Repair of Subacute Intrasubstance Rupture of the Biceps Brachii in a 16-Year-Old Patient.

A 16-year-old boy sustained a complete, closed, intrasubstance rupture of both heads of the biceps brachii after a rope swing accident. The patient was managed with open direct repair of the muscle belly. After the surgery, he underwent an extensive physical therapy regimen and regained full range of motion and strength. To our knowledge, this is the youngest reported case of a subacute intrasubstance rupture of the biceps brachii muscle treated surgically in the literature. There is no consensus in the literature regarding the optimal management of these injuries. Given the satisfactory outcome, we suggest that open direct repair of the muscle belly is a reasonable option for the pediatric population.

Mast cell deficiency attenuates progression of atherosclerosis and hepatic steatosis in apolipoprotein E-null mice.

Mast cells are important cells of the immune system and are recognized as participants in the pathogenesis of atherosclerosis. In this study, we evaluated the role of mast cells on the progression of atherosclerosis and hepatic steatosis using the apolipoprotein E-deficient (ApoE(-/-)) and ApoE(-/-)/mast cell-deficient (Kit(W-sh/W-sh)) mouse models maintained on a high-fat diet. The en face analyses of aortas showed a marked reduction in plaque coverage in ApoE(-/-)/Kit(W-sh/W-sh) compared with ApoE(-/-) after a 6-mo regimen with no significant change noted after 3 mo. Quantification of intima/media thickness on hematoxylin and eosin-stained histological cross sections of the aortic arch revealed no significant difference between ApoE(-/-) and ApoE(-/-)/Kit(W-sh/W-sh) mice. The high-fat regimen did not induce atherosclerosis in either Kit(W-sh/W-sh) or wild-type mice. Mast cells with indications of degranulation were seen only in the aortic walls and heart of ApoE(-/-) mice. Compared with ApoE(-/-) mice, the serum levels of total cholesterol, low-density lipoprotein and high-density lipoprotein were decreased by 50% in ApoE(-/-)/Kit(W-sh/W-sh) mice, whereas no appreciable differences were noted in serum levels of triglycerides or very low density lipoprotein. ApoE(-/-)/Kit(W-sh/W-sh) mice developed significantly less hepatic steatosis than ApoE(-/-) mice after the 3-mo regimen. The analysis of Th1/Th2/Th17 cytokine profile in the sera revealed significant reduction of interleukin (IL)-6 and IL-10 in ApoE(-/-)/Kit(W-sh/W-sh) mice compared with ApoE(-/-) mice. The assessment of systemic generation of thromboxane A(2) (TXA(2)) and prostaglandin I(2) (PGI(2)) revealed significant decrease in the production of PGI(2) in ApoE(-/-)/Kit(W-sh/W-sh) mice with no change in TXA(2). The decrease in PGI(2) production was found to be associated with reduced levels of cyclooxygenase-2 mRNA in the aortic tissues. A significant reduction in T-lymphocytes and macrophages was noted in the atheromas of the ApoE(-/-)/Kit(W-sh/W-sh) mice. These results demonstrate the direct involvement of mast cells in the progression of atherosclerosis and hepatic steatosis.

Human mast cells (HMC-1 5C6) enhance interleukin-6 production by quiescent and lipopolysaccharide-stimulated human coronary artery endothelial cells.

We examined the effect of intact human mast cells (HMC-1 5C6) and their selected mediators on interleukin-6 (IL-6) production and bone morphogenetic protein-2 (BMP-2) expression in human coronary artery endothelial cells (HCAEC) in the presence and absence of lipopolysaccharide (LPS). Scanning electron microscopy showed that HMC-1 5C6 cells adhere to HCAEC in cocultures. Addition of HMC-1 5C6 cells markedly enhanced the IL-6 production by quiescent and LPS-activated HCAEC even at the maximal concentration of LPS. Furthermore, mast cell-derived histamine and proteases accounted for the direct and synergistic effect of mast cells on IL-6 production that was completely blocked by the combination of histamine receptor-1 antagonist and protease inhibitors. Another novel finding is that histamine was able to induce BMP-2 expression in HCAEC. Collectively, our results suggest that endotoxin and mast cell products synergistically amplify vascular inflammation and that histamine participates in the early events of vascular calcification.

Lipopolysaccharide induces H1 receptor expression and enhances histamine responsiveness in human coronary artery endothelial cells.

Summary Histamine is a well-recognized modulator of vascular inflammation. We have shown that histamine, acting via H1 receptors (H1R), synergizes lipopolysaccharide (LPS)-induced production of prostaglandin I(2) (PGI(2)), PGE(2) and interleukin-6 (IL-6) by endothelial cells. The synergy between histamine and LPS was partly attributed to histamine -induced expression of Toll-like receptor 4 (TLR4). In this study, we examined whether LPS stimulates the H1R expression in human coronary artery endothelial cells (HCAEC) with resultant enhancement of histamine responsiveness. Incubation of HCAEC with LPS (10-1000 ng/ml) resulted in two-fold to fourfold increases in H1R mRNA expression in a time-dependent and concentration-dependent fashion. In contrast, LPS treatment did not affect H2R mRNA expression. The LPS-induced H1R mRNA expression peaked by 4 hr after LPS treatment and remained elevated above the basal level for 20-24 hr. Flow cytometric and Western blot analyses revealed increased expression of H1R protein in LPS-treated cells. The specific binding of [(3)H]pyrilamine to H1R in membrane proteins from LPS-treated HCAEC was threefold higher than the untreated cells. The LPS-induced H1R expression was mediated through TLR4 as gene silencing by TLR4-siRNA and treatment with a TLR4 antagonist inhibited the LPS effect. When HCAEC were pre-treated with LPS for 24 hr, washed and challenged with histamine, 17-, 10- and 15-fold increases in PGI(2), PGE(2) and IL-6 production, respectively, were noted. Histamine-induced enhancement of the synthesis of PGI(2), PGE(2) and IL-6 by LPS-primed HCAEC was completely blocked by an H1R antagonist. The results demonstrate that LPS, through TLR4 activation, up-regulates the expression and function of H1R and amplifies histamine-induced inflammatory responses in HCAEC.

Histamine directly and synergistically with lipopolysaccharide stimulates cyclooxygenase-2 expression and prostaglandin I(2) and E(2) production in human coronary artery endothelial cells.

Although histamine plays an essential role in inflammation, its influence on cyclooxygenases (COX) and prostanoid homeostasis is not well understood. In this study, we investigated the effects of histamine on the expression of COX-1 and COX-2 and determined their contribution to the production of PGE(2), prostacyclin (PGI(2)), and thromboxane A(2) in human coronary artery endothelial cells (HCAEC). Incubation of HCAEC monolayers with histamine resulted in marked increases in the expression of COX-2 and production of PGI(2) and PGE(2) with no significant change in the expression of COX-1. Histamine-induced increases in PGI(2) and PGE(2) production were due to increased expression and function of COX-2 because gene silencing by small interfering RNA or inhibition of the catalytic activity by a COX-2 inhibitor blocked prostanoid production. The effects of histamine on COX-2 expression and prostanoid production were mediated through H(1) receptors. In addition to the direct effect, histamine was found to amplify LPS-stimulated COX-2 expression and PGE(2) and PGI(2) production. In contrast, histamine did not stimulate thromboxane A(2) production in resting or LPS-activated HCAEC. Histamine-induced increases in the production of PGE(2) and PGI(2) were associated with increased expression of mRNA encoding PGE(2) and PGI(2) synthases. The physiological role of histamine on the regulation of COX-2 expression in the vasculature is indicated by the findings that the expression of COX-2 mRNA, but not COX-1 mRNA, was markedly reduced in the aortic tissues of histidine decarboxylase null mice. Thus, histamine plays an important role in the regulation of COX-2 expression and prostanoid homeostasis in vascular endothelium.

Chymase increases glomerular albumin permeability via protease-activated receptor-2.

Increased infiltration of the kidney by mast cells is associated with proteinuria, and interstitial fibrosis in various renal diseases. Mast cells produce serine proteases including tryptase and chymase (MCC) that act via protease-activated receptors (PARs) to induce synthesis of fibrogenic cytokines by renal cells. In the present study, we investigated direct effect of MCC and role of PARs on glomerular albumin permeability (P(alb)). Isolated rat glomeruli were incubated with MCC (0.1, 1, 10, and 100 ng/ml) for 5-30 min in presence or absence of PAR-1 and PAR-2 blocking antibodies. P(alb) was determined from the change in glomerular volume in response to an albumin oncotic gradient. The effect of direct activation of PARs on P(alb) was verified by incubating glomeruli with synthetic hexapeptide known to activate PAR-1 and PAR-2. MCC increased P(alb) of isolated rat glomeruli in a dose- and time-dependent manner. Blocking PAR-2 prevented MCC-mediated increase in P(alb). RT-PCR analysis of glomerular RNA demonstrated the presence of constitutively expressed PAR-1, -2, and -3 and low levels of PAR-4. In addition, direct activation of PAR-2 by hexapeptide SLIGKV increased P(alb) comparable to MCC, whereas PAR-1 activation by TFLLRN had no effect on P(alb). Our results document that MCC induces increase in P(alb) and that this effect is mediated through PAR-2. MCC may also play a role in renal scarring. We propose that inhibiting MCC activity or blocking the activation of PAR-2 may provide new targets for therapy in renal diseases.

Constitutive expression of functionally active protease-activated receptors 1 and 2 in human conjunctival epithelial cells.

Protease-activated receptors (PARs) are G-protein-coupled receptors which initiate inflammatory responses when activated by specific serine proteases. This study was conducted to examine whether human conjunctival epithelial cells (HCECs) express functionally active PAR1 and PAR2 using Chang conjunctival epithelial cells as in vitro model. We performed RT-PCR and immunofluorescence analyses to determine the expression of PAR1 and PAR2, and monitored the production of IL-6 after activating HCECs with PAR1 activating agents (thrombin or TFLLRN) or PAR2 activating agents (tryptase, trypsin, or SLIGKV). The results show that HCECs constitutively express PAR1 and PAR2 mRNA and proteins, and produce significant amounts of IL-6 when incubated with specific PAR-activating enzymes or agonist peptides. Thrombin- and tryptase-induced HCEC activation was blocked by PAR1 and PAR2 neutralizing antibodies, respectively, and by specific enzyme inhibitors. The constitutive expression of PAR1 and PAR2, and their activation by thrombin and tryptase, respectively, may have important implications in ocular inflammation.

Endothelial cell activation by mast cell mediators.

Mast cells are important cells of the immune system, and their secretory products regulate many vascular functions. Although considerable interest is focused on the role of mast cells and infectious agents in atherosclerosis, whether or not mast cell mediators act in concert with bacterial agents to regulate endothelial activation is not known. Here, we have described experimental techniques and presented related results to demonstrate how mast cell granule (MCG) mediators and bacterial products synergize endothelial cell inflammatory responses. The described methods outline: (1) the collection of rat peritoneal mast cells; (2) preparation of MCGs; (3) co-culture of human endothelial cells with mast cell granules; (4) determination of the regulation of endo- thelial cell inflammatory responses; (5) demonstration of the role of MCG protease and histamine in the regulation of endothelial cell function; (6) amplification of lipopolysaccharide-induced signal transduction pathways by mast cell granules; (7) elucidation of histamine-induced amplification of endothelial cell responses to Gram-negative and Gram-positive bacterial cell wall components; and (8) determination of the expression of Toll-like receptor 2 and 4. We hope the techniques described here can be used for designing experiments focusing on the regulatory role of mast cell mediators on cell functions.

Human conjunctival epithelial cells lack lipopolysaccharide responsiveness due to deficient expression of MD2 but respond after interferon-gamma priming or soluble MD2 supplementation.

Inflammatory responses to Gram-positive and Gram-negative bacterial cell wall components are initiated by Toll-like receptor 2 (TLR2) and TLR4, respectively. Therefore, the existence of functionally active TLR2 and TLR4 in human conjunctival epithelial cells (HCEC) are critical for the effective host defense against bacterial infections in the eye. We examined the ability of HCEC to respond to TLR4 ligand, lipopolysaccharide (LPS), or TLR2 ligands, lipoteichoic acid (LTA) and peptidoglycan (PGN) using the Chang conjunctival epithelial cell line and the primary conjunctival epithelial cell line (IOBA-NHC) as in vitro models. Incubation of Chang cells with LPS (1 to 1,000 ng/ml) failed to stimulate IL-6 production where as stimulation with LTA or PGN resulted in marked increases in IL-6 production. Semi-quantitative RT-PCR and immunofluorescence analyses showed that Chang cells express TLR2 and TLR4 mRNA and proteins. However, these cells expressed little or no mRNA encoding MD2, an accessory molecule required for TLR4 signaling. Incubation of Chang epithelial cells with interferon-gamma (IFNgamma), but not TNF-alpha, stimulated MD2 mRNA expression and restored LPS responsiveness. In addition, when Chang cell cultures were supplemented with soluble MD2, LPS was able to stimulate IL-6 production. The lack of LPS response, deficient expression of MD2, and induction of MD2 expression and LPS response after IFNgamma priming, were also evident in IOBA-NHC cells. These results demonstrate that HCEC lack LPS responsiveness due to deficient expression of MD2 and that the response can be restored by IFN-gamma priming or MD2 supplementation.

Histamine induces Toll-like receptor 2 and 4 expression in endothelial cells and enhances sensitivity to Gram-positive and Gram-negative bacterial cell wall components.

Histamine is a major inflammatory molecule released from the mast cell, and is known to activate endothelial cells. However, its ability to modulate endothelial responses to bacterial products has not been evaluated. In this study we determined the ability of histamine to modulate inflammatory responses of endothelial cells to Gram-negative and Gram-positive bacterial cell wall components and assessed the role of Toll-like receptors (TLR) 2 and 4 in the co-operation between histamine and bacterial pathogens. Human umbilical vein endothelial cells (HUVEC) were incubated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or peptidoglycan (PGN) in the presence or absence of histamine, and the expression and release of interleukin-6 (IL-6), and NF-kappaB translocation were determined. The effect of histamine on the expression of mRNA and proteins for TLR2 and TLR4 was also evaluated. Incubation of HUVEC with LPS, LTA and PGN resulted in marked enhancement of IL-6 mRNA expression and IL-6 secretion. Histamine alone markedly enhanced IL-6 mRNA expression in HUVEC, but it did not stimulate proportional IL-6 release. When HUVEC were incubated with LPS, LTA, or PGN in the presence of histamine marked amplification of both IL-6 production and mRNA expression was noted. HUVEC constitutively expressed TLR2 and TLR4 mRNA and proteins, and these were further enhanced by histamine. The expression of mRNAs encoding MD-2 and MyD88, the accessory molecules associated with TLR signalling, were unchanged by histamine treatment. These results demonstrate that histamine up-regulates the expression of TLR2 and TLR4 and amplifies endothelial cell inflammatory responses to Gram-negative and Gram-positive bacterial components.

Deranged aortic intima-media thickness, plasma triglycerides and granulopoiesis in Sl/Sl(d) mice.

Studies were carried out to evaluate the impact of a high-fat dietary regimen on aortic wall thickness, peripheral blood leukocyte profile, and plasma cholesterol and triglyceride levels in the mast cell-deficient Sl/Sl(d) mouse. The results demonstrated that the mean aortic wall thickness of Sl/Sl(d) mice was significantly higher than their normal littermates, and were increased in both genotypes after a 17-day high-fat regimen. In comparison with normal littermates, Sl/Sl(d) genotypes had elevated levels of plasma triglycerides with normal levels of plasma cholesterol, and the high-fat diet markedly lowered the triglyceride levels. Total peripheral blood leukocytes, the monocyte and granulocyte counts, and hemoglobin levels were significantly lower in Sl/Sl(d) mice, although the number of lymphocytes, eosinophils and basophils were the same in both genotypes. Interestingly, the high-fat diet regimen elevated leukocyte counts and the number of monocytes and granulocytes in Sl/Sl(d) mice.

Poloxamer 407-induced atherosclerosis in mice appears to be due to lipid derangements and not due to its direct effects on endothelial cells and macrophages.

Coronary heart disease secondary to atherosclerosis is still the leading cause of death in the US. Animal models used for elucidating the pathogenesis of this disease primarily involve rabbits and pigs. Previous studies from this laboratory have demonstrated intraperitoneal injections of poloxamer 407 (P-407) in both male and female mice will lead to hyperlipidemia and atherosclerosis, suggesting the use of this polymer to develop a mouse model of atherosclerosis. In order to understand the mechanism of P-407-induced hyperlipidemia and vascular lesion formation, we evaluated the direct effects of P-407 on endothelial cell and macrophage functions in vitro, and its in vivo effects on the oxidation of circulating lipids following long-term (4 month) administration. Our results demonstrated that incubation of P-407 with human umbilical vein endothelial cells in culture did not influence either cell proliferation or interleukin-6 and interleukin-8 production over a concentration range of 0-40 microM. In addition, nitric oxide production by macrophages was not affected by P-407 over a concentration range of 0-20 microM. Finally, we demonstrated that while P-407 could not induce the oxidation of LDL-C in vitro, long-term (4 month) administration of P-407 in mice resulted in elevated levels of oxidized lipids in the plasma. Thus, it is suggested that the formation of atherosclerotic lesions in this mouse model of atherosclerosis does not result from either direct stimulation of endothelial cells or macrophage activation by P-407. Instead, these data would support the premise that oxidation of lipids (perhaps low-density lipoprotein cholesterol) by an indirect mechanism following injection of P-407 may represent one of the mechanisms responsible for atheroma formation.

Signal transduction pathways in mast cell granule-mediated endothelial cell activation.

BACKGROUND: We have previously shown that incubation of human endothelial cells with mast cell granules results in potentiation of lipopolysaccharide-induced production of interleukin-6 and interleukin-8. AIMS: The objective of the present study was to identify candidate molecules and signal transduction pathways involved in the synergy between mast cell granules and lipopolysaccharide on endothelial cell activation. METHODS: Human umbilical vein endothelial cells were incubated with rat mast cell granules in the presence and absence of lipopolysaccharide, and IL-6 production was quantified. The status of c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2 activation, nuclear factor-kappaB translocation and intracellular calcium levels were determined to identify the mechanism of synergy between mast cell granules and lipopolysaccaride. RESULTS: Mast cell granules induced low levels of interleukin-6 production by endothelial cells, and this effect was markedly enhanced by lipopolysaccharide. The results revealed that both serine proteases and histamine present in mast cell granules were involved in this activation process. Mast cell granules increased intracellular calcium, and activated c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2. The combination of lipopolysaccharide and mast cell granules prolonged c-Jun amino-terminal kinase activity beyond the duration of induction by either stimulant alone and was entirely due to active proteases. However, both proteases and histamine contributed to calcium mobilization and extracellular signal-regulated kinase 1/2 activation. The nuclear translocation of nuclear factor-kappaB proteins was of greater magnitude in endothelial cells treated with the combination of mast cell granules and lipopolysaccharide. CONCLUSIONS: Mast cell granule serine proteases and histamine can amplify lipopolysaccharide-induced endothelial cell activation, which involves calcium mobilization, mitogen-activated protein kinase activation and nuclear factor-kappaB translocation.