An experimental aerosol air-agar interface mouse lymphoma assay methodology.

This work investigates a completely novel and experimental concept of exposing L5178Y cells at the air-agar-interface to mainstream cigarette smoke aerosol (Kentucky reference 3R4F). This study highlights the associated challenges of combining a suspension cell line alongside an in vitro aerosol exposure system. To achieve a monolayer, cells were 'seeded' in a concentrated cell super-mix suspension onto an RPMI/agar-matrix -base. The resulting cell suspension media was adsorbed into the agar base leaving the L5178Y cells lightly suspended on the agar surface, approximating a monolayer. Cells were deemed supportable on the agar-matrix, viable and recoverable. Using Vitrocell VC 10 exposure system and the Ames 4 exposure module, L5178Y cells were successfully exposed to a dynamic cigarette smoke aerosol, recovered and assessed for mutant frequencies, using standard assay procedures. Method development included assessment of flowing air conditions, plating efficiency and recovery of L5178Y cells from the agar-matrix surface. Positive controls MMS and B[a]P were successfully incorporated into the agar-matrix and metabolic activation was achieved by S-9 incorporation into the same agar-base-matrix. B[a]P demonstrated metabolic activation and positive response, suggesting a clear cellular interaction with the agar-matrix. Whole smoke exposed cells in the presence of metabolic activation showed a clear dose response and increasing mutant frequencies, well in excess of the controls (air and incubator) and the global evaluation factor following a 2 or 3 day expression period. This experimental concept demonstrates that L5178Y cells can be exposed to cigarette smoke aerosol, using a completely novel and a previously untested approach. Although this work successfully demonstrates the approach is viable and cells can be plated and maintained on an agar-matrix, more optimisation and robustness assessment is required before it can be considered fully adapted and used alongside other whole aerosol methodologies for the assessment of cigarette smoke and other inhaled aerosols.

A supervised machine learning approach to characterize spinal network function.

Spontaneous activity is a common feature of immature neuronal networks throughout the central nervous system and plays an important role in network development and consolidation. In postnatal rodents, spontaneous activity in the spinal cord exhibits complex, stochastic patterns that have historically proven challenging to characterize. We developed a software tool for quickly and automatically characterizing and classifying episodes of spontaneous activity generated from developing spinal networks. We recorded spontaneous activity from in vitro lumbar ventral roots of 16 neonatal [postnatal day (P)0-P3] mice. Recordings were DC coupled and detrended, and episodes were separated for analysis. Amplitude-, duration-, and frequency-related features were extracted from each episode and organized into five classes. Paired classes and features were used to train and test supervised machine learning algorithms. Multilayer perceptrons were used to classify episodes as rhythmic or multiburst. We increased network excitability with potassium chloride and tested the utility of the tool to detect changes in features and episode class. We also demonstrate usability by having a novel experimenter use the program to classify episodes collected at a later time point (P5). Supervised machine learning-based classification of episodes accounted for changes that traditional approaches cannot detect. Our tool, named SpontaneousClassification, advances the detail in which we can study not only developing spinal networks, but also spontaneous networks in other areas of the nervous system. NEW & NOTEWORTHY Spontaneous activity is important for nervous system network development and consolidation. Our software uses machine learning to automatically and quickly characterize and classify episodes of spontaneous activity in the spinal cord of newborn mice. It detected changes in network activity following KCl-enhanced excitation. Using our software to classify spontaneous activity throughout development, in pathological models, or with neuromodulation, may offer insight into the development and organization of spinal circuits.

Extreme testing of undiluted e-cigarette aerosol in vitro using an Ames air-agar-interface technique.

There is a growing consensus that e-cigarettes hold the potential for reducing the harm associated with cigarette smoking. Recently published studies have reported in vitro testing of e-cigarettes, demonstrating reduced toxicological and biological effects. Few studies however have reported the use of e-cigarettes under extreme testing conditions. To assess the full mutagenic potential of a commercially available electronic-cigarette (Vype ePen), this study investigated the delivery of aerosol under extreme conditions, using a scaled-down 35 mm plate Ames bacterial reverse mutagenicity assay. S. typhimurium strains TA98, TA100, TA97, TA104 and E. coli WP2 uvrA pKM101 with or without metabolic activation (S9), were employed. Using a modified Vitrocell VC 10 exposure system 0, 180, 360, 540, 720 or 900 puffs of undiluted e-cigarette aerosol was generated and delivered to bacterial cultures aligned to reported human consumption data. The results demonstrate that no mutagenic activity was observed in any strain under any test condition even when exposed to 900 puffs of undiluted e-cigarette aerosols +/- S9. Positive control responses were observed in all strains +/- S9. Nicotine assessments demonstrated an increased and consistent aerosol delivery, with calculated maximum doses of ∼1 mg/mL delivery of nicotine. These data demonstrate the validity of this unique testing approach and adds further information to the growing weight of evidence that e-cigarettes offer substantially reduced exposure when compared to conventional cigarette smoke. For future in vitro assessments of next generation tobacco and nicotine products, the generation, delivery and testing of undiluted aerosols can now be considered.

An inter-machine comparison of tobacco smoke particle deposition in vitro from six independent smoke exposure systems.

There are several whole smoke exposure systems used to assess the biological and toxicological impact of tobacco smoke in vitro. One such system is the Vitrocell® VC 10 Smoking Robot and exposure module. Using quartz crystal microbalances (QCMs) installed into the module, we were able to assess tobacco smoke particle deposition in real-time. We compared regional deposition across the module positions and doses delivered by six VC 10s in four independent laboratories: two in the UK, one in Germany and one in China. Gauge R&r analysis was applied to the total data package from the six VC 10s. As a percentage of the total, reproducibility (between all six VC 10s) and repeatability (error within an individual VC 10) accounted for 0.3% and 7.4% respectively. Thus Gauge R&r was 7.7%, less than 10% overall and considered statistically fit for purpose. The dose-responses obtained from the six machines across the four different locations demonstrated excellent agreement. There were little to no positional differences across the module at all airflows as determined by ANOVA (except for one machine and at three airflows only). These results support the on-going characterisation of the VC 10 exposure system and suitability for tobacco smoke exposure in vitro.

Immunoreactivity profile of peripheral blood mononuclear cells from patients with ragweed-induced allergic rhinitis.

BACKGROUND: Seasonal rhinitis is manifested by a series of nasal symptoms in response to exposure to seasonal allergens including ragweed pollen. Understanding its immunological mechanisms may help to better manage the disease. OBJECTIVE: We sought to determine comprehensively ragweed-induced cytokine and chemokine production by peripheral blood mononuclear cells from normal individuals and patients with seasonal rhinitis sensitized to ragweed pollen, and to assess its regulation by exogenous IL-10. METHODS: Cells were cultured in the presence or absence of a purified ragweed pollen extract with or without exogenous IL-10. Cytokines and chemokines were measured in the supernatant. Gene expression was evaluated using real-time quantitative reverse transcription PCR. RESULTS: Ragweed stimulation significantly increased the production of the Th2-associated cytokines IL-5, IL-9 and IL-13, the chemokines CCL17 and CCL22 and the regulatory cytokine IL-10 in allergic patients, whereas transforming growth factor-beta (TGF-beta) production was increased only in normal individuals. No difference was detected between groups in the production of the Th1 cytokine IFN-gamma or the Th1-affiliated chemokines CXCL10 and CXCL11. Exogenous IL-10 significantly suppressed spontaneous and induced production of both Th1- and Th2-associated cytokines and chemokines. CONCLUSION: Our work demonstrated that locally manifested allergic rhinitis is underlined by a systemic Th2 immune response specific to allergens. The molecular pathogenesis of allergic rhinitis may be linked to a compromised allergen-specific immune regulation, e.g., reduced spontaneous and allergen-induced TGF-beta production in patients compared with healthy controls. Our data also show that IL-10 inhibits both the effector and directional mechanisms of allergen-specific immune response, further supporting its potential therapeutic benefit in preventing and treating allergic diseases.

Mechanical stretch regulates TRPC expression and calcium entry in human myometrial smooth muscle cells.

Stretch is known to stimulate myometrial hyperplasia and hypertrophy in early pregnancy and uterine contraction at term. We propose that transduction of the stretch signal involves alteration of intracellular calcium signalling, including changes in transient receptor potential canonical (TRPC) isoform expression. The aim of the present study was to investigate the effect of prolonged mechanical (tonic) stretch in vitro on human myometrial smooth muscle cell calcium signalling and TRPC expression. Cells were cultured from myometrial biopsies, obtained from women undergoing elective Caesarean section at term, grown on Flexiplates and subjected to 25% tonic mechanical stretch for 1, 4 and 14 h. Time-matched control cells were not stretched. Mechanical stretch (14 h) increased basal calcium entry and cyclopiazonic acid (CPA)-induced calcium/Mn(2+) entry (P < 0.05) in Fura-2 loaded cells. The calcium selectivity of CPA-thapsigarin induced inward currents, measured by patch clamp electrophysiology, was also increased in stretched cells compared with control cells (P < 0.05). Real time PCR and Western blot data demonstrated that TRPC3 and TRPC4 mRNA and TRPC3 protein expression were increased by stretch (P < 0.05), respectively. These data support the hypothesis that uterine stretch modulates uterine growth and contractility in pregnancy via alterations in calcium signalling.

Physiological induction of transient receptor potential canonical proteins, calcium entry channels, in human myometrium: influence of pregnancy, labor, and interleukin-1 beta.

This study investigated gestational regulation of transient receptor potential canonical (TrpC) proteins, putative calcium entry channels in human myometrium, and the potential modulation of TrpC expression by IL-1 beta, a cytokine implicated in labor. Total RNA and proteins were isolated from myometrial biopsies obtained from NP women, pregnant women at term not in labor (TNL), or term active labor (TAL) and from primary cultured human myometrial smooth muscle cells incubated with IL-1 beta or IL-1 beta with or without nimesulide. Semiquantitative RT-PCR demonstrated significant up-regulation of TrpC1 in TAL and TNL (P < or = 0.01) and TrpC6 (P < or = 0.01) and TrpC7 (P < or = 0.05) in TAL samples. TrpC3 and TrpC4 mRNA expression was unaffected. Western blot demonstrated significant up-regulation of TrpC1 in TAL and TNL (P < or = 0.05) and TrpC3 (P < or = 0.01), TrpC4 (P < or = 0.05), and TrpC6 (P < or = 0.01) in TAL samples. IL-1 beta did not alter TrpC1, 3, 4, 6, or 7 mRNA expression; but IL-1 beta exclusively up-regulated TrpC3 protein expression (P < or = 0.05). TrpC3 up-regulation was unaffected by cyclooxygenase blockade. These data demonstrate physiological regulation of TrpC mRNA and protein and suggest an important role for TrpC proteins in human myometrium during labor.

Molecular identification and localization of Trp homologues, putative calcium channels, in pregnant human uterus.

The mechanisms underlying the switch from uterine quiescence to contractile activity in labour are not clearly understood. Increasing evidence suggests that pathways of myometrial calcium homeostasis, including store-operated calcium entry (SOCE), may play an important role. The molecular basis of the membrane-associated calcium channels contributing to SOCE in pregnant human myometrium is not known, but they are likely to be hetero- or homo-oligomeric assemblies of transient receptor potential channel (TrpC) proteins, encoded by the mammalian homologues of Drosophila Trp genes. This study has therefore determined Trp gene expression and also TrpC protein expression and localization in term pregnant human myometrial tissue and primary cultured human myometrial smooth muscle (HMSM) cells. RT-PCR amplified fragments of Trp1, Trp3, Trp4, Trp6 and Trp7. PCR products were 100% homologous to published human sequences. Western blot analysis detected TrpC1, TrpC3, TrpC4 and TrpC6 proteins, which were of expected size. Immunolocalization revealed TrpC1, TrpC3, TrpC4 and TrpC6 protein expression in myometrial tissue and HMSM cells. TrpC protein immunostaining in HMSM cells was distributed in a distinct reticular fashion. TrpC proteins may be candidate proteins forming SOCE channels in term pregnant human myometrium.

Sequence and functional characterisation of the marmoset monkey (Callithrix jacchus) prolactin receptor: comparative homology with the human long-form prolactin receptor.

This study demonstrates the cloning and in-vitro characterisation of the marmoset monkey (Callithrix jacchus) prolactin receptor cDNA. The marmoset prolactin receptor cDNA was generated by reverse transcription-polymerase chain reaction using adrenal RNA and primers designed from prolactin receptor conserved regions. Sequence analysis predicts a mature protein of 598 amino acids exclusive of the 24 amino acid signal peptide. The marmoset prolactin receptor cDNA shares 93 and 61% base pair, and 89 and 61% amino acid sequence homologies with the long form human and rat prolactin receptor cDNA, respectively. The marmoset prolactin receptor cDNA sequence retains all the receptor sequences that have been shown previously to be essential for ligand binding, structural integrity and signal transduction. Transfection of human 293 fibroblast cells with the marmoset prolactin receptor cDNA (three independent experiments) confirmed the expression of a receptor that has high binding affinity to human growth hormone (K(a)=3.6+/-0.07 nM(-1) and B(max)=7.55+/-2.06x10(-11) M) and human prolactin (K(a)=3.1+/-0.12 nM(-1) and B(max)=2.87+/-0.66x10(-11) M). Functionality of the receptor was assessed by co-transfection of 293 fibroblast cells with marmoset prolactin receptor cDNA and the Jak2 cDNA, or marmoset prolactin receptor and a Stat5 responsive element linked to the luciferase coding sequence. Incubation of the cells with 18 nM ovine prolactin resulted in rapid phosphorylation of Jak2 as ascertained by Western blotting. In addition, the marmoset prolactin receptor cDNA led to 9.06+/-0.47-fold induction of luciferase gene activity. This was comparable with the induction observed following transfection with the human prolactin receptor cDNA (8.55+/-0. 5-fold). In-vivo prolactin receptor expression in the marmoset monkey was assessed by ribonuclease protection assay and detected in a number of tissues including female reproductive organs. These data confirm the cloning and functionality of the marmoset prolactin receptor cDNA. The marmoset prolactin receptor shares a high sequence homology with the long-form human prolactin receptor, and both receptors bind hormones with comparable affinity and confer a similar intracellular response. The marmoset monkey may provide a useful tool to investigate the role of prolactin in primate reproduction.

Localization and signaling of the prolactin receptor in the uterus of the common marmoset monkey.

This study investigated the expression and signaling pathway of PRL and its receptor in the non-pregnant uterus of the common marmoset monkey. Immunohistochemistry localized PRL expression to the stromal compartment of the endometrium. Expression was minimal during the proliferative phase and was up-regulated during the mid to late secretory phase of the ovulatory cycle. In situ hybridization and immunohistochemistry localized expression of the PRL receptor to the glandular epithelium of the endometrium. Similar to that of PRL, PRL receptor expression was minimal during the proliferative phase and was dramatically up-regulated during the secretory phase. The temporal pattern of PRL receptor gene expression in the marmoset uterus across the cycle was further confirmed by ribonuclease protection assay. The roles of Janus kinase-2 (JAK2) and signal transducer and activator of transcription-1 (STAT1) in the intracellular signaling pathway of PRL were also assessed in the mid to late secretory phase. JAK2/STAT1 proteins were localized in the glandular epithelial compartment, and both proteins were temporally phosphorylated in response to PRL. Finally, the pattern of expression of the interferon regulatory factor-1 (IRF-1) gene and the effect of PRL on transcription of IRF-1 were investigated during the mid to late secretory phase. IRF-1 expression in the marmoset uterus was encoded by a protein of 48 kDa and was localized to the glandular epithelial compartment, as was observed for the PRL receptor and JAK2/STAT1 proteins. Moreover, incubation of mid to late secretory uterine tissue with PRL for 1 and 3 h resulted in 0.4 +/- 0.2- and 2.4 +/-0.5-fold (P < 0.05) inductions of the IRF-1 gene, respectively. These studies confirm the expression of both PRL and its receptor in the uterus of the marmoset monkey. Expression of both genes is up-regulated during the mid to late secretory phase of the ovulatory cycle. PRL function in the marmoset uterus is linked to the JAK/STAT signaling pathway, leading to the regulation of expression of PRL-responsive genes such as IRF-1. The site of expression of PRL, PRL receptors, and IRF-1 in the marmoset uterus suggest that PRL may influence glandular epithelial function and direct gene transcription in these cells in a paracrine fashion.

Comparative analysis of Native admissions and registrations to northwestern Ontario treatment facilities: hospital and community sectors.

OBJECTIVE: To study Native and non-Native admissions to acute psychiatric care in the northwestern region of Ontario in 1992. METHOD: To replicate a 1986 to 1987 study comparing Native to non-Native admissions to acute psychiatric care in the northwestern region of Ontario in 1992 and examine Native registrations to community mental health agencies in the first 6 months of 1993. RESULTS: The comparative analysis of hospital admissions revealed that: Natives are still being admitted at 33% more than the rate expected on the basis of population; depression appears to be underdiagnosed for Natives; they continue to be admitted mainly for reasons other than major psychiatric conditions; substance abuse and forensic history are commonly involved; they stay in hospital for twice as long as their non-Native control; they more often come from rural settings; and they are less likely to be followed by the outpatient service and more likely to be followed by the criminal justice system. The examination of registrations to community mental health agencies revealed that: the same overrepresentation of Natives; mood- and thought-presenting problems of Natives in this sector were identical to non-Natives; and their length of stay was similar. The psychiatric hospital appears to be providing acute care treatment, not for the serious psychiatric illnesses for which it is mandated, but for atypical admissions that result from economic, social and cultural dislocation. There may be underdiagnosis of atypical depression in the Native hospitalized population. When asked what they are being treated for the diagnostic profile of Natives and non-Natives is identical on mood and thought dimensions. CONCLUSION: No appreciable change has occurred over the 5 years in the way hospital psychiatric services are used by Natives. Cultural stereotypes may be influencing the diagnosis of Natives in inappropriate ways. Enhancing Native control of treatment programs and community development may provide a partial solution. Properly mandated and accountable community agencies (both generic- and culture-specific) will help reduce unnecessary hospitalization.

Record linkage in a regional mental health planning study: accuracy of unique identifiers, reliability of sociodemographics, and estimating identification error.

Assembling information about individuals over time allows health managers and researchers to describe the progression of diseases, the care history of individuals and the sequences of care episodes that potentially result in improving individuals' health status. However, current mental health statistics generally focus on sets of events rather than groups of individuals making it impossible to distinguish between two different persons being admitted and the same person being admitted twice. Accurate figures on treatment prevalence cannot be generated and multiservice users across time or across agencies will inflate the statistics used to plan needed services. The capacity to link consistently defined bits of information together is critical to developing a reliable information system. This article examines the adequacy of using unique identifier codes to accomplish linkage by focusing on one example of record linkage that incorporates mental health information from both community and institutional sectors in one region of Ontario, Canada. Findings indicate that unique "cradle to grave" identifiers do not guarantee accuracy if manual transcription is involved.

Cross-validating factors associated with discharges against medical advice.

Between six percent and 35% of psychiatric patients discharge themselves from hospital against medical advice (AMA). The discharges may prevent patients from deriving the full benefit of hospitalization and may result in rapid rehospitalization. We examined sociodemographic and clinical characteristics of 195 irregular discharges from a 237 bed psychiatric hospital over a five year period and found that AMA discharges increased over the study period to a peak of 25% in 1986. There was a strong negative correlation between AMA discharge rates and the willingness of physicians to commit patients involuntarily. Multiple discriminant analysis revealed a set of nine variables that accurately classified 78% of cases into regular or irregular discharge categories. Further analysis revealed that there are two distinct subgroups of patients who discharge themselves AMA: those who repeatedly left the hospital AMA in a regular "revolving back door" pattern and those who left AMA only once. The repeat group exceeded the one-time group in terms of prior admissions, appearances before review boards, and percentage of Natives. The repeat group also spent twice as long in hospital, and 27% were readmitted within one-week of the index AMA discharge. Less than three percent of the one-time AMA group was readmitted within a week. These results were cross-validated on a new sample of irregular discharges and matched controls.