Prolonged survival of pancreatic islet allografts mediated by adenovirus immunoregulatory transgenes.

The adenovirus (Ad) early region 3 (E3) genes code for at least four proteins that inhibit the host immune responses mediated by cytotoxic T lymphocytes and tumor necrosis factor alpha. To evaluate the potential use of these immunoregulatory viral functions in facilitating allogeneic cell transplantation, the Ad E3 genes were expressed in pancreatic beta cells in transgenic mice under control of the rat insulin II promoter. Transgenic H-2b/d (C57BL/6 x BALB/c) islets, expressing the Ad E3 genes, remained viable for at least 94 days after transplantation under the kidney capsule of BALB/c (H-2d) recipients. Nontransgenic H-2b/d control islets were rejected as anticipated between 14 and 28 days. Histological analysis of the transplanted transgenic islets revealed normal architecture. Immunohistochemical studies with antisera to islet hormones revealed the presence of both beta and non-beta islet cells, suggesting a propagation of the immunosuppressive effect of Ad proteins from beta cells to other islet cells. The use of viral genes, which have evolved to regulate virus-host interactions, to immunosupress the anti-genicity of donor transplant tissue suggests additional ways for prolonging allograft survival. In addition, these findings have implications for designing Ad vectors for gene therapy.

Systematic proteomic strategies to map the human signaling landscape

Thesis (Ph.D.)--University of Washington, 2016-05

Donor pretreatment with progenipoietin-1 is superior to granulocyte colony-stimulating factor in preventing graft-versus-host disease after allogeneic stem cell transplantation

The granulocyte colony-stimulating factor (G-CSF) and Fit-3 receptor agonist progenipoietin-1 (ProGP-1) has potent effects on dendritic cell (DC) expansion and may be an alternative to G-CSF for the mobilization of stem cells for allogeneic stem cell transplantation (SCT). We studied the ability of stem cell grafts mobilized with this agent to induce graft-versus-host disease (GVHD) to minor and major histocompatibility antigens in the well-described B6 --> B6D2F1 SCT model. ProGP-1, G-CSIF, or control diluent was administered to donor B6 mice. ProGP-1 expanded all cell lineages in the spleen, and unseparated splenocytes from these animals produced large amounts of interleukin 10 (IL-10) and transforming growth factor beta (TGFbeta) whereas the expression of T-cell adhesion molecules was diminished. Transplantation survival was 0%, 50%, and 90% in recipients of control-, G-CSF-, and ProGP-1-treated allogeneic donor splenocytes, respectively (P < .0001). Donor pretreatment with ProGP-1 allowed a 4-fold escalation in T-cell dose over that possible with G-CSF. Donor CD4 T cells from allogeneic SCT recipients of ProGP-1 splenocytes demonstrated an anergic response to host antigen, and cytokine production (interferon gamma [IFNγ], IL-4, and IL-10) was also reduced while CD8 T-cell cytotoxicity to host antigens remained intact. Neither CD11c(hi) DCs nor CD11c(dim)/B220(hi) DCs from ProGP-1-treated animals conferred protection from GVHD when added to control spleen. Conversely, when equal numbers of purified T cells from control-, G-CSF-, or ProGP-1-treated allogeneic donors were added to allogeneic T-cell-depleted control spleen, survival at day 60 was 0%, 15%, and 90%, respectively (P < .0001). The improved survival in recipients of ProGP-1 T cells was associated with reductions in systemic tumor necrosis factor alpha generation and GVHD of the gastrointestinal tract. We conclude that donor pretreatment with ProGP-1 is superior to G-CSIF for the prevention of GVHD after allogeneic SCT, primarily due to incremental affects on T-cell phenotype and function

Steatosis and liver cell apoptosis in chronic hepatitis C: A mechanism for increased liver injury

Steatosis is increasingly recognized as a cofactor influencing the progression of fibrosis in chronic hepatitis Q however, the mechanisms by which it contributes to liver injury remain uncertain. We studied 125 patients with chronic hepatitis C to assess the effect of steatosis on liver cell apoptosis and the expression of Bcl-2, Bd-x(L), Bax, and tumor necrosis factor alpha (TNF-alpha) and the relationship between liver cell apoptosis and disease severity. A significant increase in liver cell apoptosis was seen in liver sections with increasing grade of steatosis (r = 0.42; P < .0001). Hepatic steatosis and previous heavy alcohol consumption were the only two variables independently associated with the apoptotic index. Increasing steatosis was associated with decreased Bcl-2 mRNA levels and an increase in the proapoptotic Bax/Bcl-2 ratio (r = -0.32, P = .007; and r = 0.27, P = .02, respectively). In the absence of steatosis, increased liver cell apoptosis was not associated with stellate cell activation or fibrosis (r = 0.26, P = .11; r = 0.06, P = .71, respectively). In contrast, in the presence of steatosis, increasing apoptosis was associated with activation of stellate cells and increased stage of fibrosis (r = 0.35, P = .047; r = 0.33, P = .03, respectively), supporting the premise that the steatotic liver is more vulnerable to liver injury. In patients with hepatitis C virus genotype 3, there was a significant correlation between TNF-&alpha; mRNA levels and active caspase-3 (r = 0.54, P = .007). In conclusion, these observations suggest a mechanism whereby steatosis contributes to the progression of liver injury in chronic hepatitis C. Further investigation will be required to determine the molecular pathways responsible for the proapoptotic effect of steatosis and whether this increase in apoptosis contributes directly to fibrogenesis.

Regulation of antigen processing and presentation molecules in West Nile virus-infected human skin fibroblasts

Infection of humans with the West Nile flavivirus principally occurs via tick and mosquito bites. Here, we document the expression of antigen processing and presentation molecules in West Nile virus (WNV)-infected human skin fibroblast (HFF) cells. Using a new Flavivirus-specific antibody, 4G4, we have analyzed cell surface human leukocyte antigen (HLA) expression on virus-infected cells at a single cell level. Using this approach, we show that West Nile Virus infection alters surface HLA expression on both infected HFF and neighboring uninfected HFF cells. Interestingly, increased surface HLA evident on infected HFF cultures is almost entirely due to virus-induced interferon (IFN)alpha/beta because IFNalpha/beta-neutralizing antibodies completely prevent increased surface HLA expression. In contrast, RT-PCR analysis indicates that WNV infection results in increased mRNAs for HLA-A, -B, and -C genes, and HLA-associated molecules low molecular weight polypeptide-2 (LMP-2) and transporter associated with antigen presentation-1 (TAP-1), but induction of these mRNAs is not diminished in HFF cells cultured with IFNalpha/beta-neutralizing antibodies. Taken together, these data support the idea that that both cytokine-dependent and cytokine-independent mechanisms account for WNV-induced HLA expression in human skin fibroblasts. (C) 2004 Elsevier Inc. All rights reserved.

Tumour necrosis factor inhibitors

The cytokine, tumour necrosis factor-alpha (TNF-alpha) plays a key role in the pathogenesis of many chronic inflammatory and rheumatic diseases, in particular, Crohn's disease, rheumatoid arthritis, ankylosing spondylitis and psoriatic arthritis. Controlled trials have shown that the TNF inhibitors (etanercept, infliximab and adalimumab) significantly reduce symptoms and signs, improve function and quality of life, and reduce radiologically evident damage in patients with rheumatoid diseases. For reasons that are not entirely clear, etanercept does not work in Crohn's disease. Injection site and intravenous reactions and increased risk of infection (in particular, reactivation of tuberculosis) are associated with the use of these agents. Increased risk of lymphoproliferative disease, the development of lupus-like syndromes and demyelination, including optic neuritis and reactivation of multiple sclerosis, are under evaluation in long-term follow-up studies. The TNF inhibitors are expensive (about $18000 per year), and in some patients need to be given continuously to maintain benefit, even in the presence of other immunosuppressive therapy.

A role for the phagosome in cytokine secretion

Membrane traffic in activated macrophages is required for two critical events in innate immunity: proinflammatory cytokine secretion and phagocytosis of pathogens. We found a joint trafficking pathway linking both actions, which may economize membrane transport and augment the immune response. Tumor necrosis factor alpha (TNF alpha) is trafficked from the Golgi to the recycling endosome (RE), where vesicle-associated membrane protein 3 mediates its delivery to the cell surface at the site of phagocytic cup formation. Fusion of the RE at the cup simultaneously allows rapid release of TNF alpha and expands the membrane for phagocytosis.

Cytokine secretion via cholesterol-rich lipid raft-associated SNAREs at the phagocytic cup

Lipopolysaccharide-activated macrophages rapidly synthesize and secrete tumor necrosis factor alpha(TNF alpha) to prime the immune system. Surface delivery of membrane carrying newly synthesized TNF alpha is controlled and limited by the level of soluble N-ethylmaleimide-sensitive factor attachment protein receptor ( SNARE) proteins syntaxin 4 and SNAP-23. Many functions in immune cells are coordinated from lipid rafts in the plasma membrane, and we investigated a possible role for lipid rafts in TNF alpha trafficking and secretion. TNF alpha surface delivery and secretion were found to be cholesterol-dependent. Upon macrophage activation, syntaxin 4 was recruited to cholesterol-dependent lipid rafts, whereas its regulatory protein, Munc18c, was excluded from the rafts. Syntaxin 4 in activated macrophages localized to discrete cholesterol-dependent puncta on the plasma membrane, particularly on filopodia. Imaging the early stages of TNF alpha surface distribution revealed these puncta to be the initial points of TNF alpha delivery. During the early stages of phagocytosis, syntaxin 4 was recruited to the phagocytic cup in a cholesterol-dependent manner. Insertion of VAMP3-positive recycling endosome membrane is required for efficient ingestion of a pathogen. Without this recruitment of syntaxin 4, it is not incorporated into the plasma membrane, and phagocytosis is greatly reduced. Thus, relocation of syntaxin 4 into lipid rafts in macrophages is a critical and rate-limiting step in initiating an effective immune response.

Scaffold-free, Human Mesenchymal Stem Cell-Based Tissue Engineered Blood Vessels.

Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an important role in the development of organs-on-a-chip. Most TEBV construction involves scaffolding with biomaterials such as collagen gel or electrospun fibrous mesh. Hypothesizing that a scaffold-free TEBV may be advantageous, we constructed a tubular structure (1 mm i.d.) from aligned human mesenchymal cell sheets (hMSC) as the wall and human endothelial progenitor cell (hEPC) coating as the lumen. The burst pressure of the scaffold-free TEBV was above 200 mmHg after three weeks of sequential culture in a rotating wall bioreactor and perfusion at 6.8 dynes/cm(2). The interwoven organization of the cell layers and extensive extracellular matrix (ECM) formation of the hMSC-based TEBV resembled that of native blood vessels. The TEBV exhibited flow-mediated vasodilation, vasoconstriction after exposure to 1 μM phenylephrine and released nitric oxide in a manner similar to that of porcine femoral vein. HL-60 cells attached to the TEBV lumen after TNF-α activation to suggest a functional endothelium. This study demonstrates the potential of a hEPC endothelialized hMSC-based TEBV for drug screening.

Natural history of markers of collagen turnover in patients with early diastolic dysfunction and impact of eplerenone

OBJECTIVES: This study was designed to evaluate the impact of eplerenone on collagen turnover in preserved systolic function heart failure (HFPSF).

BACKGROUND: Despite growing interest in abnormal collagen metabolism as a feature of HFPSF with diastolic dysfunction, the natural history of markers of collagen turnover and the impact of selective aldosterone antagonism on this natural history remains unknown.

METHODS: We evaluated 44 patients with HFPSF, randomly assigned to control (n = 20) or eplerenone 25 mg daily (n = 24) for 6 months, increased to 50 mg daily from 6 to 12 months. Serum markers of collagen turnover and inflammation were analyzed at baseline and at 6 and 12 months and included pro-collagen type-I and -III aminoterminal peptides, matrix metalloproteinase type-2, interleukin-6 and -8, and tumor necrosis factor-alpha. Doppler-echocardiographic assessment of diastolic filling indexes and tissue Doppler analyses were also obtained.

RESULTS: The mean age of the patients was 80 +/- 7.8 years; 46% were male; 64% were receiving an angiotensin-converting enzyme inhibitor, 34% an angiotensin-II receptor blocker, and 68% were receiving beta-blocker therapy. Pro-collagen type-III and -I aminoterminal peptides, matrix metalloproteinase type-2, interleukin-6 and -8, and tumor necrosis factor-alpha increased with time in the control group. Eplerenone treatment had no significant impact on any biomarker at 6 months but attenuated the increase in pro-collagen type-III aminoterminal peptide at 12 months (p = 0.006). Eplerenone therapy was associated with modest effects on diastolic function without any impact on clinical variables or brain natriuretic peptide.

CONCLUSIONS: This study demonstrates progressive increases in markers of collagen turnover and inflammation in HFPSF with diastolic dysfunction. Despite high background utilization of renin-angiotensin-aldosterone modulators, eplerenone therapy prevents a progressive increase in pro-collagen type-III aminoterminal peptide and may have a role in management of this disease. (The Effect of Eplerenone and Atorvastatin on Markers of Collagen Turnover in Diastolic Heart Failure; NCT00505336).

Role of NleH, a type III secreted effector from attaching and effacing pathogens, in colonization of the bovine, ovine, and murine gut

The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes human and animal gut via formation of attaching and effacing lesions. EHEC strains use a type III secretion system to translocate a battery of effector proteins into the mammalian host cell, which subvert diverse signal transduction pathways implicated in actin dynamics, phagocytosis, and innate immunity. The genomes of sequenced EHEC O157:H7 strains contain two copies of the effector protein gene nleH, which share 49% sequence similarity with the gene for the Shigella effector OspG, recently implicated in inhibition of migration of the transcriptional regulator NF-kappaB to the nucleus. In this study we investigated the role of NleH during EHEC O157:H7 infection of calves and lambs. We found that while EHEC DeltanleH colonized the bovine gut more efficiently than the wild-type strain, in lambs the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. Using the mouse pathogen Citrobacter rodentium, which shares many virulence factors with EHEC O157:H7, including NleH, we observed that the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. We found no measurable differences in T-cell infiltration or hyperplasia in colons of mice inoculated with the wild-type or the nleH mutant strain. Using NF-kappaB reporter mice carrying a transgene containing a luciferase reporter driven by three NF-kappaB response elements, we found that NleH causes an increase in NF-kappaB activity in the colonic mucosa. Consistent with this, we found that the nleH mutant triggered a significantly lower tumor necrosis factor alpha response than the wild-type strain.

Treadmill walking exercise modulates bone mineral status and inflammatory cytokines in obese asthmatic patients with long term intake of corticosteroids.

Background: Obesity and asthma are an important public health problem in Saudi Arabia. An increasing body of data supports the hypothesis that obesity is a risk factor for asthma. Asthma appears to be associated with low bone mineral density (BMD) due to long-term use of corticosteroids. Studies recently showed that weight bearing exercise training can increase mineral bone density, reduce weight and improve metabolic control. Objective: The present study aimed to measure the effects of treadmill walking exercises on bone mineral status and inflammatory cytokines in obese asthmatic patients treated with long term intake of corticosteroids. Methods: Eighty obese asthmatic patients of both sexes, their age ranged from 41 to 53 years. Subjects were divided into two equal groups: training group (group A) received aerobic exercise training on treadmill for six months in addition to the medical treatment where, the control group (group B) received only the medical treatment. Results: The results of this study indicated a significant increase in BMD of the lumbar spine & the radius, serum calcium and high density lipoprotein cholesterol (HDL-c) & significant reduction in parathyroid hormone, leptin, tumor necrosis factor– alpha(TNF-α), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), low density lipoprotein cholesterol (LDL-c), triglycerides (TG) and body mass index (BMI) in group (A), while these changes were not significant in group (B).Also; there was a significant difference between both groups at the end of the study. Conclusion: Treadmill walking exercise training is an effective treatment policy to improve bone mineral status and modulates inflammatory cytokines and blood lipids profile in obese asthmatic patients with long term intake of corticosteroids.

Effects of coagulation factors and inflammatory cytokines on development of acute myocardial infarction in patients younger than 60 years

Purpose: To investigate the effects of coagulation factors and inflammatory cytokines on acute myocardial infarction (AMI) development in patients younger than 60 years. Methods: In this study, 60 patients admitted to The First Affiliated Hospital of Dalian Medical University (Dalian, China) with AMI and 30 other subjects matched with the patients for age and ethnicity but without AMI were enrolled. Blood samples were collected from the AMI patients and the control subjects after a 12-h fast. Subsequently, the levels of coagulation factors (F) II (FII), VII (FVII), VIII (FVIII), fibrinogen (Fg) and von Willebrand factor (vWF) in plasma were analyzed by enzyme-linked immunosorbent assay (ELISA). The protein expression levels of these coagulation factors were determined by Western blot analysis. Inflammatory factors including C-reactive protein (CRP), tumor necrosis factor-α (TNF-α) and interleukin- 6 (IL-6) were also measured by ELISA. Results: FII, FVII, FVIII, Fg and vWF levels in plasm were significantly higher in AMI patients compared with control subjects (p < 0.01). Furthermore, the protein expression levels of FII, FVII, FVIII, Fg and vWF were also significantly up-regulated in AMI patients compared with those in control subjects. Additionally, no significant difference was observed in CRP between AMI patients and control subjects (p > 0.05). However, TNF-α and IL-6 levels in the plasma of AMI patients were significantly higher than those in control subjects (p < 0.05). Conclusion: The results reveal that the pathogenesis of AMI in patients younger than 60 years might be closely related to the high levels of coagulation factors and inflammatory cytokines in the blood.

Elevated Hypercoagulability Markers In Hemoglobin Sc Disease.

Hemoglobin SC disease is a very prevalent hemoglobinopathy, however very little is known specifically about this condition. There appears to be an increased risk of thromboembolic events in hemoglobin SC disease, but studies evaluating the hemostatic alterations are lacking. We describe a cross-sectional observational study evaluating coagulation activation markers in adult hemoglobin SC patients, in comparison with sickle cell anemia patients and healthy controls. A total of 56 hemoglobin SC and 39 sickle cell anemia patients were included in the study, all in steady state, and 27 healthy controls. None of the patients were in use of hydroxyurea. Hemoglobin SC patients presented a significantly up-regulated relative expression of tissue factor, as well as elevations in thrombin-antithrombin complex and D-dimer, in comparison to controls (p<0.01). Hemoglobin SC patients presented lower tissue factor expression, and thrombin-antithrombin complex and D-dimer levels when compared to sickle cell anemia patients (p<0.05). Endothelial activation (soluble thrombomodulin and soluble vascular cell adhesion molecule-1), and inflammation (tumor necrosis factor-alpha) markers were both significantly elevated in hemoglobin SC patients when compared to controls, being as high as the levels seen in sickle cell anemia. Overall, in hemoglobin SC patients, higher hemolytic activity and inflammation were associated with a more intense activation of coagulation, and hemostatic activation was associated with two very prevalent chronic complications seen in hemoglobin SC disease: retinopathy and osteonecrosis. In summary, our results demonstrate that hemoglobin SC patients present a hypercoagulable state, although this manifestation was not as intense as that seen in sickle cell anemia.