CXCR2 antagonists for the treatment of pulmonary disease.

Chemokines have long been implicated in the initiation and amplification of inflammatory responses by virtue of their role in leukocyte chemotaxis. The expression of one of the receptors for these chemokines, CXCR2, on a variety of cell types and tissues suggests that these receptors may have a broad functional role under both constitutive conditions and in the pathophysiology of a number of acute and chronic diseases. With the development of several pharmacological, immunological and genetic tools to study CXCR2 function, an important role for this CXC chemokine receptor subtype has been identified in chronic obstructive pulmonary disease (COPD), asthma and fibrotic pulmonary disorders. Interference with CXCR2 receptor function has demonstrated different effects in the lungs including inhibition of pulmonary damage induced by neutrophils (PMNs), antigen or irritant-induced goblet cell hyperplasia and angiogenesis/collagen deposition caused by lung injury. Many of these features are common to inflammatory and fibrotic disorders of the lung. Clinical trials evaluating small molecule CXCR2 antagonists in COPD, asthma and cystic fibrosis are currently underway. These studies hold considerable promise for identifying novel and efficacious treatments of pulmonary disorders.

Biology and therapeutic potential of cannabinoid CB2 receptor inverse agonists.

Evidence has emerged suggesting a role for the cannabinoid CB2 receptor in immune cell motility. This provides a rationale for a novel and generalized immunoregulatory role for cannabinoid CB2 receptor-specific compounds. In support of this possibility, we will review the biology of a class of cannabinoid CB2 receptor-specific inverse agonist, the triaryl bis-sulfones. We will show that one candidate, Sch.414319, is potent and selective for the cannabinoid CB2 receptor, based on profiling studies using biochemical assays for 45 enzymes and 80 G-protein coupled receptors and ion channels. We will describe initial mechanistic studies using this optimized triaryl bis-sulfone, showing that the compound exerts a broad effect on cellular protein phosphorylations in human monocytes. This profile includes the down regulation of a required phosphorylation of the monocyte-specific actin bundling protein L-plastin. We suggest that this observation may provide a mechanism for the observed activity of Sch.414319 in vivo. Our continued analysis of the in vivo efficacy of this compound in diverse disease models shows that Sch.414319 is a potent modulator of immune cell mobility in vivo, can modulate bone damage in antigen-induced mono-articular arthritis in the rat, and is uniquely potent at blocking experimental autoimmune encephalomyelitis in the rat.

Interleukin 4 retards dissemination of a human B-cell lymphoma in severe combined immunodeficient mice.

We have examined the antitumor activity of murine interleukin 4 (IL-4) on development of a human B-cell lymphoma (Daudi) in severe combined immunodeficient (SCID) mice. The progression of Daudi cells in SCID mice was followed by histological staining and by flow cytometric analysis of CD20+ cells in spleen, liver, bone marrow, and kidneys. By day 35, CD20+ Daudi cells populate the majority of space in the bone marrow and kidney in vehicle-treated mice. Mice receiving i.p. injections of IL-4, commencing 7 or 14 days after tumor inoculation, exhibit a reduction in tumor burden as well as a decrease in CD20+ cells in both compartments. The antitumor activity of IL-4 does not appear to be due to an antiproliferative effect, since the cytokine does not alter the growth of Daudi cells in vitro, nor does it correlate with any marked cellular infiltrate in tumor-bearing tissues. In 51Cr-release assays, we observed that splenocytes from IL-4-treated mice were capable of lysing YAC-1 but not Daudi cell targets. Our findings demonstrate that: (a) systemic administration of IL-4 retards dissemination of a human B-cell lymphoma in SCID mice; and (b) antitumor activity elicited by IL-4 may not involve a direct effect on proliferation of Daudi cells or on the induction of cytolytic activity.

Influence of IL-10 on murine CFU-pre-B formation.

We have examined the ability of interleukin-10 (IL-10) to influence murine B cell development in vitro and in vivo. In vitro treatment of young adult mouse bone marrow cells with 0.5 to 10 ng/ml human IL-10 (hIL-10) produced a significant enhancement of IL-7-mediated colony-forming unit-pre-B (CFU-pre-B) formation, while IL-10 concentrations > 10 ng/ml had no net effect. IL-10 by itself was unable to stimulate pre-B cell colony formation, even at optimal concentrations. The increase in CFU-pre-B produced by IL-10 was specifically blocked by anti-hIL-10 antibody, but not by anti-stem cell factor (SCF) antibody, and was observed with both unfractionated and purified B220+ surface immunoglobulin (sIg-) bone marrow cells. CFU-pre-B from the IL-10 treatment group contained a higher percentage of CD43+B220+ blast-like cells than colonies exposed to IL-7 only. In vivo administration of 0.1 microgram hIL-10 per day to mice treated with a single sublethal dose of cyclophosphamide (CY) resulted in a dramatic and accelerated recovery of CFU-pre-B numbers as compared to vehicle-administered mice. This enhancement was seen as early as day 11 post-CY, and the number of CFU-pre-B was comparable to normal age-matched control mice by day 16. In contrast, the number of CFU-pre-B in vehicle-treated mice remained significantly lower than age-matched and IL-10-treated animals as long as day 22 post-CY. No differences in the number of pre-B and mature B cells in bone marrow or in the number of mature B cells in peripheral lymphoid organs were detected in IL-10-treated mice. Myeloid cell recovery, assessed by the CFU-granulocyte/macrophage (CFU-GM) assay and the number of marrow Mac-1+ cells, was unaffected by IL-10 treatment of CY-dosed animals. These results indicate that IL-10 enhanced IL-7-stimulated murine pre-B cell colony formation and imply a role for IL-10 in normal B lymphopoiesis.

Cerebrospinal fluid creatine kinase-BB isoenzyme activity and outcome after subarachnoid hemorrhage.

BACKGROUND: The brain is rich in creatine kinase-BB isoenzyme activity (CK-BB), which is not normally present in cerebrospinal fluid (CSF). Results of previous studies have shown that CK-BB can be detected in the CSF of patients with aneurysmal subarachnoid hemorrhage (SAH), but whether CK-BB levels correlate with patients' neurologic outcomes is unknown. OBJECTIVE: To evaluate the relationship between CSF CK-BB level and outcome after SAH. DESIGN: Prospective observational cohort. SETTING: University-affiliated tertiary care center. PATIENTS: Convenience sample of 30 patients seen for cerebral aneurysm clipping. INTERVENTIONS: We sampled and assayed CSF for CK isoenzymes a median of 3 days after SAH in 27 patients, and at the time of unruptured aneurysm clipping in 3 patients. MAIN OUTCOME MEASURES: Without knowledge of CK results, we assigned the Glasgow Outcome Scale score early (approximately 1 week) and late (approximately 2 months) after surgery. RESULTS: Higher CSF CK-BB levels were associated with higher Hunt and Hess grades at hospital admission (Spearman rank correlation, p = 0.69; P<.001), lower Glasgow Coma Scale scores at hospital admission (p = -0.72; P<.001), and worse early outcomes on the Glasgow Outcome Scale (p = -0.64; P<.001). For patients with a favorable early outcome (Glasgow Outcome Scale score, 3-5), all CK-BB levels were less than 40 U/L. With a cutoff value of 40 U/L, CK-BB had a sensitivity of 70% and a specificity of 100% for predicting unfavorable early outcome (Glasgow Outcome Scale score, 1-2). Having a CK-BB level greater than 40 U/L increased the chance of an unfavorable early outcome, from 33% (previous probability) to 100%, whereas a CK-BB level of 40 U/L or less decreased it to 13%. Similar findings were obtained when considering late outcomes. CONCLUSION: The level of CSF CK-BB may help predict neurologic outcome after SAH.

Single-dose versus conventional therapy of urinary tract infections in female adolescents.

A randomized, prospective study was done to assess the efficacy of single-dose nu conventional treatment of acute urinary tract infection (UTI) in female adolescents. Thirty-one 12- to 18-year-old female adolescents with symptoms of an acute UTI and a urine culture with greater than 10(5) organisms were treated with amoxicillin, either as a 3.0-g single dose or 250-mg three times daily for ten days. Urine cultures obtained three days after completing therapy in each group, showed bacteriologic cure rate of 69% (11/16) with single-dose treatment compared with a cure rate of 87% (13/15) in the conventional treatment group (P = .23). When patients with resistant organisms were excluded, the cure rate was 85% in both groups. Complete symptom resolution in less than two days after commencing treatment occurred in 36% of single-dose group nu none of the patients in the conventional-dose group. The finding has not been previously reported in single-dose trials. Candida vaginitis occurred in 20% of the conventional-dose group nu none of the single-dose group. All patients in the single-dose group kept their first scheduled follow-up appointment, whereas 40% in the conventional group required reminders and rescheduling. Perfect compliance with the medication regimen was reported by 27% of the patients taking ten days of medicine. Although single-dose cure rates may not be superior to conventional-dose rates, the advantages of single-dose treatment include increased compliance with medication and follow-up, decreased side effects, and more rapid resolution of symptoms.

The Avianca plane crash: an emergency medical system's response to pediatric survivors of the disaster.

OBJECTIVE: On January 25, 1990, a jetliner crashed on Long Island, New York. Twenty-two children survived the crash. The purpose of this study was to evaluate the emergency medical system's response to these pediatric survivors. METHODS: A questionnaire was sent to all local, acute care hospitals to determine their specific pediatric capabilities and to rank them as level I, II, or III pediatric centers; level I centers are tertiary care facilities. A second questionnaire was sent to all hospitals that received pediatric survivors to collect specific clinical information for each patient. Based on this clinical information a Pediatric Trauma Score (PTS) was assigned to each patient. Children with a PTS < or = 8 are considered to be at increased risk of trauma-related mortality. The assigned PTS was compared to the level of the pediatric center to which each patient was transported. RESULTS: Of 25 children on board the plane, 22 (88%) survived the crash; of 135 adults on board, 70 (52%) survived (chi 2 = 9.9, P = .002). Seven children had a PTs < or = 8; only 1 of these high-risk patients was transported directly to a level I pediatric center, and only 2 of the 5 high-risk children initially transported to level III facilities were transferred to higher level pediatric centers. CONCLUSIONS: Pediatric survivors were neither adequately triaged nor transported to appropriate facilities which could optimize their care. Possible explanations for this include: (1) unique features of the rescue operation, (2) limited pediatric training of prehospital personnel, and (3) deficiencies of the regional disaster plan. Emergency medical services systems and disaster plans can be made more responsive to children's needs by: (1) acknowledging that children have special needs requiring referral, (2) improving the training of prehospital personnel in pediatric emergency care, (3) classifying ill and injured children according to appropriate triage criteria, (4) recognizing existing tertiary care pediatric centers as the optimal location for the treatment of critically ill and injured children, and (5) designating these centers as the appropriate transport destination for critically ill and injured children.

Regulation of granulocyte colony-stimulating factor gene expression by interleukin-17.

Interleukin-17 (IL-17) has been previously reported to induce stromal cells to produce a number of hematopoietic and proinflammatory cytokines, including granulocyte colony-stimulating factor (G-CSF). Here, we have evaluated the mechanisms responsible for the augmentation of G-CSF gene expression by IL-17, using the murine 3T3 fibroblast cell line. Treatment of 3T3 cells, but not primary bone marrow-derived macrophages or murine monocyte/macrophage cell lines, resulted in increased steady-state G-CSF mRNA levels within 2-4 h and augmented G-CSF protein production. The combination of IL-17 and LPS enhanced G-CSF expression in an additive fashion. Stability studies revealed that IL-17 stabilized G-CSF mRNA levels, with a t1/2 of 4 h, compared to a t1/2 of less than 2 h in medium or LPS-treated cells. Induction of G-CSF expression in 3T3 cells by IL-17 did not appear to require tyrosine kinase activation or de novo protein synthesis. These studies indicate that post-transcriptional mechanisms play an important role in IL-17-induced G-CSF expression in fibroblasts and suggest that IL-17 may be useful for further delineating mechanisms of G-CSF gene regulation.

Mitochondrial and MB isoenzymes of creatine kinase in cerebrospinal fluid from patients with hypoxic-ischemic brain damage.

Measurements of cerebrospinal fluid (CSF) creatine kinase (CK, EC 2.7.3.2) isoenzyme activity have been used to predict outcome in patients with acute brain injury following cardiac arrest. We identified two CK isoenzymes previously unreported in CSF from 16 patients with hypoxic-ischemic brain damage. Prior to analysis, the CK in the CSF samples was reactivated with dithiothreitol. CK isoenzymes were identified using electrophoretic and immunologic methods. Total CK activity ranged from 23 to 924 U/L (mean 452). CSF-CK-BB was the predominant isoenzyme present in all cases. In addition to CSF-CK-BB, the authors identified CSF-CK-MM in 6 cases, CSF-CK-MB in 8 cases, and CSF-mitochondrial-CK in 14 cases. The presence of CSF-CK-MM was significantly related to blood contaminating the CSF (P less than 0.02). It is proposed that CSF-CK-MB results from recombination of CK-MM and CK-BB in CSF and that mitochondrial CK is released with CK-BB into the CSF from the damaged brain tissue.

Evaluation of signal transduction pathways in chemoattractant-induced human monocyte chemotaxis.

The intracellular signaling pathways involved in human monocyte chemotaxis toward a variety of chemoattractant molecules were evaluated using selected pharmacological agents. Neither phosphatidylinositol-3-kinase (P13K) or extracellular signal-regulated kinase (ERK) activity were required for monocyte migration toward monocyte chemoattractant protein-1 (MCP-1), RANTES (Regulated on Activation, Normal T cell Expressed and Secreted), macrophage inflammatory protein-1alpha (MIP-1alpha) or formyl-Met-Leu-Phe (fMLP), since pretreatment with wortmannin or LY294002, or with PD098059, had no effect on the chemotactic response. Addition of forskolin and IBMX significantly attenuated chemotaxis to each of these chemoattractants and was reversed by co-treatment with Rp-cAMP, a competitive inhibitor of cAMP-dependent protein kinase A. Incubation with the protein kinase C (PKC) inhibitor GF109203X-HCl (GF109) did not affect monocyte migration, but pretreatment of monocytes with PMA significantly impaired the response to each of these chemotactic agents. Inhibition by PMA was reversed by co-treatment with GF109, implying that heterologous PKC activation is capable of desensitizing chemokine and fMLP-induced monocyte chemotaxis. These results help to define the signalling pathways involved in human monocyte chemotaxis and suggest pharmacological approaches to evaluating the cross-desensitization of chemoattractant-induced leukocyte migration.

Evaluation of chemokine- and phlogistin-mediated leukocyte chemotaxis using an in vivo sponge model.

We have directly compared the in vivo activity of a number of chemokines and phlogistins using a modified murine in vivo sponge model in which gelatin sponges are soaked with chemoattractant and implanted in the peritoneal cavity. Sponges soaked with murine JE/MCP-1 (monocyte chemoattractant protein-1) or zymosan promoted the chemotaxis of specific leukocyte populations in a time-dependent manner, as judged by multiparameter flow cytometry, with granulocytes predominating in zymosan-soaked sponges and granulocytes and macrophages present in JE/MCP-1-soaked sponges. Smaller numbers of B, T and dendritic cells were identified as well. Eotaxin selectively chemoattracted eosinophils in this model, while MIG induced significant T cell migration relative to other chemokines. Cell migration was inhibited by administration of methotrexate, piroxicam or dexamethasone, and JE/MCP-1-mediated trafficking was impaired by treatment with anti-JE antibody or with IL-10, suggesting a role for pro-inflammatory factors in amplifying the JE/MCP-1-induced response. This amplification phase involves the production of the chemokine KC, since anti-KC antibody significantly attenuated JE/MCP-1-induced chemotaxis. These results indicate that intraperitoneally implanted chemoattractant-soaked gelatin sponges are capable of inducing a pronounced inflammatory response characterized by the selective migration of leukocyte populations, and suggest that this model may be useful for delineating the activity of novel inhibitors of leukocyte chemotaxis.

Update in medical toxicology.

This article examines some current issues in toxicologic care. First there is a review of the scope of pediatric poisonings and some aspects of initial management. Then there is a discussion of the decision-making process required to properly use gastric decontamination in the management of poisonings. Each of the common methods available--emesis, gastric lavage, activated charcoal, catharsis, and whole bowel irrigation--is discussed. Finally, several new and old antidotes are reviewed, namely naloxone, glucagon, bicarbonate, dimercaptosuccinic acid, digoxin-specific fab fragments, and flumazenil.

Computer programs that teach the interpretation of image-based laboratory tests.

OBJECTIVE: To review the effort of the University of Washington (UW) Department of Laboratory Medicine to develop and use personal computer programs to teach the interpretation of image-based clinical laboratory tests to medical technologists and other health care workers. DATA SOURCES: Professional journals and books; Software owned by and licensed by the University of Washington. STUDY SELECTION: Not applicable. DATA EXTRACTION: Not applicable. DATA SYNTHESIS: We have been developing interactive personal computer (PC) programs for teaching image-based laboratory tests to medical technologists and other health care workers. The programs, called "Laboratory Tutors," are useful for teaching microscope-based tests and tests based on electrophoresis. Our programs include ANA-Tutor, which teaches the immunofluorescence assay for anti-nuclear antibodies; Gram Stain-Tutor, which teaches the direct Gram stain; Electrophoresis-Tutor, which teaches the interpretation of agarose gel protein electrophoretic patterns; Urinalysis-Tutor, which teaches the microscopic examination of urine sediment; in addition to other programs. The tutorials are all based on high-quality digital images that were acquired and processed using digital imaging systems. They require minimal computer literacy and have a number of advantages over standard approaches to teaching image-based laboratory tests. The computer tutorials are used in UW's medical technology and medical school curriculum, where they are used as supplements to traditional instruction. CONCLUSION: Laboratory tutors are computer programs that use high resolution digital images to teach the interpretation of image-based laboratory tests. We plan to continue to develop these programs, study their educational effectiveness, and update them periodically.

Double-stranded RNA induces molecular and inflammatory signatures that are directly relevant to COPD.

Polyinosinic:polycytidylic acid (poly I:C) is a synthetic analogue of double-stranded (ds)RNA, a molecular pattern associated with viral infections, that is used to exacerbate inflammation in lung injury models. Despite its frequent use, there are no detailed studies of the responses elicited by a single topical administration of poly I:C to the lungs of mice. Our data provides the first demonstration that the molecular responses in the airways induced by poly I:C correlate to those observed in the lungs of chronic obstructive pulmonary disease (COPD) patients. These expression data also revealed three distinct phases of response to poly I:C, consistent with the changing inflammatory cell infiltrate in the airways. Poly I:C induced increased numbers of neutrophils and natural killer cells in the airways, which were blocked by CXCR2 and CCR5 antagonists, respectively. Using gene set variation analysis on representative clinical data sets, gene sets defined by poly I:C-induced differentially expressed genes were enriched in the molecular profiles of COPD but not idiopathic pulmonary fibrosis patients. Collectively, these data represent a new approach for validating the clinical relevance of preclinical animal models and demonstrate that a dual CXCR2/CCR5 antagonist may be an effective treatment for COPD patients.

The practice of adolescent medicine in staff model HMOs.

Adolescent medicine is a medical specialty that may be represented in a prepaid group practice, but has generally not been perceived as necessary. A survey of The HMO Group shows considerable variation in staffing and the centralization of adolescent health care. An adolescent medicine practice may be rapidly created and promoted through notification of patients and professionals. Adolescent medicine providers can attend to age-appropriate educational and office setting needs of their patients, and study quality assurance of adolescent care by others. The HMO may benefit from improved age-specific care and increased physician and patient satisfaction. Problems with coverage and staffing may occur. HMOs should consider using one adolescent medicine specialist for each five to six pediatricians.