Pathology teaching in different undergraduate medical curricula within and outside the United States: a pilot study.

Pathology education is taught using different curricula in the United States (USA) and abroad. We evaluate and compare the hours spent in different forms of pathology teaching such as lectures, team-based learning (TBL), problem-based learning (PBL), and other methods taught in general and systemic pathology amongst different medical schools within the USA and outside the USA. The total number of lecture hours taught in general and systemic pathology combined was greater in outside schools than within the USA (141 h vs 97.8 h, respectively). Three subjects in general pathology and six subjects in systemic pathology had a significantly greater lecture hours in outside medical schools. The greatest difference was the hours spent in labs were longer for both general and systems pathology in schools outside the USA. The overall utilization of PBL in general and systemic pathology teaching combined was much greater outside the USA compared to within the USA (average overall hours PBL - 97.2 outside vs 16.5 in the USA), however, the reverse was observed for using TBL (average overall hours TBL - 59.5 outside vs 84.5 in USA). Average hours used with other methods of teaching was also greater in outside medical schools compared to USA medical schools (80.8 h vs 44 h, respectively). Pathology teaching in both general and systemic pathology has more extensive lecture hours, laboratory hours, PBL, and other methods of teaching pathology in outside medical schools with different curricula than USA medical schools. TBL is utilized more extensively in USA medical schools.

A PKCß-LYN-PYK2 Signaling Axis Is Critical for MCP-1-Dependent Migration and Adhesion of Monocytes.

MCP-1-induced monocyte chemotaxis is a crucial event in inflammation and atherogenesis. Identifying the important signal transduction pathways that control monocyte chemotaxis can unravel potential targets for preventive therapies in inflammatory disease conditions. Previous studies have shown that the focal adhesion kinase Pyk2 plays a critical role in monocyte motility. In this study, we investigated the MCP-1-mediated activation of Pyk2 (particularly by the phosphorylation of Tyr402) in primary human peripheral blood monocytes. We showed that MCP-1 induces Src phosphorylation in a similar time frame and that the MCP-1-induced Pyk2 tyrosine phosphorylation is controlled by the Src family kinase. We also report, in this study, that PKCß, an isoform of PKC, is required for both Src and Pyk2 activation/phosphorylation in response to MCP-1 stimulation. We identified Lyn as the specific Src kinase isoform that is activated by MCP-1 and acts upstream of Pyk2 in primary monocytes. Furthermore, Lyn is found to be indispensable for monocyte migration in response to MCP-1 stimulation. Moreover, our coimmunoprecipitation studies in monocytes revealed that PKCß, Pyk2, and Lyn exist constitutively in a molecular complex. To our knowledge, our study has uncovered a novel PKCß-Lyn-Pyk2 signaling cascade in primary monocytes that regulates MCP-1-induced monocyte adhesion and migration.

MicroRNA-155 deficiency results in decreased macrophage inflammation and attenuated atherogenesis in apolipoprotein E-deficient mice.

OBJECTIVE: microRNA-155 (miR155) plays a critical role in immunity and macrophage inflammation. We aim to investigate the role of miR155 in atherogenesis. APPROACH AND RESULTS: Quantitative real-time polymerase chain reaction showed that miR155 was expressed in mouse and human atherosclerotic lesions. miR155 expression in macrophages was correlated positively with proinflammatory cytokine expression. Lentivirus-mediated overexpression of miR155 in macrophages enhanced their inflammatory response to lipopolysaccharide through targeting suppressor of cytokine signaling-1 and impaired cholesterol efflux from acetylated low-density lipoprotein-loaded macrophages, whereas deficiency of miR155 blunted macrophage inflammatory responses and enhanced cholesterol efflux possibly via enhancing lipid loading-induced macrophage autophagy. We next examined the atherogenesis in apolipoprotein E-deficient (apoE(-/-)) and miR155(-/-)/apoE(-/-) (double knockout) mice fed a Western diet. Compared with apoE(-/-) mice, the double knockout mice developed less atherosclerosis lesion in aortic root, with reduced neutral lipid content and macrophages. Flow cytometric analysis showed that there were increased number of regulatory T cells and reduced numbers of Th17 cells and CD11b+/Ly6C(high) cells in the spleen of double knockout mice. Peritoneal macrophages from the double knockout mice had significantly reduced proinflammatory cytokine expression and secretion both in the absence and presence of lipopolysaccharide stimulation. To determine whether miR155 in leukocytes contributes to atherosclerosis, we performed a bone marrow transplantation study. Deficiency of miR155 in bone marrow-derived cells suppressed atherogenesis in apoE(-/-) mice, demonstrating that hematopoietic cell-derived miR155 plays a critical role. CONCLUSIONS: miR155 deficiency attenuates atherogenesis in apoE(-/-) mice by reducing inflammatory responses of macrophages, enhancing macrophage cholesterol efflux and resulting in an antiatherogenic leukocyte profile. Targeting miR155 may be a promising strategy to halt atherogenesis.

Renal protective effects of α-calcitonin gene-related peptide in deoxycorticosterone-salt hypertension.

Deoxycorticosterone salt (DOC-salt) hypertension-induced renal damage is enhanced in α-calcitonin gene-related peptide (α-CGRP) knockout (KO) compared with wild-type (WT) mice. However, since the α-CGRP KO mice have a 15-20 mmHg higher baseline mean arterial pressure (MAP) than WT mice, they also have a higher MAP than WT mice throughout the course of DOC-salt hypertension. To determine the mechanism by which the absence of α-CGRP enhances hypertension-induced renal damage, DOC-salt hypertension was induced in telemetry probe implanted α-CGRP KO and WT mice. To equalize the blood pressure (BP) to that of DOC-salt WT mice, an additional group of DOC-salt α-CGRP KO mice was given 0.025% hydralazine to drink. The DOC-salt protocol increased the final MAP in α-CGRP KO mice to 155 ± 6 mmHg and in WT mice to 140 ± 5 mmHg. The MAP of the hydralazine-treated DOC-salt α-CGRP KO mice was 139 ± 6 mmHg. Urinary excretion of microalbumin and isoprostane, a marker for oxidative stress, was increased, and creatinine clearance was decreased in DOC-salt α-CGRP KO compared with DOC-salt WT mice. Equalization of the MAP in DOC-salt α-CGRP KO to that of DOC-salt WT mice did not significantly improve these parameters. Renal macrophage infiltration; desmin, a marker of podocyte damage; and the inflammatory cytokines TNF-α and IFN-γ and the chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α) were increased in DOC-salt α-CGRP KO mice and were not reduced by hydralazine treatment. However, BP equalization did improve the renal histopathological damage, as determined by light microscopy. Therefore, in DOC-salt hypertension in mice, the mechanism(s) of the renal protective effects of α-CGRP are both BP independent and BP dependent.

Metabolic products of soluble epoxide hydrolase are essential for monocyte chemotaxis to MCP-1 in vitro and in vivo.

Monocyte chemoattractant protein-1 (MCP-1)-induced monocyte chemotaxis is a major event in inflammatory disease. Our prior studies have demonstrated that MCP-1-dependent chemotaxis requires release of arachidonic acid (AA) by activated cytosolic phospholipase A(2) (cPLA(2)). Here we investigated the involvement of AA metabolites in chemotaxis. Neither cyclooxygenase nor lipoxygenase pathways were required, whereas pharmacologic inhibitors of both the cytochrome-P450 (CYP) and the soluble epoxide hydrolase (sEH) pathways blocked monocyte chemotaxis to MCP-1. To verify specificity, we demonstrated that the CYP and sEH products epoxyeiscosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs), respectively, restored chemotaxis in the presence of the inhibitors, indicating that sEH-derived products are essential for MCP-1-driven chemotaxis. Importantly, DHETs also rescued chemotaxis in cPLA(2)-deficient monocytes and monocytes with blocked Erk1/2 activity, because Erk controls cPLA(2) activation. The in vitro findings regarding the involvement of CYP/sEH pathways were further validated in vivo using two complementary approaches measuring MCP-1-dependent chemotaxis in mice. These observations reveal the importance of sEH in MCP-1-regulated monocyte chemotaxis and may explain the observed therapeutic value of sEH inhibitors in treatment of inflammatory diseases, cardiovascular diseases, pain, and even carcinogenesis. Their effectiveness, often attributed to increasing EET levels, is probably influenced by the impairment of DHET formation and inhibition of chemotaxis.

Influence of dietary saturated fat content on adiposity, macrophage behavior, inflammation, and metabolism: composition matters.

We examined the effects of three high-fat diets (HFD), differing in the percentage of total calories from saturated fat (SF) (6%, 12%, and 24%) but identical in total fat (40%), on body composition, macrophage behavior, inflammation, and metabolic dysfunction in mice. Diets were administered for 16 weeks. Body composition and metabolism [glucose, insulin, triglycerides, LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), total cholesterol (TC)] were examined monthly. Adipose tissue (AT) expression of marker genes for M1 and M2 macrophages and inflammatory mediators [Toll-like receptor (TLR)-2, TLR-4, MCP-1, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, suppressor of cytokine signaling (SOCS)1, IFN-γ] was measured along with activation of nuclear factor kappa-B (NFκB), c-Jun N-terminal kinase (JNK), and p38- mitogen-activated protein kinase (MAPK). AT macrophage infiltration was examined using immunohistochemistry. Circulating MCP-1, IL-6, adiponectin, and leptin were also measured. SF content, independent of total fat, can profoundly affect adiposity, macrophage behavior, inflammation, and metabolic dysfunction. In general, the 12%-SF diet, most closely mimicking the standard American diet, led to the greatest adiposity, macrophage infiltration, and insulin resistance (IR), whereas the 6%-SF and 24%-SF diets produced lower levels of these variables, with the 24%-SF diet resulting in the least degree of IR and the highest TC/HDL-C ratio. Macrophage behavior, inflammation, and IR following HFD are heavily influenced by dietary SF content; however, these responses are not necessarily proportional to the SF percentage.

Linking tumor-associated macrophages, inflammation, and intestinal tumorigenesis: role of MCP-1.

Tumor-associated macrophages are associated with poor prognosis in certain cancers. Monocyte chemoattractant protein 1 (MCP-1) is thought to be the most important chemokine for recruitment of macrophages to the tumor microenvironment. However, its role on tumorigenesis in a genetic mouse model of colon cancer has not been explored. We examined the role of MCP-1 on tumor-associated macrophages, inflammation, and intestinal tumorigenesis. Male Apc(Min/+), Apc(Min/+)/MCP-1(-/-) or wild-type mice were euthanized at 18 wk of age and intestines were analyzed for polyp burden, apoptosis, proliferation, ß-catenin, macrophage number and phenotype, markers for cytotoxic T lymphocytes and regulatory T cells, and inflammatory mediators. MCP-1 deficiency decreased overall polyp number by 20% and specifically large polyp number by 45% (P < 0.05). This was consistent with an increase in apoptotic cells (P < 0.05), but there was no change detected in proliferation or ß-catenin. MCP-1 deficiency decreased F4/80-positive cells in both the polyp tissue and surrounding intestinal tissue (P < 0.05) as well as expression of markers associated with M1 (IL-12 and IL-23) and M2 macrophages (IL-13, CD206, TGF-ß, and CCL17) (P < 0.05). MCP-1 knockout was also associated with increased cytotoxic T lymphocytes and decreased regulatory T cells (P < 0.05). In addition, MCP-1(-/-) offset the increased mRNA expression of IL-1ß and IL-6 in intestinal tissue and IL-1ß and TNF-α in polyp tissue (P < 0.05), and prevented the decrease in SOCS1 expression (P < 0.05). We demonstrate that MCP-1 is an important mediator of tumor growth and immune regulation that may serve as an important biomarker and/or therapeutic target in colon cancer.

An integrated ultrasound curriculum (iUSC) for medical students: 4-year experience.

A review of the development and implementation of a 4-year medical student integrated ultrasound curriculum is presented. Multiple teaching and assessment modalities are discussed as well as results from testing and student surveys. Lessons learned while establishing the curriculum are summarized. It is concluded that ultrasound is a well received, valuable teaching tool across all 4 years of medical school, and students learn ultrasound well, and they feel their ultrasound experience enhances their medical education.

Beta-lapachone micellar nanotherapeutics for non-small cell lung cancer therapy.

Lung cancer is the leading cause of cancer-related deaths with current chemotherapies lacking adequate specificity and efficacy. Beta-lapachone (beta-lap) is a novel anticancer drug that is bioactivated by NAD(P)H:quinone oxidoreductase 1, an enzyme found specifically overexpressed in non-small cell lung cancer (NSCLC). Herein, we report a nanotherapeutic strategy that targets NSCLC tumors in two ways: (a) pharmacodynamically through the use of a bioactivatable agent, beta-lap, and (b) pharmacokinetically by using a biocompatible nanocarrier, polymeric micelles, to achieve drug stability, bioavailability, and targeted delivery. Beta-lap micelles produced by a film sonication technique were small ( approximately 30 nm), displayed core-shell architecture, and possessed favorable release kinetics. Pharmacokinetic analyses in mice bearing subcutaneous A549 lung tumors showed prolonged blood circulation (t(1/2), approximately 28 h) and increased accumulation in tumors. Antitumor efficacy analyses in mice bearing subcutaneous A549 lung tumors and orthotopic Lewis lung carcinoma models showed significant tumor growth delay and increased survival. In summary, we have established a clinically viable beta-lap nanomedicine platform with enhanced safety, pharmacokinetics, and antitumor efficacy for the specific treatment of NSCLC tumors.

Maintenance of donor-specific chimerism despite osteopontin-associated bone fibrosis in a vascularized bone marrow transplantation model.

BACKGROUND: The role of vascularized bone allografting is not established in plastic and reconstructive surgery. The authors evaluated the contribution by osteopontin to fibrosis of allografted bone in a vascularized bone marrow transplantation model across a major histocompatibility complex barrier. METHODS: Thirty-six transplantations were performed between Brown Norway (RT1 n) donors and Lewis (RT1 l) recipients divided into three groups: group 1, isografts between Lewis rats (n = 12); group 2, allografts without treatment (n = 8); and group 3, allografts under a 7-day alphabeta-T-cell receptor/cyclosporine protocol (n = 16). Flow cytometry assessed the presence of chimerism for donor major histocompatibility complex class I (RT1 n) antigens. Immunostaining was used to determine osteopontin expression in grafted and recipient bone, and histologic examination was used to assess bone architecture. RESULTS: Early engraftment of donor bone marrow cells (RT1 n) into the recipient bone marrow compartment was achieved at posttransplantation day 7. This corresponded with osteopontin expression restricted to the endosteum of trabecular bone and was associated with the preservation of hematopoietic cells within donor bone. Cell migration between donor and recipient bone marrow compartments was confirmed by the presence of recipient cells (RT1 l) within the allografted bone and donor-origin cells (RT1 n) within the recipient bone. At posttransplantation day 63, osteopontin expression within allografted bone was associated with allograft bone fibrosis and lack of hematopoietic properties. In contrast, the recipient's contralateral bone demonstrated a highly localized osteopontin expression pattern within the endosteum and active hematopoiesis with the presence of donor-specific (RT1 n) cells and correlated with chimerism maintenance. CONCLUSIONS: These results confirm that despite up-regulation of osteopontin expression and fibrosis of allografted bone, vascularized bone marrow transplantation resulted in efficient engraftment of donor cells into the recipient's bone marrow compartment, leading to chimerism maintenance.

Period 2 mutation accelerates ApcMin/+ tumorigenesis.

Colorectal cancer risk is increased in shift workers with presumed circadian disruption. Intestinal epithelial cell proliferation is gated throughout each day by the circadian clock. Period 2 (Per2) is a key circadian clock gene. Per2 mutant (Per2(m/m)) mice show an increase in lymphomas and deregulated expression of cyclin D and c-Myc genes that are key to proliferation control. We asked whether Per2 clock gene inactivation would accelerate intestinal and colonic tumorigenesis. The effects of PER2 on cell proliferation and beta-catenin were studied in colon cancer cell lines by its down-regulation following RNA interference. The effects of Per2 inactivation in vivo on beta-catenin and on intestinal and colonic polyp formation were studied in mice with Per2 mutation alone and in combination with an Apc mutation using polyp-prone Apc(Min/+) mice. Down-regulation of PER2 in colon cell lines (HCT116 and SW480) increases beta-catenin, cyclin D, and cell proliferation. Down-regulation of beta-catenin along with Per2 blocks the increase in cyclin D and cell proliferation. Per2(m/m) mice develop colonic polyps and show an increase in small intestinal mucosa beta-catenin and cyclin D protein levels compared with wild-type mice. Apc(Min/+)Per2(m/m) mice develop twice the number of small intestinal and colonic polyps, with more severe anemia and splenomegaly, compared with Apc(Min/+) mice. These data suggest that Per2 gene product suppresses tumorigenesis in the small intestine and colon by down-regulation of beta-catenin and beta-catenin target genes, and this circadian core clock gene may represent a novel target for colorectal cancer prevention and control.

iPLA2beta: front and center in human monocyte chemotaxis to MCP-1.

Monocyte chemoattractant protein-1 (MCP-1) directs migration of blood monocytes to inflamed tissues. Despite the central role of chemotaxis in immune responses, the regulation of chemotaxis by signal transduction pathways and their in vivo significance remain to be thoroughly deciphered. In this study, we examined the intracellular location and functions of two recently identified regulators of chemotaxis, Ca(2+)-independent phospholipase (iPLA(2)beta) and cytosolic phospholipase (cPLA(2)alpha), and substantiate their in vivo importance. These enzymes are cytoplasmic in unstimulated monocytes. Upon MCP-1 stimulation, iPLA(2)beta is recruited to the membrane-enriched pseudopod. In contrast, cPLA(2)alpha is recruited to the endoplasmic reticulum. Although iPLA(2)beta or cPLA(2)alpha antisense oligodeoxyribonucleotide (ODN)-treated monocytes display reduced speed, iPLA(2)beta also regulates directionality and actin polymerization. iPLA(2)beta or cPLA(2)alpha antisense ODN-treated adoptively transferred mouse monocytes display a profound defect in migration to the peritoneum in vivo. These converging observations reveal that iPLA(2)beta and cPLA(2)alpha regulate monocyte migration from different intracellular locations, with iPLA(2)beta acting as a critical regulator of the cellular compass, and identify them as potential targets for antiinflammatory strategies.

A new rat model of maxilla allotransplantation.

We developed a rat model to test the effects of vascularized maxilla allotransplantation on composite maxillary substructures. Allograft maxilla transplantations were performed across the major histocompatibility barrier between 10 Lewis-Brown-Norway (RT1(n+l)) and 10 Lewis (RT1(l)) recipient rats under cyclosporin A monotherapy. Grafts were dissected along Le-Fort II osteotomy lines based on the common carotid artery and external jugular vein and transplanted to the anterior abdominal wall via microvascular anastomosis. Allografts were examined by tomography, flow cytometry, angiography, and histology. Three of the allografts survived up to 105 days without any signs of rejection. High level of donor-specific chimerism for T-cell and B-cell lineages was maintained in the peripheral blood. The incisors continued to grow; teeth buds, bone, cartilage, and mucosa remained intact. Moderate inflammation of the nasal, oral mucosa, and keratinous metaplasia was noted histologically. We created a maxilla allotransplantation model that allows the study of immunologic responses and demonstrates potential clinical applications based on the growth properties of the allograft.

Applications of bilateral vascularized femoral bone marrow transplantation for chimerism induction across the major histocompatibility (MHC) barrier: part II.

Bilateral vascularized bone marrow transplant (VBMT) model was designed to induce chimerism across the major histocompatibility (MHC) barrier under combined alphabeta T-cell receptor monoclonal antibody and cyclosporine A (alphabeta-TCRmAb/CsA) protocol. Seventeen transplants were performed between BN(RT1) donors and Lewis(RTI) recipients. Group I, isograft controls; Group II, allografts rejection controls; Group III, allografts under 7-day protocol of alphabeta-TCRmAb/CsA. Donor bilateral femoral bones were bilaterally anastomosed to the abdominal aorta and inferior vena cava of recipient. At day 7 posttransplantation, all bone flaps were viable. Groups I and III survived without signs of rejection. In Group III, peak level of chimerism in peripheral blood was evaluated at day 21 (24.2%), at day 63 declined to 1.5%, and was maintained at this level thereafter. Donor-derived cells were present in the bone marrow of recipients at 28.2% at day 21 posttransplant. Histology confirmed viability of bone marrow cells in isograft during the entire follow-up and up to 35 days in treatment Group III. Bilateral VBMT induced donor-specific chimerism across the MHC barrier under the immunomodulatory protocol of alphabeta-TCRmAb/CsA.

Bilateral vascularized femoral bone transplant: a new model of vascularized bone marrow transplantation in rats, part I.

We present a new model of vascularized bone marrow transplantation-bilateral vascularized femoral bone (BVFB) isograft transplant based on abdominal aorta and inferior vena cava. A total of 7 BVFB isograft transplants were performed between Lewis (RT1) rats. In the donor, both femoral bones were harvested based on the abdominal aorta and inferior vena cava. In the recipient, the harvested isograft transplants were transferred into the inguinal region (in 3 animals) and into the abdominal cavity (in 4 animals). The mean operation time was 3 hours and 35 minutes. The mean warm ischemic time was 35 minutes. The vascular pedicles of the transplants that were transferred into the inguinal region were thrombosed at day 7 posttransplantation. The vascular pedicles of transplants into the abdominal cavity were patent and the bones were viable during the follow-up period of 63 days posttransplant. We have confirmed the feasibility of BVFB transplantation based on abdominal aorta and inferior vena cava.

Biological coating for arterial stents: the next evolutionary change in stents.

PURPOSE: To describe the in vivo results of a promising new stent-graft lined with peritoneum. METHODS: Eighteen dogs underwent balloon angioplasty injury to the bilateral iliac arteries followed by placement of either an 8-mm x 5-cm glutaraldehyde-fixed bovine peritoneum- lined balloon-expandable stent (PLS) or a similarly sized, commercially prepared, polyester-lined self-expanding stent (DLS) as a control. Animals were sacrificed at 1 and 6 months. Biplanar arteriography and intravascular ultrasound were done at the time of sacrifice, and the vessels were harvested after perfusion fixation for histology/morphometry. Immunofluorescence with CD34 and factor VIII staining was used to evaluate endothelialization, while alpha-actin was used to quantify smooth muscle cell (SMC) deposition. RESULTS: At 1 month, all vessels were patent in both groups. At 6 months, 8 of 9 vessels were open in the PLS group versus 6 of 9 in the control DLS group. Vessel lumen diameter at 1 month was significantly greater in the PLS vessels compared to the DLS group at 1 cm above the stent (35.9 +/- 4.4 versus 29.4 +/- 4.7 mm2; p = 0.02) and 1 cm below the stent (37.2 +/- 7.1 versus 25.2 +/- 3.2 mm2; p = 0.005); these results persisted to 6 months. Histological morphometry demonstrated progression of neointimal hyperplasia in the DLS stent between 30 and 180 days (8.3 +/- 1.79 versus 14.9 +/- 6.6 mm2; p = 0.03), whereas the peritoneum-lined stent had no change during the same time period (4.62 +/- 0.98 versus 4.72 +/- 0.97 mm2; p = 0.85). The same patterns were true for the intima:media ratio. Immunohistochemistry demonstrated complete endothelialization at 6 months in both DLS and PLS. However, SMC staining with alpha-actin demonstrated more smooth muscle actin-positive cells in the DLS compared to the PLS (327 +/- 87 versus 262 +/- 73 counts/5 high-powered fields; p = 0.04). CONCLUSION: Peritoneum-lined stents offer a novel method to improve patency of lower extremity arterial stents.

In vivo and mechanical properties of peritoneum/fascia as a novel arterial substitute.

OBJECTIVE: This study evaluates the efficacy of bovine peritoneum/fascia as an arterial substitute. METHODS AND OUTCOME MEASURES: Twelve dogs underwent bilateral femoral artery patch angioplasty with a glutaraldehyde-fixed bovine peritoneal/fascial patch (PFA patch) on one side and polyester patch on the contralateral side. Arteriograms were performed just before vessel harvest at 1 and 6 months, and vessels were evaluated for aneurysms and inflammation. Histologic analysis included intima area, media thickness, and lumen area. Immunofluorescence for CD 34 and Factor VIII was done to evaluate endothelialization and alpha-actin for smooth muscle cell growth. Mechanical strength testing was evaluated in separate PFA patches and compared independently to a commercially available bovine pericardial patch and polyester patch. RESULTS: All vessels examined at both 1 and 6 months were patent with no arteriographic evidence of stenosis. There was no evidence of aneurysm formation in any vessel and no difference between groups in inflammatory reaction. One polyester patch at 1 month developed an infection. Microscopic evaluation of experimental vessels revealed no difference between groups in intima area at 1 month (2.1 +/- 1.2 vs 2.2 +/- 1.2 mm 2 ; P = .5) and at 6 months (1.81 +/- 1.2 vs 1.9 +/- 1.2 mm 2 ; P = .5). There was no difference in media thickness, but the PFA patch group had a greater lumen area at 1 month (8.8 +/- 2.9 vs 9.8 +/- 3.0 mm 2 ; P = .02) and 6 months (10.5 +/- 4.2 vs 11.7 +/- 5.6 mm 2 ; P = .02). Immunofluorescence for CD34 and Factor VIII demonstrated complete re-endothelialization of all patches. The polyester patch had a chronic inflammatory response, but not the PFA patch. Mechanical strength testing demonstrated that compared to pericardium, the PFA patch had superior ( P < .05) failure tension, stiffness, and suture pull-out strength, whereas extensibility, fatigue tension, relax slope, and creep tests were not different. Polyester demonstrated superior suture pull-out, stiffness, relax slope, and failure strain ( P < .05), but it was not different in failure tension and extensibility than the PFA patch. However, the PFA patch had significantly less creep (0.25 +/- 0.25 vs 4.92 +/- 0.84; P < .01). CONCLUSIONS: The PFA patch has similar clot-resistant properties to polyester and is superior to the pericardial patch in mechanical strength. It is a promising endothelial alternative for not only arterial patches but other vascular products. CLINICAL RELEVANCE: The search for an artificial, thromboresistant, and intimal hyperplasia resistant interface between blood and native blood vessels still continues. This study demonstrates the feasibility and proof of concept of the peritoneum's clot-resistant properties. When adding the underlying fascia, it serves as an ideal arterial patch. Other studies are underway evaluating its feasibility as a bypass graft and a "drug coated"-like stent lining.

Human monocytes use Rac1, not Rac2, in the NADPH oxidase complex.

Phagocyte NADPH oxidase is critical for defense against pathogens and contributes to inflammatory tissue injury. One component of the NADPH oxidase complex is the small GTP-binding protein Rac. There are two isoforms of Rac, and Rac2 is the predominant isoform in neutrophils and has been shown to be essential for NADPH oxidase activity. In primary human monocytes we report that in contrast to neutrophils, Rac1 is the predominantly expressed isoform. Upon monocyte activation by a variety of agents, we found that Rac1 dissociates from Rho GDP dissociation inhibitor (RhoGDI) and translocates to the membrane. We also found that Rac1 interacts with two other NADPH oxidase components, p67phox and p47phox, upon monocyte activation. These data indicate that Rac1, and not Rac2, is a component of the activated NADPH oxidase in monocytes. This finding suggests that it may be possible to selectively interfere with either monocyte or neutrophil NADPH oxidase activity, thereby selectively targeting chronic versus acute inflammatory processes.

Protein kinase C beta is required for human monocyte chemotaxis to MCP-1.

Monocyte chemoattractant protein 1 (MCP-1) is important in attracting monocytes to sites of inflammation. Using predominantly pharmacological approaches, prior studies have indicated that serine/threonine kinases are involved in the MCP-1-induced signaling pathways. We report here that there is substantial inhibition of MCP-1-stimulated chemotaxis of human monocytes treated with inhibitors selective for the subset of serine/threonine kinases, protein kinase C (PKC). Selective inhibitors of PKC such as GF109203X and Calphostin C both caused approximately 80% inhibition of chemotaxis. Because these pharmacological inhibitors do not specifically inhibit individual PKC isoforms, we chose to use antisense oligodeoxyribonucleotides (ODN) to specifically reduce PKC isoform expression, first by inhibiting expression of the conventional PKC family, and next by using specific antisense ODN for PKCalpha and PKCbeta. Conventional PKC-antisense ODN treatment completely and significantly inhibited monocyte chemotaxis to MCP-1, whereas sense-control ODN caused no significant inhibition. PKCbeta-antisense ODN caused 89.2% inhibition of chemotaxis at its highest dose. In contrast, PKCbeta-sense ODN and PKCalpha-antisense and -sense ODN were without effect. Further studies evaluating the calcium response that is triggered upon MCP-1 interaction with its receptor, CCR2, indicate that this response is not altered by antisense or sense ODN treatment, thus supporting our hypothesis that PKCbeta is critical for post-receptor signal transduction downstream of the immediate calcium signal. These data contribute to our developing understanding of the signal transduction pathways involved in the chemotactic response of human monocytes to MCP-1 and uniquely identify the requirement for the PKCbeta isoform in this important process.