Fredika M. Robertson

Temporal sequence of pulmonary cytokine gene expression in response to endotoxin in C3H/HeN endotoxin-sensitive and C3H/HeJ endotoxin-resistant mice.

Although previous studies suggested that tumor necrosis factor alpha (TNF‐α) was a critical cytokine responsible for the inflammation observed after exposure to endotoxin, other mediators may also play an important role in the regulation of systemic inflammatory responses independent of TNF‐α. The present study compared the temporal sequence of endotoxin‐induced TNF‐α, interleukin‐1α (IL‐1α), and interleukin‐10 (IL‐10) gene expression and cellular localization of cytokine proteins in pulmonary tissue of two strains of mice that have a genetically based differential sensitivity to endotoxin. Lung tissue and plasma were harvested from endotoxin‐sensitive C3H/HeN and endotoxin‐resistant C3H/HeJ micé at 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 12 h, and 24 h after intraperitoneal (i.p.) injection of 5 mg/kg endotoxin (Escherichia coli–derived lipopolysaccharide, serotype 0111:B4). There were significant elevations in both TNF‐α gene and IL‐2α expression immediately (15 min) after endotoxin injection in C3H/HeN mice. Although levels of TNF‐α mRNA in the two mouse strains were similar at 1‐2 h, the IL‐1α gene expression in pulmonary tissue isolated from endotoxin‐resistant mice was not comparable to the levels detected in C3H/HeN endotoxin‐sensitive mice at the same times. The most dramatic difference in endotoxin‐induced cytokine gene expression between the two strains of mice was in IL‐10 mRNA levels in pulmonary tissue isolated from endotoxin‐sensitive mice, compared to the lack of detectable increase in IL‐10 gene expression in C3H/HeJ endotoxin‐resistant mice above baseline at any time point examined. Quantitation of neutrophil infiltration into pulmonary tissue using immunochemical detection of CR‐1, a myeloid differentiation‐specific antibody, demonstrated that there was a significantly decreased inflammatory infiltrate in pulmonary tissue isolated from C3H/HeJ mice following endotoxin administration, which correlated with decreased levels of proinflammatory cytokine immunoreactive protein within pulmonary cells. Pulmonary cytokine synthesis and immunoreactive protein production did not directly correlate with either the magnitude or the temporal sequence of increases in plasma cytokine levels, suggesting that systemic levels of cytokines may not accurately reflect the cytokine response within the local tissue milieu. The present observations demonstrate that the differential synthesis and production of immunosuppressive cytokines as well as proinflammatory cytokines may be important variables in the determination of the extent of infiltration of inflammatory cells into the local pulmonary site in response to endotoxin and may significantly contribute to the determination of sensitivity or resistance to endotoxin in this murine model.

Induction of leukocyte activation by meshes surgically implanted in the peritoneal cavity

The search for surgical prosthetics that do not act to promote infection has been frustrating. However, recent experience with the implantation of polyglycolic acid mesh (PGA) used as an intestinal sling has been associated with a lack of pelvic infections. To examine the basis for these observations, 1 x 1-cm pieces of biomaterials were sewn onto the peritoneal cavity of rats. The biomaterials examined included PGA mesh, a composite mesh composed of a permanent nonabsorbable Novafil inner layer coated with a PGA outer layer, polyvinyl alcohol sponge, and silicon elastomer. Biomaterials were removed at postoperative days 1, 2, 8, and 14, and examined for bacteriostatic and bactericidal activity by standard techniques. Mesh adherent leukocytes were evaluated for their ability to oxidize dichlorofluorescein diacetate (DCFH-DA), which is fluorescent in the presence of intracellular hydrogen peroxide produced by both polymorphonuclear cells and monocytes. PGA and the composite mesh had no intrinsic bacteriostatic or bactericidal activity. However, adherent leukocytes were induced to oxidize DCFH at levels 10-fold and 5-fold, respectively, by postoperative day 14, compared with earlier time points and other biomaterials. The ability of these PGA meshes to stimulate respiratory bursts by peritoneal cells may partly be responsible for the lack of infections associated with their use.

Peritoneal leukocyte response following placement of polyglycolic acid intestinal sling in patients with rectal carcinoma

The intestinal sling procedure has been used successfully without the observance of pelvic infections. This procedure involves the implantation of polyglycolic acid (PGA) mesh to hold the bowel out of the pelvis to prevent radiation enteritis. We previously showed that PGA mesh has no intrinsic bactericidal activity. Since phagocytic leukocytes produce reactive oxygen intermediates during respiratory burst that are associated with oxygen-dependent bactericidal activity, we examined peritoneal cell types and their respiratory burst activity isolated from patients with biopsy-confirmed rectal carcinoma who underwent the intestinal sling procedure (N=12) compared with patients who did not (N=13). There was no significant difference in the cell types within the peritoneal cavity over the 7-day postoperative period examined. However, there was a significant increase in the ability of leukocytes isolated from mesh patients to produce hydrogen peroxide in the absence of an exogenous stimulus (P <0.05),as measured by flow cytometric quantitation of oxidation of the hydroperoxide-sensitive dye, 2′,7′-dichlorofluorescin diacetate (DCFH-DA). Despite the higher endogenous DCFH oxidation by leukocytes from mesh patients, the cells retained the ability to oxidize DCFH following treatment with a membrane-active stimulant for respiratory burst activity, 12-O-tetradecanoylphorbol-13-acetate. These observations suggest that PGA mesh used for the intestinal sling procedure stimulates the respiratory burst activity of peritoneal leukocytes during the postoperative period in which bacterial proliferation and colonization occur. The stimulation of reactive oxygen intermediates involved in oxygen-dependent bactericidal activity by PGA mesh may be one of the mechanisms underlying the lack of infections observed with the use of PGA mesh in contaminated settings.

Regulation of Aromatase in Breast Cancer and Correlation of Aromatase and Cyclooxygenase Gene Expression

Approximately two-thirds of newly diagnosed breast cancers (BC) in the USA are hormone-dependent BC, requiring estrogen (E) for tumor growth. E is important for the local stimulation of growing malignancies in the breast. 17β-Estradiol (E2), the most potent endogenous E, is biosynthesized from androgens by the cytochrome P450 enzyme complex called aromatase (1–3). The highest levels of aromatase enzymatic activity are present in the ovaries of premenopausal women, in the placenta of pregnant women, and in the peripheral adipose tissues of postmenopausal women and of men. Aromatase activity has also been demonstrated to be present in breast tissue in vitro (4–7). Furthermore, expression of aromatase is highest in or near breast tumor sites (5,8). The gene expressing cytochrome P450arom protein is referred to as CYP19 and is part of the cytochrome P450 superfamily. The cDNA for aromatase encodes a 55 kDa protein containing 503 amino acids (1, 9). The regulation of aromatase expression varies due to the different promotors in each tissue (10, 11); thus, aromatase biosynthesis is tissue-specific and tightly regulated (1). The increased expression of aromatase cytochrome P450arom observed in BC tissues was recently associated with a switch in the major promoter region utilized in gene expression, resulting in promoter II as the predominant promoter used in BC tissues (12,13). As a result of the use of the alternate promoter, the regulation of E biosynthesis switches from one, controlled primarily by glucocorticoids and cytokines, to another regulated through cAMP-mediated pathways (12).

Inhibition of ultraviolet light B-induced cutaneous inflammation by a specific cyclooxygenase-2 (COX-2) inhibitor

Ultraviolet B (UVB) radiation is responsible for the majority of cutaneous damage following both acute and long-term exposure, and is believed to be the most important etiologic agent in human skin cancer. UVB carcinogenesis initially induces an inflammatory response characterized by edema, dermal infiltration of leukocytes, as well as the production and release of prostaglandins, which may be critical to the observed damaging effects of UVB light on skin. Recently, a specific cyclooxygenase-2 (COX-2) inhibitor, Celecoxib, was developed, which inhibits COX-2-induced inflammation without inhibiting the cytoprotective function of cyclooxygenase-1 (COX-1). Studies have demonstrated that oral administration of Celecoxib decreased the incidence of skin and colon tumors. Recently, the process of inflammation has been linked to tumor formation. The present study examined the effects of a topical application of Celecoxib on the acute UVB-induced cutaneous inflammatory response. We show that topical Celecoxib treatment effectively reduced many parameters of UVB-mediated inflammation, including edema, dermal myeloperoxidase activity, neutrophil infiltration, and prostaglandin E2 (PGE2) levels. By inhibiting this inflammatory response, topical Celecoxib treatment could ultimately be effective in preventing tumor development and progression in the skin, which is known to result from long-term UV exposure.