL BM. This is an important finding as these two innate cell types provide critical first responder protection against invading pathogens. Our study also uncovered that levels of blood monocytes can mirror their diminished production in BM. Future studies will focus on determining the mechanism(s) that reduce the ability of CLL BM to sustain immune cell production in addition to determining if restoration of hematopoiesis is a necessary outcome for more effective therapies. DisclosuresDing:Merck: Research Funding. Kay:Pharmacyclics: Research Funding; Tolero Pharmaceuticals: Research Funding; Acerta: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Morpho-Sys: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Protective effect of Cl-amidine against CLP-induced lethal septic shock in mice.

Production of innate and adaptive immune cells from hematopoietic stem cells, and maturation of T lymphocytes are effective immune responses to fight severe microbial infection. In sepsis, this emergency myelopoiesis is damaged, leading to failure of bacterial clearance, and excessive stress-induced steroids cause immature T-lymphocyte apoptosis in thymus. We recently found that Cl-amidine, a peptidylarginine deiminase (PAD) inhibitor, improves survival in a mouse model of cecal ligation and puncture (CLP)-induced septic shock. In the present study we investigated how Cl-amidine promotes survival, focusing on protective effects of Cl-amidine on immune response. We confirmed survival-improving effect of Cl-amidine and are the first to explore the role of Cl-amidine in immune response. CLP caused bone marrow (BM) and thymus atrophy, decreased innate immune cells in BM. CLP increased levels of cytokines (IL-1β, IL-6, and TNF-α) and bacteria load in blood/liver. In primary splenocyte culture, lipopolysaccharide increased TNF-α production. In contrast, Cl-amidine attenuated these CLP and lipopolysaccharide-induced alterations. Moreover, Cl-amidine increased circulating monocytes. Collectively, our results demonstrate Cl-amidine plays protective roles by significantly decreasing BM and thymus atrophy, restoring innate immune cells in BM, increasing blood monocytes and blood/liver bacteria clearance, and attenuating pro-inflammatory cytokine production in a murine model of lethal sepsis.

Analysis of Normal Hematopoietic Stem and Progenitor Cell Contents in Childhood Acute Leukemia Bone Marrow

Childhood acute leukemias (AL) are characterized by the excessive production of malignant precursor cells at the expense of effective blood cell development. The dominance of leukemic cells over normal progenitors may result in either direct suppression of functional hematopoiesis or remodeling of microenvironmental niches, contributing to BM failure and AL-associated mortality. We undertook this study to investigate the contents and functional activity of hematopoietic stem/progenitor cells (HSPC) and their relationship to immune cell production and risk status in AL pediatric patients. Multiparametric flow cytometry of BM aspirates was performed to classify AL on the basis of lineage and differentiation stages and to analyze HSPC and immune cell frequencies. Controlled co-culture systems were conducted to evaluate functional lineage potentials of primitive cells. Statistical correlations and inter-group significant differences were established. Among 113 AL BM aspirates, 26.5% corresponded to ProB, 19.5% to PreB and 32% contain ProB and PreB differentiation stages, whereas nearly 9% of the cases were T- and 13% myeloid-lineage leukemias. We identified ProB-ALL as the subtype endowed with the highest relative contents of HSPC, whereas T-ALL and PreB-ALL showed a critically reduced size of both HSC and MLP compartments. Notably, lower cell frequencies of HSPC in ProB-ALL correlated to high-risk prognosis at disease debut. HSPC abundance at initial diagnosis may aid to predict the clinical course of ALL and to identify high-risk patients. A clearer understanding of their population dynamics and functional properties in the leukemia setting will potentially pave the way for targeted therapies.

Monoclonal antibodies protect from Staphylococcal Enterotoxin K (SEK) induced toxic shock and sepsis by USA300 Staphylococcus aureus

ABSTRACTStaphylococcus aureus is a leading infectious cause of life-threatening disease in humans, yet there is currently no vaccine to combat this bacterium. The pathogenesis of S. aureus is mediated by a diverse array of protein toxins including a large family of secreted pyrogenic superantigens. Neutralization of superantigens, including SEB and TSST-1, has proven to be protective in several animal models of toxic shock and sepsis. We demonstrate, for the first time, that a far more prevalent staphylococcal superantigen, SEK, can also induce lethal shock in mice. Additionally, we describe monoclonal antibodies (mAbs) that inhibit SEK-induced mitogenicity as well as protect against SEK-induced lethality, and enhance survival from S. aureus septicemia in murine models. MAb-4G3 (IgG2b), mAb-5G2 (IgG1), and mAb-9H2 (IgG1), all inhibit SEK-induced proliferation and cytokine production of human immune cells. We then demonstrate that passive immunization with a combination of mAb-4G3 and mAb-5G4, 2 mAbs that do not compete for epitope(s) on SEK, significantly enhance survival in a murine model of SEK-induced toxic shock (p = 0.006). In the setting of sepsis, passive immunization with this combination of mAbs also significantly enhances survival in mice after challenge with CA-MRSA strain USA300 (p = 0.03). Furthermore, septic mice that received mAb treatment in conjunction with vancomycin exhibit less morbidity than mice treated with vancomycin alone. Taken together, these findings suggest that the contribution of SEK to S. aureus pathogenesis may be greater than previously appreciated, and that adjunctive therapy with passive immunotherapy against SEs may be beneficial.

Matthias Nahrendorf: Healing Insights.

Born in Salzwedel, East Germany, Matthias Nahrendorf entered adulthood just as the Iron Curtain was lifting in Eastern Europe. He earned his medical degree and PhD in cardiovascular biology at Heidelberg University, and completed his residency plus fellowships in internal medicine and cardiovascular imaging at University of Wuerzburg. In 2004, a postdoctoral position at Massachusetts General Hospital’s Center for Molecular Imaging Research brought Nahrendorf to Boston. He is now a principal investigator at MGH’s Center for Systems Biology and director of the hospital’s Mouse Imaging Program. Nahrendorf, 45, is pursuing a deeper understanding of the consequences of ischemic injury, especially regarding how monocytes/macrophages impact acute healing and scar development,1–3 and how an imbalance in the process fuels prolonged inflammation and then heart failure. An associate professor of radiology at Harvard Medical School, Nahrendorf and his collaborators are also developing molecular imaging tools to noninvasively study regulators of heart failure biology and infarct healing processes.4–6 A long-term goal of the work is to devise new therapies for ischemic injury that reduce the action of inflammatory cells to what is necessary only to promote healthy healing, Nahrendorf says. If that could be achieved, “Ideally we wouldn’t have heart failure anymore.” I have one brother, he’s younger, and my parents. It was a pretty sheltered childhood. I grew up in Magdeburg, and the (Berlin) Wall came down when I was 18. Army service was compulsory in East Germany. So I spent one year in the army and then Germany reunited, so I was free to go wherever I wanted. My parents are both doctors. My dad is a cardiologist, my mom a pediatrician. I had a happy childhood. They were never telling me what to do. But I was obviously influenced by their jobs. They talked about …

The influence of protein malnutrition on biological and immunomodulatory aspects of bone marrow mesenchymal stem cells

Tissues that require a great supply of nutrients and possess high metabolic demands, such as lympho-hemopoietics tissues, are the first to be affected by protein malnutrition (PM). Thus, PM directly affects hemopoiesis and the production and function of immune cells. Consequently, malnourished individuals are more susceptible to infections. Mesenchymal stem cells (MSCs) have immunomodulatory properties and are important in the formation of lympho-hemopoietic stroma. Since an adequate supply of nutrients is essential to sustain stroma formation, which is mainly constituted of MSCs and differentiated cells originated from them, this study investigated whether PM would influence some biological and immunomodulatory aspects of MSCs. Two-month-old Balb/c mice were divided into control and malnourished groups receiving normoproteic or hypoproteic diets, respectively (12% and 2% of protein) for 28 days. MSCs obtained from control (MSCct) and malnourished (MSCmaln) animals were characterized. In addition, the proliferation rate and cell cycle protein expression were determined, but no differences in these parameters were observed. In order to evaluate whether PM affects the immunomodulatory properties of MSCs, the expression of NFκB and STAT-3, and the production of IL-1α, IL-1β, IL-6, IL-10, TGF-β and TNF-α by MSCs were assessed. MSCmaln expressed lower levels of NF-κB and the production of IL-1β, IL-6 and TGF-β was significantly influenced by PM. Furthermore, MSCct and MSCmaln culture supernatants affected lymphocyte and macrophage proliferation. However, MSCmaln did not reduce the production of IFN-γ nor stimulate the production of IL-10 in lymphocytes in the same manner as observed in MSCct. Overall, this study implied that PM modifies immunosuppressive properties of MSCs.

Experience of contamination during autologous cell manufacturing in cell processing facility under the Japanese Medical Practitioners Act and the Medical Care Act.

Cell therapy and regenerative medicine technologies require strict cell manufacturing procedures to be defined and addressed. Maintenance of the aseptic environment is critical to preclude extrinsic contamination risks, similar to conventional pharmaceutical manufacturing. However, intrinsic contamination risks exist in all cell manufacturing processes owing to the use of cells as the raw materials that cannot be sterilized, thus giving rise to the primary and secondary risks of cell contamination and cross-contamination, respectively. Analysis of contamination risks was conducted on experienced batches (29,858 batches) for the production of immune cells derived from autologous blood mononuclear cells under the Medical Practitioners Act and the Medical Care Act in Japan. From these batches, 0.06% (18 cases) of contamination occurred, representing low probability of contamination incidence during cell processing. Almost all the causes of these contaminations were regarded to be from the collected blood (intrinsic contamination), and subsequent cross-contaminations were prevented, considering that the secondary contamination risk can be reduced by adequate managements of operational procedures for changeover in aseptic environment.

The Molecular Mechanisms of Vitamin A Deficiency in Multiple Sclerosis.

Vitamin A, considered to be an essential nutrient, has important actions in immunological responses and the central nervous system (CNS). Neuroimmunological functions of vitamin A are mediated through its active metabolite, retinoic acid (RA). In the CNS, RA contributes to regeneration and plasticity, while also playing a key role in enhancing tolerance and reducing inflammatory responses by regulating T cell, B cell and dendritic cell populations. However, evidence has indicated lower plasma levels of vitamin A in patients with multiple sclerosis (MS). Vitamin A deficiency leads to dysregulation of immune tolerance and pathogenic immune cell production in this disease. Vitamin A may ameliorate MS pathogenesis through numerous mechanisms including a reduction in inflammatory processes by re-establishing the balance between pathogenic (Th1, Th17, Th9) and immunoprotective cells (Th2, Tregs), modulating B cell and dendritic cell function as well as increasing tolerance of autoimmunity and regeneration in the CNS. Thus, the results from the current review suggest that vitamin A can be considered as a potential treatment in MS disease management.

Differences in Innate immune cell production of IFNα and IFNβ to cell activation in the Autoinflammatory Diseases CANDLE (Chronic Atypical Neutrophilic Dermatosis with Lipodystrophy and Elevated temperature) and SAVI (STING Associated Vasculopathy with Onset in Infancy)

Abstract The Type I IFN mediated autoinflammatory diseases SAVI and CANDLE are caused by gain-of-function mutations in Stimulator of Interferon Genes (STING) and loss-of-function mutations in proteasome components. We evaluated the innate immune cellular origin of IFN Type I in SAVI and CANDLE. We examined expression of IFN genes from FACS sorted T, B, NK and monocytes in SAVI (n=5) and CANDLE (n=3) normalized to healthy controls (HC). The Interquartile Range of Type I IFN expression was 78(4–151) and 481-fold(56-38,242) of IFNB1 and 60(3–116) and 0.4-fold(0.1–39) of IFNA7/IFNA17 in CANDLE and SAVI monocytes, respectively. Monocyte depletion reduced IFN message by 90% and FACS analysis confirmed that monocytes and not dendritic cells are the main source of IFNs. The overall mean ratio of IFNβ to IFNα was 989:1 in SAVI and 1.3:1 in CANDLE monocytes. Mean IFNα plasma levels measured by pan IFNα ELISA with 31pg/ml ±58 SEM in CANDLE and 230pg/ml ±112 SEM in SAVI. IP10 serum levels were 2215pg/ml ±702 SEM and 3632pg/ml ±860 SEM in CANDLE and SAVI, respectively. STING pathway activation with cGAMP in the presence of cycloheximide (CHX), a blocker of translational elongation, led to IFNB1 but not IFNA1, 2, 7, 17, 21 while CHX + epoxomicin, a proteasome inhibitor led to transcription of IFNAs and IFNB1 suggesting secondary upregulation of IFNAs in SAVI. Compared to healthy controls, CANDLE and SAVI had increased left-shifted immature CD13lo CD16+ neutrophils (9 and 4-fold, respectively), as well as higher expression of the activation marker CD36+ (6.9 and 6.9-fold, respectively) in total granulocytes. In conclusion, Type I IFN is mainly produced in monocytes and analyses of the IFN signaling in CANDLE and SAVI will allow us to develop improved targeted therapies.

Pseudomonas aeruginosa and Its Bacterial Components Influence the Cytokine Response in Thymocytes and Splenocytes.

Infections with Pseudomonas aeruginosa may cause many different diseases. The spectrum of such infections in general includes inflammation and bacterial sepsis. Hospital-acquired pneumonia, naturally resistant to a wide range of antibiotics, is associated with a particularly high mortality rate in mechanically ventilated patients. The pathogenesis of P. aeruginosa is complex and mediated by several virulence factors, as well as cell-associated factors. We have previously demonstrated that stimulation with different bacteria triggers the cytokine response of thymocytes. In this study, we investigated the effect of P. aeruginosa and its different components on the cytokine production of immature and mature immune cells. We found that the induced cytokine pattern in the thymus and the spleen after infections with P. aeruginosa is primarily mediated by lipopolysaccharide (LPS) of the outer cell membrane, but other components of the bacterium can influence the cytokine secretion as well. Stimulation with heat-killed P. aeruginosa and LPS does not influence the amount of cytokine-producing CD4(+) T cells but instead suppresses the emergence of Th17 cells. However, stimulation with P. aeruginosa or its components triggers the interleukin-17 (IL-17) response both in thymocytes and in splenocytes. We conclude that infections with P. aeruginosa affect the cytokine secretion of immature and mature cells and that IL-17 and Th17 cells play only a minor role in the development of pathological systemic inflammatory disease conditions during P. aeruginosa infections. Therefore, other inflammatory immune responses must be responsible for septic reactions of the host.