N-terminal or signal peptide sequence engineering prevents truncation of human monoclonal antibody light chains.

Monoclonal antibodies (mAbs) contain short N-terminal signal peptides on each individual polypeptide that comprises the mature antibody, targeting them for export from the cell in which they are produced. The signal peptide is cleaved from each heavy chain (Hc) and light chain (Lc) polypeptide after translocation to the ER and prior to secretion. This process is generally highly efficient, producing a high proportion of correctly cleaved Hc and Lc polypeptides. However, mis-cleavage of the signal peptide can occur, resulting in truncation or elongation at the N-terminus of the Hc or Lc. This is undesirable for antibody manufacturing as it can impact efficacy and can result in product heterogeneity. Here, we describe a truncated variant of the Lc that was detected during a routine developability assessment of the recombinant human IgG1 MEDI8490 in Chinese hamster ovary cells. We found that the truncation of the Lc was caused due to the use of the murine Hc signal peptide together with a lambda Lc containing an SYE amino acid motif at the N-terminus. This truncation was not caused by mis-processing of the mRNA encoding the Lc and was not dependent on expression platform (transient or stable), the scale of the fed-batch culture or clonal lineage. We further show that using alternative signal peptides or engineering the Lc SYE N-terminal motif prevented the truncation and that this strategy will improve Lc homogeneity of other SYE lambda Lc-containing mAbs. Biotechnol. Bioeng. 2017;114: 1970-1977. © 2017 Wiley Periodicals, Inc.

An observational study investigating the impact of simulated patients in teaching communication skills in preclinical dietetic students.

BACKGROUND: Simulated patients (SPs) are often used in dietetics for the teaching and assessment of communication skills. The present study aimed to determine the impact of a SP encounter on communication skills in undergraduate preclinical dietetic students in the context of the resources required for delivering this educational strategy. METHODS: This observational study collected assessment data from four cohorts of third-year dietetic students to examine the effect of participation in SP-embedded Objective Structured Clinical Exams. Students completed two SP interviews, 2 weeks apart, and communication skills were measured on both occasions. A subgroup of students received a video of their SP encounter. Differences between the two SP interview scores were compared to assess the impact of the SP encounter on communication skills. The required staff and resources were described. RESULTS: Data were collected involving 215 students. Out of 30 marks, there was a modest mean (SD) improvement in communication skills from the first to the second SP interview of 2.5 (4.2) (P < 0.01). There was an association between student ability and improvement in communication skills, with failing students demonstrating the greatest improvement between SP encounters. There were no observed benefits for the subset of students who received videos. CONCLUSIONS: Providing repeat SP interview opportunities results in only modest improvement in communication skills for most students. The use of SPs needs to be considered in context of the substantial costs and resources involved and tailored to student ability.

Pain and dementia: a diagnostic challenge.

PURPOSE: The aim was to present current knowledge about pain assessment in people with dementia and to discuss special challenges and possible solutions. METHODS: A literature search in MEDLINE® was performed. RESULTS: Due to the changing demographics of an aging population, an increasing number of people with dementia is expected. Many of these people will simultaneously suffer pain. Under-detection and under-treatment of pain in persons suffering from dementia is often described. As dementia progresses, the ability of the sufferer to verbally communicate his/her pain is often compromised, complicating the task of recognizing and treating pain. To improve pain recognition in dementia, many pain assessment tools have been developed. However, psychometric properties have to date been insufficiently examined. IMPLICATIONS: Self-report ratings should be performed as long as justifiable. Behavioural pain assessment tools should be used in advanced dementia despite their current imperfections: in particular, the PAINAD for daily use and the PACSLAC at longer intervals. All available additional information about pain should be considered.

Endothelin: a novel peptide in the posterior pituitary system.

Endothelin (ET), originally characterized as a 21-residue vasoconstrictor peptide from endothelial cells, is present in the porcine spinal cord and may act as a neuropeptide. Endothelin-like immunoreactivity has now been demonstrated by immunohistochemistry in the paraventricular and supraoptic nuclear neurons and their terminals in the posterior pituitary of the pig and the rat. The presence of ET in the porcine hypothalamus was confirmed by reversed-phase high-pressure liquid chromatography and radioimmunoassay. Moreover, in situ hybridization demonstrated ET messenger RNA in porcine paraventricular nuclear neurons. Endothelin-like immunoreactive products in the posterior pituitary of the rat were depleted by water deprivation, suggesting a release of ET under physiological conditions. These findings indicate that ET is synthesized in the posterior pituitary system and may be involved in neurosecretory functions.

Computer assisted age progression.

A computer assisted method for altering the perceived age of a human face is presented. Our technique is based on calculating a trajectory or axis within a multi-dimensional space that captures the changes in large scale facial structure, shading and complexion associated with aging. Fine facial details associated with increasing age, such as wrinkles, are added to the aged face using a variation on a standard image processing technique called high boost filtering. The method is successfully applied to two-dimensional photographic images exhibiting uncontrolled variations in pose and illumination. Unlike our previous work on automated age progression, here the objective is to allow a certain degree of manual control over the process by the adjustment of three key progression-control-parameters. In the future this work may form the basis for a software tool to be used by forensic artists.

Geriatric syndromes as predictors of adverse outcomes of hospitalization.

AIMS: The aim of this study was to determine the association between the common geriatric syndromes and predefined adverse outcomes of hospitalization and to identify the most important independent predictors of adverse outcomes using information gained within 24 h of admission in older general medical patients. METHODS: A prospective longitudinal cohort study of patients aged > or =75 years admitted to the rapid assessment medical unit in a teaching hospital was carried out. The role of geriatric syndromes in predicting outcomes was examined in univariate and multivariate models. The outcome measures were (i) length of hospital stay (LOS) of 28 days or more, (ii) institutionalization or change in residential care status to a more dependent category at discharge or during 3 months post-discharge, (iii) unplanned readmissions during 3 months and (iv) mortality in hospital or 3 months post-discharge. RESULTS: The presence of geriatric syndromes was significantly associated with increased LOS and institutionalization or change in residential care status to a more dependent category. The factors most predictive of these outcomes were impaired pre-admission functional status in activities of daily living, recurrent falls, urinary incontinence and supported living arrangements. The geriatric syndromes appeared less important in predicting unplanned readmission and death. CONCLUSION: The presence of geriatric syndromes in older general medical patients is an important determinant of adverse outcomes of hospitalization, particularly of LOS and admission to residential care. The predictors most useful for screening patients for these outcomes, within 24 h of admission, appear to be the presence of certain pre-existing geriatric syndromes before admission.

Relationship between self-reported cognitive difficulties, objective neuropsychological test performance and psychological distress in chronic pain.

BACKGROUND: Persons with chronic pain often report problems with cognitive abilities, such as memory or attention. There is limited understanding of whether objective performance is consistent with subjective reports, and how psychological factors contribute. We aimed to investigate these relationships in a group of patients expressing cognitive concerns, and evaluate the utility of self-report tools for pain management settings. METHOD: Participants with chronic pain (n = 41) completed standardized neuropsychological tests, and self-report measures of cognitive functioning, pain, mood and sleep, as part of a broader study investigating cognitive performance in pain. RESULTS: Average neuropsychological test performance was subtly below normative means (within one standard deviation). Twenty-five percent of the sample scored substantially below age-adjusted norms on one or more objective tests. There were moderate-to-large associations between objective performance (e.g. Trail-Making B) and subjective cognitive complaints (e.g. Everyday Memory Questionnaire - Revised), controlling for age and education level. This was moderated by anxiety, such that subjective-objective relationships were particularly strong in those with higher anxiety. Poorer test performance was associated with higher pain intensity and catastrophizing. Subjective-objective cognition relationships remained after controlling for catastrophizing. CONCLUSION: Patients' self-reported cognitive concerns concurred with objectively measured performance, independent of age, education and catastrophizing. Moreover, those with severe anxiety were more accurate in predicting their cognitive performance. The findings highlight some interesting cognition-mood relationships, and suggest that easy-to-administer questionnaires, such as the Everyday Memory Questionnaire - Revised and the Behavior Rating Inventory of Executive Function - Adult Version, may be useful to capture cognitive concerns in clinical settings. SIGNIFICANCE: Cognitive concerns in chronic pain reflected objective neurocognitive performance. This was moderated by anxiety, such that self-reported cognition was more consistent with objective performance in those with high anxiety. Our findings suggest that reported cognitive concerns should be heeded, and self-report measures may be used clinically to facilitate dialogue about cognitive functioning.

Financial and recovery worry one year after traumatic injury: A prognostic, registry-based cohort study.

BACKGROUND: Levels of stress post-injury, especially after compensable injury, are known to be associated with worse long-term recovery. It is therefore important to identify how, and in whom, worry and stress manifest post-injury. This study aimed to identify demographic, injury, and compensation factors associated with worry about financial and recovery outcomes 12 months after traumatic injury. METHODS: Participants (n = 433) were recruited from the Victorian Orthopaedic Trauma Outcomes Registry and Victorian State Trauma Registry after admission to a major trauma hospital in Melbourne, Australia. Participants completed questionnaires about pain, compensation experience and psychological wellbeing as part of a registry-based observational study. RESULTS: Linear regressions showed that demographic and injury factors accounted for 11% and 13% of variance in financial and recovery worry, respectively. Specifically, lower education, discharge to inpatient rehabilitation, attributing fault to another and having a compensation claim predicted financial worry. Worry about recovery was only predicted by longer hospital stay and attributing fault to another. In all participants, financial and recovery worry were associated with worse pain (severity, interference, catastrophizing, kinesiophobia, self-efficacy), physical (disability, functioning) and psychological (anxiety, depression, PTSD, perceived injustice) outcomes 12 months post-injury. In participants who had transport (n = 135) or work (n = 22) injury compensation claims, both financial and recovery worry were associated with sustaining permanent impairments, and reporting negative compensation system experience 12 months post-injury. Financial worry 12 months post-injury was associated with not returning to work by 3-6 months post-injury, whereas recovery worry was associated with attributing fault to another, and higher healthcare use at 6-12 months post-injury. CONCLUSIONS: These findings highlight the important contribution of factors other than injury severity, to worry about finances and recovery post-injury. Having a compensation claim, failure to return to work and experiencing pain and psychological symptoms also contribute to elevated worry. As these factors explained less than half of the variance in worry, however, other factors not measured in this study must play a role. As worry may increase the risk of developing secondary mental health conditions, timely access to financial, rehabilitation and psychological supports should be provided to people who are not coping after injury.

Location, location, location: Variation in sensitivity to pain across the body.

BACKGROUND: There is evidence that sensitivity to noxious stimuli differs between the sexes and across the body, but few studies have investigated differences in the perception and experience of acute pain stimuli across the body in healthy individuals. METHODS: We recruited 52 healthy participants, aged 18-36 (50% men) and administered 39, 42 and 45 °C stimuli at four body sites bilaterally to examine differences in the experience of pain intensity and unpleasantness between body sites via an 11-point numerical rating scale. RESULTS: Noxious and innocuous thermal heat stimuli were perceived as significantly more intense when delivered to the wrist (M = 3.98, SD = 1.93) and back (M = 4.07, SD = 1.98) compared to the shoulder (M = 3.45, SD = 1.91) and leg (M = 3.46, SD = 1.87). Pain unpleasantness ratings yielded similar findings; stimuli were perceived as more unpleasant when administered to the wrist (M = 2.83, SD = 1.93) and lower back (M = 3.04, SD = 2.11) compared to the shoulder (M = 2.63, SD = 1.85) and leg (M = 2.26, SD = 1.82). CONCLUSIONS: These findings suggest that painful thermal stimuli delivered to the wrist and back are perceived as more intense and unpleasant compared with other body sites in healthy persons. These differences may be due to variations in receptor density, or the relative importance of these sites for daily living and survival. SIGNIFICANCE: Moreover, these insights are helpful for the design of studies investigating pain experience in healthy persons in experimental or clinical settings. WHAT DOES THIS STUDY ADD?: We tested sensitivity to acute suprathreshold thermal stimulations across a range of body sites to investigate for potential variability. We found significant differences in the perceived intensity and unpleasantness of noxious and innocuous thermal stimuli at the wrist and lower back, compared with the shoulder and leg. These results suggest that pain experience is driven by receptor density or the relative functional importance of these sites.

Prenatal exposure to cannabis and maternal and child health outcomes: a systematic review and meta-analysis.

OBJECTIVE: To assess the effects of use of cannabis during pregnancy on maternal and fetal outcomes. DATA SOURCES: 7 electronic databases were searched from inception to 1 April 2014. Studies that investigated the effects of use of cannabis during pregnancy on maternal and fetal outcomes were included. STUDY SELECTION: Case-control studies, cross-sectional and cohort studies were included. DATA EXTRACTION AND SYNTHESIS: Data synthesis was undertaken via systematic review and meta-analysis of available evidence. All review stages were conducted independently by 2 reviewers. MAIN OUTCOMES AND MEASURES: Maternal, fetal and neonatal outcomes up to 6 weeks postpartum after exposure to cannabis. Meta-analyses were conducted on variables that had 3 or more studies that measured an outcome in a consistent manner. Outcomes for which meta-analyses were conducted included: anaemia, birth weight, low birth weight, neonatal length, placement in the neonatal intensive care unit, gestational age, head circumference and preterm birth. RESULTS: 24 studies were included in the review. Results of the meta-analysis demonstrated that women who used cannabis during pregnancy had an increase in the odds of anaemia (pooled OR (pOR)=1.36: 95% CI 1.10 to 1.69) compared with women who did not use cannabis during pregnancy. Infants exposed to cannabis in utero had a decrease in birth weight (low birth weight pOR=1.77: 95% CI 1.04 to 3.01; pooled mean difference (pMD) for birth weight=109.42 g: 38.72 to 180.12) compared with infants whose mothers did not use cannabis during pregnancy. Infants exposed to cannabis in utero were also more likely to need placement in the neonatal intensive care unit compared with infants whose mothers did not use cannabis during pregnancy (pOR=2.02: 1.27 to 3.21). CONCLUSIONS AND RELEVANCE: Use of cannabis during pregnancy may increase adverse outcomes for women and their neonates. As use of cannabis gains social acceptance, pregnant women and their medical providers could benefit from health education on potential adverse effects of use of cannabis during pregnancy.

Cytokine induction by the immunomodulators imiquimod and S-27609.

Imiquimod (R-837, S-26308) and the analogue S-27609 were evaluated for cytokine induction in human blood cells. Both compounds induced interferon-alpha (IFN), tumor necrosis factor-alpha (TNF), interleukin (IL)-1 beta, and IL-6 with S-27609 being 5 to 10 times more potent. Imiquimod and S-27609 also induced IL-1 alpha, IL-1 receptor antagonist, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and macrophage inflammatory protein-1 alpha. The profile of cytokines induced by imiquimod and S-27609 was different from those seen with lipopolysaccharide and polyinosinic-polycytidylic acid. Kinetic studies with both imiquimod and S-27609 revealed induction of cytokines as early as 1-4 h after stimulation. Although most of the cytokines produced by S-27609 were secreted, significant concentrations of IL-1 alpha and IL-1 beta remained intracellular. Monocytes were largely responsible for the cytokines produced. Finally, S-27609-induced mRNA expression for TNF, IFN, and IL-8, and this induction did not require protein synthesis. Taken together, these studies extend previous findings by showing induction of additional cytokines and providing insight into the mechanism of cytokine induction by these molecules.

Affective, sensory and empathic sharing of another's pain: The Empathy for Pain Scale.

BACKGROUND: Through two studies, we introduce and validate the Empathy for Pain Scale (EPS), which characterizes the phenomenology of empathy for pain, including the vicarious experience of pain when seeing others in pain. METHODS: In study 1, 406 individuals completed the EPS and Interpersonal Reactivity Index (IRI). In the EPS, four painful scenarios (witnessing surgery, patient recovering from surgery, assault and accidental injury) were rated for 12 emotional, empathic and sensory responses. In study 2, 59 participants completed the same questionnaires and then watched and rated videos of sporting injuries. RESULTS: In study 1, we identified three factors of the EPS with principal component analysis, which were validated with confirmatory factor analysis: affective distress; vicarious pain; and empathic concern. The EPS demonstrated good psychometric properties, re-test reliability (n = 105) and concurrent validity. In study 2, we validated the EPS against empathic reactions to the pain of others as displayed in video clips depicting sporting injuries and showed that the scale has unique utility to characterize empathic reactions to pain above general trait empathy measures. Both studies showed that the affective distress and empathic concern subscales of the EPS correlated with measures of cognitive and affective empathy from the IRI, whereas the vicarious pain subscale was only correlated with the personal distress IRI subscale. CONCLUSIONS: The EPS is a psychometrically sound new scale that characterizes empathy for pain and vicarious pain. The EPS offers valuable insight to the phenomenological profile of the affective, empathic and sensory dimensions of empathy for pain.

Cytokine induction in hairless mouse and rat skin after topical application of the immune response modifiers imiquimod and S-28463.

ALDARA (imiquimod cream 5%) recently became available for the treatment of genital and perianal warts; however, the topical mechanism of action of imiquimod is not fully understood. Imiquimod, and its analogs R-842, S-27609, and S-28463, are potent anti-viral and anti-tumor agents in animal models. Much of the biologic activity of these compounds can be attributed to the induction of cytokines, including interferon-alpha, tumor necrosis factor-alpha, interleukins-1, -6, -8, and others. This study was performed to characterize the response of mice and rats to topical application of imiquimod and S-28463 and also to evaluate these agents in cultures of murine and human skin cells. Topical administration of imiquimod or S-28463 to the flanks of hairless mice and rats leads to increases in local concentrations of interferon and tumor necrosis factor in the skin. The concentrations of interferon and tumor necrosis factor were higher at the site of drug application than in skin from the contralateral flank or skin from untreated animals. Interferon-alpha mRNA levels were also elevated in the skin of mice after topical application of either imiquimod or S-28463. In vitro, both imiquimod and S-28463 induced increases in interferon and tumor necrosis factor in cultures of cells isolated from hairless mouse skin. Imiquimod also increased interleukin-8 concentrations in human keratinocyte and fibroblast cultures, whereas S-28463 induced increases in tumor necrosis factor in fibroblast cultures. These results demonstrate that imiquimod and S-28463 stimulate production of cytokines in the skin after topical application, which may play a major role in its activity in genital wart patients.

Localization of 7B2, neuromedin B, and neuromedin U in specific cell types of rat, mouse, and human pituitary, in rat hypothalamus, and in 30 human pituitary and extrapituitary tumors.

The distributions of three novel peptides, 7B2, neuromedin B, and neuromedin U, in rat, mouse, and human pituitaries, rat hypothalamus, and 30 human pituitary tumors were investigated with immunocytochemistry. Immunoreactivity for 7B2 was present in rat, mouse, and human gonadotropes, in intermediate lobe cells and posterior lobe nerve fibers in rats and mice, in rat hypothalamus (particularly in the median eminence), and in eight human pituitary gonadotropinomas. In gonadectomized rats, larger, more numerous LH beta- and 7B2-immunoreactive gonadotropes were seen than in controls. Extractable 7B2-like immunoreactivity was elevated but not significantly so in gonadectomized rat pituitaries [males: castrated, 37.4 +/- 4.3 (mean +/- SE); controls, 26.9 +/- 4.3; females: ovariectomized, 27.2 +/- 2.7; controls, 19.1 +/- 2.2 pmol/gland]. Neuromedin B immunoreactivity was found in normal rat and mouse thyrotropes and weakly in "thyroidectomy" cells in hypothyroid rats, in which extractable pituitary neuromedin B was significantly depleted (thyroidectomized, 87.0 +/- 14.0; methimazole-treated, 82.0 +/- 11.4; control, 230.7 +/- 25.6 fmol/gland). Hyperthyroid rat pituitaries showed increased TSH beta and neuromedin B immunoreactivities and neuromedin B content (TRH-treated, 385.2 +/- 30.2; T4-treated, 352.6 +/- 20.2; control, 230.7 +/- 25.6 fmol/gland). Neuromedin U immunoreactivity occurred in corticotropes of all species, in rat and mouse intermediate lobe, and throughout the rat hypothalamus, with immunoreactive cell bodies in the arcuate nucleus. Neuromedin U-immunoreactive cells were present in six of six human pituitary and five of six human extrapituitary corticotropinomas. In adrenalectomized rats, corticotropes were larger and more numerous than in controls, but extractable anterior pituitary neuromedin U-like immunoreactivity was not raised (adrenalectomized, 3.30 +/- 0.45; control, 3.32 +/- 0.27 pmol/gland). Our findings suggest that 7B2, neuromedin B, and neuromedin U may be involved in pituitary function.

The distribution of nine peptides in rat spinal cord with special emphasis on the substantia gelatinosa and on the area around the central canal (lamina X).

The comparative distribution of nine peptides was examined in the L4 segment of the rat cord using the peroxidase antiperoxidase technique. The peptides examined were substance P, neurotensin, cholecystokinin, methionine-enkephalin, oxytocin, neurophysin, adrenocorticotrophin, thyrotropin releasing hormone, and vasoactive intestinal polypeptide. No transport blocking agents were used and in spite of this cell bodies containing substance P, neurotensin, cholecystokinin, and methionine-enkephalin were observed. All peptides except for thyrotropin releasing hormone were observed in fibers in laminae I and II. All peptides were present in the area around the central canal, lamina X. Each peptide had its own characteristic distribution within fibers in the gray and white matter.

Ontogeny of peptide- and amine-containing neurones in motor, sensory, and autonomic regions of rat and human spinal cord, dorsal root ganglia, and rat skin.

The developmental patterns of neurofilament triplet proteins, peptide and amine immunoreactivities were compared in motor (ventral spinal cord), sensory (dorsal spinal cord, dorsal root ganglia, epidermis), and autonomic (intermediolateral cell columns, dermis) regions in the rat and human. In the rat, neurofilament triplet proteins first appeared in motoneurones (embryonic day 13). In the youngest human fetuses studied (6 weeks), immunoreactivity was present throughout the spinal cord. Peptides and amines occurred later. Calcitonin gene-related peptide, galanin, somatostatin, neuropeptide Y and its C-flanking peptide (CPON) were the first to appear localized to motoneurones (embryonic days 15-17 rat; fetal weeks 6-14 human). Numbers of immunoreactive motoneurones decreased toward birth, but immunoreactive fibers increased in the ventral horn with enkephalin, thyrotrophin-releasing hormone, and the monoaminergic markers 5-hydroxytryptamine and tyrosine hydroxylase (all presumably of supraspinal origin) the last to appear perinatally. In the dorsal horn, particularly in the rat, a transient expression of substance P-, somatostatin-, and neuropeptide Y/CPON-immunoreactive cells was detected (embryonic days 15-17). A pronounced increase of calcitonin gene-related peptide-, galanin-, somatostatin- and substance P- immunoreactive fibers was found perinatally in both species. This coincided with an increased detection of cells in the dorsal root ganglia containing these peptides and the earliest appearance of calcitonin gene-related peptide-, somatostatin-, and substance P-immunoreactive fibers in the rat epidermis. Few antigens were localized to the intermediolateral cell columns before embryonic day 20 (rat), fetal week 20 (human), with thyrotrophin-releasing hormone-, 5-hydroxytryptamine-, tyrosine hydroxylase-, and vasoactive intestinal polypeptide-immunoreactive nerves appearing perinatally. In the rat dermis, tyrosine hydroxylase-immunoreactive fibers (sympathetic fibers) and fibers immunoreactive for neuropeptide Y/CPON and vasoactive intestinal polypeptide were detected from postnatal day 1. In conclusion, 1) peptide and amine immunoreactivity develops in motor before sensory or autonomic regions, 2) many peptide-containing cells are transient in fetal life, and 3) central terminals of dorsal root ganglion cells express peptides before terminals in the skin.

The distribution and origin of a novel brain peptide, neuropeptide Y, in the spinal cord of several mammals.

The distribution of neuropeptide Y [NPY]-immunoreactive material was examined in the spinal cord and dorsal root ganglia of rat, guinea-pig, cat, marmoset, and horse. Considerable concentrations of NPY and similar distribution patterns of immunoreactive nerve fibres were found in the spinal cord of all species investigated. The dorsal root ganglia of the cat and the horse contained numerous immunoreactive nerve fibres, but in these species, as in the other three studied [rat, guinea-pig, marmoset], no positively stained cell bodies were found. Neuropeptide Y-immunoreactive nerves were observed at all levels of the spinal cord, being most concentrated in the dorsal horn. In the rat, guinea-pig, and marmoset, there was a marked increase of NPY-immunoreactive fibres in the lumbosacral regions of the spinal cord, and this was reflected by a considerable increase of extractable NPY. Estimations of NPY-immunoreactive material in the various regions of the rat spinal cord were as follows: cervical, 13.8 +/- 1.0; thoracic, 21.1 +/- 2.5; lumbar, 16.3 +/- 2.9; sacral, 92.4 +/- 8.5 pmol/gm wet weight of tissue +/- SEM. In the ventral portion of the guinea-pig spinal cord they were as follows: cervical, 7.1 +/- 1.2; thoracic, 8.2 +/- 3.6; lumbar, 22.6 +/- 7.0; sacral, 36.7 +/- 9.5 pmol/gm wet weight of tissue +/- SEM. Analysis of spinal cord extracts by reverse phase high performance liquid chromatography [HPLC] demonstrated that NPY-immunoreactive material elutes in the position of pure NPY standard. No changes in the concentration and distribution of the NPY-like material in the rat spinal cord were observed following a variety of surgical and pharmacological manipulations, including cervical rhizotomy, sciatic nerve section and ligation, and local application of capsaicin [50 mM] to one sciatic nerve. It is therefore suggested that most of the NPY-immunoreactive material in the spinal cord is derived either from intrinsic nerve cell bodies or from supraspinal tracts.

Novel peptide pancreastatin: its occurrence and codistribution with chromogranin A in the central nervous system of the pig.

The distribution of pancreastatin immunoreactivity was investigated in porcine brain, spinal cord, dorsal root ganglia, and pituitary. In the brain, immunoreactive cell bodies were present in many areas including the cortex, basal ganglia, hippocampus, thalamus, hypothalamus, mesencephalic reticular formation, cerebellum, and medulla oblongata. Immunoreactive fibres were most abundant in the globus pallidus, stria terminalis, entopeduncular nucleus, hippocampus, and in the substantia nigra. In the spinal cord, immunoreactive cells were found in laminae IV-IX. Immunoreactive fibres were concentrated in the dorsal horn. Pancreastatin immunoreactivity was localised to fibres and small cells (5-10% of the total) in the dorsal root ganglia. In the posterior pituitary, many immunoreactive fibres were present and in the anterior lobe subsets of gonadotrophs and thyrotrophs were pancreastatin-immunoreactive. The localisation of pancreastatin showed a parallel distribution with chromogranin A. Coexistence of pancreastatin with calcitonin gene-related peptide (CGRP) immunoreactivity in cell bodies in the spinal cord, including motoneurones, and with CGRP or galanin immunoreactivities in dorsal root ganglion cells was also noted. The differential pattern of pancreastatin immunostaining was reflected in the extractable levels of peptide with highest concentrations in the cortex (55.8 +/- 6.0 pmol/g wet weight, mean +/- S.E.M.), thalamus (60.0 +/- 5.0 pmol/g), hypothalamus (54.4 +/- 6.5 pmol/g), and anterior pituitary (2,714 +/- 380 pmol/g). Characterisation of pancreastatin immunoreactivity in the hypothalamus and pituitary by gel permeation and high-pressure liquid chromatography revealed multiple molecular forms, one of which was indistinguishable from natural porcine pancreastatin. The widespread distribution of pancreastatin immunoreactivity suggests this peptide may play a part in several neuroendocrine, autonomic, somatic, and sensory functions, and its colocalisation with chromogranin A is consistent with a precursor-product relationship.

Cellular requirements for cytokine production in response to the immunomodulators imiquimod and S-27609.

Imiquimod (R-837) and its analog, S-27609, belong to a class of imidazoquinolinamines that have potent antitumor and antiviral effects in animals. Much of their biologic activity is a result of the induction of cytokines, including interferon-alpha (IFN-alpha), tumor necrosis factor alpha (TNF), and others. In this study, the cells responsible for S-27609- and imiquimod-induced cytokine production were characterized. E rosette+ T cells were not the major cell population responsible for IFN-alpha and TNF in response to S-27609 or imiquimod. In contrast, E rosette- cells and unseparated PBMC produced similar concentrations of IFN-alpha and TNF in response to S-27609 and imiquimod. Elimination of monocytes by treatment with the lysosomotropic agent L-leucine methyl ester (LME) or depletion using antibody to CD14 and immunomagnetic beads abrogated IFN-alpha and TNF production induced by S-27609, imiquimod, or LPS but not poly(I)/(C). LME treatment also abolished interleukin (IL)-1 alpha, IL-beta, IL-6, and IL-8 production stimulated by S-27609 and imiquimod. Removal of HLA-DR+ or CD36+ monocytes also caused a significant reduction in S-27609- and imiquimod-induced IFN-alpha and TNF. Elimination of B cells, NK cells, and dendritic cells did not significantly reduce cytokine induction in response to S-27609. Thus, the cell population responsible for the majority of cytokine release in human PBMC in response to S-27609 and imiquimod is a E rosette-, CD14+, CD36+, HLA-DR+ monocyte.

The effect of age on A delta- and C-fibre thermal pain perception.

It has been suggested that ageing may have a differential effect on C fibre-mediated protopathic/tonic pain versus epicritic/phasic pain perception mediated by A delta fibres. The present study attempted to independently assess age-related changes in the function of A delta- and C-nociceptive fibres by examining CO2 laser-induced thermal pain thresholds before, during and after a compression block of the superficial radial nerve in 15 young and 15 healthy elderly adult subjects. Nerve block efficacy was monitored via measures of cold, warm and mechanical threshold, and simple reaction time. During nerve compression block, reaction time and mechanical threshold increased, cold sensation became impaired while warm sensation remained unaffected throughout the test in both groups. With respect to pain sensitivity, young adults exhibited significant increases in thermal pain threshold during A-fibre block while pain threshold remained relatively stable across the 3 test periods in the elderly group. It would appear that elderly adults rely predominantly on C-fibre input when reporting pain whereas younger adults utilise additional input from A delta fibres. Subsequent analysis revealed that during pre- and post-block periods, older adults exhibited a significant elevation in thermal pain threshold; however, when A delta-fibre function was impaired and only C-fibre information was available, both groups responded similarly. These findings support the notion of a differential age-related change in A-fibre-mediated epicritic pain perception versus C-fibre-mediated protopathic pain.