Crossing the Antarctica: Exploring the Effects of Appetite-Regulating Hormones and Indicators of Nutrition Status during a 93-Day Solo-Expedition.

Future deep space astronauts must maintain adequate nutrition despite highly stressful, isolated, confined and dangerous environments. The present case-study investigated appetite regulating hormones, nutrition status, and physical and emotional stress in a space analog condition: an explorer conducting a 93-day unsupported solo crossing of Antarctica. Using the dried blood spot (DBS) method, the subject drew samples of his blood on a regular basis during the expedition. The DBSs were later analyzed for the appetite regulating hormones leptin and adiponectin. Energy intake and nutritional status were monitored by analysis of albumin and globulin (including their ratio). Interleukin-6 (IL-6) was also analyzed and used as an energy sensor. The results showed a marked reduction in levels of the appetite-reducing hormone, leptin, and the appetite stimulating hormone, adiponectin, during both extreme physical and psychological strain. Nutrition status showed a variation over the expedition, with below-normal levels during extreme psychological strain and levels abutting the lower bounds of the normal range during a phase dominated by extreme physical hardship. The IL-6 levels varied substantially, with levels above the normal range except during the recovery phase. It was concluded that a daily intake of 5058 to 5931 calories seemed to allow recovery of both appetite and nutritional status between extreme physical and psychological hardship during a long Arctic expedition. Furthermore, IL-6 may be a sensor in the muscle-liver, muscle-fat and muscle-brain crosstalk. These results may help guide nutrition planning for future astronaut crews, mountaineers and others involved in highly demanding missions.

Analysis of Inflammatory Mediators in Newborn Dried Blood Spot Samples by Chip-Based Immunoaffinity Capillary Electrophoresis.

A chip-based immunoaffinity capillary electrophoresis (ICE) system has been developed for measuring inflammatory mediators in dried blood samples routinely taken from newborn babies. A defined area of each dried blood spot was removed from the sample card and its contents eluted. The recovered eluates were injected into the chip and the analytes of interest isolated by the immunoaffinity disk within the chip. The captured analytes were labeled in-situ with a red light-emitting laser dye and electro-eluted into the chip separation channel. Electrophoretic separation of all of the analytes was achieved within 2.0 min with quantification of each peak being performed by online LIF detection and integration of each peak area. The degree of accuracy and precision achieved by the chip-based system is comparable to conventional immunoassays and the system is robust enough to be applied to the analysis of clinical samples.

Recent advances in CE and microchip-CE in clinical applications: 2014 to mid-2017.

CE and microchip CE (ME) are powerful tools for the analysis of a number of different analytes and have been applied to a variety of clinical fields and human samples. This review will present an overview of the most recent applications of these techniques to different areas of clinical medicine during the period of 2014 to mid-2017. CE and ME have been applied to clinical chemistry, drug detection and monitoring, hematology, infectious diseases, oncology, endocrinology, neonatology, nephrology, and genetic screening. Samples examined range from serum, plasma, and urine to lest utilized materials such as tears, cerebral spinal fluid, sweat, saliva, condensed breath, single cells, and biopsy tissue. Examples of clinical applications will be given along with the various detection systems employed.

Measurement of Inflammatory Chemokines in Micro-dissected Tissue Biopsy Samples by Chip-Based Immunoaffinity Capillary Electrophoresis.

To aid in the biochemical analysis of human skin biopsies, a chip-based immunoaffinity capillary electrophoresis (ICE) system has been developed for measuring inflammatory chemokines in micro-dissected areas of the biopsy. Following isolation of the areas of interest, the tissue was solubilized and the analytes of interest were isolated by the immunoaffinity disk within the chip. The captured analytes were labeled in situ with a 635 nm light-emitting laser dye and electro-eluted into the chip separation channel. Electrophoretic separation of all of the analytes was achieved in 2.5 min with quantification of each peak being performed by online LIF detection and integration of each peak area. The degree of accuracy and precision achieved by the chip-based system is comparable to conventional immunoassays and the system is robust enough to be applied to the analysis of clinical samples. Further, with the expanding array of antibodies that are commercially available, this chip-based system can be applied to a wide variety of different biomedical and clinical analyses.

Isolation of a circulating CD45-, CD34dim cell population and validation of their endothelial phenotype.

Accurately detecting circulating endothelial cells (CECs) is important since their enumeration has been proposed as a biomarker to measure injury to the vascular endothelium. However, there is no single methodology for determining CECs in blood, making comparison across studies difficult. Many methods for detecting CECs rely on characteristic cell surface markers and cell viability indicators, but lack secondary validation. Here, a CEC population in healthy adult human subjects was identified by flow cytometry as CD45-, CD34dim that is comparable to a previously described CD45-, CD31bright population. In addition, nuclear staining with 7-aminoactinomycin D (7-AAD) was employed as a standard technique to exclude dead cells. Unexpectedly, the CD45-, CD34dim, 7-AAD- CECs lacked surface detectable CD146, a commonly used marker of CECs. Furthermore, light microscopy revealed this cell population to be composed primarily of large cells without a clearly defined nucleus. Nevertheless, immunostains still demonstrated the presence of the lectin Ulex europaeus and von Willebrand factor. Ultramicro analytical immunochemistry assays for the endothelial cell proteins CD31, CD34, CD62E, CD105, CD141, CD144 and vWF indicated these cells possess an endothelial phenotype. However, only a small amount of RNA, which was mostly degraded, could be isolated from these cells. Thus the majority of CECs in healthy individuals as defined by CD45-, CD34dim, and 7-AAD- have shed their CD146 surface marker and are senescent cells without an identifiable nucleus and lacking RNA of sufficient quantity and quality for transcriptomal analysis. This study highlights the importance of secondary validation of CEC identification.

A structural framework for a near-minimal form of life: mass and compositional analysis of the helical mollicute Spiroplasma melliferum BC3.

Spiroplasma melliferum is a wall-less bacterium with dynamic helical geometry. This organism is geometrically well defined and internally well ordered, and has an exceedingly small genome. Individual cells are chemotactic, polar, and swim actively. Their dynamic helicity can be traced at the molecular level to a highly ordered linear motor (composed essentially of the proteins fib and MreB) that is positioned on a defined helical line along the internal face of the cell's membrane. Using an array of complementary, informationally overlapping approaches, we have taken advantage of this uniquely simple, near-minimal life-form and its helical geometry to analyze the copy numbers of Spiroplasma's essential parts, as well as to elucidate how these components are spatially organized to subserve the whole living cell. Scanning transmission electron microscopy (STEM) was used to measure the mass-per-length and mass-per-area of whole cells, membrane fractions, intact cytoskeletons and cytoskeletal components. These local data were fit into whole-cell geometric parameters determined by a variety of light microscopy modalities. Hydrodynamic data obtained by analytical ultracentrifugation allowed computation of the hydration state of whole living cells, for which the relative amounts of protein, lipid, carbohydrate, DNA, and RNA were also estimated analytically. Finally, ribosome and RNA content, genome size and gene expression were also estimated (using stereology, spectroscopy and 2D-gel analysis, respectively). Taken together, the results provide a general framework for a minimal inventory and arrangement of the major cellular components needed to support life.

HIV-1 Vpr induces adipose dysfunction in vivo through reciprocal effects on PPAR/GR co-regulation.

Viral infections, such as HIV, have been linked to obesity, but mechanistic evidence that they cause adipose dysfunction in vivo is lacking. We investigated a pathogenic role for the HIV-1 accessory protein viral protein R (Vpr), which can coactivate the glucocorticoid receptor (GR) and co-repress peroxisome proliferator-activated receptor γ (PPARγ) in vitro, in HIV-associated adipose dysfunction. Vpr circulated in the blood of most HIV-infected patients tested, including those on antiretroviral therapy (ART) with undetectable viral load. Vpr-mediated mechanisms were dissected in vivo using mouse models expressing the Vpr transgene in adipose tissues and liver (Vpr-Tg) or infused with synthetic Vpr. Both models demonstrated accelerated whole-body lipolysis, hyperglycemia and hypertriglyceridemia, and tissue-specific findings. Fat depots in these mice had diminished mass, macrophage infiltration, and blunted PPARγ target gene expression but increased GR target gene expression. In liver, we observed blunted PPARα target gene expression, steatosis with decreased adenosine monophosphate-activated protein kinase activity, and insulin resistance. Similar to human HIV-infected patients, Vpr circulated in the serum of Vpr-Tg mice. Vpr blocked differentiation in preadipocytes through cell cycle arrest, whereas in mature adipocytes, it increased lipolysis with reciprocally altered association of PPARγ and GR with their target promoters. These results delineate a distinct pathogenic sequence: Vpr, released from HIV-1 in tissue reservoirs after ART, can disrupt PPAR/GR co-regulation and cell cycle control to produce adipose dysfunction and hepatosteatosis. Confirmation of these mechanisms in HIV patients could lead to targeted treatment of the metabolic complications with Vpr inhibitors, GR antagonists, or PPARγ/PPARα agonists.

Detection of cerebral spinal fluid-associated chemokines in birth traumatized premature babies by chip-based immunoaffinity CE.

A major concern in treating premature infants with birth-associated head trauma is the rapid determination of reliable biomarkers of neuroinflammation. To this end a chip-based immunoaffinity CE device has been applied to determine the concentrations of inflammation-associated chemokines in samples of cerebral spinal fluid collected from such subjects. The chip utilizes replaceable immunoaffinity disks, to which reactive antibody fragments (FAb) of six antichemokine-specific antibodies were immobilized. Following injection of a sample into the device, the analytes were captured by the immobilized FAbs, labeled in situ with a red laser dye, chemically released and separated by CE. Each resolved peak was measured on-line by LIF detection and the results compared to standard curves produced by running known chemokine standards through the immunoaffinity system. The complete processing of a sample took 10 min with separation of all six analytes being achieved in less than 2 min. The system compared well to commercial ELISA, analysis of the results by linear regression demonstrating r(2) values in the range of 0.903-0.978, and intra and interassay CV of the migration times and the measured peak areas being less than 2.3 and 5%, respectively. Application of the system to analysis of cerebrospinal fluid from head traumatized babies clearly indicated the group with mild trauma versus those with severe injury. Additionally, CE analysis demonstrated that the severe trauma group could be divided into individuals with good and poor prognosis, which correlated with the clinical finding for each patient.

Psychological hardiness predicts neuroimmunological responses to stress.

Psychological hardiness characterizes people who remain healthy under psychosocial stress. The present exploratory study investigates possible links between hardiness and several immune and neuroendocrine markers: IL-6, IL-12, IL-4, IL-10, & neuropeptide-Y. A total of 21 Norwegian navy cadets were studied in the context of a highly stressful military field exercise. Blood samples were collected midway, and again late in the exercise when stress levels were highest. Psychological hardiness (including commitment, control, and challenge) was measured two days before the exercise. While all subjects scored high in hardiness, some were high only in commitment and control, but relatively low in challenge. These "unbalanced" hardiness subjects were also more stress reactive, showing suppressed proinflammatory cytokines (IL-12), increased anti-inflammatory cytokines (IL-4, IL-10), and lower neuropeptide-Y levels as compared to the hardiness-balanced group. This study thus shows that being high in hardiness with a balanced profile is linked to more moderate and healthy immune and neuroendocrine responses to stress.

Stromal factors SDF1α, sFRP1, and VEGFD induce dopaminergic neuron differentiation of human pluripotent stem cells.

Human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons hold potential for treating Parkinson's disease (PD) through cell replacement therapy. Generation of DA neurons from hESCs has been achieved by coculture with the stromal cell line PA6, a source of stromal cell-derived inducing activity (SDIA). However, the factors produced by stromal cells that result in SDIA are largely undefined. We previously reported that medium conditioned by PA6 cells can generate functional DA neurons from NTera2 human embryonal carcinoma stem cells. Here we show that PA6-conditioned medium can induce DA neuronal differentiation in both NTera2 cells and the hESC I6 cell line. To identify the factor(s) responsible for SDIA, we used large-scale microarray analysis of gene expression combined with mass spectrometric analysis of PA6-conditioned medium (CM). The candidate factors, hepatocyte growth factor (HGF), stromal cell-derived factor-1 α (SDF1α), secreted frizzled-related protein 1 (sFRP1), and vascular endothelial growth factor D (VEGFD) were identified, and their concentrations in PA6 CM were established by immunoaffinity capillary electrophoresis. Upon addition of SDF1α, sFRP1, and VEGFD to the culture medium, we observed an increase in the number of cells expressing tyrosine hydroxylase (a marker for DA neurons) and ßIII-tubulin (a marker for immature neurons) in both the NTera2 and I6 cell lines. These results indicate that SDF1α, sFRP1, and VEGFD are major components of SDIA and suggest the potential use of these defined factors to elicit DA differentiation of pluripotent human stem cells for therapeutic intervention in PD.

Maternal immune activation by LPS selectively alters specific gene expression profiles of interneuron migration and oxidative stress in the fetus without triggering a fetal immune response.

Maternal immune activation (MIA) is a risk factor for the development of schizophrenia and autism. Infections during pregnancy activate the mother's immune system and alter the fetal environment, with consequential effects on CNS function and behavior in the offspring, but the cellular and molecular links between infection-induced altered fetal development and risk for neuropsychiatric disorders are unknown. We investigated the immunological, molecular, and behavioral effects of MIA in the offspring of pregnant Sprague-Dawley rats given an intraperitoneal (0.25 mg/kg) injection of lipopolysaccharide (LPS) on gestational day 15. LPS significantly elevated pro-inflammatory cytokine levels in maternal serum, amniotic fluid, and fetal brain at 4 h, and levels decreased but remained elevated at 24 h. Offspring born to LPS-treated dams exhibited reduced social preference and exploration behaviors as juveniles and young adults. Whole genome microarray analysis of the fetal brain at 4 h post maternal LPS was performed to elucidate the possible molecular mechanisms by which MIA affects the fetal brain. We observed dysregulation of 3285 genes in restricted functional categories, with increased mRNA expression of cellular stress and cell death genes and reduced expression of developmentally-regulated and brain-specific genes, specifically those that regulate neuronal migration of GABAergic interneurons, including the Distal-less (Dlx) family of transcription factors required for tangential migration from progenitor pools within the ganglionic eminences into the cerebral cortex. Our results provide a novel mechanism by which MIA induces the widespread down-regulation of critical neurodevelopmental genes, including those previously associated with autism.

Aberrant heart rate and brainstem brain-derived neurotrophic factor (BDNF) signaling in a mouse model of Huntington's disease.

Huntington's disease (HD) is associated with profound autonomic dysfunction including dysregulation of cardiovascular control often preceding cognitive or motor symptoms. Brain-derived neurotrophic factor (BDNF) levels are decreased in the brains of HD patients and HD mouse models, and restoring BDNF levels prevents neuronal loss and extends survival in HD mice. We reasoned that heart rate changes in HD may be associated with altered BDNF signaling in cardiovascular control nuclei in the brainstem. Here we show that heart rate is elevated in HD (N171-82Q) mice at presymptomatic and early disease stages, and heart rate responses to restraint stress are attenuated. BDNF levels were significantly reduced in brainstem regions containing cardiovascular nuclei in HD mice and human HD patients. Central administration of BDNF restored the heart rate to control levels. Our findings establish a link between diminished BDNF expression in brainstem cardiovascular nuclei and abnormal heart rates in HD mice, and suggest a novel therapeutic target for correcting cardiovascular dysfunction in HD.

Assessment of chemokine profiles in human skin biopsies by an immunoaffinity capillary electrophoresis chip.

Atopic dermatitis is a skin condition resulting in a skin rash from exposure to environmental factors. Skin biopsies taken from patients suffering from atopic dermatitis were micro-dissected and analyzed using a microchip-based immunoaffinity CE system for the presence of CXCL1, CXCL5 and CXCL8 and CCL1, CCL3 and CCL5 chemokines. Disposable immunoaffinity disks with immobilized antibodies were used to capture the CXC and CC chemokines from the homogenized skin samples. The captured analytes were then labeled with AlexaFluor 633, eluted from the disk and separated by CE. The labeled chemokines were identified and quantified by laser induced fluorescence. The total analysis time was less than 40min, including the biopsy microdissection, pre-analysis preparation of the sample and the ICE-CHIP analysis, which took less than 10min with inter- and intra-assay CV's below 6.4%. Microchip-based immunoaffinity CE could distinguish between normal skin biopsies and those with inflammation. Patients with neutrophil cellular infiltrates by histopathology showed increased concentrations of CXCL1, CXCL5 and CXCL8 while increases of CCL1, CCL3 and CCL5 corresponded to the patient group demonstrating monocytic and T-lymphocyte infiltration by histopathology. This system demonstrates the ability to identify and quantify immunochemical analytes in frozen sections taken from clinical histopathology samples.

Validity and reliability of perinatal biomarkers of adiposity after storage as dried blood spots on paper.

OBJECTIVE: To validate use of chip-based immunoaffinity capillary electrophoresis on dried blood spot samples (DBSS) to measure obesity-related hormones. METHODS: Chip-based immunoaffinity capillary electrophoresis was used to measure adiponectin, leptin and insulin in capillary serum and DBSS in pregnant women and infant heelstick at birth and six weeks. Concordance of measurements was determined with Pearson's correlation and Bland-Altman plots. RESULTS: We report high concordance between results obtained from serum and DBSS. CONCLUSIONS: Ease of sample collection and storage makes DBSS an optimal method for use in studies involving neonates and young children, as well as studies conducted in areas where freezer storage is not available.

Strong viremia control in vaccinated macaques does not prevent gradual Th17 cell loss from central memory.

It has been proposed that microbial translocation might play a role in chronic immune activation during HIV/SIV infection. Key roles in fighting bacterial and fungal infections have been attributed to Th17 and Tc17 cells. Th17 cells can be infected with HIV/SIV, however whether effective vaccination leads to their maintenance following viral challenge has not been addressed. Here we retrospectively investigated if a vaccine regimen that potently reduced viremia post-challenge preserved Th17 and Tc17 cells, thus adding benefit in the absence of sterilizing protection. Rhesus macaques were previously vaccinated with replication-competent Adenovirus recombinants expressing HIVtat and HIVenv followed by Tat and gp140 protein boosting. Upon SHIV(89.6P) challenge, the vaccines exhibited a significant 4 log reduction in chronic viremia compared to sham vaccinated controls which rapidly progressed to AIDS [39]. Plasma and cryopreserved PBMC samples were examined pre-challenge and during acute and chronic infection. Control macaques exhibited a rapid loss of CD4(+) cells, including Th17 cells. Tc17 cells tended to decline over the course of infection although significance was not reached. Immune activation, assessed by Ki-67 expression, was associated with elevated chronic viremia of the controls. Significantly increased plasma IFN-γ levels were also observed. No increase in plasma LPS levels were observed suggesting a lack of microbial translocation. In contrast, vaccinated macaques had no evidence of immune activation within the chronic phase and preserved both CD4(+) T-cells and Tc17 cells in PBMC. Nevertheless, they exhibited a gradual, significant loss of Th17 cells which concomitantly displayed significantly higher CCR6 expression over time. The gradual Th17 cell decline may reflect mucosal homing to inflammatory sites and/or slow depletion due to ongoing low levels of SHIV replication. Our results suggest that potent viremia reduction during chronic SHIV infection will delay but not prevent the loss of Th17 cells.

Immunoaffinity capillary electrophoresis: a new versatile tool for determining protein biomarkers in inflammatory processes.

Many diseases caused by inflammatory processes can progress to a chronic state causing deterioration in the quality of life and a poor prognosis for long-term survival. To address inflammatory diseases effectively, early detection and novel therapeutics are required. However, this can be challenging, in part because of the lack of early predictive biomarkers and the limited availability of adequate technologies capable of the identification/characterization of key predictive biomarkers present in biological materials, especially those found at picomolar concentrations and below. This review highlights the need for state-of-the art methodologies, with high-sensitivity and high-throughput capabilities, for determination of multiple biomarkers. Although many new biomarkers have been discovered recently, existing technology has failed to successfully bring this advancement to the patient's bedside. We present an overview of the various advances available today to extend the discovery of predictive biomarkers of inflammatory diseases; in particular, we review the technology of immunoaffinity capillary electrophoresis (IACE), which combines the use of antibodies as highly selective capture agents with the high resolving power of capillary electrophoresis. This two-dimensional hybrid technology permits the quantification and characterization of several protein biomarkers simultaneously, including subtle structural changes such as variants, isoforms, peptide fragments, and post-translational modifications. Furthermore, the results are rapid, sensitive, can be performed at a relatively low cost, without the introduction of false positive or false negative data. The IACE instrumentation can have relevance to medical, pharmaceutical, environmental, military, cultural heritage (authenticity of art work), forensic science, industrial and research fields, and in particular as a point-of-care biomarker analyzer in translational medicine.

Goitrogenic anions, thyroid-stimulating hormone, and thyroid hormone in infants.

BACKGROUND: Environmental exposure of infants to perchlorate, thiocyanate, nitrate, might interfere with thyroid function. U.S. women with higher background perchlorate exposure have higher thyroid-stimulating hormone (TSH) and lower thyroxine (T4). There are no studies with individual measures of thyroid function and these goitrogens available in infants. OBJECTIVE: We examined the association of urinary perchlorate, nitrate, iodide, and thiocyanate with urinary T4 and TSH in infants and whether that association differed by sex or iodide status. METHODS: We used data and samples from the Study of Estrogen Activity and Development, which assessed hormone levels of full-term infants over the first 12 months of life. The study included 92 full-term infants between birth and 1 year of age seen up to four times. Perchlorate, thiocyanate, nitrate, and iodide were measured in 206 urine samples; TSH and T4 and were measured in urines and in 50 blood samples. RESULTS: In separate mixed models, adjusting for creatinine, age, sex, and body mass index, infants with higher urinary perchlorate, nitrate or thiocyanate had higher urinary TSH. With all three modeled, children with higher nitrate and thiocyanate had higher TSH, but higher perchlorate was associated with TSH only in children with low iodide. Unexpectedly, exposure to the three chemicals was generally associated with higher T4. CONCLUSIONS: The association of perchlorate exposure with increased urinary TSH in infants with low urinary iodide is consistent with previous findings. Higher thiocyanate and nitrate exposure were also associated with higher TSH in infants.

Age and energy intake interact to modify cell stress pathways and stroke outcome.

OBJECTIVE: Age and excessive energy intake/obesity are risk factors for cerebrovascular disease, but it is not known if and how these factors affect the extent of brain damage and outcome in ischemic stroke. We therefore determined the interactions of age and energy intake on the outcome of ischemic brain injury, and elucidated the underlying mechanisms. METHODS: We utilized a novel microchip-based immunoaffinity capillary electrophoresis technology to measure a panel of neurotrophic factors, cytokines, and cellular stress resistance proteins in brain tissue samples from young, middle-aged, and old mice that had been maintained on control or energy-restricted diets prior to middle cerebral artery occlusion and reperfusion. RESULTS: Mortality from focal ischemic stroke was increased with advancing age and reduced by an intermittent fasting (IF) diet. Brain damage and functional impairment were reduced by IF in young and middle-aged mice, but not in old mice. The basal and poststroke levels of neurotrophic factors (brain-derived neurotrophic factor and basic fibroblast growth factor), protein chaperones (heat shock protein 70 and glucose regulated protein 78), and the antioxidant enzyme heme oxygenase-1 were decreased, whereas levels of inflammatory cytokines were increased in the cerebral cortex and striatum of old mice compared with younger mice. IF coordinately increased levels of protective proteins and decreased inflammatory cytokines in young, but not in old mice. INTERPRETATION: Reduction in dietary energy intake differentially modulates neurotrophic and inflammatory pathways to protect neurons against ischemic injury, and these beneficial effects of IF are compromised during aging, resulting in increased brain damage and poorer functional outcome.

Analysis of neurotrophins in human serum by immunoaffinity capillary electrophoresis (ICE) following traumatic head injury.

Neurotrophins, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and beta-nerve growth factor (beta-NGF), play an active role in the development, maintenance and survival of cells of the central nervous system (CNS). Previous research has indicated that a decrease in concentrations of these neurotrophins is often associated with cell death and ultimately patient demise. However, much of the research conducted analyses of samples taken directly from the CNS, i.e., of samples that are not readily available in clinical trauma centers. In an attempt to obtain a method for evaluating neurotrophins in a more readily accessible matrix, i.e., serum, a precise and accurate immunoaffinity capillary electrophoresis (ICE) method was developed and applied to measure neurotrophins in serum from patients with various degrees of head injury. The five neurotrophins of interest were extracted and concentrated by specific immunochemically immobilized antibodies, bound directly to the capillary wall, and eluted and separated in approximately 10min. NT-3, BDNF, CNTF and beta-NGF showed a marked decrease in concentration as the severity of the head injury increased: mild versus severe: 91% decrease for NT-3; 93 % decrease for BDNF; 93 % decrease for CNTF; and a 87 % decrease for beta-NGF. This decrease in concentration is consistent with the neuro-protective roles that neurotrophins play in the maintenance and survival of neuronal cells. The results obtained by the ICE method were closely comparable with those generated by a commercially available ELISA method.