Haem is associated with thrombosis in neonates and infants undergoing cardiac surgery for congenital heart disease.

BACKGROUND: Haem levels are associated with thrombosis in a variety of diseases, as well as being a contributing cause of thrombotic events in animal models. MATERIALS AND METHODS: We retrospectively analyzed samples from 39 children who underwent cardiac surgery with cardiopulmonary bypass, including 15 children who developed a postoperative thrombosis and 24 controls. RESULTS: Patients who developed thrombosis postoperatively had statistically significant higher average haem levels over time (presurgery to 12 h postsurgery) compared to patients who did not develop thrombosis. CONCLUSION: Higher cell-free total haem levels are associated with a higher risk of thrombosis in a paediatric cardiac surgical cohort.

A review of the available clinical therapies for vulvodynia management and new data implicating proinflammatory mediators in pain elicitation.

Localised provoked vulvodynia (LPV) is a common, chronic, and disabling condition: patients experience profound pain and a diminished quality of life. The aetiologic origins of vulvodynia are poorly understood, yet recent evidence suggests a link to site-specific inflammatory responses. Fibroblasts isolated from the vestibule of LPV patients are sensitive to proinflammatory stimuli and copiously produce pain-associated proinflammatory mediators (IL-6 and PGE2 ). Although LPV is a multifactorial disorder, understanding vulvar inflammation and targeting the inflammatory response should lead to treatment advances, especially for patients exhibiting signs of inflammation. NFκB (already targeted clinically) or other inflammatory components may be suitable therapeutic targets. TWEETABLE ABSTRACT: Vulvodynia is a poorly understood, prevalent, and serious women's health issue requiring better understanding to improve therapy.

Ionizing radiation induces myofibroblast differentiation via lactate dehydrogenase.

Pulmonary fibrosis is a common and dose-limiting side-effect of ionizing radiation used to treat cancers of the thoracic region. Few effective therapies are available for this disease. Pulmonary fibrosis is characterized by an accumulation of myofibroblasts and excess deposition of extracellular matrix proteins. Although prior studies have reported that ionizing radiation induces fibroblast to myofibroblast differentiation and collagen production, the mechanism remains unclear. Transforming growth factor-ß (TGF-ß) is a key profibrotic cytokine that drives myofibroblast differentiation and extracellular matrix production. However, its activation and precise role in radiation-induced fibrosis are poorly understood. Recently, we reported that lactate activates latent TGF-ß through a pH-dependent mechanism. Here, we wanted to test the hypothesis that ionizing radiation leads to excessive lactate production via expression of the enzyme lactate dehydrogenase-A (LDHA) to promote myofibroblast differentiation. We found that LDHA expression is increased in human and animal lung tissue exposed to ionizing radiation. We demonstrate that ionizing radiation induces LDHA, lactate production, and extracellular acidification in primary human lung fibroblasts in a dose-dependent manner. We also demonstrate that genetic and pharmacologic inhibition of LDHA protects against radiation-induced myofibroblast differentiation. Furthermore, LDHA inhibition protects from radiation-induced activation of TGF-ß. We propose a profibrotic feed forward loop, in which radiation induces LDHA expression and lactate production, which can lead to further activation of TGF-ß to drive the fibrotic process. These studies support the concept of LDHA as an important therapeutic target in radiation-induced pulmonary fibrosis.

Anticancer Role of PPARgamma Agonists in Hematological Malignancies Found in the Vasculature, Marrow, and Eyes.

The use of targeted cancer therapies in combination with conventional chemotherapeutic agents and/or radiation treatment has increased overall survival of cancer patients. However, longer survival is accompanied by increased incidence of comorbidities due, in part, to drug side effects and toxicities. It is well accepted that inflammation and tumorigenesis are linked. Because peroxisome proliferator-activated receptor (PPAR)-gamma agonists are potent mediators of anti-inflammatory responses, it was a logical extension to examine the role of PPARgamma agonists in the treatment and prevention of cancer. This paper has two objectives: first to highlight the potential uses for PPARgamma agonists in anticancer therapy with special emphasis on their role when used as adjuvant or combined therapy in the treatment of hematological malignancies found in the vasculature, marrow, and eyes, and second, to review the potential role PPARgamma and/or its ligands may have in modulating cancer-associated angiogenesis and tumor-stromal microenvironment crosstalk in bone marrow.

Targeting cyclooxygenase-2 in hematological malignancies: rationale and promise.

There is much interest in the potential use of Cox-2 selective inhibitors in combination with other cancer therapeutics. Malignancies of hematopoietic and non-hematopoietic origin often have increased expression of cyclooxygenase-2 (Cox-2), a key modulator of inflammation. For example, hematological malignancies such as chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma and multiple myeloma often highly express Cox-2, which correlates with poor patient prognosis. Expression of Cox-2 enhances survival and proliferation of malignant cells, while negatively influencing anti-tumor immunity. Hematological malignancies expressing elevated levels of Cox-2 potentially avoid immune responses by producing factors that enhance angiogenesis and metastasis. Cellular immune responses regulated by natural killer cells, cytotoxic T lymphocytes, and T regulatory cells are also influenced by Cox-2 expression. Therefore, Cox-2 selective inhibitors have promising therapeutic potential in patients suffering from certain hematological malignancies.

Release of biologically active CD154 during collection and storage of platelet concentrates prepared for transfusion.

BACKGROUND: Millions of platelet transfusions are given each year. Transfusion reactions occur in as many as 30% of patients receiving unmodified platelet transfusions. The cause of some transfusion reactions remains unclear. The current paradigm suggests that platelet concentrates (PC) contain proinflammatory mediators that are released by white blood cells during collection, processing and storage. CD154 (CD40 ligand, CD40L) is a potent inflammatory mediator, normally sequestered inside the resting platelet, that is known to translocate to the platelet membrane and be shed into plasma in response to agonist activation. We hypothesized that platelet-soluble CD154 (sCD154) is 'spontaneously' released by transfused platelets and plays a major role in transfusion reactions. OBJECTIVES: To determine the time course and biological properties of CD154 translocation and release during collection and storage of platelets for transfusion. METHODS: We measured surface and sCD154 in platelets prepared by the platelet-rich plasma method or apheresis by fluorescence-activated cell sorting and enzyme-linked immunosorbent assay, respectively. The specific biological activity of platelet sCD154 was assayed by stimulation of the CD154/CD40 pathway in known CD40-positive cells with PC-derived supernatants. RESULTS AND CONCLUSIONS: We demonstrate that PCs prepared for transfusion have high levels of membrane-bound CD154 and sCD154, with maximum levels being seen 72 h after platelet collection. Importantly, we show that platelet-derived sCD154 potently stimulates CD40-positive cells. We propose that platelet-derived CD154 is a key 'cytokine' responsible for adverse reactions associated with platelet transfusions. Improved methods of platelet collection and/or storage, which limit CD154 expression, could reduce the risks of transfusion reaction.

The aryl hydrocarbon receptor is a regulator of cigarette smoke induction of the cyclooxygenase and prostaglandin pathways in human lung fibroblasts.

Cigarette smoking can lead to chronic lung inflammation and lung cancer. Chronic inflammation, associated with expression of cyclooxygenase-2 (COX-2) and prostaglandins, predisposes to malignancy. We recently demonstrated that human lung fibroblasts are activated by cigarette smoke to express COX-2 and prostaglandin E(2) (PGE(2)). Little is known about the mechanism whereby smoke activates human lung fibroblasts to produce proinflammatory mediators. Herein, we report the central role of the aryl hydrocarbon receptor (AHR) in cigarette smoke extract (CSE)-induced COX-2, microsomal PGE(2) synthase (mPGES), and PGE(2) production in human lung fibroblasts. Western blot analysis revealed that primary strains of human lung fibroblasts express AHR and aryl hydrocarbon nuclear translocator protein, supporting the possibility that smoke activates lung fibroblasts through this pathway. Experiments were subsequently performed to determine whether the AHR was activated by CSE. Immunocytochemistry and EMSA analysis revealed that CSE induced nuclear translocation of the AHR in human lung fibroblasts. CSE decreased protein levels of the AHR, consistent with AHR ligand-induced proteosome-mediated degradation. CSE also induced mPGES-1 and COX-2 protein and increased PGE(2) production. Treatment of human fibroblasts with AHR antagonists in the presence of CSE inhibited AHR nuclear translocation as well as COX-2, mPGES-1, and PGE(2) production. These data indicate that the AHR pathway plays an important role in cigarette smoke-mediated COX-2 and PG production in human lung fibroblasts and may contribute to tobacco-associated inflammation and lung disease.

Role of CXCR2 in cigarette smoke-induced lung inflammation.

It has been hypothesized that the destruction of lung tissue observed in smokers with chronic obstructive pulmonary disease and emphysema is mediated by neutrophils recruited to the lungs by smoke exposure. This study investigated the role of the chemokine receptor CXCR2 in mediating neutrophilic inflammation in the lungs of mice acutely exposed to cigarette smoke. Exposure to dilute mainstream cigarette smoke for 1 h, twice per day for 3 days, induced acute inflammation in the lungs of C57BL/6 mice, with increased neutrophils and the neutrophil chemotactic CXC chemokines macrophage inflammatory protein (MIP)-2 and KC. Treatment with SCH-N, an orally active small molecule inhibitor of CXCR2, reduced the influx of neutrophils into the bronchoalveolar lavage (BAL) fluid. Histological changes were seen, with drug treatment reducing perivascular inflammation and the number of tissue neutrophils. beta-Glucuronidase activity was reduced in the BAL fluid of mice treated with SCH-N, indicating that the reduction in neutrophils was associated with a reduction in tissue damaging enzymes. Interestingly, whereas MIP-2 and KC were significantly elevated in the BAL fluid of smoke exposed mice, they were further elevated in mice exposed to smoke and treated with drug. The increase in MIP-2 and KC with drug treatment may be due to the decrease in lung neutrophils that either are not present to bind these chemokines or fail to provide a feedback signal to other cells producing these chemokines. Overall, these results demonstrate that inhibiting CXCR2 reduces neutrophilic inflammation and associated lung tissue damage due to acute cigarette smoke exposure.

The CD40-CD40 ligand system: a potential therapeutic target in atherosclerosis.

Atherosclerosis is a leading cause of cardiovascular disease in the westernized world. This review highlights emerging evidence linking atherosclerosis to the CD40-CD40 ligand (CD154) pathway. Recently, atherosclerosis has been associated with chronic inflammation, linking it to the immune system. This novel viewpoint may serve as an additional target for therapeutic intervention. CD40 and CD154 are highly expressed in atherosclerotic human plaques. Recent data from preclinical animal models of atherosclerosis show that disruption of the CD40-CD154 pathway can prevent atherosclerotic progression and may reverse established lesions. Blockade of the CD40-CD154 pathway by biologicals or small molecules may prove valuable in the treatment of atherosclerosis.

Fibroblast heterogeneity: existence of functionally distinct Thy 1(+) and Thy 1(-) human female reproductive tract fibroblasts.

Little is known about fibroblasts from the female reproductive tract, much less whether or not functional subsets exist. Fibroblasts are key as sentinel cells for recruiting white blood cells and for wound healing. The purpose of this research was to evaluate the possibility that functional subsets of fibroblasts exist in the human female reproductive tract. The strategy used was to define fibroblast subpopulations based on their surface expression of the Thy 1 antigen. In situ staining of human myometrium and endometrium showed heterogeneous staining for Thy 1. Freshly derived strains of fibroblasts from the myometrium and endometrium also demonstrated heterogeneous Thy 1 expression. For the first time, using magnetic beading and fluorescence-activated cell sorting, human myometrial fibroblasts were successfully separated into functionally unique Thy 1(+) and Thy 1(-) subsets. Both subsets produced the proinflammatory cytokines interleukin (IL)-6 and IL-8 after IL-1beta stimulation, but only the Thy 1(+) subset produced MCP-1. Furthermore, only Thy 1(+) fibroblasts up-regulated CD40 surface expression with IL-1beta or interferon-gamma treatment. Engagement of CD40 in the Thy 1(+) subpopulation induced IL-6, IL-8, and MCP-1. The discovery of functional subsets of reproductive tract fibroblasts now permits assessment of their roles in the normal functions of the reproductive tract and in disease states such as adhesions and menorrhagia.

IL-8 production in human lung fibroblasts and epithelial cells activated by the Pseudomonas autoinducer N-3-oxododecanoyl homoserine lactone is transcriptionally regulated by NF-kappa B and activator protein-2.

The destructive pulmonary inflammation associated with Pseudomonas aeruginosa colonization is caused, in part, by the production of the chemokine IL-8, which recruits neutrophils into the lung. The Pseudomonas autoinducer, N-3-oxododecanoyl homoserine lactone (3-O-C12-HSL), is a small lipid-soluble molecule that is essential in the regulation of many P. aeruginosa virulence factors, but little is known about how it affects eukaryotic cells. In this report we demonstrate that 3-O-C12-HSL is a potent stimulator of both IL-8 mRNA and protein from human fibroblasts and epithelial cells in vitro. The IL-8 produced from these 3-O-C12-HSL-stimulated cells was found to be functionally active by inducing the chemotaxis of neutrophils. To determine a mechanism for this IL-8 induction, deletion constructs of the IL-8 promoter were examined. It was found that the DNA region between nucleotides -1481 and -546 and the transcription factor NF-kappaB were essential for the maximal induction of IL-8 by 3-O-C12-HSL. This was confirmed by EMSAs, where 3-O-C12-HSL induced a shift with both AP-2 and NF-kappaB consensus DNA. The activation of NF-kappaB and subsequent production of IL-8 were found to be regulated by a mitogen-activated protein kinase pathway. These findings support the concept that the severe lung damage that accompanies P. aeruginosa infections is caused by an exuberant neutrophil response stimulated by 3-O-C12-HSL-induced IL-8. Understanding the mechanisms of 3-O-C12-HSL activation of lung structural cells may provide a means to help control lung damage during infections with P. aeruginosa.

The relationship of CD5+ B lymphocytes to macrophages: insights from normal biphenotypic B/macrophage cells.

For decades, numerous investigators have reported derivation of macrophage-like cells from CD5(+) pre-B cell lymphomas. Recently, it has become clear that biphenotypic CD5(+) B/macrophage cells are not a spurious result of malignancy. Indeed, the existence of normal biphenotypic cells with CD5(+) B lymphocyte and macrophage characteristics has been demonstrated in the mouse. This review considers normal B/macrophage cell function in an evolutionary context where a primitive, flexible cell type could perform dual roles in adaptive and innate immunity.

Expression of stromal-derived factor-1 is decreased by IL-1 and TNF and in dermal wound healing.

Stromal-derived factor-1 (SDF-1) is a CXC chemokine that is believed to be constitutively expressed by stromal cells of numerous tissues. In this report, we demonstrate that dermal fibroblasts and vessels of noninflamed tissues express SDF-1. Unexpectedly, we found that expression of SDF-1 is regulated by inflammation. Expression of SDF-1 by primary cultures of human gingival fibroblasts is potently inhibited by activated macrophages via secretion of IL-1alpha and TNF-alpha. Levels of SDF-1 mRNA also decrease in acutely inflamed mouse dermal wounds. We propose that SDF-1 functions as a homeostatic regulator of tissue remodeling, whose expression stabilizes existing dermal architecture.

Cd40 expression in uterine tissues: a key regulator of cytokine expression by fibroblasts.

CD40 is a cell surface receptor initially discovered on cells of the hemopoietic lineage. Its primary role on immune cells is to enhance their activation and hence their production of cytokines and immunomodulatory molecules. Recently, CD40 has also been detected on human fibroblasts. An emerging view of the fibroblast is that it is far more than a structural cell, being capable of intimate interaction with cells of the immune system. In fibroblasts from several tissues, the engagement of CD40 with its ligand (CD40L) resulted in the secretion of proinflammatory molecules such as interleukin-6 (IL-6) and IL-8. Currently, there are few data about the presence of the CD40-CD40L system in female reproductive tissues. This study investigates the expression of CD40 by human endometrium, myometrium, and cervix both in situ and in tissue explant-derived fibroblasts. CD40 was detected mainly in the perivascular region of endometrium, myometrium, and cervix. Light staining for CD40 was observed in stromal elements. Additionally, the basal epithelium of cervix expressed CD40. Fibroblastic cells derived from all three sources express low levels of CD40, and this is up-regulated with interferon-gamma treatment (500 U/mL; 72 h). When activated with interferon-gamma and CD40L, the fibroblasts secreted increased amounts of IL-6, IL-8, and MCP-1. These data suggest that the CD40-CD40L system may provide a link between the resident structural cells of these reproductive tissues and the infiltrating immune cells or activated platelets that may express CD40L. The possible interaction of CD40 with CD40L may be particularly important during events such as menstruation and cervical ripening, where up-regulation of the proinflammatory molecules IL-6 and IL-8 is viewed as critical for these processes. In addition, dysregulation of this system may be a contributory factor to problems such as menstrual dysfunction and preterm labor.

Peroxisome proliferator activator receptor-gamma agonists and 15-deoxy-Delta(12,14)(12,14)-PGJ(2) induce apoptosis in normal and malignant B-lineage cells.

The research described herein evaluates the expression and functional significance of peroxisome proliferator activator receptor-gamma (PPAR-gamma) on B-lineage cells. Normal mouse B cells and a variety of B lymphoma cells reflective of stages of B cell differentiation (e.g., 70Z/3, CH31, WEHI-231, CH12, and J558) express PPAR-gamma mRNA and, by Western blot analysis, the 67-kDa PPAR-gamma protein. 15-Deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), a PPAR-gamma agonist, has a dose-dependent antiproliferative and cytotoxic effect on normal and malignant B cells as shown by [(3)H]thymidine and 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assays. Only PPAR-gamma agonists (thiazolidinediones), and not PPAR-alpha agonists, mimicked the effect of 15d-PGJ(2) on B-lineage cells, indicating that the mechanism by which 15d-PGJ(2) negatively affects B-lineage cells involves in part PPAR-gamma. The mechanism by which PPAR-gamma agonists induce cytotoxicity is via apoptosis, as shown by annexin V staining and as confirmed by DNA fragmentation detected using the TUNEL assay. Interestingly, addition of PGF(2alpha), which was not known to affect lymphocytes, dramatically attenuated the deleterious effects of PPAR-gamma agonists on B lymphomas. Surprisingly, 15d-PGJ(2) induced a massive increase in nuclear mitogen-activated protein kinase activation, and pretreatment with PGF(2alpha) blunted the mitogen-activated protein kinase activation. This is the first study evaluating PPAR-gamma expression and its significance on B lymphocytes. PPAR-gamma agonists may serve as a counterbalance to the stimulating effects of other PGs, namely PGE(2), which promotes B cell differentiation. Finally, the use of PGs, such as 15d-PGJ(2), and synthetic PPAR-gamma agonists to induce apoptosis in B-lineage cells may lead to the development of novel therapies for fatal B lymphomas.

PPAR-gamma-mediated regulation of normal and malignant B lineage cells.

Prostaglandins of the E-series stimulate B lymphocytes by enhancing immunoglobulin-class switching and antibody production. Little is known about whether or not other prostaglandins affect B lineage cells and perhaps counterbalance the stimulatory effects of PGE2. PGD2 is a major product of cyclooxygenase in bone marrow and in macrophages, suggesting a role for this lipid product in immunological responses. PGD2 undergoes dehydration to the biologically active prostaglandin 15-deoxy-delta 12,14-PGJ2 (15d-PGJ2) that binds to the nuclear receptor known as peroxisome proliferator-activated receptor gamma (PPAR-gamma). We found that normal mouse B cells and a variety of B lymphoma cells (e.g., 70Z/3, WEHI-231, CH12, and J558) express PPAR-gamma mRNA and the 67-kDa PPAR-gamma protein. 15d-PGJ2 had a dose-dependent antiproliferative/cytotoxic effect on normal and malignant B cells, as shown by 3H-thymidine and MTT assays. Only PPAR-gamma agonists (i.e., thiazolidinediones) mimicked the effect of 15d-PGJ2 on B lineage cells, indicating that the mechanism by which 15d-PGJ2 negatively affects B lineage cells involves PPAR-gamma. The mechanism whereby PPAR-gamma agonists induced cytotoxicity is via apoptosis, as shown by Annexin V assay. PPAR-gamma agonists may serve as a counterbalance to the stimulating effects of PGE2, which promotes B-cell differentiation. The use of prostaglandins, such as 15d-PGJ2, and synthetic PPAR-gamma agonists to induce apoptosis in B lineage cells may lead to the development of therapies for fatal PGE2-resistant B lymphomas.