PF-04886847 (an inhibitor of plasma kallikrein) attenuates inflammatory mediators and activation of blood coagulation in rat model of lipopolysaccharide (LPS)-induced sepsis.

The plasma kallikrein-mediated proteolysis regulates both thrombosis and inflammation. Previous study has shown that PF-04886847 is a potent and competitive inhibitor of kallikrein, suggesting that it might be useful for the treatment of kallikrein-kinin mediated inflammatory and thrombotic disorders. In the rat model of lipopolysaccharide (LPS) -induced sepsis used in this study, pretreatment of rats with PF-04886847 (1 mg/kg) prior to LPS (10 mg/kg) prevented endotoxin-induced increase in granulocyte count in the systemic circulation. PF-04886847 significantly reduced the elevated plasma 6-keto PGF1α levels in LPS treated rats, suggesting that PF-04886847 could be useful in preventing hypotensive shock during sepsis. PF-04886847 did not inhibit LPS-induced increase in plasma TNF-α level. Pretreatment of rats with PF-04886847 prior to LPS did not attenuate endotoxin-induced decrease in platelet count and plasma fibrinogen levels as well as increase in plasma D-dimer levels. PF-04886847 did not protect the animals against LPS-mediated acute hepatic and renal injury and disseminated intravascular coagulation (DIC). Since prekallikrein (the zymogen form of plasma kallikrein) deficient patients have prolonged activated partial thromboplastin time (aPTT) without having any bleeding disorder, the anti-thrombotic property and mechanism of action of PF-04886847 was assessed. In a rabbit balloon injury model designed to mimic clinical conditions of acute thrombotic events, PF-04886847 reduced thrombus mass dose-dependently. PF-04886847 (1 mg/kg) prolonged both aPTT and prothrombin time (PT) in a dose-dependent manner. Although the findings of this study indicate that PF-04886847 possesses limited anti-thrombotic and anti-inflammatory effects, PF-04886847 may have therapeutic potential in other kallikrein-kinin mediated diseases.

Differing requirement for inducible nitric oxide synthase activity in clearance of primary and secondary Cryptococcus neoformans infection.

The role of nitric oxide in resistance to cryptococcal infection was investigated. Mice deficient in inducible nitric oxide synthase (INOS) did not survive a primary intratracheal infection as did INOS-replete control mice. Despite adequate recruitment of host cells and generation of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha at the site of infection, INOS-deficient mice failed to clear yeast from their lungs by five weeks of infection, in contrast to wild-type mice. INOS-deficient mice also had higher yeast brain burdens than did control mice after a primary intracerebral infection. Therefore, generation of nitric oxide is required for resistance to primary cryptococcal infection. However, INOS-deficient mice vaccinated subcutaneously and rechallenged intravenously had lung and brain yeast burdens equivalent to those of vaccinated controls, and therefore expressed effective acquired immunity to Cryptococcus neoformans. Cells harvested from infected INOS-deficient mice by bronchoalveolar lavage acted as anti-cryptococcal effectors in vitro at an effector:target ratio of 100:1, provided IFN-gamma was present, but did not inhibit yeast proliferation at a 10:1 effector:target ratio as cells from wild-type mice did. Therefore, INOS activity is important for anti-cryptococcal function of effectors of immunity during the primary response, but not for the generation or expression of secondary immunity to C. neoformans.

Effect of bisphosphonates on surface hydrophobicity and phosphatidylcholine concentration of rodent gastric mucosa.

Bisphosphonates are a family of chemically related zwitterionic molecules that are used clinically to retard bone resorption in individuals with osteoporosis and associated skeletal diseases. Inflammation and ulceration of the upper gastrointestinal tract by a mechanism that relates to a topical irritant action is associated with the consumption of some bisphosphonates. In the present study, we investigated the effects of three bisphosphonate molecules, pamidronate, alendronate, and risedronate on the surface hydrophobicity and phosphatidylcholine (PC) concentration of the antral mucosa. We also examined how these surface changes related to mucosal injury in an established rat model, in which the test compounds were administered in combination with indomethacin. We initially determined that a combination of pamidronate (300 mg/kg) and indomethacin (40 mg/kg) induced a significant reduction in mucosal surface hydrophobicity and macroscopic lesion formation by 15 min and mucosal PC concentration by 30 min, with the magnitude of these changes increasing over the 4-hr study period. An equivalent dose of alendronate or risedronate in combination with indomethacin produced modest or no macroscopic injury, respectively, to the antral mucosa over the 4-hr study, although the bisphosphonates clearly induced surface injury and some glandular necrosis when examined at the light microscopic level. These bisphosphonates also induced modest decreases in antral surface hydrophobicity and mucosal PC concentration that appeared to be related to their injurious potential. In conclusion, the variable toxicity of bisphosphonates to the antral mucosa appears to be associated with their ability to compromise the surface hydrophobic phospholipid barrier of the tissue, with pamidronate > > > alendronate > risedronate. This bisphosphonate effect on the surface barrier may trigger the development of mucosal injury and possible ulceration.

Gastric damage in the rat with nitrogen-containing bisphosphonates depends on pH.

BACKGROUND: The use of nitrogen-containing bisphosphonates (N-BPs) has been reported to be associated with gastrointestinal intolerance. The fasted, indomethacin-treated rat provides a model for assessing the gastrointestinal effects of these compounds. AIMS: The aims of this study were to elucidate the effect of pH on N-BP-induced gastric damage, and to evaluate the structure-activity relationship between N-BP anti-resorptive and gastric effects. METHODS: Fasted rats were dosed concomitantly with indomethacin (40 mg/kg, subcutaneously) and an N-BP (pamidronate, alendronate, or risedronate at 150 or 300 mg/kg, orally), with the N-BP dosing solutions adjusted to pH 2, 4 or 7. The aminopentane and aminohexane N-BPs (150, 225 or 300 mg/kg, orally) were only tested at pH 4 only. RESULTS: Nitrogen-containing bisphosphonate-induced gastric damage was pH-dependent, with increased damage at increasing pH. CONCLUSIONS: Gastric damage potential did not correlate with bone anti-resorptive effects, and the more potent anti-resorptive N-BPs were not necessarily more damaging to the stomach.

B cells are essential for vaccination-induced resistance to virulent Toxoplasma gondii.

T lymphocytes and gamma interferon (IFN-gamma) are known mediators of immune resistance to Toxoplasma gondii infection, but whether B cells also play an important role is not clear. We have investigated this issue using B-cell-deficient (muMT) mice. If vaccinated with attenuated T. gondii tachyzoites, muMT mice are susceptible to a challenge intraperitoneal infection with highly virulent tachyzoites that similarly vaccinated B-cell-sufficient mice resist. Susceptibility is evidenced by increased numbers of parasites at the challenge infection site and by extensive mortality. The susceptibility of B-cell-deficient mice does not appear to be caused by deficient T-cell functions or diminished capacity of vaccinated and challenged B-cell-deficient mice to produce IFN-gamma. Administration of Toxoplasma-immune serum, but not nonimmune serum, to vaccinated B-cell-deficient mice significantly prolongs their survival after challenge with virulent tachyzoites. Vaccinated mice lacking Fc receptors or the fifth component of complement resist a challenge infection, suggesting that neither Fc-receptor-dependent phagocytosis of antibody-coated tachyzoites nor antibody-dependent cellular cytotoxicity nor antibody-and-complement-dependent lysis of tachyzoites is a crucial mechanism of resistance. However, Toxoplasma-immune serum effectively inhibits the infection of host cells by tachyzoites in vitro. Together, the results support the hypothesis that B cells are required for vaccination-induced resistance to virulent tachyzoites in order to produce antibodies and that antibodies may function protectively in vivo by blocking infection of host cells by tachyzoites.

Decreased resistance to primary intravenous Cryptococcus neoformans infection in aged mice despite adequate resistance to intravenous rechallenge.

It is often stated that impaired immune functions in the aged underlie their greater susceptibility to infections. Indeed, in many experimental settings, T-cell responses in aged mice have been shown to be deficient compared with those from young adults. Nonetheless, there are very few examples where a greater susceptibility to infection in aged mice has been demonstrated to result from impaired T-cell function. The clinical importance of understanding the basis for increased susceptibility to infection that accompanies advanced age dictates a need for experimental models with which to study the effect that aging has on immunological resistance to infection. This study was undertaken to investigate whether aged mice were less resistant than young adult control mice to infection with the fungus Cryptococcus neoformans. After a primary intravenous challenge with yeast, aged mice died sooner and developed higher organ burdens of yeast than did young adults. Deficient in vitro responses were observed in T cells from aged mice; however, greater susceptibility to intravenous infection appeared not to result from less effective T-cell-dependent resistance in vivo. In fact, T-cell-replete aged mice were more susceptible to intravenous cryptococcal infection than were T-cell-depleted young adults. Furthermore, aged mice were as resistant to primary pulmonary challenge with Cryptococcus as were young adults. Similarly, vaccinated aged mice were as resistant to rechallenge as were young adult counterparts. Therefore, despite demonstrably deficient in vitro responses of T cells from aged mice, their T-cell-dependent resistance to C. neoformans is as effective as that of young adults.

Nonclinical model for assessing gastric effects of bisphosphonates.

Gastrointestinal intolerance has been associated with amino bisphosphonate therapy in the clinic. The objective of this study was to develop a model for assessing bisphosphonate-induced gastric damage that may aid in the development of future bisphosphonate therapies. Rats were dosed concomitantly with indomethacin (40 mg/kg, subcutaneously) and an amino or pyridinyl bisphosphonate (orally at. 150, 225 or 300 mg/kg). The bisphosphonates studied were pamidronate and alendronate (primary amino bisphosphonates) and risedronate and NE-97221 (pyridinyl bisphosphonates). Macroscopically, alendronate induced significantly (P < 0.05) more antral damage (both lesion length and number) than pamidronate and risedronate at 225 and 300 mg/kg, and more than NE-97221 at 300 mg/kg. NE-97221 induced significantly more antral damage (lesion length) than risedronate at 225 mg/kg and a greater number of lesions compared to pamidronate and risedronate at 225 and 300 mg/kg. The model was validated histologically, and macroscopic findings correlated with histologic evidence of antral mucosal necrosis and inflammatory infiltration of the lamina propria. The calcium chelators EGTA and EDTA did not induce gastric damage in this model when dosed according to the same protocol as the nitrogen-containing bisphosphonates. This suggests that calcium chelation does not account for the gastric effects in this model. The fasted, indomethacin-treated rat provides a novel nonclinical model to assess gastric effects of bisphosphonates, which may aid in the development of future bisphosphonate therapies. These data suggest that when expressed on an actual or anticipated clinical dose basis for osteoporosis (pamidronate, 150 mg; alendronate, 5-10 mg; risedronate and NE-97221, 5 mg), primary amino bisphosphonates may have a greater potential for inducing gastric damage than do pyridinyl bisphosphonates.

CR3-dependent resistance to acute Toxoplasma gondii infection in mice.

Studies were performed to determine whether resistance to acute Toxoplasma gondii infection in mice depends on a mechanism involving CR3, the type 3 complement receptor. Nineteen of 22 mice (86%) given multiple injections of the anti-CR3 monoclonal antibody, 5C6, prior to and after intraperitoneal inoculation of cysts of the ordinarily mildly virulent ME49 strain of T. gondii died within 8 to 12 days, whereas control antibody-treated mice survived. All (five of five) anti-CR3-treated BALB/c mice infected via the natural peroral route died within 8 days of infection. Flow cytometric analysis of cells recovered from peritoneal lavages of anti-CR3-treated T. gondii-infected mice revealed that the percentage of Thy-1+ CD4- CD8- cells was reduced to about 50% of that of control antibody-treated mice and to about 20% of the number of such cells in controls. The numbers of macrophages, polymorphonuclear leukocytes, and lymphocytes recovered from the peritoneal cavities of T. gondii-infected mice were all reduced in anti-CR3-treated mice to about 40% of those of controls. In addition, anti-CR3-treated mice had less than 25% of the induced NK cell activity of the controls, and gamma interferon was reduced to undetectable levels. Thus, the rapid death of anti-CR3-treated mice was probably caused by impaired preimmune defenses. Histological examination of anti-CR3-treated T. gondii-infected mice revealed extensive liver pathology compared with that of infected mice given a control antibody or uninfected mice given anti-CR3. The inflammation, degeneration, and necrosis in most of the anti-CR3-treated mice were severe enough to account for the observed mortalities.

Characterization of the effects of tebufelone on hepatic cytochromes P450 in the beagle dog.

Tebufelone (1-[3,5-bis(1,1-dimethylethyl)-4-hydroxy-phenyl]-hex-5-yne-1-one) is an investigational ditertiary butylphenol nonsteroidal anti-inflammatory drug. The purpose of the present study was to assess the effects of tebufelone on hepatocyte ultrastructure and hepatic cytochromes p450 (P450s) in the beagle dog after 2 weeks of oral administration at dose levels of 0, 5, 15, 50, and 100 mg/kg/day (N = 1/sex/dose level). Hepatic tissue was obtained at necropsy for histologic, ultrastructural, and biochemical evaluation. Hepatocellular hypertrophy was observed in only a single tebufelone-treated dog (50 mg/kg). Electron microscopic evaluation, however, revealed marked dose-dependent increases in smooth endoplasmic reticulum in all of the tebufelone treatment groups. Biochemical indicators suggested that tebufelone produced mixed effects on hepatic P450s. p-Nitroanisole O-demethylase and, to a greater extent, ethoxyresorufin O-deethylase activities were decreased with increasing tebufelone dose. The precise mechanism by which tebufelone decreased ethoxyresorufin O-deethylase activity in dogs in unknown, but it was not by competitive inhibition, P450 inactivation, or reduced CYP1A expression. Tebufelone treatment increased NADPH-dependent cytochrome c reductase, total P450, and indicators of CYP2B11 (chloramphenicol covalent binding and immunochemically determined 2B11) and CYP3A12 (erythromycin N-demethylase, triacetyloleandomycin spectral complex formation, testosterone 6 beta-hydroxylase, and immunochemically determined 3A12). The largest increase in the 2B11 and 3A12 markers occurred in the 50 or 100 mg/kg treatment groups. The greatest increase in CYP2B11 markers produced by tebufelone treatment ranged from 2- to 3-fold, whereas the increase in CYP3A12 markers ranged from 5- to 10-fold. The changes in hepatic ultrastructure and increases in CYP2B11 and CYP3A12 markers produced by tebufelone in dogs are similar to that reported for phenobarbital.

Resistance to Cryptococcus neoformans is associated with an inflammatory response to Toxoplasma gondii in the central nervous system of mice.

We have studied the resistance of Toxoplasma gondii-infected mice to subsequent infection with Cryptococcus neoformans. Mice infected with the moderately virulent ME49 strain of T. gondii are resistant to proliferation of yeast cells in their brains after intravenous inoculation of the serotype A C. neoformans strain 184. The resistance serves to limit proliferation of yeast cells that colonize the brain. Maximal levels of resistance correlate not with maximal systemic specific anti-Toxoplasma resistance but rather with high levels of inflammatory response, presumably to parasites released from cysts in the brain. Resistance is localized, as mice infected with ME49 show only limited resistance in their lungs after intratracheal instillation of yeast cells, but there is substantial protection against development of cerebral cryptococcosis.

Preimmune resistance to Toxoplasma gondii in aged and young adult mice.

Aged individuals are more susceptible to certain infections than are young adults. To investigate the relative resistance capabilities of aged and young adult mice, responses that are induced within the first week of a Toxoplasma gondii infection, which are known to be involved in preimmune resistance, were compared in young adult and aged mice. Aged mice did not differ reproducibly from young adults in numbers of induced Thy-1+ CD4- CD8- cells or interferon-gamma levels. Numbers of induced CD4+ and CD8+ T cells, associated with acquired immunity, were as high in aged mice as in young adults. Natural killer cell activity, although induced to a high level, was lower in aged mice. Aged mice thus are capable of inducing a mechanism of preimmune resistance to T. gondii and presumably other infectious agents. Nonetheless, aged mice died within 8-12 days after intraperitoneal or peroral inoculation of 500 T. gondii cysts, whereas young adult mice survived. Causes other than an age-related impairment in preimmune resistance mechanisms are apparently responsible for the increased susceptibility of aged mice to T. gondii infection.

Toxoplasma gondii: effect of sublethal irradiation on host resistance in mice.

The effect of sublethal whole-body irradiation (500 rad) on the resistance of C57BL/6 mice to infection with Toxoplasma gondii was studied. Mice irradiated 1 day before or 4 days after infection via the intraperitoneal or peroral route with cysts of the mildly virulent ME49 strain of T. gondii died sooner than did unirradiated controls. The time to death of irradiated mice was suggestive of impaired acquired immunity. Irradiated mice infected intraperitoneally with T. gondii cysts exhibited reduced levels of Thy-1+CD4-CD8- cells, less natural killer cell activity against YAC-1 targets, and lower levels of IFN-gamma than controls. Numbers of CD4+ and CD8+ cells were also lower in infected, irradiated mice. Irradiated mice immunized with a vaccine strain of T. gondii, and later challenged with a highly virulent strain, were less well protected than unirradiated controls. Irradiation appears to impair early, CD4(+)- and CD(8+)-independent resistance to T. gondii, as well as acquisition of immunity.

Role of tumor necrosis factor and gamma interferon in acquired resistance to Cryptococcus neoformans in the central nervous system of mice.

Although naive C.B-17 and BALB/cBy mice die of meningoencephalitis within 5 weeks of intravenous infection with an opportunistic strain of Cryptococcus neoformans, immunized mice express an acquired, CD4+ T-cell-dependent immunity and survive an intravenous infection. Infusion of lymphocytes from immune mice into severe combined immunodeficiency (SCID) mice renders these mice more resistant to cryptococcal brain infection than uninfused controls. We have investigated the role of gamma interferon (IFN-gamma) and tumor necrosis factor (TNF) in acquired resistance to C. neoformans. Neutralization of either IFN-gamma or TNF impaired resistance of immune BALB/cBy or C.B-17 mice to cryptococci. At 10 days postinfection, there were approximately 10 times as many yeast cells in the brains of mice treated with either anticytokine antibody as in the brains of mice treated with control antibody. Simultaneous neutralization of IFN-gamma and TNF further exacerbated infection. Neutralization of IFN-gamma or TNF also impaired resistance in immune lymphocyte-infused SCID mice, resulting in significantly higher yeast burdens in brains of cytokine-neutralized mice than in brains of controls. Concurrent neutralization of IFN-gamma and TNF rendered SCID recipients of immune cells equivalent to uninfused SCID mice with respect both to brain yeast burdens at 10 days and to survival. Anti-TNF treatment alone also curtailed survival. Histological examination of the brains of cytokine-neutralized mice revealed deficiencies in ability to focus inflammatory cells at brain lesions. These data demonstrate that both IFN-gamma and TNF are important mediators of acquired resistance to cryptococcal meningoencephalitis.

Measurement of estrogen and progesterone receptors in abnormal human parathyroid tissue.

Estrogen and/or progestin administration to postmenopausal women with primary hyperparathyroidism lowers serum calcium. We measured cytosolic estrogen receptors (ER) and progesterone receptors (PR) by classical hormone-receptor binding techniques in parathyroid tissue removed from 10 men and 20 women, and ER by immunocytochemistry in tissue from an additional one man and seven women in order to ascertain whether these agents might exert a direct effect upon tissue responsible for hyperparathyroidism. ER were negative (< 3.1 fmol bound estradiol/10 mg tissue) in all 8 adenomas and 4 of 5 secondary hyperplasias removed from men, and from women in 19 of 22 adenomas, 2 of 3 secondary hyperplasias, and 3 of 4 primary hyperplasias. PR were negative (< 10.1 fmol bound progesterone/10 mg tissue) in 7 of 8 adenomas and all 5 secondary hyperplasias removed from men, and from women in 20 of 22 adenomas, all 3 secondary hyperplasias, and all 4 primary hyperplasias. For immunocytochemical studies, quick-frozen specimens were analyzed with a monoclonal antibody (Abbott Laboratory) directed at nuclear ER. All eight samples--five adenoma and three primary hyperplasia--were negative. We conclude that abnormal human parathyroid tissues have nondetectable levels of ER and PR. It is unlikely that estrogen and progesterone exert a direct, ER, or PR-mediated effect upon parathyroid tissue.

Characterization of a simplified method of cryopreserving human parathyroid tissue.

Cryopreservation of human parathyroid tissue plays an important role in managing difficult parathyroid disease. It also can permit investigators to conduct experiments without dependence on the operating room schedule. Availability of cryopreservation has been limited by the perceived need for expensive, complex equipment. We adapted a simple method of freezing cell suspensions to freezing human parathyroid tissue. Vials containing human parathyroid in culture media, dimethylsulfoxide, and patient serum were placed in a plastic rack in a metal pan containing prechilled (4 degrees C) ethanol and placed in a -70 degrees C freezer. We compared viability (trypan blue dye exclusion by collagenase dispersed cells) of tissue frozen in this manner to that of tissue frozen in a programmable liquid nitrogen freezer at 1 degrees C per minute, a cooling rate recommended for human parathyroid tissue. The viability of 30 patients' samples cooled in liquid nitrogen (average length of storage 5 months) was 74% +/- 15% and that of 64 patients' samples cooled in ethanol (average length of storage 26 months) was 71% +/- 15%. Viability of 19 samples of fresh tissue was 79% +/- 10%. Neither method had a statistically significant correlation between length of storage and viability. Successful cryopreservation with simplified technology may expand the availability of parathyroid tissue to meet both clinical and investigative requirements.