Novel phase I trial design to evaluate the addition of cediranib or selumetinib to preoperative chemoradiotherapy for locally advanced rectal cancer: the DREAMtherapy trial.

BACKGROUND: The DREAMtherapy (Dual REctal Angiogenesis MEK inhibition radiotherapy) trial is a novel intertwined design whereby two tyrosine kinase inhibitors (cediranib and selumetinib) were independently evaluated with rectal chemoradiotherapy (CRT) in an efficient manner to limit the extended follow-up period often required for radiotherapy studies. PATIENTS AND METHODS: Cediranib or selumetinib was commenced 10 days before and then continued with RT (45 Gy/25#/5 wks) and capecitabine (825 mg/m2 twice a day (BID)). When three patients in the cediranib 15-mg once daily (OD) cohort were in the surveillance period, recruitment to the selumetinib cohort commenced. This alternating schedule was followed throughout. Three cediranib (15, 20 and 30 mg OD) and two selumetinib cohorts (50 and 75 mg BID) were planned. Circulating and imaging biomarkers of inflammation/angiogenesis were evaluated. RESULTS: In case of cediranib, dose-limiting diarrhoea, fatigue and skin reactions were seen in the 30-mg OD cohort, and therefore, 20 mg OD was defined as the maximum tolerated dose. Forty-one percent patients achieved a clinical or pathological complete response (7/17), and 53% (9/17) had an excellent clinical or pathological response (ECPR). Significantly lower level of pre-treatment plasma tumour necrosis factor alpha (TNFα) was found in patients who had an ECPR. In case of selumetinib, the 50-mg BID cohort was poorly tolerated (fatigue and diarrhoea); a reduced dose cohort of 75-mg OD was opened which was also poorly tolerated, and further recruitment was abandoned. Of the 12 patients treated, two attained an ECPR (17%). CONCLUSIONS: This novel intertwined trial design is an effective way to independently investigate multiple agents with radiotherapy. The combination of cediranib with CRT was well tolerated with encouraging efficacy. TNFα emerged as a potential predictive biomarker of response and warrants further evaluation.

Tumourigenic non-small-cell lung cancer mesenchymal circulating tumour cells: a clinical case study.

BACKGROUND: Over the past decade, numerous reports describe the generation and increasing utility of non-small-cell lung cancer (NSCLC) patient-derived xenografts (PDX) from tissue biopsies. While PDX have proven useful for genetic profiling and preclinical drug testing, the requirement of a tissue biopsy limits the available patient population, particularly those with advanced oligometastatic disease. Conversely, 'liquid biopsies' such as circulating tumour cells (CTCs) are minimally invasive and easier to obtain. Here, we present a clinical case study of a NSCLC patient with advanced metastatic disease, a never smoker whose primary tumour was EGFR and ALK wild-type. We demonstrate for the first time, tumorigenicity of their CTCs to generate a patient CTC-derived eXplant (CDX). PATIENTS AND METHODS: CTCs were enriched at diagnosis and again 2 months later during disease progression from 10 ml blood from a 48-year-old NSCLC patient and implanted into immunocompromised mice. Resultant tumours were morphologically, immunohistochemically, and genetically compared with the donor patient's diagnostic specimen. Mice were treated with cisplatin and pemetrexed to assess preclinical efficacy of the chemotherapy regimen given to the donor patient. RESULTS: The NSCLC CDX expressed lung lineage markers TTF1 and CK7 and was unresponsive to cisplatin and pemetrexed. Examination of blood samples matched to that used for CDX generation revealed absence of CTCs using the CellSearch EpCAM-dependent platform, whereas size-based CTC enrichment revealed abundant heterogeneous CTCs of which ∼80% were mesenchymal marker vimentin positive. Molecular analysis of the CDX, mesenchymal and epithelial CTCs revealed a common somatic mutation confirming tumour origin and showed CDX RNA and protein profiles consistent with the predominantly mesenchymal phenotype. CONCLUSIONS: This study shows that the absence of NSCLC CTCs detected by CellSearch (EpCAM(+)) does not preclude CDX generation, highlighting epithelial to mesenchymal transition and the functional importance of mesenchymal CTCs in dissemination of this disease.

A caspase-3 'death-switch' in colorectal cancer cells for induced and synchronous tumor apoptosis in vitro and in vivo facilitates the development of minimally invasive cell death biomarkers.

Novel anticancer drugs targeting key apoptosis regulators have been developed and are undergoing clinical trials. Pharmacodynamic biomarkers to define the optimum dose of drug that provokes tumor apoptosis are in demand; acquisition of longitudinal tumor biopsies is a significant challenge and minimally invasive biomarkers are required. Considering this, we have developed and validated a preclinical 'death-switch' model for the discovery of secreted biomarkers of tumour apoptosis using in vitro proteomics and in vivo evaluation of the novel imaging probe [(18)F]ML-10 for non-invasive detection of apoptosis using positron emission tomography (PET). The 'death-switch' is a constitutively active mutant caspase-3 that is robustly induced by doxycycline to drive synchronous apoptosis in human colorectal cancer cells in vitro or grown as tumor xenografts. Death-switch induction caused caspase-dependent apoptosis between 3 and 24 hours in vitro and regression of 'death-switched' xenografts occurred within 24 h correlating with the percentage of apoptotic cells in tumor and levels of an established cell death biomarker (cleaved cytokeratin-18) in the blood. We sought to define secreted biomarkers of tumor apoptosis from cultured cells using Discovery Isobaric Tag proteomics, which may provide candidates to validate in blood. Early after caspase-3 activation, levels of normally secreted proteins were decreased (e.g. Gelsolin and Midkine) and proteins including CD44 and High Mobility Group protein B1 (HMGB1) that were released into cell culture media in vitro were also identified in the bloodstream of mice bearing death-switched tumors. We also exemplify the utility of the death-switch model for the validation of apoptotic imaging probes using [(18)F]ML-10, a PET tracer currently in clinical trials. Results showed increased tracer uptake of [(18)F]ML-10 in tumours undergoing apoptosis, compared with matched tumour controls imaged in the same animal. Overall, the death-switch model represents a robust and versatile tool for the discovery and validation of apoptosis biomarkers.

Sustained tumour eradication after induced caspase-3 activation and synchronous tumour apoptosis requires an intact host immune response.

Effective anticancer treatments often result in the induction of large amounts of tumour cell death. In vivo, such dying tumour cells are a potential source of antigens for T-cell stimulation. Although apoptosis is generally considered nonimmunogenic, recent evidence suggests that some anticancer therapies that induce apoptosis can elicit antitumour immune responses. Here, a doxycycline-inducible, constitutively active caspase-3 ('death switch') was constructed in a murine tumour model to explore the impact of the host immune response to rapid, synchronous and substantial tumour cell apoptosis. In vitro, up to 80% of tumour cells underwent apoptotic cell death within 24 h and death was accompanied by the release of potential 'danger signal' molecules HMGB1 and HSP90. In vivo, death switch induction provoked rapid, pronounced tumour regression in immune-competent and immune-deficient mice, but sustained tumour eradication was observed only in immune-competent mice. Moreover, the majority of mice that were tumour free after death switch induction were protected from further tumour rechallenge. In addition, long-term remission after induction of the death switch was completely abrogated following depletion of CD8 T cells. These data suggest that sustained tumour eradication after substantial tumour apoptosis requires an antitumour host immune response that prevents tumour relapse. In many patients, cancer therapies produce encouraging initial responses that are only short lived. These results provide new insights that may have important implications for further development of strategies that result in long-term tumour clearance after initially effective anticancer treatment.

Right thing at a wrong time? Adenosine A3 receptors and cerebroprotection in stroke.

The involvement of adenosine A3 receptors in normal and pathologic functions of the brain remains to be defined. Previous studies have shown that chronic preischemic administration of the agonist [N6-(3-iodobenzyl)-5'-N-methylcarboxoamidoadenosine or IB-MECA) results in a significant protection of neurons in selectively vulnerable brain regions and in an equally significant reduction of the subsequent mortality. Acute administration of the drug, on the other hand, resulted in a pronounced worsening of these parameters. We now report that the effect of administration of IB-MECA depends on the timing of treatment with respect to the onset of the focal insult, and provide the first data supporting speculation that treatment with adenosine A3 receptor agonists may decrease the infarct size following focal brain ischemia. Treatment with IB-MECA administered 20 min prior to transient middle cerebral ischemia (MCAOt = 30 min) resulted in a significant increase of the infarct size (p < 0.01), whereas administration 20 min after ischemia resulted in statistically significant decrease of the infarct volume. Postischemic treatment results in improved neuronal preservation, decreased intensity of reactive gliosis, and pronounced reduction of microglial infiltration. The data indicate that the effects of adenosine A3 receptor stimulation depend on the differential impact of these receptors on both neuronal and non-neuronal elements of the cerebral tissue, for example, astrocytes, microglia, and vasculature.

Topographic organization and neurochemical identity of dorsal raphe neurons that project to the trigeminal somatosensory pathway in the rat.

The primary goals of this study were to: 1) examine the distribution of neurons within the dorsal raphe (DR) nucleus that project to cortical and subcortical sites along the trigeminal somatosensory pathway in rat; 2) determine the extent to which different regions within this ascending sensory system receive collateral projections from the same DR neuron; and 3) identify the putative transmitters contained within these DR projection neurons. Long-Evans hooded rats received pressure injections of various combinations of retrograde fluorescent tracers; into the whisker-related regions of the primary somatosensory cortex (barrel field cortex [BC]), ventral posterior medial thalamus (VPM), and principal nucleus of the trigeminal complex (PrV). The distribution of retrogradely labeled neurons within the DR was examined by fluorescence microscopy. The major finding was that cortically projecting neurons were located within the midline regions of the rostral portion of the DR, whereas cells projecting to subcortical trigeminal somatosensory structures were distributed bilaterally in the lateral wing regions of the DR as well as in the midline portions of the nucleus. Single neurons that send axon collaterals to multiple cortical and subcortical trigeminal somatosensory targets were observed in the dorsomedian and ventromedian regions of the DR. DR neurons that projected to cortical and subcortical sites contained serotonin but not tyrosine hydroxylase, the marker enzyme for catecholamine transmitters. Taken together, these findings provide further evidence of neurochemical specificity and functional anatomical organization within the DR efferent projection system.

Elevation of nitrotyrosine and nitrate concentrations in cystic fibrosis sputum.

Nitric oxide (NO) is increased in the exhaled air of some patients with inflammatory lung disorders, but not in others. NO may combine with superoxide to form peroxynitrite, which lowers NO gas concentrations, increases formation of nitrate, and increases nitration of tyrosine residues on proteins. We hypothesized that superoxide released from neutrophils in the lower respiratory tract of cystic fibrosis (CF) results in increased nitrate and nitrotyrosine levels in sputum. In order to test this hypothesis, exhaled NO was collected from 5 stable adult CF subjects and from 5 nonsmoking normal controls. Consistent with previous reports, exhaled NO concentrations were not increased in CF exhaled air (22.6 +/- 1.5 ppb vs. 28.6 +/- 1.5 ppb in normals, P > 0.05). Sputum was collected from 9 adult CF subjects and the same 5 normal controls and evaluated for nitrite, nitrate, and nitrotyrosine. Nitrate and nitrotyrosine levels, but not nitrite, were significantly elevated in CF. Recently, myeloperoxidase has also been implicated as a mechanism of nitrotyrosine formation. Therefore, myeloperoxidase was measured and found to be elevated in the CF sputum (64.2 +/- 35.9 vs. 0.73 +/- 0.16 U/mL, P < 0.001), and was found to correlate with concentrations of nitrotyrosine (r = 0.87, P < 0.05). However, in vitro studies with myeloperoxidase and murine lung epithelial cells did not demonstrate a reduction of NO gas with nitrotyrosine or an increase in nitrate formation. These data demonstrate that nitrate and nitrotyrosine are elevated in the sputa of CF subjects and suggest increased production of NO in the lower respiratory tract of CF patients, despite the relatively low exhaled NO levels. Pediatr Pulmonol. 2000; 30:79-85. Published 2000 Wiley-Liss, Inc.

Losartan: a review of its use, with special focus on elderly patients.

UNLABELLED: Losartan is an orally active, nonpeptide, selective angiotensin subtype 1 (AT1) receptor antagonist. It provides a more specific and complete blockade of the actions of angiotensin II than renin or ACE inhibitors. Short term (up to 12 weeks' duration) clinical trials have shown losartan to be as effective at lowering blood pressure (BP) [causes a decrease in BP < or = 26/20 mm Hg] in elderly patients with hypertension as recommended dosages of captopril, atenolol, enalapril, felodipine and nifedipine. In patients with isolated systolic hypertension (ISH) the efficacy of losartan was similar to that of atenolol. The addition of hydrochlorothiazide to losartan therapy provides greater antihypertensive efficacy, equivalent to that seen with captopril plus hydrochlorothiazide. Preliminary evidence also indicates that losartan therapy contributes to the regression of left ventricular hypertrophy associated with chronic hypertension. Exercise capacity is increased by losartan in patients with either asymptomatic or symptomatic heart failure. Results from the Losartan Heart Failure Survival or ELITE II (Evaluation of Losartan in the Elderly II) study indicate that there was no statistically significant difference between losartan and captopril in reducing overall deaths or in reducing sudden cardiac death and/or resuscitated cardiac arrest in patients with heart failure. Other than ELITE II, little conclusive long term mortality and morbidity data exist for losartan. Additional long term trials to evaluate the survival benefits of losartan in elderly patients with hypertension, renal disease or after an acute myocardial infarction are currently in progress. In elderly patients with hypertension, the incidence of treatment-related adverse events associated with once daily losartan (alone or in combination with hydrochlorothiazide) [19 to 27%] was similar to felodipine (23%) and nifedipine (21%), however, losartan tended to be better tolerated than captopril (11 vs 16%). Losartan was also better tolerated than atenolol in patients with ISH (10.4 vs 23%). In patients with heart failure the renal tolerability of losartan was similar to that of captopril, but losartan was associated with a lower withdrawal rate because of adverse events. No dosage adjustment is required in elderly or in patients with mild to moderate renal dysfunction, and the risk of first-dose hypotension is low. CONCLUSIONS: comparative data have shown losartan to be as effective as other antihypertensive agents in the treatment of elderly patients with hypertension. Treatment with losartan is therefore an option for first-line therapy in all patients with hypertension, particularly those who are not well managed with or who are intolerant of their current therapy. Morbidity and mortality data from the Losartan Heart Failure Survival (ELITE II) study show that losartan has potential in the treatment of heart failure.

Inhibition of MIP-1alpha-induced human neutrophil and monocyte chemotactic activity by reactive oxygen and nitrogen metabolites.

Peroxynitrite, formed by the reaction between nitric oxide (NO) and superoxide, has been implicated in the pathogenesis of numerous disease processes. Several studies have shown that peroxynitrite-induced protein nitration may compromise enzyme and protein function. We hypothesized that peroxynitrite may regulate cytokine function during inflammation. To test this hypothesis, the neutrophil and monocyte chemotactic responses of macrophage inflammatory protein-1alpha (MIP-1alpha) incubated with and without peroxynitrite were evaluated. Peroxynitrite attenuated neutrophil chemotactic activity (NCA) and monocyte chemotactic activity (MCA) by MIP-1alpha in a dose-dependent manner (P < .05). The inhibitory effects were not significant on NCA and MCA induced by leukotriene B4 or complement-activated serum incubated with peroxynitrite. The reducing agents deferoxamine, dithiothreitol, and exogenous L-tyrosine abrogated the NCA and MCA inhibition by peroxynitrite. Papa-NONOate, an NO donor, or a combination of xanthine and xanthine oxidase to generate superoxide, did not show an inhibitory effect on NCA and MCA induced by MIP-1alpha. In contrast, 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, elicited a concentration-dependent reduction in NCA and MCA induced by MIP-1alpha. Consistent with its capacity to reduce NCA and MCA, peroxynitrite treatment reduced MIP-1alpha binding to neutrophils and monocytes. Nitrotyrosine was detected in the MIP-1alpha incubated with peroxynitrite. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of MIP-1alpha binding to neutrophils and monocytes and a reduction in NCA and MCA. These data demonstrate that peroxynitrite modulates the inflammatory cell migration by MIP-1alpha, and they suggest that oxidants may play an important role in the regulation of MIP-1alpha-induced inflammatory cell chemotaxis.

Reactive nitrogen and oxygen species attenuate interleukin- 8-induced neutrophil chemotactic activity in vitro.

Peroxynitrite, formed by the reaction between nitric oxide and superoxide, has been shown to induce protein nitration, which compromises protein function. We hypothesized that peroxynitrite may regulate cytokine function during inflammation. To test this hypothesis, the neutrophil chemotactic activity (NCA) of interleukin-8 (IL-8) incubated with peroxynitrite was evaluated. Peroxynitrite attenuated IL-8 NCA in a dose-dependent manner (p < 0.01) but did not significantly reduce NCA induced by leukotriene B(4) or complement-activated serum. The reducing agents, dithionite, deferoxamine, and dithiothreitol, reversed and exogenous L-tyrosine abrogated the peroxynitrite-induced NCA inhibition. Papa-NONOate [N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1, 2-dialase or sodium nitroprusside, NO donors, or a combination of xanthine and xanthine oxidase to generate superoxide did not show an inhibitory effect on NCA induced by IL-8. In contrast, small amounts of SIN-1, a peroxynitrite generator, caused a concentration-dependent inhibition of NCA by IL-8. Consistent with its capacity to reduce NCA, peroxynitrite treatment reduced IL-8 binding to neutrophils. Nitrotyrosine was detected in the IL-8 incubated with peroxynitrite by enzyme-linked immunosorbent assay. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of IL-8 binding to neutrophils and a reduction in NCA and suggest that oxidants may play an important role in regulation of IL-8-induced neutrophil chemotaxis.

Effects of reactive oxygen and nitrogen metabolites on eotaxin-induced eosinophil chemotactic activity in vitro.

Peroxynitrite, an oxidant generated by the interaction between superoxide and nitric oxide (NO), has been implicated in the etiology of numerous disease processes. Several studies have shown that peroxynitrite-induced protein nitration may compromise enzyme and protein function. We hypothesized that peroxynitrite may regulate cytokine function during inflammation. To test this hypothesis, the eosinophil chemotactic responses of eotaxin incubated with and without peroxynitrite were evaluated. Peroxynitrite attenuated eotaxin-induced eosinophil chemotactic activity (ECA) in a dose-dependent manner (P < 0.05). The inhibitory effects were not significant on ECA induced by leukotriene B(4) or complement-activated serum incubated with peroxynitrite. The reducing agents deferoxamine and dithiothreitol reversed the ECA inhibition by peroxynitrite, and exogenous L-tyrosine abrogated the inhibition by peroxynitrite. PAPA-NONOate (an NO donor) or a combination of xanthine and xanthine oxidase to generate superoxide did not show an inhibitory effect on ECA induced by eotaxin. In contrast, 3-morpholinosydnonimine, a peroxynitrite generator, caused a concentration-dependent inhibition of ECA by eotaxin. Consistent with its capacity to reduce ECA, peroxynitrite treatment reduced eotaxin binding to eosinophils. Nitrotyrosine was detected in the eotaxin incubated with peroxynitrite. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of eotaxin binding to eosinophils and a reduction in ECA. These data demonstrate that peroxynitrite modulates the eosinophil migration by eotaxin, and suggest that oxidants may play an important role in regulation of eotaxin-induced eosinophil chemotaxis.

Effects of reactive oxygen and nitrogen metabolites on MCP-1-induced monocyte chemotactic activity in vitro.

Peroxynitrite, an oxidant generated by the interaction between superoxide and nitric oxide (NO), can nitrate tyrosine residues, resulting in compromised protein function. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that attracts monocytes and has a tyrosine residue critical for function. We hypothesized that peroxynitrite would alter MCP-1 activity. Peroxynitrite attenuated MCP-1-induced monocyte chemotactic activity (MCA) in a dose-dependent manner (P < 0.05) but did not attenuate leukotriene B4 or complement-activated serum MCA. The reducing agents dithionite, deferoxamine, and dithiothreitol reversed the MCA inhibition by peroxynitrite, and exogenous L-tyrosine abrogated the inhibition by peroxynitrite. PAPA-NONOate, an NO donor, or superoxide generated by xanthine and xanthine oxidase did not show an inhibitory effect on MCA induced by MCP-1. The peroxynitrite generator 3-morpholinosydnonimine caused a concentration-dependent inhibition of MCA by MCP-1. Peroxynitrite reduced MCP-1 binding to monocytes and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite, with subsequent inhibition of MCP-1 binding to monocytes, and suggest that peroxynitrite may play a role in regulation of MCP-1-induced monocyte chemotaxis.

Ofloxacin otic solution: a review of its use in the management of ear infections.

UNLABELLED: Ofloxacin is a synthetic fluoroquinolone antibacterial agent that inhibits the supercoiling activity of bacterial DNA gyrase, halting DNA replication. Ofloxacin has been formulated as a 0.3% otic solution for the treatment of ear infections. Topical administration of ofloxacin otic solution 0.3% produces very high concentrations of drug in the ear, thus broadening the spectrum of activity of ofloxacin greatly, to cover most common ear pathogens. Results of clinical trials indicate that ofloxacin otic solution 0.3% is as effective as topical neomycin/polymixin B/hydrocortisone preparations in the treatment of otitis externa (clinical cure rate >80% in adults and >95% in children for both treatments) and oral amoxicillin/clavulanic acid in the treatment of otitis media in the presence of tympanostomy tubes in children (clinical cure rates 76 and 69% for ofloxacin and amoxicillin/clavulanic acid, respectively). It is also effective in the treatment of chronic suppurative otitis media in adolescents and adults with perforated tympanic membranes (75 to 91% clinical cure rate). Because of the limited systemic absorption after topical administration, ofloxacin otic solution 0.3% is well tolerated. Adverse events were usually classed as mild to moderate, with < or =2% considered severe. The most frequent adverse events were bitter taste (5%), primarily in patients with non-intact tympanic membranes, and pruritus (2%). The incidence of adverse events with ofloxacin otic solution 0.3% was similar to that with other ototopical preparations and significantly less than that with oral amoxicillin/clavulanic acid. Unlike comparative ototopical antibacterials, ofloxacin was not ototoxic or chondrotoxic in animal studies. In addition, no ototoxicity was detected in clinical studies in humans. CONCLUSIONS: Ofloxacin otic solution 0.3% is clinically effective in the treatment of otitis externa and otitis media in patients with tympanic membrane perforations or tympanostomy tubes. The high concentrations achieved with this ototopical solution render it active against a broad spectrum of organisms. It is well tolerated, avoiding many systemic adverse events, and is not associated with ototoxicity. As the first ototopical agent approved for use in patients with non-intact tympanic membranes, ofloxacin otic solution 0.3% provides a valuable advantage over current treatment alternatives.

Effects of reactive oxygen and nitrogen metabolites on RANTES- and IL-5-induced eosinophil chemotactic activity in vitro.

Eosinophils and increased production of nitric oxide (NO) and superoxide, components of peroxynitrite, have been implicated in the pathogenesis of a number of allergic disorders including asthma. Peroxynitrite induced protein nitration may compromise enzyme and protein function. We hypothesized that peroxynitrite may modulate eosinophil migration by modulating chemotactic cytokines. To test this hypothesis, the eosinophil chemotactic responses of regulated on activation, normal T cell expressed and secreted (RANTES) and interleukin (IL)-5 incubated with and without peroxynitrite were evaluated. Peroxynitrite-attenuated RANTES and IL-5 induced eosinophil chemotactic activity (ECA) in a dose-dependent manner (P < 0.05) but did not attenuate leukotriene B4 or complement-activated serum ECA. The reducing agents deferoxamine and dithiothreitol reversed the ECA inhibition by peroxynitrite, and exogenous L-tyrosine abrogated the inhibition by peroxynitrite. PAPA-NONOate, a NO donor, or superoxide generated by lumazine or xanthine and xanthine oxidase, did not show an inhibitory effect on ECA. The peroxynitrite generator, 3-morpholinosydnonimine, caused a concentration-dependent inhibition of ECA. Peroxynitrite reduced RANTES and IL-5 binding to eosinophils and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of RANTES and IL-5 binding to eosinophils and suggest that peroxynitrite may play a role in regulation of eosinophil chemotaxis.

Origin, distribution, and morphology of galaninergic fibers in the rodent trigeminal system.

The neuropeptide galanin (Gal) is found throughout the central nervous system. Of particular interest is the fact that Gal is present within the majority of noradrenergic locus coeruleus (LC) neurons. However, very few, if any, Gal-immunoreactive fibers have been identified in many of the major efferent targets of LC, including sensory neocortex and dorsal thalamus. The goal of the present study was to examine the Gal fiber innervation of the rodent trigeminal somatosensory system and its connection to the LC. Our results show that at least two different morphological profiles of Gal-immunoreactive fibers are present within relay nuclei along the ascending trigeminal pathway. Numerous small caliber Gal-immunoreactive fibers with bouton-like swellings were noted within the barrel cortex, the ventroposterior medial (VPM) nucleus, the posterior medial (POm) nucleus, the zona incerta (ZI), the reticular nucleus (nRT) of the thalamus, and the principal (PrV) and spinal (SpV) nuclei of the trigeminal complex. Immunoreactive fibers were prevalent in, but not restricted to, layer I of the barrel cortex. Within the somatosensory thalamus, the density of Gal-immunoreactive fibers was higher in POm than in VPM. Laminae I and II of SpV and the nRT and ZI also contained dense, large-diameter Gal-immunoreactive fibers. These large-diameter Gal-immunoreactive fibers did not co-contain dopamine beta-hydroxylase (DBH). In contrast, virtually every small-caliber Gal-immunoreactive fiber colocalized with DBH. To determine whether Gal-immunoreactive fibers originated from LC, we combined immunohistochemical procedures with fluorescent tracing techniques. After retrograde tracer injections into several trigeminal relay nuclei, we observed that approximately 50% of the labeled LC neuronal population was immunoreactive for Gal. Our results suggest an extensive Gal-immunoreactive fiber innervation of the rodent trigeminal system, much of which may originate from LC neurons in the brainstem.

Insulin aspart.

Insulin aspart is a recombinant analogue of human insulin. Following subcutaneous insulin injection (0.15 to 0.2 U/kg), significantly higher serum insulin concentrations are achieved in a shorter time with insulin aspart than with human insulin. The subsequent decline in serum insulin concentrations is also more rapid with insulin aspart. In healthy individuals undergoing euglycaemic glucose clamp testing, glucose infusion rates were higher and reached maximum concentrations significantly earlier after insulin aspart than after human insulin. Interindividual variability in pharmacodynamic and pharmacokinetic parameters with insulin aspart was generally less than that with human insulin, whereas the intraindividual variability in these parameters was similar after each insulin. In patients with type 1 diabetes postprandial glucose excursions were less pronounced with insulin aspart than human insulin. Daytime glucose control was better and minimum glucose levels during the night were not as low with insulin aspart as with human insulin. In diabetic patients treated with insulin aspart there was generally a lower frequency of hypoglycaemic events than in patients treated with human insulin.

Expression of CD1D mRNA transcripts in human choriocarcinoma cell lines and placentally derived trophoblast cells.

Human placental trophoblast is critically involved in mediating maternal tolerance of the fetal semiallograft. Genes encoding highly polymorphic major histocompatibility complex (MHC) class I and class II antigens that could provoke maternal immune rejection responses are silenced in trophoblast. However, several MHC class I or class I-related products exhibiting reduced or negligible polymorphism are expressed and assumed to be functionally involved in maintaining pregnancy. The CD1 gene family encodes non-polymorphic MHC class I-like products that have the unusual ability to present non-peptide antigens to T cells. One member, CD1D, is expressed in certain epithelial cells and interacts with a specific T-cell subset that may promote the development of Th2-mediated responses believed to be associated with pregnancy. In this study we examined the expression of CD1D in human trophoblast cell lines and placentally derived trophoblast cells by reverse transcriptase-polymerase chain reaction using CD1D-specific oligonucleotide primers. We have found that CD1D mRNA transcripts are expressed in trophoblast cells and cell lines. We have also identified a novel alternatively spliced CD1D mRNA transcript lacking exon 4. Exon 4-intact and exon 4-deficient CD1D transcripts appear to be differentially expressed in different trophoblast and non-trophoblast cell populations. Our studies suggest that at least one member of the CD1 family is transcribed in human trophoblast.

Labour-associated changes in the regulation of production of immunomodulators in human amnion by glucocorticoids, bacterial lipopolysaccharide and pro-inflammatory cytokines.

Parturition is associated with changes in the production of inflammatory mediators by gestational tissues. An explant system was established to study the change in response of human amnion to various regulating factors during labour. Disks of tissue (6 mm) were excised from amnion membranes obtained either at term by Caesarian section before labour (n = 5-6) or after spontaneous vaginal delivery (n = 3-7). After 24 h equilibration in media, the tissues were treated with interleukin 1 beta (10 ng ml-1), tumour necrosis factor alpha (100 ng ml-1), lipopolysaccharide (5 micrograms ml-1) and dexamethasone (1 mumol l-1) or an appropriate vehicle control for 24 h (n = 3 wells per treatment). Media were harvested and interleukin 10, interleukin 6 and prostaglandin E2 concentrations were determined by immunoassay. In tissues taken both before and after the onset of labour, basal interleukin 10 production by amnion explants was near to the limit of detection. Basal production rates of PGE2 by amnion explants were significantly higher (P < 0.0012; Mann-Whitney U test) in tissues taken during labour than in tissues taken before the onset of labour, while interleukin 6 production was not significantly altered by labour. Production rates of interleukin 6 and prostaglandin E2 were significantly increased by interleukin 1 beta, tumour necrosis factor alpha and lipopolysaccharide in explants from tissues taken during and before labour, while the responsiveness of interleukin 10 production to these treatments was inconsistent. Dexamethasone had no effect on interleukin 6 production by amnion explants, but significantly inhibited prostaglandin E2 production, although this inhibition was approximately 30% lower in tissues obtained after the onset of labour. These results support the presence of inflammatory positive feedback cycles, coincident with a deficiency of an anti-inflammatory factor within gestational tissue, which may be involved in the progression or maintenance of labour.

Superoxide released from neutrophils causes a reduction in nitric oxide gas.

Exhaled nitric oxide (NO) is increased in some inflammatory airway disorders but not in others such as cystic fibrosis and acute respiratory distress syndrome. NO can combine with superoxide (O-2) to form peroxynitrite, which can decompose into nitrate. Activated polymorphonuclear neutrophils (PMNs) releasing O-2 could account for a reduction in exhaled NO in disorders such as cystic fibrosis. To test this hypothesis in vitro, we stimulated confluent cultures of LA-4 cells, a murine lung epithelial cell line, to produce NO. Subsequently, human PMNs stimulated to produce O-2 were added to the LA-4 cells. A gradual increase in NO in the headspace above the cultures was observed and was markedly reduced by the addition of PMNs. An increase in nitrate in the culture supernatant fluids was measured, but no increase in nitrite was detected. Superoxide dismutase attenuated the PMN effect, and xanthine/xanthine oxidase reproduced the effect. No changes in epithelial cell inducible NO synthase protein or mRNA were observed. These data demonstrate that O-2 released from PMNs can decrease NO by conversion to nitrate and suggest a potential mechanism for modulation of NO levels in vivo.

Labor-associated changes in interleukin-10 production and its regulation by immunomodulators in human choriodecidua.

Parturition is associated with increased production of proinflammatory mediators by gestational tissues. Interleukin-10 (IL-10) is an antiinflammatory cytokine produced by human chorion, decidual, and trophoblast tissues. To study the effects of immunomodulators on IL-10, IL-6, and PGE2 production by human choriodecidua before and after labor, an organ explant system was established. Tissue disks (6 mm) were excised from choriodecidual membranes obtained at term by cesarean section before labor (n=6-7) or after spontaneous vaginal delivery (n=7-8). After 24-h equilibration in medium, the tissues were treated with IL-1beta (10 ng/mL), tumor necrosis factor-alpha (100 ng/mL), lipopolysaccharide (5 microg/mL), dexamethasone (1 micromol/ L), or an appropriate vehicle control (n=3 wells/treatment) for 24 h. Media were harvested, and IL-10, IL-6, and PGE2 concentrations were determined by immunoassay. Basal choriodecidual production rates of IL-10 were significantly decreased with labor (P < 0.001), whereas PGE2 and IL-6 production rates increased. The production of all three substances was increased by IL-1beta, tumor necrosis factor-alpha, and lipopolysaccharide, but inhibited by dexamethasone. In contrast to PGE2 and IL-6, there was significantly increased responsiveness of IL-10 production to inflammatory stimuli after labor, but decreased responsiveness to the inhibitory effects of dexamethasone. These data indicate that IL-10 could play a role in modulating or promoting resolution of the inflammatory processes associated with labor at term and with intrauterine infection-associated preterm labor.