Walter Long

A Monoclonal Antibody to High-Molecular Weight Kininogen Is Therapeutic in a Rodent Model of Reactive Arthritis

We reported that high-molecular weight kininogen is proangiogenic by releasing bradykinin and that a monoclonal antibody to high-molecular weight kininogen, C11C1, blocked its binding to endothelial cells. We now test if this antibody can prevent arthritis and systemic inflammation in a Lewis rat model. We studied 32 animals for 16 days. Group I (negative control) received saline intraperitoneally. Group II (disease-treated) received peptidoglycan-polysaccharide simultaneously with C11C1. Group III (disease-untreated) received peptidoglycan-polysaccharide simultaneously with isotype-matched mouse IgG. Group IV (disease-free-treated) and group V (disease-free isotype-treated) received saline and C11C1 or mouse IgG. Analysis of joint diameter changes showed a decrease in the C11C1 disease-treated group compared to the disease-untreated group. The hind paw inflammatory score showed a decrease in the intensity and extent of inflammation between the disease-untreated and the C11C1 disease-treated group. Prekallikrein, high-molecular weight kininogen, factor XI, and factor XII were decreased in the disease-untreated group compared to the C11C1 disease-treated group. T-kininogen was increased in the disease-untreated group when compared with the C11C1 disease-treated group. Disease-free groups IV and V did not show any sign of inflammation at any time. This study shows that monoclonal antibody C11C1 attenuates plasma kallikrein-kinin system activation, local and systemic inflammation, indicating therapeutic potential in reactive arthritis.

Production and detection of coliphage T4 endonuclease V polyclonal and monoclonal antibodies using staphylococcal protein-A hybrid proteins

To facilitate the production of antibodies against endonuclease V, a pyrimidine dimer-specific DNA glycosylase produced in bacteriophage T4-infected Escherichia coli, we constructed plasmids containing protein-A-endonuclease V fusion genes under control of the E. coli tac promoter. Induction with isopropyl-beta-D-thiogalactopyranoside produced large amounts of fusion proteins, which could easily be purified on human IgG agarose columns. The affinity-purified fusion proteins were injected into rabbits and mice to produce polyclonal and monoclonal antibodies, and also used for the screening of the monoclonal antibodies. These antibodies recognized endonuclease V on immunoblots, and also inhibited the DNA-glycosylase activity in vitro. Epitope mapping of monoclonal antibodies showed that they all (6/6) recognized determinants in the C-half of endonuclease V. A convenient way to detect primary antibodies on nitrocellulose was also developed using a crude protein extract containing protein-A-beta-galactosidase fusion protein and subsequent detection with a mixture of dyes.

Improved Exercise Capacity and Reduced Systemic Inflammation After Adenoviral-Mediated SERCA-2a Gene Transfer

BACKGROUND We hypothesized that sarcoplasmic reticulum Ca2+ ATPase pump (SERCA-2a) gene delivery would have beneficial effects upon exercise capacity and markers of inflammation in the setting of heart failure. MATERIALS AND METHODS A pressure-overload model of experimental heart failure was used in rats. Following a decrease in fractional shortening of >or=25%, animals underwent intracoronary adenoviral-mediated gene transfection using SERCA-2a. Heart failure animals were randomized to receive the SERCA-2a gene, the beta galactosidase (control) gene, or followed without any further intervention. Exercise and hemodynamic testing were performed, and myocardial and systemic markers of inflammation were assayed after 7 and 21 d. RESULTS Animals receiving Ad.SERCA-2a showed an increase in exercise tolerance (499.0 +/- 14.9 versus 312.8 +/- 10.5 s, P < 0.0001) relative to Ad.Gal group. Groups treated with Ad.SERCA-2a had significantly decreased serum levels of the inflammatory markers interleukin-1, interleukin-6, and tumor necrosis factor-alpha compared with Ad.Gal-treated animals. Serum levels of atrial natriuretic peptide were decreased in animals receiving Ad.SERCA-2a compared with animals receiving Ad.Gal at day 7 (0.35 +/- 0.03 versus 0.52 +/- 0.11 pg/mL, P = 0.001). Myocardial levels of the proapoptotic protein bax were reduced in Ad.SERCA-2a -treated animals compared with those receiving Ad.Gal at day 7 (protein level/actin: 0.24 +/- 0.05 versus 0.33 +/- 0.04, P = 0.04) and day 21 (protein level/actin: 0.61 +/- 0.04 versus 0.69 +/- 0.01, P = 0.001). CONCLUSIONS Genetic modulation of heart failure using the SERCA-2a gene was associated with improvement in cardiac function and exercise capacity as well as improvements in heart-failure associated inflammatory markers.

Effect of Adenoviral-Mediated Transfer of Transforming Growth Factor-β1 on Colonic Anastomotic Healing

PURPOSETransforming growth factor-β1 plays a central role in colonic repair. We examined the temporal effect of vector-mediated transfer of transforming growth factor-β1 on colonic anastomotic healing.METHODSMale Sprague-Dawley rats (n = 24) underwent transection of the distal colon and single-layer anastomosis. Proximal to the anastomosis, the colon was again transected and a colostomy was matured proximally. The distal colon was intubated with a silicone catheter, tunneled along subcutaneous tissues, and connected to a swivel apparatus for postoperative luminal infusion. Rats were randomized into four groups (n = 6 each). Two control groups received 1010 plaque-forming units of a Type 5 E1-deleted adenovirus carrying the bacterial β-galactosidase gene either immediately following surgery or on postoperative Day 3. The treatment groups received transforming growth factor-β1 with the same viral construct at parallel time points. On postoperative Day 6, anastomotic bursting pressure and site were determined in situ with the anastomotic tissue subsequently harvested and analyzed by enzyme-linked immunosorbent assay for β-galactosidase and transforming growth factor-β1.RESULTSWhen compared with its corresponding control, the group that received the transforming growth factor-β1 gene on postoperative day 3 had a significantly higher bursting pressure (mmHg; 119 ± 16 vs. 160 ± 12, mean ± SD; P = 0.001). While the majority of colons (5/6) from the control group burst at the anastomosis, none of the colons in the group that received transforming growth factor-β1 on day 3 burst at the anastomotic site (P = 0.007). β-Galactosidase levels (pg/ml) in anastomotic tissue were significantly increased in both control groups when compared with their respective treatment groups (101 ± 43 vs. 38 ± 30, P = 0.01 when infused the day of surgery and 243 ± 92 vs. 50 ± 30, P = 0.009 when infused on day 3). Anastomotic levels of transforming growth factor-β1 were also increased in the group receiving the transforming growth factor-β1 gene on day 3 (214 ± 66 vs. 135 ± 24, P = 0.02).CONCLUSIONSGene transfer into the healing colonic anastomosis can be effectively achieved via intraluminal administration of adenoviral vectors. Transfer of transforming growth factor-β1 increased the strength of colonic anastomoses when given at Day 3 but not at Day 0, demonstrating its diverse effects in the wound healing sequence. Thus, gene transfer of transforming growth factor-β1 may avoid the need for a diverting stoma in cases of rectal surgery and impaired healing resulting from chemotherapy or radiation.

Adherence of streptococci to cells infected with herpesvirus

The attachment of streptococci and staphylococci to cells infected with influenza virus has been previously reported and has been correlated with the increased incidence of these bacterial infections during flu epidemics. The present work was initiated to determine whether infection with herpes simplex virus (HSV) might lead to preferential bacterial attachment. HEp-2 cells were grown in monolayer and infected with HSV, Type I or Type II. Twenty-four hours later the cells were incubated with suspensions of various organisms, including Group A and B streptococci, Staphylococcus aureus, Propionibacterium acnes, and Candida albicans. After incubation for one hour, the cells were washed and fixed. Bacterial adherence and virus infection were assessed by scanning electron microscopy (SEM) as well as conventional light and transmission electron microscopy (TEM). Only Group A streptococci adhered to virus-infected cells, both to cells infected with HSV, Type I and those infected with HSV, Type II. SEM and TEM revealed bacteria attaching to cells with budding virus particles. Preincubation of infected cells with anti-HSV serum prevented bacterial adherence. These findings suggest that infection of oral epithelium with HSV might lead to superinfection with Group A streptococci.

Host cell reactivation of uv- and X-ray-damaged herpes simplex virus by epstein-barr virus (EBV)-transformed lymphoblastoid cell lines☆

Abstract The efficacy of using an infected centers assay, employing herpes simplex virus-infected, Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) as components, to study host cell reactivation has been explored. Herpes simplex virus type 1 (HSV-1) was shown through the infected centers assay to have detectable but varying ability to lytically infect LCLs established from chromosomal breakage syndromes or closely related genetic disorders. The rate of HSV inactivation by ultraviolet (uv) irradiation was faster in LCLs established from Cockaynes syndrome than in normal LCLs, and faster still in LCLs established from xeroderma pigmentosum. These results indicate that Cockaynes syndrome, while having what appears to be quantitatively normal levels of uv-induced DNA repair replication, shows decreased ability to host cell reactivate uv-damaged HSV. In direct contrast, X-irradiated HSV showed identical survival when assayed on normal LCLs or LCLs established from ataxia telangiectasia showing increased sensitivity to X irradiation as measured by colony formation. Through the infected centers assay, it has also been possible to demonstrate low levels of multiplicity reactivation of mutagen-damaged HSV in permanently proliferating LCLs.

Effects of Nucleoside Analogs on Epstein-Barr Virus-Induced Transformation of Human Umbilical Cord Leukocytes and Epstein-Barr Virus Expression in Transformed Cells

Two methods of assay, measuring (i) stimulation of host cell DNA synthesis by [3H]thymidine incorporation and (ii) morphological transformation in microtiter plates, were employed to determine what effect treatment during infection with adenine arabinoside or 5-iodo-5′-amino-2′,5′-dideoxyuridine has on Epstein-Barr virus (EBV) (B95-8)-induced transformation of human umbilical cord leukocytes. It was found that adenine arabinoside inhibited EBV-induced transformation in a dose-dependent manner in both assays, beginning at drug concentrations (<2 μg/ml) which had little effect on either spontaneous or phytohemagglutinin-induced host cell DNA synthesis. Adenine arabinoside was more effective in inhibiting morphological transformation than EBV-induced host DNA synthesis. Adenine arabinoside treatment was also effective in reducing both EB viral capsid antigen expression and production of biologically active extracellular transforming virus in EBV-transformed cells. In contrast, 5-iodo-5′-amino-2′,5′-dideoxyuridine, which inhibited herpes simplex virus replication, had little effect on EBV-induced transformation as measured by either method of assay. However, 5-iodo-5′-amino-2′,5′-dideoxyuridine was found to be effective in inhibiting viral capsid antigen expression and production of extracellular transforming virus in EBV-transformed cells.

Effects of S-adenosylhomocysteine and Analogs on Epstein-Barr Virus (EBV)-Induced Transformation, EBV DNA Methylation and Gene Expression

EBV DNA becomes increasingly methylated with time following cell transformation (1) while hypomethylation has been associated with expression of viral genes (2). S-adenosylhomocysteine (SAH), a product of the S-adenosylmethionine transmethylation reaction, acts as a competitive inhibitor of DNA methyltransferases (3). In the present study SAH and analogs, sinefungin (SF) and 5′-deoxy-5′-S-isobutyladenosine (SIBA), as well as 5-azacytidine, which inhibits methylation of nascent DNA (4), were utilized in studies to determine the role of DNA methylation in the regulation of Epstein-Barr virus (EBV) functions such as transformation of human peripheral blood lymphocytes (PBL) and gene expression in lymphoblastoid cell lines. SF and SIBA, but not SAH, inhibited EBV-induced transformation of human PBLs. Indirect immunofluorescence and flow cytometric analyses revealed a significant increase in viral capsid antigen expression in lymphoblastoid cell lines following exposure to the analogs SF and SIBA, but not SAH.