Physician, Heal Thy Community

What if the opioid epidemic and the chronic health problems we encounter in West Virginia and beyond are a symptom of a deeper problem? How can we create healthcare to address the root issues here, and then spread these solutions globally

Alveolar macrophages lack CCR2 expression and do not migrate to CCL2

Background: The recruitment of mononuclear cells has important implications for tissue inflammation. Previous studies demonstrated enhanced CCR1 and CCR5 expression and decreased CCR2 expression during in vitro monocyte to macrophage differentiation. To date, no study examined the in vivo differences in chemokine receptor expression between human peripheral blood monocytes and alveolar macrophages. Methods: We examined the expression of these receptors in human peripheral blood monocytes and alveolar macrophages using microarray analysis, reverse-transcriptase PCR, flow cytometry and migration analyses. Results: In contrast to peripheral blood monocytes, alveolar macrophages did not express the CCL2 receptor, CCR2, and did not migrate toward CCL2. In contrast, monocytes and freshly isolated resident alveolar macrophages both migrated towards CCL3. However, up to 6-fold more monocytes migrated toward equivalent concentrations of CCL3 than did alveolar macrophages from the same donor. While peripheral blood monocytes expressed the CCL3 receptor, CCR1, alveolar macrophages expressed the alternate CCL3 receptor, CCR5. The addition of anti-CCR5 blocking antibodies completely abrogated CCL3-induced migration in alveolar macrophages, but did not affect the migration of peripheral blood monocytes. Conclusion: These data support the specificity of CCL2 to selectively drive monocyte, but not alveolar macrophage recruitment to the lung and CCR5 as the primary macrophage receptor for CCL3

NAC and DTT promote TGF-β1 monomer formation: demonstration of competitive binding

TGF-β plays an important role in the genesis and progression of pulmonary fibrosis. We sought to determine the role of mononuclear phagocytes in the activation of TGF-β and found that freshly isolated peripheral blood monocytes spontaneously released TGF-β. Stimulating these monocytes with GM-CSF or LPS, but not MCSF, augmented the activation of TGF-β. In human monocytes, the free thiol compounds DTT and NAC decreased the activity of TGF-β, without affecting TGF-β mRNA transcription. Both NAC and DTT lessened the biological activity of recombinant active TGF-β in a cell-free system. We found that NAC and DTT reduced dimeric active TGF-β from a 25 kDa protein to 12.5 kDa inactive monomer. This conversion was reversed using the oxidizing agent diamide. Diamide also restored biological activity to NAC or DTT-treated TGF-β. Reduction of TGF-β to monomers could competitively inhibit active dimeric TGF-β and block intracellular signaling events. Our observations suggest that modulation of the oxidative state of TGF-β may be a novel therapeutic approach for patients with pulmonary fibrosis

Mesenchymal Stem Cells for Cardiac Regeneration: Translation to Bedside Reality

Cardiovascular disease (CVD) is the leading cause of death worldwide. According to the World Health Organization (WHO), an estimate of 17.3 million people died from CVDs in 2008 and by 2030, the number of deaths is estimated to reach almost 23.6 million. Despite the development of a variety of treatment options, heart failure management has failed to inhibit myocardial scar formation and replace the lost cardiomyocyte mass with new functional contractile cells. This shortage is complicated by the limited ability of the heart for self-regeneration. Accordingly, novel management approaches have been introduced into the field of cardiovascular research, leading to the evolution of gene- and cell-based therapies. Stem cell-based therapy (aka, cardiomyoplasty) is a rapidly growing alternative for regenerating the damaged myocardium and attenuating ischemic heart disease. However, the optimal cell type to achieve this goal has not been established yet, even after a decade of cardiovascular stem cell research. Mesenchymal stem cells (MSCs) in particular have been extensively investigated as a potential therapeutic approach for cardiac regeneration, due to their distinctive characteristics. In this paper, we focus on the therapeutic applications of MSCs and their transition from the experimental benchside to the clinical bedside

Two-dimensional gel proteome reference map of blood monocytes

BACKGROUND: Blood monocytes play a central role in regulating host inflammatory processes through chemotaxis, phagocytosis, and cytokine production. However, the molecular details underlying these diverse functions are not completely understood. Understanding the proteomes of blood monocytes will provide new insights into their biological role in health and diseases. RESULTS: In this study, monocytes were isolated from five healthy donors. Whole monocyte lysates from each donor were then analyzed by 2D gel electrophoresis, and proteins were detected using Sypro Ruby fluorescence and then examined for phosphoproteomes using ProQ phospho-protein fluorescence dye. Between 1525 and 1769 protein spots on each 2D gel were matched, analyzed, and quantified. Abundant protein spots were then subjected to analysis by mass spectrometry. This report describes the protein identities of 231 monocyte protein spots, which represent 164 distinct proteins and their respective isoforms or subunits. Some of these proteins had not been previously characterized at the protein level in monocytes. Among the 231 protein spots, 19 proteins revealed distinct modification by protein phosphorylation. CONCLUSION: The results of this study offer the most detailed monocyte proteomic database to date and provide new perspectives into the study of monocyte biology

Using an agent-based model to analyze the dynamic communication network of the immune response

<p>Abstract</p> <p>Background</p> <p>The immune system behaves like a complex, dynamic network with interacting elements including leukocytes, cytokines, and chemokines. While the immune system is broadly distributed, leukocytes must communicate effectively to respond to a pathological challenge. The Basic Immune Simulator 2010 contains agents representing leukocytes and tissue cells, signals representing cytokines, chemokines, and pathogens, and virtual spaces representing organ tissue, lymphoid tissue, and blood. Agents interact dynamically in the compartments in response to infection of the virtual tissue. Agent behavior is imposed by logical rules derived from the scientific literature. The model captured the agent-to-agent contact history, and from this the network topology and the interactions resulting in successful versus failed viral clearance were identified. This model served to integrate existing knowledge and allowed us to examine the immune response from a novel perspective directed at exploiting complex dynamics, ultimately for the design of therapeutic interventions.</p> <p>Results</p> <p>Analyzing the evolution of agent-agent interactions at incremental time points from identical initial conditions revealed novel features of immune communication associated with successful and failed outcomes. There were fewer contacts between agents for simulations ending in viral elimination (<it>win</it>) versus persistent infection (<it>loss</it>), due to the removal of infected agents. However, early cellular interactions preceded successful clearance of infection. Specifically, more Dendritic Agent interactions with TCell and BCell Agents, and more BCell Agent interactions with TCell Agents early in the simulation were associated with the immune <it>win </it>outcome. The Dendritic Agents greatly influenced the outcome, confirming them as hub agents of the immune network. In addition, unexpectedly high frequencies of Dendritic Agent-self interactions occurred in the lymphoid compartment late in the <it>loss </it>outcomes.</p> <p>Conclusions</p> <p>An agent-based model capturing several key aspects of complex system dynamics was used to study the emergent properties of the immune response to viral infection. Specific patterns of interactions between leukocyte agents occurring early in the response significantly improved outcome. More interactions at later stages correlated with persistent inflammation and infection. These simulation experiments highlight the importance of commonly overlooked aspects of the immune response and provide insight into these processes at a resolution level exceeding the capabilities of current laboratory technologies.</p

Asc-Dependent and Independent Mechanisms Contribute to Restriction of Legionella Pneumophila Infection in Murine Macrophages

The apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc) is an adaptor molecule that mediates inflammatory and apoptotic signals. Legionella pneumophila is an intracellular bacterium and the causative agent of Legionnaire's pneumonia. L. pneumophila is able to cause pneumonia in immuno-compromised humans but not in most inbred mice. Murine macrophages that lack the ability to activate caspase-1, such as caspase-1−/− and Nlrc4−/− allow L. pneumophila infection. This permissiveness is attributed mainly to the lack of active caspase-1 and the absence of its down stream substrates such as caspase-7. However, the role of Asc in control of L. pneumophila infection in mice is unclear. Here we show that caspase-1 is moderately activated in Asc−/− macrophages and that this limited activation is required and sufficient to restrict L. pneumophila growth. Moreover, Asc-independent activation of caspase-1 requires bacterial flagellin and is mainly detected in cellular extracts but not in culture supernatants. We also demonstrate that the depletion of Asc from permissive macrophages enhances bacterial growth by promoting L. pneumophila-mediated activation of the NF-κB pathway and decreasing caspase-3 activation. Taken together, our data demonstrate that L. pneumophila infection in murine macrophages is controlled by several mechanisms: Asc-independent activation of caspase-1 and Asc-dependent regulation of NF-κB and caspase-3 activation

An In Silico Modeling Approach to Understanding the Dynamics of Sarcoidosis

BACKGROUND: Sarcoidosis is a polygenic disease with diverse phenotypic presentations characterized by an abnormal antigen-mediated Th1 type immune response. At present, progress towards understanding sarcoidosis disease mechanisms and the development of novel treatments is limited by constraints attendant to conducting human research in a rare disease in the absence of relevant animal models. We sought to develop a computational model to enhance our understanding of the pathological mechanisms of and predict potential treatments of sarcoidosis. METHODOLOGY/RESULTS: Based upon the literature, we developed a computational model of known interactions between essential immune cells (antigen-presenting macrophages, effector and regulatory T cells) and cytokine mediators (IL-2, TNFα, IFNγ) of granulomatous inflammation during sarcoidosis. The dynamics of these interactions are described by a set of ordinary differential equations. The model predicts bistable switching behavior which is consistent with normal (self-limited) and "sarcoidosis-like" (sustained) activation of the inflammatory components of the system following a single antigen challenge. By perturbing the influence of model components using inhibitors of the cytokine mediators, distinct clinically relevant disease phenotypes were represented. Finally, the model was shown to be useful for pre-clinical testing of therapies based upon molecular targets and dose-effect relationships. CONCLUSIONS/SIGNIFICANCE: Our work illustrates a dynamic computer simulation of granulomatous inflammation scenarios that is useful for the investigation of disease mechanisms and for pre-clinical therapeutic testing. In lieu of relevant in vitro or animal surrogates, our model may provide for the screening of potential therapies for specific sarcoidosis disease phenotypes in advance of expensive clinical trials

The Human TPR Protein TTC4 Is a Putative Hsp90 Co-Chaperone Which Interacts with CDC6 and Shows Alterations in Transformed Cells

BACKGROUND: The human TTC4 protein is a TPR (tetratricopeptide repeat) motif-containing protein. The gene was originally identified as being localized in a genomic region linked to breast cancer and subsequent studies on melanoma cell lines revealed point mutations in the TTC4 protein that may be associated with the progression of malignant melanoma. METHODOLOGY/PRINCIPLE FINDINGS: Here we show that TTC4 is a nucleoplasmic protein which interacts with HSP90 and HSP70, and also with the replication protein CDC6. It has significant structural and functional similarities with a previously characterised Drosophila protein Dpit47. We show that TTC4 protein levels are raised in malignant melanoma cell lines compared to melanocytes. We also see increased TTC4 expression in a variety of tumour lines derived from other tissues. In addition we show that TTC4 proteins bearing some of the mutations previously identified from patient samples lose their interaction with the CDC6 protein. CONCLUSIONS/SIGNIFICANCE: Based on these results and our previous work with the Drosophila Dpit47 protein we suggest that TTC4 is an HSP90 co-chaperone protein which forms a link between HSP90 chaperone activity and DNA replication. We further suggest that the loss of the interaction with CDC6 or with additional client proteins could provide one route through which TTC4 could influence malignant development of cells

MiR-155 Induction by F. novicida but Not the Virulent F. tularensis Results in SHIP Down-Regulation and Enhanced Pro-Inflammatory Cytokine Response

The intracellular Gram-negative bacterium Francisella tularensis causes the disease tularemia and is known for its ability to subvert host immune responses. Previous work from our laboratory identified the PI3K/Akt pathway and SHIP as critical modulators of host resistance to Francisella. Here, we show that SHIP expression is strongly down-regulated in monocytes and macrophages following infection with F. tularensis novicida (F.n.). To account for this negative regulation we explored the possibility that microRNAs (miRs) that target SHIP may be induced during infection. There is one miR that is predicted to target SHIP, miR-155. We tested for induction and found that F.n. induced miR-155 both in primary monocytes/macrophages and in vivo. Using luciferase reporter assays we confirmed that miR-155 led to down-regulation of SHIP, showing that it specifically targets the SHIP 3′UTR. Further experiments showed that miR-155 and BIC, the gene that encodes miR-155, were induced as early as four hours post-infection in primary human monocytes. This expression was dependent on TLR2/MyD88 and did not require inflammasome activation. Importantly, miR-155 positively regulated pro-inflammatory cytokine release in human monocytes infected with Francisella. In sharp contrast, we found that the highly virulent type A SCHU S4 strain of Francisella tularensis (F.t.) led to a significantly lower miR-155 response than the less virulent F.n. Hence, F.n. induces miR-155 expression and leads to down-regulation of SHIP, resulting in enhanced pro-inflammatory responses. However, impaired miR-155 induction by SCHU S4 may help explain the lack of both SHIP down-regulation and pro-inflammatory response and may account for the virulence of Type A Francisella