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Metrology in clinical chemistry aims to ensure the equivalence of measurement results from different in-vitro diagnostic measurement devices (IVD MD) for use in healthcare. The metrological traceability of measurement results to higher-order references is the cornerstone to achieving equivalent results. However, other fundamentals are also needed, including the commutability of reference materials and external quality assessment (EQA) materials for monitoring the equivalence of measurement results at the end-user level. This manuscript summarizes the findings and opinions expressed at the Joint Community for Traceability in Laboratory Medicine (JCTLM) workshop held on December 4-5, 2023. The workshop explored the relationship between EQA/proficiency testing and metrological traceability to higher-order references. EQA monitors the equivalence of measurement results from end-user IVD MDs. The workshop discussed the role and challenges of using EQA to improve and maintain the equivalence of measurement results. It also elucidated current developments in establishing the clinical suitability of laboratory results expressed as analytical performance specifications (APS)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Reporting a measurement procedure and its analytical performance following method evaluation in a peer-reviewed journal is an important means for clinical laboratory practitioners to share their findings. It also represents an important source of evidence base to help others make informed decisions about their practice. At present, there are significant variations in the information reported in laboratory medicine journal publications describing the analytical performance of measurement procedures. These variations also challenge authors, readers, reviewers, and editors in deciding the quality of a submitted manuscript. The International Federation of Clinical Chemistry and Laboratory Medicine Working Group on Method Evaluation Protocols (IFCC WG-MEP) developed a checklist and recommends its adoption to enable a consistent approach to reporting method evaluation and analytical performance characteristics of measurement procedures in laboratory medicine journals. It is envisioned that the LEAP checklist will improve the standardisation of journal publications describing method evaluation and analytical performance characteristics, improving the quality of the evidence base that is relied upon by practitioners., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Introduction: The future health of the offspring can be influenced by longstanding maternal anxiety and depression disorders during pregnancy. The present study aimed to explore the effect of psychiatric disorders during pregnancy on placental epigenetics., Methods: We measured DNA methylation patterns in term-placentas of women either suffering longstanding anxiety and depression symptoms (Index group, with overt symptoms), or a healthy population (Control, none/only mild symptoms). Whole genome DNA methylation profiling was performed using the TruSeq® Methyl Capture EPIC Library Prep Kit (Illumina, San Diego, CA, USA) for library preparation and NGS technology for genomic DNA sequencing., Results: The results of high-throughput DNA methylation analysis revealed that the Index group had differential DNA methylation at epigenome-wide significance (p < 0.05) in 226 genes in the placenta. Targeted enrichment analyses identified hypermethylation of genes associated with psychiatric disorders (BRINP1, PUM1), and ion homeostasis (COMMD1), among others. The ECM (extracellular matrix)-receptor interaction pathway was significantly dysregulated in the Index group compared to the Control. In addition, DNA methylation/mRNA integration analyses revealed that four genes with key roles in neurodevelopment and other important processes (EPB41L4B, BMPR2, KLHL18, and UBAP2) were dysregulated at both, DNA methylation and transcriptome levels in the Index group compared to Control., Discussion: The presented results increase our understanding of how maternal psychiatric disorders may affect the newborn through placental differential epigenome, suggesting DNA methylation status as a biomarker when aiming to design new preventive techniques and interventions., Competing Interests: Declaration of competing interest None of the authors have any conflicts of interest to declare., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

When increasing the quality in clinical laboratories by decreasing measurement uncertainty, reliable methods are needed not only to quantify the performance of measuring systems, but also to set goals for the performance. Sigma metrics used in medical laboratories for documenting and expressing levels of performance, are evidently totally dependent on the "total permissible error" used in the formulas. Although the conventional biological variation (BV) based model for calculation of the permissible (or allowable) total error is commonly used, it has been shown to be flawed. Alternative methods are proposed, mainly also based on the within-subject BV. Measurement uncertainty models might offer an alternative to total error models. Defining the limits for analytical quality still poses a challenge in both models. The aim of the present paper is to critically discuss current methods for establishing performance specifications by using the measurement of sodium concentrations in plasma or serum. Sodium can be measured with high accuracy but fails by far to meet conventional performance specifications based on BV. Since the use of sodium concentrations is well established for supporting clinical care, we question the concept that quality criteria for sodium and similar analytes that are under strict homeostatic control are best set by biology., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: The biological (CV I ), preanalytical (CV PRE ), and analytical variation (CV A ) are inherent to clinical laboratory testing and consequently, interpretation of clinical test results., Methods: The sum of the CV I , CV PRE , and CV A , called diagnostic variation (CV D ), was used to derive clinically acceptable analytical performance specifications (CAAPS) for clinical chemistry measurands. The reference change concept was applied to clinically significant differences (CD) between two measurements, with the formula CD = z*√2* CV D . CD for six measurands were sought from international guidelines. The CAAPS were calculated by subtracting variances of CV I and CV PRE from CV D . Modified formulae were applied to consider statistical power (1-β) and repeated measurements., Results: The obtained CAAPS were 44.9% for urine albumin, 0.6% for plasma sodium, 22.9% for plasma pancreatic amylase, and 8.0% for plasma creatinine (z = 3, α = 2.5%, 1-β = 85%). For blood HbA 1c and plasma low-density lipoprotein cholesterol, replicate measurements were necessary to reach CAAPS for patient monitoring. The derived CAAPS were compared with analytical performance specifications, APS, based on biological variation., Conclusions: The CAAPS models pose a new tool for assessing APS in a clinical laboratory. Their usability depends on the relevance of CD limits, required statistical power and the feasibility of repeated measurements., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Uncertainty is an inseparable part of all kinds of measurements performed in clinical laboratories. Accreditation standards including the ISO/IEC 17025:2017 and ISO 15189:2012 require that laboratories have routines for calculating the measurement uncertainty of reported results. Various guidelines such as CLSI EP29, Nordest 537, and ISO 20914:2019 have proposed methods for this purpose. However, due to the conceived complexity of the proposed calculation methods, these guidelines have not been generally and effectively applied in clinical laboratories. High workload and measurand heterogeneity favor a pragmatic utilitarian approach. The purpose of this paper is to describe such an approach, including its advantages and disadvantages. Measurement uncertainty should include the most influential factors affecting patients' test results. Since patients' samples for the same measurand can be analyzed in one laboratory or several laboratories using different measuring systems, the measurement uncertainty should be calculated using results obtained from analyzing the same internal quality control material if commutable or patients pooled/split samples., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The galanin family currently consists of four peptides, namely galanin, galanin-message associated peptide, galanin-like peptide and alarin. Unlike galanin that signals through three different G protein-coupled receptors; GAL 1 , GAL 2 , and GAL 3 , binding at its N-terminal end, the cognate receptors for other members of the galanin family are currently unknown. Research using short N-terminal galanin fragments generated either by enzymatic cleavage or solid-phase synthesis has revealed differences in their receptor binding properties exerting numerous biological effects distinct from galanin(1-29) itself. Our studies on tissue extracts derived from rat small intestine and bovine gut using chromatographic techniques and sensitive galanin(1-16)-specific radioimmunoassay revealed the presence of immunoreactive compounds reacting with antiserum against galanin(1-16) distributed in distinct elution volumes. These results suggested a possible presence of short N-terminal galanin fragments also in vivo. Moreover, employing immunoaffinity chromatography and reverse-phase high performance liquid chromatography (HPLC) followed by mass spectrometry allowed specific enrichment of these immunoreactive compounds from rat tissues and identification of their molecular structure. Indeed, our study revealed presence of several distinct short N-terminal galanin sequences in rat tissue. To prove their receptor binding, four of the identified sequences were synthetized, namely, galanin(1-13), galanin(1-16), galanin(1-20), galanin(6-20), and tested on coronal rat brain sections competing with 125 I-labeled galanin(1-29). Our autoradiographs confirmed that galanin(1-13), galanin(1-16), and galanin(1-20) comprehensively displaced 125 I-galanin(1-29) but galanin(6-20) did not. Here we show, for the first time, that short N-terminal galanin fragments occur naturally in rat tissues and that similar or identical galanin sequences can be present also in tissues of other species., Biological Significance: This study is first to provide an evidence of the presence of short N-terminal galanin fragments in vivo in a biological system and provides further foundations for the previous studies using synthetized short N-terminal galanin fragments., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Aim: To evaluate the effects of almost continuous skin-to-skin contact (SSC) on salivary cortisol, parental stress, parental depression, and breastfeeding., Study Design: This is a randomised study engaging families of late preterm infants (32-35 weeks gestation). Salivary cortisol reactivity was measured in infants during a nappy change at one month corrected age, and in infants and mothers during still-face at four month corrected age. Both parents completed the Swedish Parenthood Stress Questionnaire (SPSQ) at one month and the Edinburgh Postnatal Depression Scale (EPDS) at one and four months. Ainsworth's sensitivity scale was used to control for parental sensitivity., Subjects: Thirty-seven families from two different neonatal care units in Sweden, randomised to either almost continuous SSC or standard care (SC)., Results: Infants randomised to SSC had a lower salivary cortisol reactivity at one month (p=0.01). There was a correlation between the mothers' and the preterm infants' salivary cortisol levels at four months in the SSC group (ρ=0.65, p=0.005), but not in the SC group (ρ=0.14, p=0.63). Fathers in SSC scored lower on the SPSQ sub-scale spouse relationship problems compared to fathers in SC (p<0.05)., Conclusions: Almost continuous SSC decreases infants' cortisol reactivity in response to handling, improves the concordance between mothers' and infants' salivary cortisol levels, and decreases fathers' experiences of spouse relationship problems., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The recent claim that stimulated platelets activate the intrinsic pathway of coagulation by the release of polyphosphates has been considered a breakthrough in hemostasis research. In little more than 3 years, the original publication by Müller et al has been cited >100 times. However, none of the citing articles has sought to independently validate this potentially paradigm-shifting concept. To this end, we performed extensive experimentation in vitro and in vivo in an attempt to verify the claim that factor XII (FXII) is primarily activated by stimulated platelets. In contrast to the original assertion, platelet-derived polyphosphates were found to be weak activators of FXII, with a FXIIa-generating activity of <10% compared with equivalent concentrations of kaolin. Using different coagulation assays, it was shown that platelet contribution to whole blood coagulation was unrelated to the generation of activated FXII in vitro. Additionally, key results used to verify the hypothesis in the original study in vivo were found to be irreproducible. We conclude that platelet-derived polyphosphates are not physiologically relevant activators of FXII.

Injury to neurons results in up-regulation of galanin in some central and peripheral systems, and it has been suggested that this neuropeptide may play a protective and trophic role, primarily mediated by galanin receptor 2 (GalR2). The objective of the present study was to investigate galanin, GalR1, GalR2 and GalR3 gene expression in the female rat brain 7 days after a 60-min unilateral occlusion of the middle cerebral artery followed by reperfusion. Quantitative real-time PCR was employed in punch-biopsies from the locus coeruleus, somatosensory cortex and dorsal hippocampal formation, including sham-operated rats as controls. Galanin gene expression showed a ∼2.5-fold increase and GalR1 a ∼1.5-fold increase in the locus coeruleus of the ischemic hemisphere compared to the control side. Furthermore, the GalR1 mRNA levels decreased by 35% in somatosensory cortex of the ischemic hemisphere. Immunohistochemical analysis indicated a depletion of galanin from cell bodies and dendrites in the locus coeruleus after middle cerebral artery occlusion. The present results suggest that a stroke-induced forebrain lesion up-regulates synthesis of galanin and GalR1 in the locus coeruleus, a noradrenergic cell group projecting to many forebrain areas, including cortex and the hippocampal formation. These results support the notion that galanin may play a role in the response of the central nervous system to injury., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Several studies have shown that injury to the central and peripheral nervous system can increase expression of galanin, a 29 amino acid neuropeptide. Moreover, there is evidence that galanin, especially through its galanin receptor 2 (GalR2) receptor, plays a neuroprotective role in different injury models. However, direct studies of a possible neuroprotective effect of galanin in experimental stroke models are lacking. Galanin, a GalR2/3 agonist or artificial CSF was continuously infused intracerebroventricularly (i.c.v.) in naïve female rats after a 60min transient and focal occlusion of the middle cerebral artery. The animals were sacrificed, and the ischemic lesion was visualized using 2,3,5-triphenyltetrazolium hydrochloride (TTC) staining. The lesion was 98% larger after i.c.v. administration of the GalR2/3 agonist (2.4nmol/day) seven days after occlusion compared to artificial CSF (p=0.023). No statistically significant differences were found after seven days in the groups treated with galanin in three different concentrations (0.24, 2.4 and 24nmol/day; p=0.939, 0.715 and 0.977, respectively). There was no difference in the size of the ischemic lesions measured after three days in the galanin-treated group (2.4nmol/d) compared to artificial CSF (p=0.925). The present results show, surprisingly, that a GalR2/3 agonist doubled the size of the ischemic lesion. Whether this effect primarily reflects the properties of the current model, species, gender and/or the mode of galanin administration, e.g. causing desensitization, or whether galanin indeed lacks neuroprotective effect of its own, remains to be corroborated., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Objective: Vasoactive intestinal peptide (VIP) is a 28-amino acid peptide widely expressed in the body and binding three types of receptors: VPAC(1)-R, VPAC(2)-R and PAC(1)-R. Based on beneficial effects of VIP and VPAC(1)-R agonists in mouse models of several chronic inflammatory disorders, we hypothesized that activation of VIP receptors would prevent atherosclerosis development in apolipoprotein E-deficient mice., Methods and Results: Contrary to our hypothesis, administration of a VPAC(1)-R agonist, (Ala(11,22,28))-VIP aggravated atherosclerotic lesion development in the aortic root of these mice compared to control mice. This was accompanied by a significant increase in the expression of MHC class II protein I-A(b), and suggests enhanced inflammatory activity in the vessel wall. The amount of macrophage-specific CD68 staining as well as serum cholesterol and triglyceride levels did not change as a result of the (Ala(11,22,28))-VIP treatment, i.e. the treatment resulted in significant changes in lipid accumulation in the lesions without changing the number of macrophages or systemic lipid levels. Interestingly, administration of VIP did not alter the course of the disease., Conclusion: Despite beneficial effects in murine models of several inflammatory disorders, VPAC(1)-R activation aggravates atherosclerotic lesion formation in apolipoprotein E-deficient mice through enhanced inflammatory activity in the vessel wall., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Estrogen alters excitability and changes synaptic morphology in the rat hippocampal formation. We have compared, by means of radioimmunoassay and in situ hybridization, the effects of short-term treatment with 17beta-estradiol on neuropeptide Y (NPY) in the brain of ovariectomized mice. A highly significant reduction in concentrations of NPY-like immunoreactivity (LI) was observed in the hippocampal formation, some cortical areas and the caudate nucleus 1h after administration of 17beta-estradiol as compared to the control group. In contrast, NPY transcript levels increased in the hippocampal formation (dentate gyrus) and the caudate nucleus, possibly representing a compensatory increase of NPY synthesis following increased estradiol-induced NPY release. These data suggest that 17beta-estradiol, via membrane-related mechanisms, increases NPY release and synthesis in forebrain areas involved in cognition, mood and motor functions.

Background: Increased intracranial pressure induces neurogenic pulmonary edema (NPE), potentially explaining why only lungs from less than 20% of brain dead organ donors can be used for transplantation. This study investigated the underlying mechanisms of NPE, focusing on neuropeptides, which potently induce vasoconstriction, vasodilatation, and neurogenic inflammation., Methods: Brain death was induced in 10 pigs by increasing the intracranial pressure. Eight additional pigs served as controls. Neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), and substance P were analyzed in plasma, bronchoalveolar lavage (BAL) fluid, and homogenized lung tissue 6 hours after brain death. Pulmonary oxygen exchange was estimated using partial pressure of arterial oxygen (Pao2)/fraction of inspired oxygen (Fio2), and pulmonary edema by wet/dry weight ratio., Results: Brain death induced a decrease in Pao(2)/Fio2 (p < 0.001) and increased the wet/dry weight of both apical (p = 0.01) and basal lobes (p = 0.03). NPY and CGRP concentrations were higher in the BAL fluid of brain-dead animals compared with controls (p = 0.02 and p = 0.02) and were positively correlated with the wet/dry weight ratio. NPY content in lung tissue was lower in brain-dead animals compared with controls (p = 0.04) and was negatively correlated with the wet/dry weight ratio. There were no differences in substance P concentrations between the groups., Conclusion: NPY was released from the lung tissue of brain-dead pigs, and its concentration was related to the extent of pulmonary edema. NPY may be one of several crucial mediators of neurogenic pulmonary edema, raising the possibility of treatment with NPY-antagonists to increase the number of available lung donors.

The effects of hypothermia on galanin concentrations and the relation between ischemic brain lesions, hypothermia and galanin concentrations in a transient and focal rat stroke model were investigated in order to elucidate whether hypothermia-induced alterations in galanin concentrations could constitute a part of the established neuroprotective effect of hypothermia. Female rats were allocated to normothermia (37 degrees C) or hypothermia (33 degrees C) treatments during a 60 min microclip middle cerebral artery occlusion. The ischemic lesions were visualized after observation periods of 2 or 7 days and the concentration of galanin measured by radioimmunoassay in extracts of punch biopsies from both the lesioned and the contralateral control hemisphere. Hypothermia-induced an overall increase in the concentrations of immunoreactive galanin (p<0.001). The elevated galanin levels were predominantly found in the non-ischemic control hemisphere, in the hippocampus, thalamus and the posterior part of parietal cortex. The galanin concentrations were lower in the ischemic hemisphere in both the normo- and hypothermic animals compared to the corresponding contra lateral intact hemisphere (p=0.049). The factor of time, 2 respectively 7 days, did not show any significant difference regarding the galanin concentrations (p=0.844). Multivariate analyses of variance revealed significant effect of ischemia on the size of the ischemic brain lesions (p=0.001) but no overall effect of temperature when data from both 2 and 7 days observation periods were analyzed together. The ischemic lesions were generally larger at 33 degrees after 2 days (p=0.230). Prolonged observation time of 7 days resulted in a significant reduction of the ischemic brain lesion (p=0.011) with smaller ischemic lesions in the hypothermic group. Our data support the notion that hypothermia-induced increase in the tissue concentrations of galanin in the brain are the result of changes from optimal homeostatic conditions - the hypothermia-induced stress - rather than the ischemia/re-perfusion lesion induced changes in galanin concentrations. Hypothermia-induced elevation in galanin concentration is therefore not likely to be amongst the major protective mechanisms of hypothermia.

Background: Due to the occurrence of sleep disturbances and fatigue in chronic fatigue syndrome (CFS), an investigation was performed to examine if there is an abnormal excretion of gamma-aminobutyric acid (GABA) and/or its structural analogue beta-alanine in the urine from CFS patients. Both GABA and beta-alanine are inhibitory neurotransmitters in the mammalian central nervous system., Methods: The 24 h urine excretion of GABA and beta-alanine was determined by isotope dilution gas chromatography mass spectrometry in 33 CFS patients and 43 healthy controls. The degree of symptoms in both patients and controls was measured by grading of three typical CFS symptoms using a Visual Analogue Scale., Results: Men had a significantly higher excretion of both beta-alanine and GABA than women. Comparing CFS patients with healthy controls showed no significant difference in excretion of neither beta-alanine nor GABA. No correlation was found between the excretion of beta-alanine or GABA and any of the three characteristic CFS symptoms measured. However, two female and two male CFS patients excreted considerably higher amounts of beta-alanine in their 24 h urine samples than control subjects., Conclusions: Increased excretion of beta-alanine was found in a subgroup of CFS patients, indicating that there may be a link between CFS and beta-alanine in some CFS patients.

The distribution of galanin mRNA-expressing cells and galanin-immunoreactive (IR) cell bodies and processes was studied in the brain of mice overexpressing galanin under the PDGF-B promoter (GalOE mice) and of wild type (WT) mice, both in colchicine-treated and non-treated animals. A widespread ectopic expression of galanin (both mRNA and peptide) was found, that is when neither transcript nor peptide could be seen in WT mice, not even after colchicine treatment. However, in some regions, such as claustrum, basolateral amygdala, thalamus, CA1 pyramidal cells, and Purkinje cells only galanin mRNA could be detected. The highest levels of galanin expression were observed in the forebrain structures (the mitral cells of the olfactory bulb, throughout the cortex, granular and pyramidal cell layers of the hippocampus), in the mesencephalon (nucleus ruber), in the cerebellum (lateral cerebellar nucleus), in the pons (sensory and motor nuclei of the trigeminal nerve), within the medulla oblongata (facial, prepositus and spinal trigeminal nuclei). High densities of galanin-IR fibers were found in the axonal terminals of the lateral olfactory tract, hippocampal and presumably cerebellar mossy fiber system, in several thalamic and hypothalamic regions and the lower brain stem.

17Beta-estradiol induced an increase in tissue concentrations of galanin in the hippocampal formation of ovariectomized rats. This increase was dose- and time dependent, and occurred already 60 min after steroid administration and was not blocked by Tamoxifen). There was also an increase in galanin in the pro-estrous phase in regularly cycling rats. The estrogen-induced rapid increase may at least in part be due to decreased release of galanin as demonstrated by in vivo microdialysis studies. Thus, sex steroid hormones may influence signalling molecules in brain areas of importance for cognitive functions.

The aim of the present study was to investigate the influence of an exercise program on neuropeptide concentrations, disease activity, impairments and disabilities in rheumatoid arthritis (RA). Eleven females (median age 60 years, median disease duration 6.5 years, ARA functional classes I or II) exercised 30 min daily for 4 weeks. The urine concentrations of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) and neuropeptide Y-like immunoreactivity (NPY-LI) were analyzed 1 week prior to exercise start, at exercise start, after 2 and 4 weeks of exercise, and after a 4-week follow-up period. Measurements of disease activity, aerobic capacity, grip force, limb muscle function, and activities of daily living (ADL) were also undertaken. The results indicate a decrease (md 5.64 pM to md 3.48 pM, P

Over the last decade, several patients afflicted with xerostomia have been treated with acupuncture. Their salivary flow rates increased significantly and the improvement lasted during a long observation period. We also found that the release of several neuropeptides in the saliva of healthy subjects can be increased by acupuncture stimulation. The concentration of vasoactive intestinal polypeptide increased significantly in the saliva of xerostomic patients after acupuncture treatment. The release of the neuropeptide calcitonin gene-related peptide (CGRP) was investigated in the saliva of xerostomic patients in order to elucidate further the mechanisms of the effect of sensory stimulation (acupuncture) on the salivary secretion. CGRP-like immunoreactivity was measured with radioimmunoassay (RIA) before and after a double series of acupuncture treatment, in stimulated saliva of 14 patients who suffered from xerostomia. The results showed that the concentration of CGRP increased significantly (P<0.001) in the saliva of these patients after the end of acupuncture treatment as compared to base-line levels. Taking into consideration the influence of CGRP on the salivary flow, as well as its trophic effect, we concluded that the increased release of CGRP could be one of the factors that affect positively the salivary flow rates of xerostomic patients who were treated with acupuncture., (Copyright 1999 Harcourt Publishers Ltd.)

We have shown in earlier studies that xerostomia can be treated successfully with acupuncture. We also found that acupuncture stimulation can increase the concentration of neuropeptides in the saliva of healthy subjects. In this study, the concentration of the neuropeptide vasoactive intestinal polypeptide (VIP) was measured in the saliva of xerostomic patients in connection with acupuncture treatment (AP). Patients suffering from xerostomia caused by irradiation treatment, Sjögren's syndrome and other systemic disorders had been treated with acupuncture. Some of these patients showed an increase of their salivary flow rates after the AP was completed. Seventeen patients out of 65 were chosen due to their ability to produce enough saliva for the radio immunoassay (RIA) analyses to be conducted prior to the start of AP. VIP-like immunoreactivity (VIP-LI) was measured in the chewing stimulated saliva of these patients before and after the whole AP (24 sessions of 30 min each). The results showed that there was a significant increase of the concentration of VIP after the AP as compared to the measurements made before the start of the treatment (p<0.05). We concluded that the increase of neuropeptide VIP might be one of the mechanisms behind the positive effect of acupuncture on the salivary flow rates of the xerostomic patients.

It has recently been hypothesized that both the sensory and sympathetic nervous system contribute to the inflammatory reaction. A scalding model was developed in anaesthetized rats to investigate the contribution of neuropeptides in heat-induced edema localized to the hindpaw. After immersing the paw in water at 60 degrees C for 10, 20, 30 and 60 s, edemic reactions were registered as change of paw volume in a plethysmograph and hindpaw perfusates collected to measure the content of neuropeptides by radioimmunoassay. A scalding period of 30 s induced the most prominent edemic reaction. There was a marked increase of the sensory neuropeptide neurokinin A and the sympathetic related transmitter neuropeptide Y in hindpaw perfusates after scalding. The effect of peripheral nerve ligation on edemic reaction and on the release of neuropeptides was investigated in rats scalded for 30 s at 60 degrees C. There was a significant decrease of edema formation in the scalded nerve ligated paw as compared with the scalded paw on the non-ligated side. Neurokinin A was not detected in nerve ligated rats before or after scalding, whereas mononeuropathic rats showed increased concentrations of neuropeptide Y. The present results indicate that the sensory as well as the sympathetic nervous system, possibly through the release of neuropeptides, may contribute to scald-induced edema.

The effects of experimentally induced ischaemia after free-flap surgery on concentrations of neuropeptide Y (NPY), neurokinin A (NKA), substance P (SP) and calcitonin gene-related peptide (CGRP)-like immunoreactivity (-LI) were studied in flap tissue and in different regions of the rat brain (striatum, hippocampus, pituitary, hypothalamus, frontal and occipital cortex). Ten days after the operation, CGRP-LI and NKA-LI were decreased in the ischaemic tissue but increased in the surrounding tissue. In the brain, CGRP-LI was increased in five of six regions analysed, with the exception of the striatum. SP-LI and NKA-LI were increased in the pituitary and hippocampus, but decreased in other brain regions. Changes of CGRP-LI in the brain correlated positively with the CGRP-LI concentrations in the surrounding flap tissue and the CGRP-LI concentrations in the ischaemic flap tissue with the extent of flap survival. The results of the present study suggest that higher concentrations of CGRP-LI are related to tissue survival and that endogenous CGRP has a regulatory effect in ischaemia.

The present study was performed to explore the modulatory potential of different endogenous opioid systems on transmission of presumed nociceptive information at the spinal cord level in thermally injured rats. Thermal injury was performed by dipping the left paw into water 60 degrees C for 20 s. This induced a significant bilateral decrease in hindpaw withdrawal latency HWL to pressure. Intrathecal administration of 10 nmol of CGRP8-37 induced a significant bilateral increase in HWL in the thermally injured group and in the intact controls. The effect of different opioid receptor antagonists on the increased latency to withdrawal response induced by intrathecal injection of 10 nmol of CGRP8-37 was explored in the thermally injured rats. The effect was reversed by intrathecal injection of 40 and 80 nmol of: b-funaltrexamine (mu opioid receptor antagonist) and naltrindole (delta opioid receptor antagonist), but not by norbinaltorphimine (kappa opioid receptor antagonist). The results of the present study show that intrathecal CGRP8-37 increases hindpaw withdrawal latency in thermally injured rats, an effect reduced by a mu as well as by a delta opioid receptor antagonist.

In recent studies we have shown that xerostomia (dry mouth) can be treated successfully with sensory stimulation (acupuncture). The increase of saliva secretion lasted often for at least one year. Some neuropeptides have been found to influence the secretion of saliva. The aim of this study was to investigate the mechanisms behind the effect of acupuncture on salivary secretion by measuring the release of neuropeptides in saliva under the influence of sensory stimulation. VIP-like immunoreactivity (VIP-LI), NPY-LI, SP-LI, CGRP-LI and NKA-LI were analysed in the saliva of eight healthy subjects. Manual acupuncture and acupuncture with low-frequency electrical stimulation (2 Hz) were used. The saliva was collected during 20 minutes before the start of acupuncture stimulation, then during 20 minutes while the needles were in situ and then for another 20 minutes after the needles were removed. Four different saliva sampling techniques were used: whole resting saliva, whole saliva stimulated by paraffin-chewing, whole saliva stimulated by citric acid (1%), and parotid saliva, also stimulated with citric acid (1%). The results showed significant increases in the release of CGRP, NPY and VIP both during and after acupuncture stimulation, especially in connection with electro-acupuncture. SP showed only few increases, mainly in connection with electro-acupuncture, whereas NKA generally was unaffected by the acupuncture stimulation. The sensory stimulation-induced increase in the release of CGRP, NPY and VIP in the saliva could be an indication of their role in the improvement of salivary flow rates in xerostomic patients who had been treated with acupuncture.

The present study was performed to explore the effects of intrathecal administration of calcitonin gene-related peptide8-37 (CGRP(8-37)) on the hindpaw withdrawal latency (HWL) to pressure in rats with one thermally injured hindpaw. Furthermore, the interaction of CGRP(8-37)and naloxone was studied. Thermal injury was performed by dipping the left paw into 60 degrees C for 20 s. This induced a significant increase in the volume of the left hindpaw (P<0.001) and significant bilateral decreases of the latency of hindpaw withdrawal response to mechanical stimulation (Left: P<0.001; right: P<0.05). Intrathecal administration of 10, 20 and 40 nmol of CGRP(8-37), but not of 1 or 5 nmol, induced a significant bilateral increase in HWLs (P<0.001). The effect of CGRP(8-37) was partly reversed by intrathecal injection of naloxone at a dose of 32 and 64 microg respectively. Using radioimmunoassay, we found a significant bilateral increase in the concentration of CGRP-like immunoreactivity in the perfusate of both hindpaws 24 h after unilateral thermal injury (left: P< 0.001; right: P< 0.05). There was also an increase in the amount of CGRP-like immunoreactivity in the cerebrospinal fluid (P< 0.001), but not in plasma. The results indicate that CGRP plays a role in the transmission of nociceptive information in the spinal cord of thermally injured rats. Furthermore, our findings suggest that opioids can modulate CGRP-related effects in the spinal cord.

The object of this study was to examine whether eating behavior, food preference, gastric emptying, and gut hormone patterns are altered after jejunoileal bypass (JIB) in patients with severe obesity. Eight obese [mean (+/- SD) body mass index (BMI; in kg/m2) 42.9 +/- 4] subjects were studied prospectively before and 9 mo after JIB with eight age- and sex-matched normal-weight control subjects. Total energy intake, data from the universal eating monitor (VIKTOR), eating motivation measured by visual analog scales, a food-preference checklist, a forced-choice list, solid-phase gastric emptying, and postprandial concentrations of cholecystokinin, motilin, and neurotensin were studied. BMI was reduced by 29% after JIB. Compared with normal subjects, the JIB patients showed a reduced desire to eat, decreased hunger, and reduced prospective consumption before a test meal. After surgery, obese subjects selected fewer food items and showed a reduced preference for high-carbohydrate and high-fat items before a test meal. There was a trend from an accelerated toward a decelerated eating pattern in obese subjects after JIB. After JIB, gastric emptying of obese subjects was slowed and similar to that in control subjects. Obese subjects had lower postprandial cholecystokinin concentrations that were lower than those of control subjects both before and after JIB. Postprandial concentrations of neurotensin were higher after JIB. We conclude that after JIB, the desire to eat and preference for high-carbohydrate and high-fat items is reduced, resulting in decreased energy intake. That gastric emptying is prolonged and gut hormone patterns are altered with low postprandial plasma cholecystokinin and high neurotensin plasma concentrations may at least partly account for these observations.

Background: Little is known about factors related to prognosis in patients with carcinoid disease. In this study we have tried to identify such factors., Patients and Methods: We have evaluated 301 consecutive carcinoid patients (256 midgut, 39 foregut and six hindgut) referred during 15 years for medical treatment with respect to tumor distribution, hormone production, prognostic factors and survival., Results: Survival was significantly shorter in midgut carcinoid patients with > or = 5 liver metastases or with high levels of urinary 5-hydroxyindoleacetic acid, plasma chromogranin A or neuropeptide K. By univariate analysis, these variables together with the presence of carcinoid syndrome were related to a higher risk of dying. In multivariate analyses, performed in the 71 patients with full information on all variables, advanced age and plasma chromogranin A > 5000 micrograms/l were independent predictors of overall survival., Conclusions: Poor prognostic factors for midgut carcinoid patients were multiple liver metastases, presence of carcinoid syndrome and high levels of the tumor markers studied. In this study the only independent predictors of bad prognosis in midgut, carcinoid patients were advanced age, which however is inherently related to overall survival, and plasma chromogranin A > 5000 micrograms/l. Thus, chromogranin A may prove to be an important prognostic marker for patients with carcinoid tumors.

Five neuropeptides: Substance P (SP), Neurokinin A (NKA), Calcitonin Gene-Related Peptide (CGRP), Neuropeptide Y (NPY) and Vasoactive Intestinal Polypeptide (VIP), were measured in the saliva of eight subjects. The saliva was collected using different stimulation techniques: whole resting saliva, whole paraffin stimulated saliva, whole citric acid stimulated saliva and parotid saliva of different secretion rates -0.25 mL/min, 0.50 mL/min and 1.00 mL/min, also stimulated by citric acid. The neuropeptides were analysed by radioimmunoassay. The results showed that the concentration of all neuropeptides decreased significantly, two- to four-fold (CGRP up to 16-fold) in whole saliva, when the salivary secretion rates increased six- to eight-fold due to stimulation. However, the amounts of all neuropeptides released over time into the whole saliva increased two- to five-fold (ten-fold for CGRP) as the volumes of saliva increased due to chewing-stimulation as compared to resting saliva or citric acid stimulated saliva. There was also more CGRP in the resting saliva than in the citric acid stimulated saliva. The concentration of CGRP in the parotid saliva decreased three- to ten-fold when the salivary flow increased, whereas the concentration of NKA increased three- to four-fold and that of NPY almost two-fold under the same conditions. The concentrations of SP and VIP did not change in the different flows of parotid saliva. The release of all neuropeptides in the parotid saliva over time showed significant increases (3-14-fold) when the secretion rates increased except CGRP, which showed no changes at all. We concluded that neuropeptides are continuously released into the saliva. Their amounts increase with stimulation, but they are diluted by the increased volume of saliva, and they are also affected by the mode of stimulation-muscular activity leads to a greater release than citric acid stimulation. As the neuropeptides play an important role in the control of salivary secretory mechanisms, their normal occurrence and release are of fundamental importance for the understanding of the function of the salivary glands.

It has been recently shown that pretreatment with a low dose of Freund's adjuvant decreases the severity of adjuvant arthritis in rats. To study the involvement of the central and peripheral nervous systems in the pretreatment effect, concentrations of substance P (SP)-, neurokinin A (NKA)-, calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)-like immunoreactivities (-LI) were measured in the cerebrospinal fluid, plasma and synovial fluid 2 and 24 h after a single s.c. injection of 0.05 mg Freund's adjuvant. Increased concentrations of CGRP-LI were found in the cerebrospinal fluid, plasma and synovial fluid. NPY-LI was decreased in the cerebrospinal fluid while NKA-LI was decreased in plasma. In the synovial fluid, SP-LI was increased at 24 h and NKA-LI was increased at 2 h following treatment. Our results indicate that part of pretreatment effect of low dose of subcutaneous Freund's adjuvant in the rat may be attributed to neurogenic mechanisms.

Substance P(SP)-, neurokinin A (NKA)-, calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)-like immunoreactivity (-LI) was studied in rats' cerebrospinal fluid (CSF), plasma and perfusates (PF) from the temporomandibular joints (TMJs) at 2, 6 and 24 h following 0.01 ml injection of 2% carrageenan (CAR) into the right TMJ. SP-, NKA-, CGRP- and NPY-LI were significantly increased in both TMJ perfusates of the treated groups compared to controls. Generally an injection with CAR into the right TMJ induced a similar influence of the concentration of SP-, NKA-, CGRP- and NPY-LI in the CSF, plasma and PF at 2, 6 and 24 h following injection. However, the most pronounced changes in neuropeptide-LI occurred intra-articularly in the joint fluid, which indicates that both the sensory and sympathetic nervous system are activated in this joint following osteoarthritis induction by carrageenan.

This is a study of neurokinin A (NKA)-, calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)- like immunoreactivity(-LI) in the cerebrospinal fluid (CSF), plasma and perfusates (PF) from the temporomandibular joints (TMJs) of the rat during acute inflammation. Substance P (10(-5) M, 0.01 ml) was injected into the right TMJ of the rat. The TMJs of the control rats, were injected with 0.01 ml saline. CSF, plasma and PF from TMJs were taken at 2, 6 and 24 hrs following injection. The neuropeptide-LI level was analysed by specific radioimmunoassays and compared with control values. Unilateral injection of SP into the rat TMJ resulted in a general increase in the concentration of NKA-, CGRP- and NPY-LI in the TMJ PF at 2, 6 and 24 hrs following injection. In the CSF NKA- and CGRP-LI was increased leaving the NPY-LI unaffected. In general no changes in peptide concentrations were seen in plasma. The results indicate that SP directly or indirectly induces a local release of peptides through an action at sensory and sympathetic neurons.

The occurrence of the neuropeptides vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) in ankle joints and dorsal root ganglia (L2-L6) was analyzed in normal and arthritic Lewis rats. In addition the effect of capsaicin pretreatment was investigated. The study included 92 rats consisting of 4 groups, 23 rats in each; normal rats, normal rats given capsaicin, arthritic rats and arthritic rats pretreated with capsaicin. The localization of the neuropeptides was assessed by immunohistochemistry and the tissue concentrations were determined by radioimmunoassay (RIA). In the arthritic rats, there was a slight increase in NPY immunoreactive nerve fibres in the ankle joint synovium and bone marrow, as compared to normal rats. Notably, there was an intense fluorescence and significant increase (p < 0.01, 41%) in the number of NPY-positive megakaryocytes in the tibial bone marrow of arthritic rats. RIA showed that the concentration of NPY-like immunoreactivity (LI) was increased by 50% in the ankle joint. Pretreatment with capsaicin did not affect the increased level of NPY-LI in the ankle joint of arthritic rats. The concentration of NPY-LI in the dorsal root ganglia was not altered in arthritic rats, nor was it affected by the capsaicin treatment. No NPY immunoreactive cells could be detected in the dorsal root ganglia. The number of VIP immunoreactive nerve fibres observed in ankle joints of arthritic and normal rats did not differ. However, RIA measurements showed an 11% increase in the VIP concentration in arthritic rats, which was unaffected by capsaicin treatment. In dorsal root ganglia, RIA disclosed a 21% increase in VIP-LI, although no VIP-positive cells could be detected. Capsaicin treatment did not affect the increased concentration of VIP-LI in the dorsal root ganglia.

Glutamine supplementation to non-lipid parenteral nutrition has been demonstrated to attenuate villus atrophy and increase mucosal DNA content in the rat. This study was performed in order to determine the effects of glutamine supplementation to a balanced TPN mixture (including lipids) on epithelial cell kinetics using autoradiography. Male Sprague-Dawley rats were used. Group 1 (control) received food and an intravenous saline infusion. Group 2 received an intravenous TPN mixture including lipids but without glutamine. The same TPN mixture, glutamine replacing an isonitrogenous amount of non-essential amino acids, was given to Group 3. Animals were fed for 7 days, whereafter blood and intestinal samples were taken 1 h after injection of tritiated thymidine. Microscopy of specimens from proximal jejunum revealed a significant reduction in the number of cells in crypts and villi in both TPN groups (2 and 3) compared to orally fed animals (p < 0.001). Epithelial cell numbers were not significantly different in Group 2 and 3. Similarly, the labelling index (number of labelled cells/number of crypt cells) was not affected by glutamine administration. In plasma, glucagon concentrations in Group 2 (TPN without glutamine) seemed to decrease compared to Group 1 and 3 (p = 0.06). In this study, glutamine supplementation did not affect apithelial atrophy or cell proliferation. It is concluded, that the effects of glutamine on mucosal atrophy and renewal in jejunum may depend on the composition of the TPN mixture supplied during parenteral feeding.

The effect of acid inhibition on gastric endocrine cells was investigated in Praomys (Mastomys) natalensis. Long-term treatment (1 to 32 weeks) with an irreversible histamine 2-receptor blocker (loxtidine) caused a sustained increase in plasma gastrin levels, which was accompanied by a gradual increase in histamine and histidine decarboxylase activity of the gastric oxyntic mucosa. The density of endocrine cells in the oxyntic mucosa increased gradually, doubled by 8 weeks, and was three times that of controls after 24 weeks of treatment. Hyperplastic changes in the endocrine cell population were evident after 2 to 8 weeks in all animals, whereas dysplastic or neoplastic lesions were observed in half the animals after 16, 24, and 32 weeks of treatment. Gross tumors in the oxyntic mucosa were observed in 1/4 of the animals treated for 24 or 32 weeks. Proliferating cells were identified as enterochromaffinlike cells because they were argyrophilic and immunopositive for chromogranin A and histamine. The results demonstrate that histamine 2-receptor blockade initiated by loxtidine promotes a rapid development of enterochromaffinlike cell tumors in Mastomys and suggest a critical role for gastrin in the formation of these tumors. However, the rate and frequency by which carcinoid tumors appeared in Mastomys after acid inhibition was much greater than that reported in other species, indicating that several factors, including hormonal and genetic factors, are important in the development of gastric endocrine tumors.

Neuropeptide Y (NPY), a regulatory peptide in both the central and peripheral nervous systems, has recently been found in neuroendocrine tumors as well as in the bone marrow of rat and certain autoimmune mice, but not in human bone marrow. To investigate a possible role for NPY in the human hematopoietic system, we have prospectively studied NPY-like immunoreactivity in plasma (P-NPY-LI) and NPY mRNA in bone marrow from children with acute leukemia. Northern blot showed high levels of NPY mRNA in bone marrow and peripheral lymphoblasts from children with B-cell precursor leukemia. In situ hybridization showed NPY mRNA in malignant B-cell precursor lymphoblasts. No NPY mRNA was detected in the bone marrow of children with T-cell leukemia. P-NPY-LI was higher (P less than .001) in 51 children with leukemia (200:50 to 385 pmol/L, median:interquartile range) compared to 51 age-matched healthy controls (37:20 to 52 pmol/L). P-NPY-LI was higher (P less than .001) in those with favorable clinical risk classification. Elevated P-NPY-LI, compared with the upper age-adjusted reference limit, was only found in children with B-cell precursor leukemia (31 of 40), whereas all children with B-cell, T-cell, or myeloid leukemia (n = 11) had normal P-NPY-LI (P less than .001). During the 2- to 46-month follow-up, children with elevated P-NPY-LI had better (P less than .001) outcome compared to those with normal P-NPY-LI (79.4% v 34.6% probability for event-free survival).

Calcitonin gene-related peptide (CGRP), but not substance P (SP), was found to inhibit edema-promoting actions of inflammatory mediators (histamine, leukotrine B4, 5-hydroxytryptamine) in vivo in the hamster cheek pouch, human skin, and rat paw. The effect of CGRP was present in the low nanomolar dose range, and it was mimicked by activation of sensory nerves with capsaicin which caused release of endogenous CGRP-like immunoreactivity (IR). The findings provide new information on the potential impact of sensory nerve activation during inflammatory processes by indicating that sensory nerves may play an anti-inflammatory role.

To investigate the hypothesis that calcitonin gene-related peptide (CGRP), a potent vasodilator, is an important physiological defence against fluid overload, plasma CGRP concentrations were measured in various degrees of fluid overload in 26 haemodialysis patients, for whom diuresis, mediated by atrial natriuretic peptide (ANP), is not a possible defence mechanism. Plasma CGRP concentrations were positively correlated with the degree of fluid excess (r = 0.815, p = 0.0001) and were significantly higher in 5 patients with severe fluid overload than in those less severely affected (143 [SE 14] vs 52 [11] pmol/l; p less than 0.001). CGRP may be an effective defence against complications of fluid overload since it can increase capitance by vasodilatation.

Neuropeptides in primary afferent neurons have been found to be engaged in the immediate type of hypersensitivity. However, their role in the delayed form of hypersensitivity is not yet established. The hypothesis that substance P (SP), neurokinin A (NKA), and calcitonin gene-related peptide (CGRP) are involved in delayed hypersensitivity was tested in oxazolone-induced, murine ear allergic contact dermatitis. Concentrations of immunoreactive SP, NKA, and CGRP were measured in extracts of the eczema ears (n = 26), whereas extracts of the opposite ears were used as controls. The SP, NKA, and CGRP contents in the treated ears were on the average 28% (p = 0.001), 32% (p = 0.004), and 15% (p = 0.016), respectively, lower than in the control ears. Lower peptide concentrations in the eczema ears indicate increased release of the peptides because the peptides are rapidly metabolized locally when released and only replenished by axonal transport from the cell bodies. Our results indicate that peptides released from primary afferent neurons play a role in the delayed type of hypersensitivity reactions.

Substance P (SP)-, neurokinin A (NKA)-, neurotensin (NT)-, neuropeptide Y (NPY)- and vasoactive intestinal polypeptide (VIP)-like immunoreactivity (Ll) were measured and characterized by specific radioimmunoassays (RIA) and reverse phase high performance liquid chromatography (HPLC) in extracts of rat brain. Concentrations of SP-Ll, NKA-Ll and NT-Ll in brains of decapitated animals were 59, 49 and 64 percent lower compared to those found in animals sacrificed by focused microwave irradiation (MW). In contrast, no difference in brain NPY-Ll and VIP-Ll levels was found between animals killed by MW and decapitation. HPLC chromatograms of SP-, NKA-, NT- and NPY-Ll showed the same immunoreactive components in extracts of brains from both groups of animals. Thus, no additional immunoreactive components were formed by MW compared to those found after decapitation. The present findings may reflect an MW-induced inhibition of peptidase activity or, perhaps, a more efficient extraction of certain neuropeptides following MW treatment. The results imply that the traditional methods of sacrifice may result in the measurement of spuriously low tissue concentrations of some peptides, e.g. tachykinins, in brain.