Edward R. Sherwood

Effects of aging on mortality, hypothermia, and cytokine induction in mice with endotoxemia or sepsis.

Aging is accompanied by an altered stress response that underlies increased susceptibility of the elderly patients to physiological stress such as infection and sepsis. In the present study, we investigated the effects of aging on mortality, hypothermia, and cytokine induction in mouse models of intra-abdominal sepsis and endotoxemia. Systemic inflammation associated with either cecal ligation/puncture (CLP) or injection with bacterial endotoxin, lipopolysaccharide (LPS), resulted in a significantly elevated mortality rate in aged (24 months) compared to young (4 months) mice. The aged mice also showed profound hypothermia during these inflammatory stresses; the severity of hypothermia at the early phase of sepsis or endotoxemia could predict the mortality of individual animals. The stress-mediated induction of interleukin-1beta, interleukin-6, and interleukin-10 (IL-1beta, IL-6, and IL-10) in the circulating blood tended to be higher with aging in both CLP and LPS models, and in particular, the induction of IL-6 was significantly augmented with aging. The serum level of IL-6 showed a strong correlation with degrees of hypothermia. In the heart and lungs, the induction of mRNA for IL-6 and IL-10 was also significantly enhanced with aging. These results clearly demonstrate an age-associated increase in mortality, hypothermia, and induction of IL-6 during endotoxemia and sepsis.

Differential Cytokeratin Expression in Normal, Hyperplastic and Malignant Epithelial Cells From Human Prostate

Studies were undertaken to define the expression of cytokeratins in normal, hyperplastic and malignant epithelial cells from human prostate. Cytokeratin (CK) polypeptides, separated by two-dimensional electrophoresis, were identified by immunoblotting with CK-specific monoclonal antibodies. CK polypeptides 5, 7, 8, 15, 18 and 19 were identified in fresh normal and hyperplastic prostate. Expression of CK 15 has not been previously reported in human prostate. Analysis of central and peripheral zone tissues from human prostate did not reveal qualitative differences in CK expression between these areas. Epithelial cells harvested from fresh BPH tissue by percoll gradient centrifugation and propagated in vitro using selective culture techniques showed alterations in CK expression compared to intact human prostate. Specifically, CKs 6, 14, 16 and 17 were noted in cultured BPH epithelial cells but not fresh normal prostate or BPH tissue. Immunoblot analysis of the established prostate cancer cell lines PC3, DU145 and LNCAP showed expression of CKs 8 and 18 but not CKs 5, 7 and 15 which were observed in benign prostate. These studies further characterize CK expression in benign and malignant human prostate and provide insights which may be useful in differentiating normal, hyperplastic and malignant epithelial cells in the human prostate gland.

Epidermal growth factor receptor activation in androgen-independent but not androgen-stimulated growth of human prostatic carcinoma cells

These studies were undertaken to assess the relative expression and autocrine activation of the epidermal growth factor receptor (EGFR) in normal and transformed prostatic epithelial cells and to determine whether EGFR activation plays a functional role in androgen-stimulated growth of prostate cancer cells in vitro. EGFR expression was determined by Western blot analysis and ELISA immunoassays. Immunoprecipitation of radiophosphorylated EGFR and evaluation of tyrosine phosphorylation was used to assess EGFR activation. The human androgen-independent prostate cancer cell lines PC3 and DU145 exhibited higher levels of EGFR expression and autocrine phosphorylation than normal human prostatic epithelial cells or the human androgen-responsive prostate cancer cell line LNCaP. PC3 and DU145 cells also showed higher levels of autonomous growth under serum-free defined conditions. Normal prostatic epithelial cells expressed EGFR but did not exhibit detectable levels of EGFR phosphorylation when cultured in the absence of exogenous EGF. Addition of EGF stimulated EGFR phosphorylation and induced proliferation of normal cells. LNCaP cells exhibited autocrine phosphorylation of EGFR but did not undergo significant proliferation when cultured in the absence of exogenous growth factors. A biphasic growth curve was observed when LNCaP cells were cultured with dihydrotestosterone (DHT). Maximum proliferation occurred at 1 nM DHT with regression of the growth response at DHT concentrations greater than 1 nM. However, neither EGFR expression nor phosphorylation was altered in LNCaP cells after androgen stimulation. In addition, DHT-stimulated growth of LNCaP cells was not inhibited by anti-EGFR. These studies show that autocrine activation of EGFR is a common feature of prostatic carcinoma cells in contrast to normal epithelial cells. However, EGFR activation does not appear to play a functional role in androgen-stimulated growth of LNCaP cells in vitro.

Epidermal growth factor-related peptides and the epidermal growth factor receptor in normal and malignant prostate

SummaryEpidermal growth factor (EGF) and transforming growth factor-alpha (TGFα) are two closely related peptides that interact with cell-surface epidermal growth factor receptors (EGFR) to induce receptor tyrosine phosphorylation and activation of intracellular signal-transduction pathways. EGF appears to be the predominant EGF-related growth factor in the normal prostate and in benign prostatic hyperplasia (BPH). Evidence indicates that EGF and TGFα are important for maintainence of the structural and functional integrity of the benign prostatic epithelium. The EGF-related peptides are primarily localized to the secretory epithelium of the benign prostate, and their production and secretion is augmented by the presence of circulating androgens. EGFR are located in the basal/neuroendocrine (NE) compartment of the benign prostate and exhibit relatively androgen-independent expression. The EGF-related peptides and EGFR are also present in neoplastic prostatic tissues. There is currently no direct evidence to implicate EGFR activation in the pathogenesis of BPH. However, the EGF-related peptides appear to play a functional role in the growth of prostatic carcinoma cells, with TGFα being the predominant growth factor. Numerous investigators have demonstrated the functional significance of a TGFα/EGFR-mediated autocrine growth pathway in cultured prostatic carcinoma cells. Studies of cultured prostate cancer cells, but not normal epithelial cells, demonstrate constitutive activation of EGFR. Androgen-independent cancer cells exhibit more EGFR expression and phosphorylation than do androgen-responsive prostate cancer cells. Most studies indicate that EGFR do not play a functional role in androgen-stimulated growth of prostate cancer cells. Several studies have correlated EGFR expression with increased nuclear size and tumor dedifferentiation. Future studies should focus on determining both the prognostic significance of EGFR expression and whether manipulation of EGFR-mediated growth can be exploited for therapeutic benefit in human prostate cancer.

Endotoxin-Induced Gamma Interferon Production: Contributing Cell Types and Key Regulatory Factors

ABSTRACT Gamma interferon (IFN-γ) is an important mediator of endotoxin (lipopolysaccharide [LPS])-induced immune responses. However, the specific cell types that produce IFN-γ in response to LPS and the cellular factors that regulate LPS-induced IFN-γ production have not been fully determined. The present studies were undertaken to characterize the cell populations that produce IFN-γ after LPS challenge in the spleens of mice and to determine the regulatory factors that modulate LPS-induced production of IFN-γ. Our studies show that the levels of splenic IFN-γ mRNA and protein production peak at 6 and 8 h, respectively, after systemic LPS challenge. Approximately 60% of IFN-γ-producing cells are natural killer (NK) cells (CD3−DX5+) and 25% are NKT cells (CD3+DX5+). Most of the remaining IFN-γ-producing cells are T cells (CD3+DX5−), macrophages, and dendritic cells. Functionally, interleukin-12 (IL-12) is the major IFN-γ-stimulating factor after LPS challenge, with costimulation provided by IL-15, IL-18, and B7 proteins. IL-10 is a major inhibitor of LPS-induced IFN-γ production. Unlike intact heat-killed gram-negative and gram-positive bacteria, the class II major histocompatibility complex did not play a functional role in LPS-induced IFN-γ production. LPS is a potent stimulus for splenic IL-10, IL-12 p40, and IL-15 mRNA expression, whereas IL-12 p35 and IL-18 mRNAs, as well as B7 proteins, are constitutively expressed in the mouse spleen. Of the factors studied, IL-18 serves as the most potent costimulus with IL-12 for IFN-γ production, followed by IL-15 and B7 proteins. These data demonstrate that NK cells and NKT cells are the most abundant IFN-γ-producing cells in the mouse spleen after LPS challenge and that IL-10 and IL-12 are key functional regulators of LPS-induced IFN-γ production.

Enhancement of interleukin-1 and interleukin-2 production by soluble glucan

Soluble glucan, a beta-1,3-linked polyglucose, is a biologic response modifier effective in the therapy of experimental neoplasia, infectious diseases and immunosuppression. Interleukin-1 (IL-1) and interleukin-2 (IL-2) are endogenous immunomodulators which are essential for effective immune responsiveness. In view of its broad spectrum of immunobiological activity, the ability of glucan to enhance the production of IL-1 and IL-2 was evaluated. Splenic IL-1 and IL-2 secretion as well as plasma IL-1 and IL-2 levels were determined in Sprague-Dawley rats receiving glucan (100 mg/kg, i.p.) at intervals ranging from 12 days to 1 h prior to collection of splenocytes and plasma. Glucan (100 mg/kg) was also injected either s.c., i.p. or i.v. on days -4, -3 and -2 prior to harvesting splenocytes on day 0. Splenic macrophage IL-1 production was initially elevated 12 h following glucan injection and was maintained for a 5 day period. IL-2 secretion by splenic lymphocytes was enhanced 6 h post-glucan and remained elevated for an additional 9 days. Plasma IL-1 activity was elevated 12 h post-injection, while IL-2 activity in plasma was enhanced at 1 h post-glucan. Peak IL-1 and IL-2 activity in plasma occurred 9 and 12 days, respectively, following glucan administration. With regard to route of administration, IV glucan was most effective in inducing lymphokine production. This study demonstrates that: (1) glucan will enhance IL-1 and IL-2 production and (2) elevations in lymphokine production can be maintained up to 12 days post-glucan.

Intranasal dexmedetomidine premedication is comparable with midazolam in burn children undergoing reconstructive surgery.

Preoperative anxiety and emergence delirium in children continue to be common even with midazolam premedication. Midazolam is unpleasant tasting even with a flavored vehicle and as a result, patient acceptance is sometimes poor. As an alternative, we evaluated dexmedetomidine administered intranasally. Dexmedetomidine an alpha-2 adrenergic agonist is tasteless, odorless, and painless when administered by this route. Alpha-2 adrenergic agonists produce sedation, facilitate parental separation, and improve conditions for induction of general anesthesia, while preserving airway reflexes. Institutional review board approval was obtained to study 100 pediatric patients randomized to intranasal dexmedetomidine (2 μg/kg) or oral midazolam (0.5 mg/kg) administered 30 to 45 minutes before the surgery. Subjects received general anesthesia with oxygen, nitrous oxide, isoflurane, and analgesics (0.05–0.1 mg/kg morphine or 0.1 mg/kg methadone). Nurses and anesthetists were blinded to the drug administered and evaluated patients for preoperative sedation, conditions for induction of general anesthesia, emergence from anesthesia, and postoperative pain. Responses of 100 patients (50 dexmedetomidine and 50 midazolam) were analyzed. Dexmedetomidine (P = .003) was more effective than midazolam at inducing sleep preoperatively. Dexmedetomidine and midazolam were comparable for conditions at induction (P > 0.05), emergence from anesthesia (P > 0.05), or postoperative pain (P > 0.05). Both drugs were equieffective in these regards. In pediatric patients, dexmedetomidine 2 μg/kg administered intranasally and midazolam 0.5 mg/kg administered orally produced similar conditions during induction and emergence of anesthesia. Intranasal administration of dexmedetomidine is more effective at inducing sleep and in some circumstances offers a useful alternative to oral midazolam in children.

Role of the MDR-1-Encoded Multiple Drug Resistance Phenotype in Prostate Cancer Cell Lines

The treatment of advanced metastatic prostate cancer by hormone manipulation or orchiectomy is frequently followed by the appearance of hormone-insensitive and highly chemoresistant tumor cells. In this study we have investigated the contribution of the P-glycoprotein-mediated drug efflux (multidrug-resistance; MDR) to the cellular resistance of prostate carcinoma-derived cell lines to diverse cytotoxic drugs by detection of P-glycoprotein (P-gp) measurement of P-gp-mediated drug transport and reversal of MDR by chemosensitizers. The in vitro chemosensitivity of three prostate cancer cell lines (PC-3, DU-145 and LNCaP) to doxorubicin was measured in a thymidine incorporation proliferation assay. Growth of the partially hormone-sensitive cell line LNCaP is inhibited by low doses of doxorubicin (IC50:27 ng./ml.), but PC-3 and DU-145 are highly resistant to the drug, with IC50 values of 10 micrograms./ml. and 7.5 micrograms./ml., respectively. The chemosensitivity of the PC-3 and DU-145 cells is increased in response to 1 microM. verapamil, 1 micrograms./ml. cyclosporine A and 2 microM. tamoxifen, which are known to partially reverse the MDR phenotype in other resistant tumors. A verapamil-sensitive drug efflux has been demonstrated for the PC-3 and Du-145, but not for the LNCaP, cell lines, using flow cytometric measurements of the P-gp substrate rhodamine 123 efflux from preloaded cells. In agreement with the functional measurements, the expression of the P-glycoprotein was detected in the PC-3 and Du-145 cell lines in Western blots using the monoclonal C 219 antibody. In conclusion, the chemoresistant and hormone-insensitive PC-3 and Du-145 cell lines express P-gp and exhibit verapamil-sensitive drug efflux, indicative of MDR. However, the low MDR-reversal rates observed in these cell lines in response to chemosensitizers in clinically achievable concentrations (approximately 2- to 3-fold reversal), point to non-MDR-associated cellular mechanisms as dominant factors of chemoresistance in prostate cancer.

Enhancement of Dendritic Cell Production by Fms-Like Tyrosine Kinase-3 Ligand Increases the Resistance of Mice to a Burn Wound Infection

Fms-like tyrosine kinase-3 ligand (Flt3L) is a hemopoietic cytokine that stimulates the production of dendritic cells. This study evaluated the ability of Flt3L-enhanced dendritic cell production to increase the resistance of mice to a burn wound infection with Pseudomonas aeruginosa, a common source of infections in burn patients that have impaired immunity and are susceptible to opportunistic microorganisms. Treatment of mice with Flt3L for 5 days caused a significant increase in dendritic cell numbers in the spleen and significantly increased survival upon a subsequent burn wound infection. Improved survival in Flt3L-treated mice was associated with limited bacterial growth and spread within the burn wounds and a decrease in systemic dissemination of P. aeruginosa. Resistance to burn wound infection could also be conferred to recipient mice by the adoptive transfer of dendritic cells that had been isolated from spleens of Flt3L-treated mice. Adoptive transfer of the same number of splenic dendritic cells from nontreated mice did not confer resistance to burn wound infection. These data indicate that Flt3L can increase the resistance of mice to a P. aeruginosa burn wound infection through both stimulation of dendritic cell production and enhancement of dendritic cell function.

Diminished bacterial clearance is associated with decreased IL-12 and interferon-gamma production but a sustained proinflammatory response in a murine model of postseptic immunosuppression.

After a major illness or injury, immune status in critically ill patients may fluctuate between a marked proinflammatory response and an immunosuppressed state. Postinflammatory immunosuppression can result in increased susceptibility to infection. Alterations of cytokine production, such as suppression of IFNγ and elevation of the anti-inflammatory cytokine IL-10, are believed to contribute to postinflammatory immunosuppression. We examined antimicrobial immunity in mice that had previously been subjected to a sublethal cecal ligation and puncture (CLP) as a model of major injury. Mice were challenged with Pseudomonas aeruginosa (5 × 107 CFU i.v.) on day 5 after CLP or sham surgery. Bacterial clearance in mice after CLP was impaired and associated with decreased production of IFNγ and increased production of IL-10 in the early response to the Pseudomonas challenge. Pseudomonas-induced production of the IFNγ-inducing factor IL-12 was also decreased in post-CLP mice. However, splenocytes from post-CLP mice remained responsive to exogenous stimulation with the IFNγ-inducing cytokines IL-12, IL-15, and IL-18 as well as T-cell receptor activation. Furthermore, production of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 were as high, or higher, in the post-CLP group compared with sham mice after P. aeruginosa challenge. Blockade of IL-10 did not reverse IL-12 and IFNγ suppression in splenocytes from post-CLP mice. These studies show that suppressed bacterial clearance in post-CLP mice is associated with decreased production of IFNγ and IL-12 and with increased production of IL-10 and proinflammatory cytokines.