Alveolar and airway sites of nitric oxide inflammation in treated asthma

The goal of this study was to identify airway and alveolar site(s) of inflammation using exhaled nitric oxide (NO) as a marker in treated patients with asthma, including response to oral corticosteroids, and correlate these sites with expiratory airflow limitation. In 53 (24 male) patients with asthma, age 43 +/- 23 years (mean +/- SD) and all on inhaled corticosteroids, post 180 microg aerosolized albuterol, FEV(1) was 74 +/- 23% predicted and FEV(1)/FVC was 68 +/- 11%. Exhaled NO at 100 ml/second was 27 +/- 23 ppb (p < 0.001 compared with normal, 12 +/- 15 ppb). Bronchial NO maximal flux was 2.4 +/- 3.1 nl/second (p < 0.001 compared with normal, 0.85 +/- 0.55). Alveolar NO concentration was 7.0 +/- 7.4 ppb (p = 0.01 compared with the normal value, 3.2 +/- 2.0 ppb). There was no significant correlation between FEV(1) % predicted or lung elastic recoil and NO bronchial flux or alveolar concentration. However, there was a weak but significant correlation between NO bronchial flux and alveolar concentration (Spearman r = 0.50, p < 0.001). In 10 subjects with asthma on inhaled corticosteroids, 5 days of 30 mg prednisone resulted in isolated significant decreases in NO alveolar concentration, from 13 +/- 10 to 4 +/- 4 ppb (p = 0.002). Despite treatment, including inhaled corticosteroids, patients with asthma may have ongoing separate airway and alveolar sites of NO inflammation, the latter responsive to oral corticosteroids.     

Misdiagnosing cystic fibrosis in the era of gene analysis: case reports

We present a scenario of how gene analysis plays a confusing role in the diagnosis of cystic fibrosis (CF). One of the two siblings we are presenting here was initially misdiagnosed as having CF, based on the two CF gene mutations identified by gene analysis. A CF gene study on the other sibling years later, however, led to further investigation and eventually to a change of diagnosis. As interesting and important as gene analysis is in CF, one must always look at each patient in the big picture. Included in the picture, in addition to the state-of-the-art genotype, is the phenotype, or (to simplify) the back-to-basic clinical manifestations.     

The use of the hydrodynamic HBV animal model to study HBV biology and anti-viral therapy.

A simple reproducible and versatile small animal model for hepatitis B virus (HBV) infection is still unavailable. We have generated a simple transient liver-targeted transgenic mouse. Hydrodynamics tail vein injection of a head-to-tail dimer of adw HBV genome (pHBVadwHTD) into immunocompetent mice generated HBsAg and HBeAg expression in both serum and hepatocytes, followed by seroconversion. The injection of pHBVadwHTD into SCID mice generated prolonged HBsAg and HBeAg antigenemia and HBV viremia. Our results demonstrate that hydrodynamic injection of naked DNA could support the generation of HBV particles. We used this model for the assessment of anti-viral agents. Administration of our human monoclonal antibodies, HBV-Ab17(XTL) and HBV-Ab19(XTL), as well as Lamivudine (3TC) treatment suppressed HBV viremia. The model presented herein supports long and stable expression of HBV and will enable determination of various biological questions related to HBV life cycle, mutants and could enhance the development of anti-viral reagents.     

A Randomized,Placebo-Controlled Study to Investigate the Safety,Tolerability, and Pharmacokinetics of 3 Weeks of Orally Administered Gefapixant in Healthy Younger and Older Adults

Purpose

Patients with chronic cough are typically female and have a mean age of?~?60 years. However, initial pharmacokinetic (PK) characterization of the P2X3-receptor antagonist gefapixant, developed to treat refractory or unexplained chronic cough, was performed in healthy participants who were predominantly younger adult males. The objective of this Phase 1 study was to assess the safety, tolerability, and PK of gefapixant in younger (18–55 years) and older (65–80 years) males and females.

Methods

A randomized, double-blind, placebo-controlled study was conducted. Healthy adult participants were stratified into 4 cohorts by age and sex (younger males/females and older males/females) and randomized 4:1 (younger adults) or 3:1 (older adults) to receive gefapixant 300 mg twice daily (BID) for 1 week, followed by gefapixant 600 mg BID for 2 weeks or placebo. Safety, tolerability, and PK were assessed.

Results

Of 36 randomized and treated participants, 28 (100%) receiving gefapixant and 6 (75%) receiving placebo reported?≥?1 adverse event (AE). The most common treatment-related AEs in the gefapixant group were taste related. Predefined renal/urologic AEs were reported by 7 (25%) participants receiving gefapixant (all mild to moderate in severity). Gefapixant exposure was generally lower in younger males compared with younger females and older adults; however, differences may have been due to estimated glomerular filtration rate.

Conclusion

The safety profile of gefapixant 300–600 mg BID was generally consistent with previous studies. Additional characterization of gefapixant PK as a function of age and sex using population PK modeling is warranted.

     

Interleukin-10 is a growth factor for human myeloma cells by induction of an oncostatin M autocrine loop

We have a previously reported that interleukin-10 (IL-10) is a potent but IL-6-unrelated growth factor for freshly explanted myeloma cells (Lu et al, Blood 85:2521, 1995). We have also shown that exogenous IL- 10 supported the growth of XG-1 and XG-2 human myeloma cell lines (HMCL) through an IL-6-independent mechanism. (Lu et al, Blood 85:2521, 1995). Because the IL-10 receptor does not involve the gp 130 IL-6 transducer, we have attempted to elucidate the mechanisms of IL-10 action on myeloma cells. Our results indicate that the myeloma cell growth factor activity of IL-10 was abrogated by an antibody to the gp 130 IL-6 transducer, indicating that it was mediated through one of the gp 130-activating cytokines. We found that myeloma cells from XG-1 and XG-2 HMCL and from 5 of 6 patients' tumoral samples produced oncostatin M (OM) constitutively but failed to produce IL-6, IL-11 and leukemia- inhibitory factor (LIF). The autocrine OM was inactive in the absence of IL-10 due to lack of a functional OM receptor on myeloma cells. IL- 10, by inducing the receptor for LIF (LIFR), produced a functional autocrine OM loop in XG-1 and XG-2 cells and in primary myeloma cells from 2 patients. We also found that some myeloma cell lines (XG-4, XG- 6, and XG-7) an fresh myeloma cells from 3 of 6 patients produced an autocrine IL-10 and that these cells constitutively expressed LIFR. One HMCL (XG-7) produced IL-10, OM, and IL-6 an expressed LIFR. The XG-7 cells used OM and IL-6 as autocrine growth factors. We have previously shown that IL-10 could induce IL-11 receptor in myeloma cells and confer on them sensitivity to IL-11 (Lu et al, FEBS Lett 377:515, 1995). Taken together, these results show that IL-10 is a key cytokine for inducing the expression of LIFR and IL-11R and possibly another uncharacterized OM coreceptor on myeloma cells and that OM and IL-10 might be produced by myeloma cells. They also emphasize that all myeloma cell growth factors reported to data involve an activation of the gp130 IL-6 transducer.     

Autocrine insulin-like growth factor-II stimulation of tumor cell migration is a progression step in human hepatocarcinogenesis

The protumorigenic insulin-like growth factor (IGF)-II is highly expressed in a significant fraction of human hepatocellular carcinomas (HCC). However, a functional dissection that clarifies the contribution of IGF-II-binding receptors in tumor progression and a respective molecular characterization of IGF-II signaling has not been performed. Therefore, expression of IGF-II and its receptors IGF-receptor type I (IGF-IR) and insulin receptor (IR) was efficiently blocked using small interfering RNA (siRNA) in HCC cells. Despite functional IR-signaling, oncogenic IGF-II effects such as tumor cell viability, proliferation, and anti-apoptosis were solely transmitted by IGF-IR. Although IGF-II signaling was previously not described in the context of HCC cell migration, the IGF-II-dependent expression profile displayed a high percentage of genes involved in cell motility and adhesion. Indeed, IGF-II overexpression promoted HCC cell migration, especially in synergy with hepatocyte growth factor (HGF). The therapeutic relevance of IGF-II/IGF-IR signaling was tested in vitro and in a murine xenograft transplantation model using the IGF-IR inhibitor picropodophyllin (PPP). IGF-IR inhibition by small molecule treatment efficiently reduced IGF-II-dependent signaling and all protumorigenic properties of the IGF-II/IGF-IR pathway. CONCLUSION: In human HCC cells, IGF-IR but not IR is involved in oncogenic IGF-II signaling. Autocrine stimulation of IGF-II induces HCC motility by integration of paracrine signals for full malignant competence. Thus, activation of IGF-II/IGF-IR signaling is likely a progression switch selected by function that promotes tumor cell dissemination and aggressive tumor behavior.     

Human cytomegalovirus increases constitutive production of interleukin- 6 and leukemia inhibitory factor by bone marrow stromal cells

Human cytomegalovirus (CMV) infection is often associated with myelosuppression and acute inflammatory reaction in immunocompromised patients. We have previously documented that CMV exposure of bone marrow (BM) stromal cells reduces the capacity of these cells to support hematopoiesis because of a decreased production of colony- stimulating factors. This study examines the potential role of CMV on constitutive and lipopolysaccharide (LPS)-stimulated production of cytokines involved in inflammatory reaction, interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) by BM stromal cells. The release of IL- 6 was already detectable 2 hours post CMV-infection (2.5-fold increase in production) and the cumulative production of IL-6 after 5 days of infection was 23 +/- 1.2 ng/mL (ninefold increase in production). CMV was also able to induce a time-dependent production of LIF that was maximal 8 hours after CMV infection (2.5-fold increase in production). Concomitantly, there was no detectable release of granulocyte colony- stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF) by CMV-infected stromal cells. The similar IL-6 and LIF production in the presence of polymyxin B ruled out the possibility that this increase could be caused by contamination of the viral stock by endotoxin. In addition, ultraviolet-inactivated virus behaved similarly to live virus and caused the release of IL-6 and LIF. However, heat-inactivated CMV was unable to induce IL-6 and LIF secretion by BM stromal cells. The production of IL-6 and LIF was also evaluated after stimulation by LPS. After 5 days of CMV exposure, the LPS-stimulated production of IL-6 and LIF was significantly lower than uninfected controls. This LPS-induced release of cytokine production was found to be dependent of viral replication. The experiments have shown that CMV is a potent inducer of IL-6 and LIF with differential effect on constitutive and LPS- stimulated cytokine production by stromal cells; we suggest that CMV induction of IL-6 and LIF during the first hours of infection could play a role in CMV-induced inflammatory reaction. Moreover, our results show that human CMV can disturb the balanced cytokine network involved in the regulation of hematopoiesis.     

Modulation of megakaryocytopoiesis by thrombopoietin: the c-Mpl ligand

We have further characterized the biological activities, mechanism of action, and target cell populations of recombinant human and murine thrombopoietin (rhTPO and rmTPO) in in vitro human and murine model systems. Alone, hTPO or mTPO stimulated the maturation of immature murine megakaryoblasts as measured in a single cell assay. The combination of hTPO or mTPO and interleukin-6 (IL-6) resulted in a further increase in megakaryocyte differentiation in this system. Murine TPO stimulated mouse megakaryocyte progenitor development. Human megakaryocyte progenitor development was potentiated by hTPO alone and further augmented in the presence of the early-acting cytokines (IL-3) or kit ligand/stem cell factor (KL/SCF). To further define the mechanism of action of TPO, neutralization studies were performed with antisera to IL-3, granulocyte-macrophage colony-stimulating factor (GM- CSF), IL-1 beta, and IL-11. No diminution in TPO activity was observed in the presence of these antisera. Moreover, because adhesive interactions are known to modulate hematopoiesis, we studied whether hTPO might alter such interactions between human bone marrow (BM) megakaryocytes and human BM stromal fibroblasts. No changes were observed in either megakaryocyte expression of the surface molecules lymphocyte function-associated antigen-1, very late activation antigen- 4, or intercellular adhesion molecule-1 or the adhesion of megakaryocytes to stromal fibroblasts after treatment with the growth factor. Furthermore, no induction of secretion of the cytokines IL-1 alpha, IL-1 beta, GM-CSF, IL-6, granulocyte-CSF, tumor necrosis factor- alpha, transforming growth factor-beta 1, or transforming growth factor- beta 2 by primary human BM megakaryocytes was noted after treatment of the cells with hTPO. To address whether TPO affects very primitive hematopoietic progenitors, we studied the residual cells from the BMs of mice treated with high doses of 5-fluorouracil. Although no effect of mTPO alone was noted on the viability or replication of such primitive murine progenitor populations, the triple combination of IL-3 + KL/SCF + TPO stimulated growth of megakaryocyte progenitors. These results indicate that TPO is a highly lineage-specific growth factor whose primary biological effects are likely to be direct modulation of the growth and maturation of committed megakaryocyte precursors and immature megakaryoblasts.     

Reciprocal changes in parathyroid hormone and thyroid function after radioiodine treatment of hyperthyroidism

Hyperthyroidism is associated with negative calcium balance, normal to increased serum calcium concentrations, and decreased cortical bone mass. There is no agreement concerning serum PTH levels in such patients. In this study, we measured serum PTH concentrations using a newly developed sensitive 2-site immunoradiometric assay in 17 hyperthyroid patients before and after radioiodine therapy. The mean serum PTH and calcium concentrations were 28 +/- 15 (+/- SD) ng/L (normal range, 12-65 ng/L) and 2.4 +/- 0.5 mmol/L (normal range, 2.1-2.6 mmol/L) before therapy. After therapy serum PTH concentrations increased in 16 of the 17 patients. The increase in serum PTH was greater in the 9 patients who became hypothyroid rapidly (29 +/- 15 to 75 +/- 29 ng/L) compared with that in the 8 patients who became euthyroid gradually (26 +/- 16 to 45 +/- 24 ng/L). Serum PTH rose along with TSH as the patients became hypothyroid after radioiodine, and both serum PTH and TSH fell when L-T4 therapy was given. The reciprocal changes in serum PTH concentrations and thyroid function over time suggest a strong association of bone mineral metabolism and thyroid status.