Secretion Of Soluble Mediators Research Articles

Endocan measurement for the postmortem diagnosis of sepsis

The vascular endothelium has been shown to play a pivotal role in the pathophysiology of sepsis through the expression of surface proteins and secretion of soluble mediators. Endocan (endothelial cell-specific molecule-1), a 50-kDa dermatan sulfate proteoglycan, is expressed by endothelial cells in lung and kidney and can be detected at low levels in the serum of healthy subjects. Increased concentrations were described in patients with sepsis, severe sepsis and septic shock compared to healthy individuals, with serum concentrations related to the severity of illness. In the present study, we investigated endocan, procalcitonin and C-reactive protein in postmortem serum from femoral blood in a series of sepsis-related fatalities and control individuals who underwent medicolegal investigations. Endocan was also measured in pericardial fluid. Two study groups were prospectively formed, a sepsis-related fatalities group and a control group. The sepsis-related fatalities group consisted of sixteen forensic autopsy cases with documented clinical diagnosis of sepsis in vivo. The control group consisted of sixteen forensic autopsy cases with various noninfectious causes of death. Postmortem serum endocan concentrations were significantly higher in the sepsis group, with values ranging from 0.519 ng/ml to 6.756 ng/ml. In the control group, endocan levels were undetectable in eleven out of sixteen cases. The results of the data analysis revealed similar endocan concentrations in the pericardial fluid of both studied groups. Endocan can be considered a suitable biological parameter for the detection of sepsis-related deaths in forensic pathology routine.

Stem Cells and Pulmonary Fibrosis: Cause or Cure?

Pulmonary fibrosis is a feature of a number of important lung diseases, and alveolar epithelial injury plays a key role in their pathogenesis. Traditionally, type II alveolar epithelial cells have been viewed as the progenitor cells of the alveolar epithelium; however, recent studies have identified a number of other progenitor and stem cell populations that may participate in alveolar epithelial repair. These studies suggest that the injury microenvironment plays a role in regulation of progenitor cell populations. In human idiopathic pulmonary fibrosis, epithelial abnormalities including altered cell cycling characteristics, hyperplasia, and metaplasia are observed, suggesting that dysregulation of epithelial progenitor cells contributes to the characteristic aberrant repair process. Reactivation of developmental signaling pathways such as the Wnt-β-catenin pathway is implicated in the dysregulation of these cells, and targeting these pathways may provide opportunities for therapeutic intervention. There has been a great deal of interest in the delivery of exogenous stem cells as a therapeutic strategy, and various stem and progenitor cell populations have improved outcomes in animal lung fibrosis models. The contributions of these cells to alveolar epithelial regeneration have been variable, and secretion of soluble mediators has been implicated in the beneficial effects. It remains to be seen whether the promising results seen in the preclinical studies will translate to human disease, and the first studies using mesenchymal stem cells in clinical trials for fibrotic lung disease are underway. Strategies using other stem cell populations hold promise, but currently these are a lot further from the bedside.

Celecoxib decreases the production of cytokines by osteoarthritic cartilage and synovium without affecting cartilage matrix degradation

Purpose: Recently, we reported a coculture model with osteoarthritic cartilage and synovial tissue that is more representative for osteoarthritis (OA) and useful to study mechanisms of action of OA therapies. Celecoxib, a COX-2 inhibitor, is frequently used for the treatment of osteoarthritis. However, its effect on cartilage metabolism has been the subject of much debate. Moreover, it is unclear how celecoxib affects the levels of soluble mediators in the joint. In the current study we investigate the effect of celecoxib on cartilage matrix turnover and the secretion of soluble mediators by cartilage and synovium in a coculture of OA cartilage and synovial tissue. Methods: OA cartilage and OA synovium explants were cultured alone or in coculture for 21 days with the addition of celecoxib at 0.1, 1.0 and 10 μM. To study cartilage matrix turnover glycosaminoglycan (GAG) content, production and release were determined. ELISAs for IL-1α, IL-1ß, IL-4, IL-6, IL-8, IL-10, IL-13, OSM, MCP1, NGF, leptin, adiponectin and PGE2 were performed on conditioned medium of day 4. For statistical analysis, univariate analysis of variance was performed, with a randomized block design to correct for inter-donor variability. P values < 0.05 were considered significantly different. Results: Both osteoarthritic cartilage and synovial tissue produced PGE2 and celecoxib inhibited the production in a dose dependent manner (P < 0.05; Fig. 1a). Celecoxib, irrespective of the concentration used, did not show an effect on GAG content, release and production in the coculture (Fig. 1b). Celecoxib at a concentration of 10 μM decreased the production of IL-1α, IL-1ß, IL-6, IL-10, OSM and NGF (P < 0.05). No effect was seen on leptin, adiponectin, IL-8 and MCP1. IL-13 and IL-4 were not detected (Fig. 1c). Conclusion: This study shows that celecoxib decreased the production of multiple soluble mediators associated with inflammation and pain in OA. Although the production of multiple pro-inflammatory cytokines and PGE2 was decreased, no effect on cartilage matrix turnover was seen. The decreased production of mediators associated with pain in OA, such as NGF, IL-6 and PGE2 is in line with the effect on pain observed in patients treated with celecoxib.

Treatment of Acute Graft-Versus-Host Disease by Means of Multipotent Mesenchymal Stromal Cells: Role of Immunomodulating Soluble Factors Expressed by These Cells in Vitro

Abstract 4554 BackgroundSevere graft-versus-host disease (GvHD) is a life-threatening complication after allogeneic hematopoietic-stem cell transplantation (allo-HSCT). Steroids are the first-line treatment for established GvHD with a response rate of 30–50%. The efficacy of multipotent mesenchymal stromal cells (MMSCs) in the treatment of steroid-resistant GvHD was reported to be 71–94%. Although it has been shown that the immunomodulatory effect of MMSCs mainly occurs through the secretion of soluble mediators, the exact mechanism of their action remains controversial. AimThe aim of the study was to investigate the influence of immunomodulating factors expressed by donor's MMSCs in vitro on the efficacy of MMSCs in the treatment of steroid-resistant GvHD. IL-6, IL-10, colony stimulating factor 1 (CSF1), indoleamine 2, 3 dioxygenase (IDO1), prostaglandin E synthase (PTEGS) and complement factor H (CFH) were measured as factors playing substantial role in GvHD development. MethodsFive patients with steroid resistant acute GvHD were treated with bone marrow derived MMSCs from hematopoietic stem cells donors. MMSCs were infused intravenously at the dose 1×106 per kg of body weight. The efficiency of GvHD therapy by means of MMSCs was scored as: complete response – 3, partial response - 2, clinical improvement – 1, no response – 0.MMSCs were cultured in aMEM with 4% human platelet lysate for 2–5 passages. Total RNA was extracted from MMSCs at 2–3 passages by standard protocol. Relative level of gene expression was estimated by the real-time PCR with previous reverse transcription in MMSCs from 31 donors and determined by normalizing the expression of each target gene to b-actin and GAPDH, calculated using ΔΔCt method for each MMSCs sample. ResultsCharacteristics of patients, donors and graft MMSCs are shown in the table 1.Table 1Characteristics of patients, donors, graft MMSCs and therapy scorePatients Diagnosis/sex/ageCML/ f/39ALL/ m/27AML/ f/20AML*/ f/51AML*/ f/51CML/ m/41Mean value (MMSCs from 31 donors)Correlation between therapy score and RELDonors sex/agef/41f/24f/16m/46m/46m/56Therapy score121320GenesRelative expression level (REL)IL-60.190.682.270.480.487.082.57±0.98−0.76IL-100.170.00.10.690.691.622.73±0.6−0.37IDO10.340.00.860.170.170.330.36±0.13−0.49PTGES5.0365.165.2615.4415.440.8910.07±3.10.45CSF13.870.411.230.740.743.782.08±0.39−0.78CFH4.965.930.561.541.540.171.98±0.360.24m – male, f - female, CML – chronic myeloid leukemia, ALL – acute lymphoid leukemia, AML – acute myeloid leukemia, *in this patient acute GvHD developed twice, after allo-HSCT and after donor’s lymphocyte transfusion for relapse.Correlation between relative level of gene expression and efficacy of MMSCs infusion was investigated. The existence of inverse negative relationship between IL-6, CSF1 expression level and treatment score was revealed. The increased level of IL-6 in donor’s MMSCs in patients with low and no response (treatment score 1 – 0) could be related to main functions of this factor in inflammation. Increased level of CSF1 in donors’ MMSCs could further enhance macrophage activation resulting in GvHD progression instead of inhibition. The augmentation of PTGES expression could be associated with improvement of response to MMSCs therapy, but the correlation was not found. There were no relationship between the expression levels of IL-10, IDO1 and CFH and efficiency of MMSCs infusion.In a patient with no response the relative expression level of all studied factors was altered in comparison with average level of their expression in MMSCs from studied donors: IL-6 and CSF1 increased about 2 fold, while the expression level of IL-10, CHF and PTGES decreased 1.7, 12 and 11 fold correspondingly. So not all MMSCs fit to GvHD therapy that could be associated with their characteristics. ConclusionsThe data demonstrate the influence of IL-6, CSF1 and PTGES expression on the efficacy of MMSCs in the treatment of steroid-resistant GvHD. As most clinical trials involve non-relative allogeneic MMSCs, analysis of expression level of IL6, CSF1 and PTGES in available MMSCs before infusion could predict the efficiency of acute GvHD therapy and permit the choice of the most proper samples. For successful clinical use of MMSCs further investigation of their properties on cellular and molecular levels are needed.Disclosures: No relevant conflicts of interest to declare. Disclosures:No relevant conflicts of interest to declare.

Mesenchymal Stem Cells in Osteoarticular Diseases

Multipotent mesenchymal stromal cells or mesenchymal stem cells (MSCs) are mainly isolated from bone marrow or fat tissue. Owing to their potential for multilineage differentiation towards bone, cartilage and fat tissue, they were initially evaluated in innovative strategies for tissue engineering. More recently, they have gained interest for their immunomodulatory properties and have been tested in various clinical trials that aim to modulate the host immune response in graft-versus-host disease or autoimmune diseases. MSC-mediated immunomodulation occurs through the secretion of soluble mediators. The clinical applications of MSCs for rheumatic diseases focus on their potential to promote tissue repair/regeneration and prevent inflammation. This article will focus on the mechanisms by which MSCs might exhibit a therapeutic potential in rheumatology. Special attention is given to their potential for innovative future strategies.

Biological Therapy for Ulcerative Colitis: What is after Anti-TNF

Ulcerative Colitis (UC) is an idiopathic chronic inflammation. Its etiology is still largely unknown. Environmental and genetic factors in combination with the microbial flora or specific microorganisms are thought to trigger the gut inflammation, leading to the activation of the intestinal immune response. Immune and non-immune cells create a cross talk via the secretion of soluble mediators and expression of cell adhesion molecules, resulting in further cell activation. Mediators such as cytokines and chemokines play a key role in cell recruitment and polarization, intercellular signal amplification or activation and differentiation. Lack of balance between pro-inflammatory and anti-inflammatory cytokines is crucial in the pathogenesis of IBD. Conventional therapy of UC quite commonly fails to avoid complications or colectomy and the therapeutic armamentarium remains still limited. New therapeutic options, such as, biological therapy, gene therapy, hematopoietic stem cell transplantation, and leucoapheresis, have been investigated recently. Biological therapy is focused on different targets involved in the inflammatory process. Several new biological drugs have been introduced in the last decade or are under investigation for the treatment of IBD. They include anti TNF-α agents, anti adhesion molecules, anti IL-12/23, anti IL-6R and more. We review the recent advances in biological therapy for UC treatment beyond the anti-TNFs.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&amp;beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1β(1, 2, 3). Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1β upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system (4, 5, 6, 7). A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1β to its mature, bioactive form (4, 5, 6, 8, 9, 10). The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture (2). The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Platelets: versatile modifiers of innate and adaptive immune responses to transplants

Over the last decade there has been mounting experimental data demonstrating that platelets contribute to acute vascular inflammation and atherosclerosis. This review focuses on recent findings that link platelets to inflammatory responses of relevance to transplants. Although it has been known that platelets modify vascular inflammation by secretion of soluble mediators and release of microparticles, new aspects of these mechanisms are being defined. For example, platelet-derived CCL5 not only functions in homomers, but also forms more potent heteromers with platelet factor 4 (PF4; CXCL4). This heteromer formation can be inhibited with small molecules. New findings also demonstrate heterologous interactions of platelet microparticles with leukocytes that may increase their range of impact. By attaching to neutrophils, platelet microparticles appear to migrate out of blood vessels and into other compartments where they stimulate secretion of cytokines. Contact of platelets with extracellular matrix also can result in cleavage of hyaluronan into fragments that serve as an endogenous danger signal. Recent findings have expanded the range of interactions by which platelets can modify innate and adaptive immune responses to transplants.

IL-6-Dependent PGE2 Secretion by Mesenchymal Stem Cells Inhibits Local Inflammation in Experimental Arthritis

BackgroundBased on their capacity to suppress immune responses, multipotent mesenchymal stromal cells (MSC) are intensively studied for various clinical applications. Although it has been shown in vitro that the immunomodulatory effect of MSCs mainly occurs through the secretion of soluble mediators, the mechanism is still not completely understood. The aim of the present study was to better understand the mechanisms underlying the suppressive effect of MSCs in vivo, using cells isolated from mice deficient in the production of inducible nitric oxide synthase (iNOS) or interleukin (IL)-6 in the murine model of collagen-induced arthritis.Principal FindingsIn the present study, we show that primary murine MSCs from various strains of mice or isolated from mice deficient for iNOS or IL-6 exhibit different immunosuppressive potential. The immunomodulatory function of MSCs was mainly attributed to IL-6-dependent secretion of prostaglandin E2 (PGE2) with a minor role for NO. To address the role of these molecules in vivo, we used the collagen-induced arthritis as an experimental model of immune-mediated disorder. MSCs effectively inhibited collagen-induced inflammation during a narrow therapeutic window. In contrast to wild type MSCs, IL-6-deficient MSCs and to a lesser extent iNOS-deficient MSCs were not able to reduce the clinical signs of arthritis. Finally, we show that, independently of NO or IL-6 secretion or Treg cell induction, MSCs modulate the host response by inducing a switch to a Th2 immune response.SignificanceOur data indicate that MSCs mediate their immunosuppressive effect via two modes of action: locally, they reduce inflammation through the secretion of anti-proliferative mediators, such as NO and mainly PGE2, and systemically they switch the host response from a Th1/Th17 towards a Th2 immune profile.

Airway Epithelial NF-κB Activation Promotes Allergic Sensitization to an Innocuous Inhaled Antigen

Activation of NF-κB in airway epithelium is observed in allergic asthma and is induced by inhalation of numerous infectious and reactive substances. Many of the substances that activate NF-κB in the airway epithelium are also capable of acting as adjuvants to elicit antigen-specific sensitization to concomitantly inhaled protein, thereby circumventing the inherent bias of the lung to promote tolerance to innocuous antigens. We have used a transgenic mouse inducibly expressing a constitutively active mutant of the inhibitor of nuclear factor κB (IκB) kinase β ((CA)IKKβ) that activates NF-κB only in nonciliated airway epithelial cells to test whether activation of this intracellular signaling pathway in this specific cell type is sufficient to establish a pulmonary environment permissive to the development of allergic sensitization to inhaled protein. When airway epithelial (CA)IKKβ was transiently expressed in antigen-naive mice only during initial inhalation of ovalbumin, the mice became allergically sensitized to the antigen. As a consequence, subsequent inhalation of ovalbumin alone led to an allergic asthma-like response that included airway hyperresponsiveness to methacholine, eosinophilia, mucus expression, elevated serum levels of antigen-specific IgE and IgG1, and splenic CD4(+) T cells that secreted T helper type 2 and type 17 cytokines in response to in vitro antigen restimulation. Furthermore, CD11c(+) cells in the mediastinal lymph nodes (MLN) of (CA)IKKβ-expressing mice displayed significantly elevated levels of activation markers. These data implicate airway epithelial NF-κB activation as a critical modulator of the adaptive immune response to inhaled antigens via the secretion of soluble mediators that affect the capacity of CD11c(+) cells to undergo maturation and promote antigen-specific allergic responses.

Mesenchymal stem cells immunosuppressive properties: is it specific to bone marrow-derived cells?

Based on their capacity to suppress immune responses, multipotent mesenchymal stromal cells (MSCs) are intensively studied for regenerative medicine. Moreover, MSCs have paracrine effects, including immunomodulation that occurs through the secretion of soluble mediators, including nitric oxide or interleukin-6, transforming growth factor-beta, human leukocyte antigen G5, and prostaglandin E2. MSCs in the bone marrow are in close contact with T and B cells and regulate immunological memory by organizing defined numbers of dedicated survival niches for plasma cells and memory T cells in the bone marrow. All of these biological effects are probably shared by all stromal cells, including fibroblasts and stem cells isolated from exfoliated deciduous teeth. The therapeutical implications are discussed.

Emerging Biologics in the Treatment of Inflammatory Bowel Disease: What is Around the Corner?

Inflammatory bowel diseases (IBD) are idiopathic chronic inflammations: the etiology of Crohn's disease (CD) and ulcerative colitis (UC) is still largely unknown. Environmental and genetic factors in combination with the microbial flora or specific microorganisms trigger an event, leading to the activation of an intestinal immune response. Immune and non-immune cells create a cross talk via the secretion of soluble mediators and expression of cell adhesion molecules, resulting in further cell activation. Mediators such as cytokines and chemokines play a role in cell recruitment and polarization, intercellular signal amplification or activation and differentiation. Considering these aspects, medical management of inflammatory bowel disease has changed considerably over the past decade. Advances in biotechnology has allowed for the introduction of many biologic therapies, other than anti-TNF therapies. Many of these drugs showed clinical benefit for induction and maintenance therapy, both in UC and CD. Although numerous, at present only monoclonal anti-TNF antibodies are currently available worldwide. Other biological agents have been tested or are under evaluation. This paper systematically reviews the mechanism-of-action, efficacy, short-term and, where available, long-term safety of biological agents that have been approved for the treatment of IBD or are under evaluation which target different molecules other than tumor necrosis factor alpha (TNF-alpha).

Dendritic cells facilitate accumulation of IL-17 T cells in the kidney following acute renal obstruction

Acute urinary obstruction causes interstitial inflammation with leukocyte accumulation and the secretion of soluble mediators. Here we show that unilateral ureteral ligation caused a progressive increase in renal F4/80(+) and F4/80(-) dendritic cells, monocytes, neutrophils and T-cells 24-72 h following obstruction. Depletion of dendritic cells by clodronate pretreatment showed these cells to be the most potent source of tumor necrosis factor and other pro-inflammatory mediators in the obstructed kidney. F4/80(+) dendritic cells and T-cells co-localized in the cortico-medullary junction and cortex of the obstructed kidney. Cytokine secretion patterns and surface phenotypes of T-cells from obstructed kidneys were found to include interferon-gamma-secreting CD4(+) and CD8(+) memory T-cells as well as interleukin 17 (IL-17)-secreting CD4(+) memory T-cells. Depletion of the intra-renal dendritic cells prior to ligation did not numerically reduce T-cells in obstructed kidneys but attenuated interferon-gamma and IL-17-competent T-cells. Our study shows that intra-renal dendritic cells are a previously unidentified early source of proinflammatory mediators after acute urinary obstruction and play a specific role in recruitment and activation of effector-memory T-cells including IL-17-secreting CD4(+) T-cells.

048 Interleukin-1β Regulates the Secretion of but not Expression of Receptors for Prostaglandin E2

Background: Activation of the cyclooxygenase-2 (COX-2) pathway and production of prostaglandin E2 (PGE2), have been linked to dermal wound healing in fetal and adult tissue. Activation of this pathway has been previously linked to interleukin (IL)-1β expression. This study addresses the mechanism by which IL-1β regulates both production of and response to PGE2 in cultured human fetal and adult dermal fibroblasts. Hypothesis: IL-1β regulates fetal and adult dermal fibroblast PGE2 signaling. Methods: Fetal and adult dermal fibroblasts were isolated from human skin explants. Secretion of soluble mediators was assayed using ELISA kits. Expression of COX-2 as well as the various PGE2 receptors (EP1, EP2, EP3, EP4) was analyzed at both the mRNA and protein levels. Results: Compared to adult fibroblasts, fetal fibroblasts produced decreased levels of IL-6 and IL-8, but similar levels of PGE2. IL-1β1 caused up-regulation of COX-2 and increased PGE2 secretion in both fetal and adult fibroblasts. However, administration of IL-1β, tumor necrosis factor-α or interferon-γ did not significantly alter the mRNA or protein levels of any PGE2 receptor subtype. Discussion: Our hypothesis is partially supported by the results. IL-1β causes up-regulation of COX-2 and increased PGE2 secretion in fetal and adult dermal fibroblasts, but does not alter the expression of PGE2 receptors. Therefore, proinflammatory mediator-induced changes in both fetal and adult skin fibroblasts appear to act through increased production of the signal molecules and not through increased or altered receptor expression for PGE2. This finding raises the possibility of increased autocrine signaling in the context of dermal inflammation, such as that associated with a wound healing response. Acknowledgments: The authors would like to thank Children’s Hospital of Pittsburgh for providing funding for this research.

Toll-Like Receptors in Human Infectious Diseases

Toll-like receptors (TLRs) have emerged as critical players in immunity. They are capable of sensing organisms ranging from protozoa to bacteria, fungi or viruses upon detection of the pathogen as well as recognizing endogenous ligands, and triggering transduction pathways. Following activation of the innate immune system, strong inflammatory signals are generated inducing inflammation and activation of the adaptive immune response. However, the deregulation of TLRs signaling pathways may be conducive to the pathogenesis of many infectious diseases. Therefore, innate and adaptive immunity are not simply sequential and complementary mechanisms of resistance to pathogen, they regulate each other through cellular contacts and the secretion of soluble mediators. Herein, we summarize recent findings on TLRs signaling in infectious diseases and how pathogens have developed strategies to evade these pathways. In this context, a potential modulation of the innate immune response could have therapeutic benefit through the development of new drugs as well as vaccination strategies to be employed in infectious diseases.

Cutting Edge: Low-Affinity, Smith Antigen-Specific B Cells Are Tolerized by Dendritic Cells and Macrophages

Polyclonal B cell activation promotes immunity without the loss of tolerance. Our data show that during activation of the innate immune system, B cell tolerance to Smith Ag Sm is maintained by dendritic cells (DCs) and macrophages (MPhi). TLR4-activated myeloid DCs and MPhi, but not plasmacytoid or lymphoid DCs, repressed autoreactive B cells through the secretion of soluble mediators, including IL-6. Although IL-6 promotes plasma cell differentiation of B cells acutely stimulated by Ag, we show that it repressed cells that were chronically exposed to self-Ag. This mechanism of tolerance was not limited to Smith Ag-specific B cells as hen egg lysozyme- and p-azophenylarsonate-specific B cells were similarly affected. Our data define a tolerogenic role for MPhi and DCs in regulating autoreactive B cells during activation of the innate immune system.

Evaluation of the frequency of virus-specific CD8+ T cells by cytokine flow cytometry.

Since its introduction over 25 years ago, flow cytometry has evolved to be one of the most important methods in cellular diagnostics and research. Within recent years, new technology has dramatically expanded the range of parameters that can be analyzed by this technology. These include, for example, the production or secretion of soluble mediators (1-4 ), cell proliferation (5-8), and the detection of molecules expressed at very low density (9,10). One of the most prominent recent developments, however, is the analysis of antigen-specific T cells. The problems encountered when analyzing antigen-specific T cells are manyfold. One of the major problems is the fact that their frequencies tend to be rather low. Recent data show that, in certain situations (e.g., acute immune responses against viruses or bacteria), the actual frequency of specific CD8 cells can be much higher than previously estimated by limiting dilution assays; however, in most other responses, the frequencies of specific T cells are clearly below these values. Frequencies as low as 1CH-10—5 have been reported (11,12).KeywordsHuman Immunodeficiency VirusMajor Histocompatibility ComplexSurface StainingTetramer StainingLimit Dilution AssayThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Ultraviolet radiation-induced tolerance.

Ultraviolet (UV) radiation is one of the most important environmental factors. Besides its well-known advantages and its indispensable effects on human life, UV light, particularly the middle wavelength range (290-320 nm [UVB]), can harm human health by inducing skin cancer, premature skin aging, inflammation, and cell death (1-4). Over the last 25 years, it has become apparent that exposure to UVB radiation may also suppress immune reactions. Review of the numerous studies on the immunosuppressive properties of UV radiation led to two quite surprising observations: 1) that UV exposure can have systemic immunologic consequences 2) that UV radiation can induce antigen-specific tolerance. This paper briefly reviews the phenomenon of UV-induced tolerance.

Endothelial cells: role in infection and inflammation.

Infection and resulting sepsis continue to be important causes of morbidity and mortality in surgical patients. Although much has been learned about the pathogens and the leukocyte responses to these pathogens, we are only beginning to understand the role of the host in these pathologies. The endothelium is a dynamic participant in cellular and organ function rather than a static barrier as it was once believed. Emerging evidence implicates the endothelium as a central effector in the inflammatory response. Through the expression of surface proteins and secretion of soluble mediators, the endothelium controls vascular tone and permeability, regulates coagulation and thrombosis, and directs the passage of leukocytes into areas of inflammation. Derangements in these normal functions may contribute significantly to a maladaptive inflammatory response leading to systemic inflammation and multiple organ failure.

In situ pulmonary vascular perfusion for improved recovery of pulmonary intravascular macrophages

The microcirculation contains mononuclear phagocytes, with features characteristic of macrophages, adhered to luminal capillary surfaces by intercellular adhesion plaques. These pulmonary intravascular macrophages may play an important role in regulating lung vascular tone and capillary permeability, and may modulate capillary endothelial cell growth and replication by the secretion of soluble mediators (i.e., arachidonate metabolites, cytokines). This study describes a technique which utilizes in situ lung perfusion to remove intravascular macrophages in large numbers from the microcirculation of porcine lung ( n = 26). This technique yielded 3.8 ± 0.5 × 10 8 (mean ± SEM) mononuclear cells which were highly phagocytic toward particulate carbon (phagocytic index, 80 ± 6%). Harvested mononuclear phagocytes reestablished intercellular adhesion plaques when placed on small vessel porcine pulmonary artery endothelial cell monolayers and exhibited histochemical characteristics typical of monocyte/macrophage lineage cells. Mononuclear cells obtained from lung microcirculation displayed size heterogeneity varying from 10.4 to 16.5 μm in diameter. Both large and small cell populations phagocytosed particulate carbon. Morphometric studies performed on collagenase-treated lung demonstrated that in situ perfusion removed significant numbers of intravascular macrophages in lung capillaries. The technique described permits the rapid removal of anchored mononuclear phagocytes from lung capillaries with minimal postmortem delay.