New effects of caffeine on corticotropin-releasing hormone (CRH)-induced stress along the intrafollicular classical hypothalamic-pituitary-adrenal (HPA) axis (CRH-R1/2, IP3 -R, ACTH, MC-R2) and the neurogenic non-HPA axis (substance P, p75NTR and TrkA) in ex vivo human male androgenetic scalp hair follicles.

BACKGROUND: Human hair is highly responsive to stress, and human scalp hair follicles (HFs) contain a peripheral neuroendocrine equivalent of the systemic hypothalamic-pituitary-adrenal (HPA) stress axis. Androgenetic alopecia (AGA) is supposed to be aggravated by stress. We used corticotropin-releasing hormone (CRH), which triggers the HPA axis, to induce a stress response in human ex vivo male AGA HFs. Caffeine is known to reverse testosterone-mediated hair growth inhibition in the same hair organ culture model. OBJECTIVES: To investigate whether caffeine would antagonize CRH-mediated stress in these HFs. METHODS: HFs from balding vertex area scalp biopsies of men affected by AGA were incubated with CRH (10-7 mol L-1 ) with or without caffeine (0·001% or 0·005%). RESULTS: Compared to controls, CRH significantly enhanced the expression of catagen-inducing transforming growth factor-ß2 (TGF-ß2) (P < 0·001), CRH receptors 1 and 2 (CRH-R1/2) (P < 0·01), adrenocorticotropic hormone (ACTH) (P < 0·001) and melanocortin receptor 2 (MC-R2) (P < 0·001), and additional stress-associated parameters, substance P and p75 neurotrophin receptor (p75NTR ). CRH inhibited matrix keratinocyte proliferation and expression of anagen-promoting insulin-like growth factor-1 (IGF-1) and the pro-proliferative nerve growth factor receptor NGF-tyrosine kinase receptor A (TrkA). Caffeine significantly counteracted all described stress effects and additionally enhanced inositol trisphosphate receptor (IP3 -R), for the first time detected in human HFs. CONCLUSIONS: These findings provide the first evidence in ex vivo human AGA HFs that the stress mediator CRH induces not only a complex intrafollicular HPA response, but also a non-HPA-related stress response. Moreover, we show that these effects can be effectively antagonized by caffeine. Thus, these data strongly support the hypothesis that stress can impair human hair physiology and induce hair loss, and that caffeine may effectively counteract stress-induced hair damage and possibly prevent stress-induced hair loss.

Light wine consumption is associated with a lower odd for cardiovascular disease in chronic kidney disease.

AIMS: To examine the association between wine consumption and the prevalence of chronic kidney disease (CKD) and cardiovascular disease (CVD). DATA SYNTHESIS: We performed a cross-sectional logistic regression analysis of National Health and Nutrition Examination Survey (NHANES) in participants 21 years of age or older from 2003 to 2006 in a large representative study of the U.S. POPULATION: Wine consumption was categorized as none (0 glass per day), light (<1 glass per day), or moderate (≥1 glasses per day). Prevalent CKD was defined as a urine albumin/creatinine ratio (UACR) ≥30 mg/g or estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2. CVD was defined as history of CVD including angina, myocardial infarction, or stroke. Only 27 (0.5%) individuals reported moderate wine consumption, whereas 57.5% and 42% reported abstinence and light wine consumption, respectively. Light wine consumption was associated with a lower prevalence of CKD as opposed to abstinence in unadjusted analysis. After adjusting for demographics and CVD risk factors light wine consumption was associated with lower prevalence of CKD defined as UACR ≥30 mg/g but not with low eGFR. Furthermore, light wine consumption was associated with significantly lower rates of CVD in the general population and in subjects with CKD. The adjusted odd of CVD for those with light wine consumption was 0.72 (CI 0.52-0.99, p = 0.046) for the subjects with CKD. CONCLUSION: These data suggest that light wine consumption (compared to abstinence) is associated with lower prevalence of CKD and a lower odd of CVD in those with CKD in the U.S.

BMP-2-coated mineral coated microparticles improve bone repair in atrophic non-unions.

Atrophic non-unions are a major clinical problem. Mineral coated microparticles (MCM) are electrolyte-coated hydroxyapatite particles that have been shown in vitro to bind growth factors electrostatically and enable a tuneable sustained release. Herein, we studied whether MCM can be used in vivo to apply Bone Morphogenetic Protein-2 (BMP-2) to improve bone repair of atrophic non-unions. For this purpose, atrophic non-unions were induced in femurs of CD-1 mice (n = 48). Animals either received BMP-2-coated MCM (MCM + BMP; n = 16), uncoated MCM (MCM; n = 16) or no MCM (NONE; n = 16). Bone healing was evaluated 2 and 10 weeks postoperatively by micro-computed tomographic (µCT), biomechanical, histomorphometric and immunohistochemical analyses. µCT revealed more bone volume with more highly mineralised bone in MCM + BMP femurs. Femurs of MCM + BMP animals showed a significantly higher bending stiffness compared to other groups. Histomorphometry further demonstrated that the callus of MCM + BMP femurs was larger and contained more bone and less fibrous tissue. After 10 weeks, 7 of 8 MCM + BMP femurs presented with complete osseous bridging, whereas NONE femurs exhibited a non-union rate of 100 %. Of interest, immunohistochemistry could not detect macrophages within the callus, indicating a good biocompatibility of MCM. In conclusion, the local application of BMP-2-coated MCM improved bone healing in a challenging murine non-union model and, thus, should be of clinical interest in the treatment of non-unions.

Catalytic reduction of NO2 with hydrogen on Pt field emitter tips: kinetic instabilities on the nanoscale.

The catalytic reduction of NO(2) with hydrogen on a Pt field emitter tip is investigated using both field electron microscopy (FEM) and field ion microscopy (FIM). A rich variety of nonlinear behavior and unusually high catalytic activity around the {012} facets are observed. Our FEM investigations reveal that the correlation function exhibits damped oscillations with a decaying envelope, showing that molecular noise will influence the dynamics of the oscillations. The dependence of the oscillatory period on the P(H(2))/P(NO(2)) pressure ratios is analyzed. Similar patterns are reported under FIM conditions. Corresponding density functional theory (DFT) calculations for the adsorption of NO(2) on Pt{012} in the presence of an external electric field are performed in order to gain an atomistic understanding of the underlying nonlinear phenomena.

ABC-transporter gene-polymorphisms are potential pharmacogenetic markers for mitoxantrone response in multiple sclerosis.

Escalation therapy with mitoxantrone (MX) in highly active multiple sclerosis is limited by partially dose-dependent side-effects. Predictors of therapeutic response may result in individualized risk stratification and MX dosing. ATP-binding cassette-transporters ABCB1 and ABCG2 represent multi-drug resistance mechanisms involved in active cellular MX efflux. Here, we investigated the role of ABC-gene single nucleotide polymorphisms (SNPs) for clinical MX response, corroborated by experimental in vitro and in vivo data. Frequencies of ABCB1 2677G>T, 3435C>T and five ABCG2-SNPs were analysed in 832 multiple sclerosis patients (Germany, Spain) and 264 healthy donors. Using a flow-cytometry-based in vitro assay, MX efflux in leukocytes from individuals with variant alleles in both ABC-genes (designated genotype ABCB1/ABCG2-L(ow), 22.2% of patients) was 37.7% lower than from individuals homozygous for common alleles (ABCB1/ABCG2-H(igh), P < 0.05, 14.8% of patients), resulting in genotype-dependent MX accumulation and cell death. Addition of glucocorticosteroids (GCs) inhibited MX efflux in vitro. ABC-transporters were highly expressed in leukocyte subsets, glial and neuronal cells as well as myocardium, i.e. cells/tissues potentially affected by MX therapy. In vivo significance was further corroborated in experimental autoimmune encephalomyelitis in Abcg2(-/-) animals. Using a MX dose titrated to be ineffective in wild-type animals, disease course and histopathology in Abcg2(-/-) mice were strongly ameliorated. Retrospective clinical analysis in MX monotherapy patients (n = 155) used expanded disability status scale, relapse rate and multiple sclerosis functional composite as major outcome parameters. The clinical response rate [overall 121 of 155 patients (78.1%)] increased significantly with genotypes associated with decreasing ABCB1/ABCG2-function [ABCB1/ABCG2-H 15/24 (62.5%) responders, ABCB1/ABCG2-I(ntermediate) 78/98 (79.6%), ABCB1/ABCG2-L 28/33 (84.8%), exact Cochran-Armitage test P = 0.039]. The odds ratio for response was 1.9 (95% CI 1.0-3.5) with each increase in ABCB1/ABCG2 score (from ABCB1/ABCG2-H to -I-, and -I to -L). In 36 patients with severe cardiac or haematological side effects no statistically relevant difference in genotype frequency was observed. However, one patient with biopsy proven cardiomyopathy only after 24 mg/m2 MX exhibited a rare genotype with variant, partly homozygous alleles in 3 ABC-transporter genes. In conclusion, SNPs in ABC-transporter genes may serve as pharmacogenetic markers associated with clinical response to MX therapy in multiple sclerosis. Combined MX/GC-treatment warrants further investigation.

Identification of distinct pathological signatures induced by patient-derived α-synuclein structures in nonhuman primates.

Dopaminergic neuronal cell death, associated with intracellular α-synuclein (α-syn)-rich protein aggregates [termed "Lewy bodies" (LBs)], is a well-established characteristic of Parkinson's disease (PD). Much evidence, accumulated from multiple experimental models, has suggested that α-syn plays a role in PD pathogenesis, not only as a trigger of pathology but also as a mediator of disease progression through pathological spreading. Here, we have used a machine learning-based approach to identify unique signatures of neurodegeneration in monkeys induced by distinct α-syn pathogenic structures derived from patients with PD. Unexpectedly, our results show that, in nonhuman primates, a small amount of singular α-syn aggregates is as toxic as larger amyloid fibrils present in the LBs, thus reinforcing the need for preclinical research in this species. Furthermore, our results provide evidence supporting the true multifactorial nature of PD, as multiple causes can induce a similar outcome regarding dopaminergic neurodegeneration.

Altered innate immune response of plasmacytoid dendritic cells in multiple sclerosis.

Plasmacytoid dendritic cells (pDCs) are of crucial importance in immune regulation and response to microbial factors. In multiple sclerosis (MS), pDCs from peripheral blood showed an immature phenotype, but its role in susceptibility to MS is not determined. Because infectious diseases are established triggers of exacerbations in MS, in this study we have characterized the expression of Toll-like receptors (TLR) and the maturation and functional properties of peripheral blood pDCs from clinically stable, untreated MS patients in response to signals of innate immunity. After stimulation of TLR-9, interferon (IFN)-alpha production by pDCs was significantly lower in MS (n = 12) compared to healthy controls (n = 9). In an allogenic two-step co-culture assay we found an impaired effect of TLR-9 stimulation on IFN-gamma expression of autologous naive T cells in MS patients (n = 4). In peripheral blood mononuclear cells, TLR-9 stimulation with type A CpG ODN resulted in a higher expression of TLR-1, -2, -4, -5 and -8 in MS patients (n = 7) compared with healthy controls (n = 11). These findings suggest an altered innate immune response to microbial stimuli in MS patients and may help understanding of why common infectious agents trigger MS attacks.

C2H2 interaction with Ni nanocrystals: towards a better understanding of carbon nanotubes nucleation in CVD synthesis.

We present a study of the early stages of carbon nanotubes nucleation in CVD synthesis by combining field ion/electron emission microscopy (FIM/FEM) and atom-probe investigation (AP) of the nickel-carbon interaction. Acetylene decomposition on Ni tips at 873K is observed to induce additional step formation on an initially facetted (polyhedral) crystal. Carbon-enriched steps are then observed to act as preferential nucleation centers of graphene sheets formation. Atom-probe experiments reveal C(2) and C(3) species and frequency dependent studies demonstrate that the origin of these species is different from C(1). Experiments provide clear evidence for the crucial role of carbon-enriched steps as nucleation sites of graphene sheets on the Ni surface.

Surface segregation of Au-Pd alloys in UHV and reactive environments: quantification by a catalytic atom probe.

The surface composition of an Au-62at%Pd alloy has been studied by means of a catalytic atom probe (CAP) before and after exposures to nitric oxide (NO) at temperatures ranging from 300 to 573K for 20min. Subsequent CAP analysis at 100K revealed a considerable surface enrichment in Pd (to approximately 80at%) after exposure at 573K. This is correlated with the occurrence of NO dissociation, and the formation of strong Pd-O bonds at the surface. Blank experiments in ultra-high vacuum reflect the surface composition of the bulk material, in excellent agreement with electron microprobe analysis. At 573K, no detectable surface segregation occurs in the absence of NO adsorption for the times and temperatures studied. However, classical Metropolis Monte-Carlo simulations performed with a semi-empirical potential on the Au(40)Pd(60) (111), (110) and (100) systems show surface enrichment of gold at equilibrium. This suggests that the temperatures of the clean surface segregation experiments are too low to reach equilibrium within times of the order of hours.

Inhibitory effects of glucocorticoids on collagen synthesis by mouse sponge granulomas and granuloma fibroblasts in culture.

The basis for the glucocorticoid-mediated decrease in tissue collagen was studied in mouse granulomas and in primary granuloma fibroblast cultures. Injection of mice for 12 days with dexamethasone (0.35 mg/kg body weight) resulted in a 50--70% inhibition of collagen synthesis and accumulation in polyvinyl sponge-induced granulomas whereas total protein synthesis was inhibited by only about 25%. The decreased collagen content of the granuloma was accounted for by both a reduced fibroblast number and diminished synthesis per cell. Growth rates, total protein synthesis and collagen synthesis were the same in granuloma fibroblast cultures derived from control or steroid-treated mice. However, addition of 3.10(-7) M hydrocortisone to the culture medium caused a 30--50% inhibition of both collagen and non-collagen protein synthesis in firbroblasts from either source. These inhibitory effects were dose- and time-dependent with a lag time of 12--24 h. Prolyl hydroxylase activity was reduced both in sponge granulomas from glucocorticoid-treated mice and in hydrocortisone-treated fibroblast cultures. However, protein synthesis was inhibited to the same extent as the inhibition of prolyl hydroxylase activity and there was no effect on peptidyl prolyl hydroxylation. These results indicate that the glucocorticoid-induced reduction of collagen synthesis and accumulation observed in mouse granulomas and primary granuloma fibroblast cultures is not specific for this protein. Furthermore, glucocorticoid-induced inhibition of collagen synthesis cannot be attributed to underhydroxylation of collagen prolyl residues.

Impact of the Asp299Gly polymorphism in the toll-like receptor 4 (tlr-4) gene on disease course of multiple sclerosis.

In multiple sclerosis patients, infection is often associated with disease deterioration. Lipopolysaccharide (LPS) from gram-negative bacteria signals via the toll-like receptor 4 (TLR-4) pathway. Therefore, we investigated the role of an Asp299Gly mutation in the TLR-4 receptor in 890 MS patients with multiple sclerosis and 350 healthy controls. No association of different genotypes with MS susceptibility, MS subtypes, or disease severity was found. In vitro LPS stimulation studies showed a significantly lower proliferation of PBMCs from donors heterozygous for the Asp299Gly mutation in comparison to PBMCs from individuals with the wild-type genotype (p=0.01). However, these functional changes seem not to have any impact on the clinical presentation of MS patients with different TLR-4 genotypes.

Effect of X-rays on the surface chemical state of Al2O3, V2O5, and aluminovanadate oxide.

The surface composition of Al(2)O(3), V(2)O(5), and aluminovanadate oxide, "V-Al-O", was studied by X-ray photoelectron spectroscopy (XPS), using Mg K(alpha) to reveal time-dependent irradiation damage of samples. Spectral parameters such as peak intensity and width and absolute and relative peak binding energies were evaluated along with the Auger parameter. Irradiation of Al(2)O(3) was found to cause partial dehydration of the surface hydroxide film, while sputter-cleaned alumina turned out to be resistant to X-rays. In V(2)O(5), a small fraction of V(4+) species was seen to form during X-ray exposure. X-ray induced damage in Al(2)O(3) and V(2)O(5) was compared to that caused by bombardment with 500 eV argon ions. The V-Al-O material which is used as a precursor of oxynitride catalysts for ammoxidation turned out to be most susceptible and could be damaged by low X-ray doses. An appreciable reduction from the V(5+) to the V(4+) formal oxidation state (the latter increases from 20 to 45% after 150 min time of exposure to Mg K(alpha) at 150 W) was found along with the decomposition of aluminum hydroxide which is believed to act as an amorphous support in this catalyst. Gas-phase analysis during X irradiation demonstrated desorption of oxygen and water molecules. X-ray induced damage is believed to be caused by electron-hole pair generation and Auger decay rather than by thermal effects since the sample surface temperature increased only slightly.

Soluble vascular cell adhesion molecule (VCAM) is associated with treatment effects of interferon beta-1b in patients with secondary progressive multiple sclerosis.

BACKGROUND: Subcutaneous IFNbeta-1b (Betaferon) is an established immunomodulatory treatment for relapsing remitting MS and active secondary progressive multiple sclerosis (SPMS). It modulates cytokine and adhesion molecule expression but long term in vivo effects of IFNbeta-1b on the immune system are not known in multiple sclerosis. OBJECTIVE: To address the effects of IFNbeta-1b on serum levels for soluble adhesion molecules and cytokine receptors from MS patients. METHODS: Serial blood samples were obtained from 40 patients of the frequent MRI subgroup (20 patients each from the placebo and the IFNbeta-1b treatment group), participating in the European multi-center clinical trial with IFNbeta-1b for secondary progressive MS, at regular intervals for up to 36 months. Soluble adhesion molecules (sVCAM, sICAM-1, sL-Selectin) as well as TNF-receptor I and II were analysed in the serum of patients by enzyme linked immunosorbent assays (ELISAs). Monthly brain MRI was performed in 34 of these patients (16 patients from the placebo and 18 from the IFNbeta-1b group) during months 1-6 and 19-24 to monitor disease activity as assessed by newly occurring gadolinium (Gd) enhancing lesions. RESULTS: An early and significant increase in sVCAM and sTNF-RII serum levels was detected in 16 out of 20 patients (80 %) treated with subcutaneous IFNbeta-1b already at month 1 but was absent in all but one patient during placebo treatment (p < 0.01). Raised sVCAM and TNF RII serum levels during months 1-6 inversely correlated with less MRI activity in the 19-24 months treatment interval in the IFNa-1b treatment group ( p = 0.0093 for TNF-RII; p = 0.047 for VCAM). CONCLUSIONS: sVCAM and sTNF RII levels in the serum of SPMS patients are increased during IFNbeta-1b therapy and may at least in part explain some of the treatment effects, like reduced immune cell transmigration.

New approaches to nanoparticle sample fabrication for atom probe tomography.

Due to their unique properties, nano-sized materials such as nanoparticles and nanowires are receiving considerable attention. However, little data is available about their chemical makeup at the atomic scale, especially in three dimensions (3D). Atom probe tomography is able to answer many important questions about these materials if the challenge of producing a suitable sample can be overcome. In order to achieve this, the nanomaterial needs to be positioned within the end of a tip and fixed there so the sample possesses sufficient structural integrity for analysis. Here we provide a detailed description of various techniques that have been used to position nanoparticles on substrates for atom probe analysis. In some of the approaches, this is combined with deposition techniques to incorporate the particles into a solid matrix, and focused ion beam processing is then used to fabricate atom probe samples from this composite. Using these approaches, data has been achieved from 10-20 nm core-shell nanoparticles that were extracted directly from suspension (i.e. with no chemical modification) with a resolution of better than ± 1 nm.

Autoantibodies to BP180 associated with bullous pemphigoid release interleukin-6 and interleukin-8 from cultured human keratinocytes.

Bullous pemphigoid is an inflammatory subepidermal blistering disease that is associated with auto- antibodies to the keratinocyte surface protein, BP180. In addition to the binding of autoantibodies, the infiltration of inflammatory cells is necessary for blister formation. Cytokines, including interleukin-6 and interleukin-8, have been implicated in the disease process of both human and experimental murine bullous pemphigoid. This study was aimed at testing the hypothesis that the binding of anti-BP180 antibodies to their target antigen triggers a signal transduction event that results in the secretion of these pro-inflammatory cytokines. Consistent with this hypothesis, treatment of cultured normal human epidermal keratinocytes with bullous pemphigoid IgG, but not control IgG, led to increased levels of interleukin-6 and interleukin-8, but not interleukin-1alpha, interleukin-1beta, tumor necrosis factor-alpha, interleukin-10, or monocyte chemoattractant protein-1, in the culture medium. This effect was concentration- and time-dependent and was abolished by depleting the bullous pemphigoid IgG of reactivity to two distinct epitopes on the BP180 NC16A domain. Upregulation of interleukin-6 and interleukin-8 was found at both protein and mRNA levels. In addition, bullous pemphigoid IgG did not induce the release of interleukin-6 and interleukin-8 from BP180-deficient keratinocytes obtained from a patient with generalized atrophic benign epidermolysis bullosa. These data indicate that bullous pemphigoid-associated autoantibodies to the human BP180 ectodomain trigger a signal transducing event that leads to expression and secretion of interleukin-6 and interleukin-8 from human keratinocytes.

Effects of oncostatin M on human cerebral endothelial cells and expression in inflammatory brain lesions.

Oncostatin M (OSM) is a member of the interleukin (IL)-6 cytokine family and modulates inflammatory responses. Here we investigated the role of OSM as an immunoregulatory factor for human cerebral endothelial cells (HCEC). Using RT-PCR we detected transcripts of the receptor components involved in OSM signaling, gp130, OSM receptor (OSMR)-beta, and leukemia inhibitory factor receptor (LIFR), in HCEC. A parallel FACS analysis revealed surface expression of gp130 and OSMR-beta, but not of LIFR on these cells. Functionally, OSM upregulated intercellular adhesion molecule-1, but did not induce vascular cell adhesion molecule-1 in HCEC. Further, OSM upregulated IL-6 and monocyte chemoattractant protein (MCP)-1, whereas IL-8 was unaffected. Combined application of tumor necrosis factor (TNF)-alpha and OSM synergistically enhanced IL-6 and MCP-1 production, but downregulated TNF-alpha-induced IL-8. As OSM regulated molecules relevant in inflammatory brain diseases, we investigated its expression in normal and pathological human brains. OSM was detected by immunohistochemistry in brains from multiple sclerosis patients in microglia, reactive astrocytes, and infiltrating leukocytes, whereas in normal brains and noninflammatory neurological diseases. immunoreactivity was absent from the parenchyma. These data suggest that immunoregulatory functions in human cerebral endothelial cells may be a mechanism by which OSM participates in the pathophysiology of inflammatory brain disease.

Site-directed mutagenesis reveals the importance of disulfide bridges and aromatic residues for structure and proliferative activity of human interleukin-4.

Mutant proteins (muteins) of human Interleukin-4 (IL4) were constructed by means of in vitro mutagenesis. The muteins were expressed in E. coli, submitted to a renaturation and purification protocol and analysed for biological activity. Exchange of the cysteines at either position 46 or 99 which form one of the three disulfide bridges resulted in a nearly complete loss of biological activity and an unstable protein. The exchange of tyrosine 124 also inactivated the protein, while a mutation of tyrosine 56 left some residual activity. Exchange of the other four cysteines or of the single tryptophane had smaller effects.

Influenza vaccination in MS: absence of T-cell response against white matter proteins.

BACKGROUND: Natural infections bear the risk of triggering MS bouts, whereas epidemiologic studies have not delineated an increased risk for disease activity after influenza virus vaccination. OBJECTIVE: To examine influenza A virus-specific and myelin protein-reactive T-cell frequencies by interferon gamma (IFNgamma)-enzyme-linked immunospot and the response of these cells by IFNgamma-reverse transcription (RT) PCR after immunization and any incidental upper respiratory tract infection (URI) in 12 patients with MS (seven with a relapsing-remitting course; five with a secondary progressive course; Kurtzke Expanded Disability Status Scale [EDSS] score from 1.0 to 6.5, without immunosuppressive treatment) and 28 healthy volunteers. RESULTS: A cellular immune response against influenza A virus was mounted in both populations at 2 weeks after vaccination. Patients with MS showed a higher relative increase (p = 0.008) than controls with respect to the number of influenza-specific T cells. Mean antibody responses against influenza A virus were increased in both populations after 2 weeks (p < 0.01). Despite these virus-specific reactions, no increase in T-cell frequencies responsive to human myelin basic protein (MBP) or recombinant human myelin oligodendrocyte protein (MOG) was observed after immunization, arguing against a general immune stimulation by influenza vaccination. In contrast, MBP-specific T-cell responses became detectable in several individuals after febrile infection. CONCLUSION: These data support the clinical observations that influenza vaccination is effective and safe in patients with MS with respect to cellular immunoreactivity against two main CNS myelin proteins.

Quantification of cytokine mRNA expression by RT PCR in samples of previously frozen blood.

In order to facilitate cytokine mRNA detection in blood cells, we have developed a highly reproducible and easily performed RNA isolation method for use with whole blood. Previously frozen human whole blood samples were lysed in guanidine thiocyanate solution to isolate total RNA. After reverse transcription a PCR method was applied to detect beta-actin and cytokine mRNA expression (interleukin-(IL)2, IL4, IL10, tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma)). The presence of cDNA was confirmed by agarose gel electrophoresis and quantitated on-line using sequence-specific fluorochrome labeled internal oligonucleotide probes. This quantitative method is based on the cleavage of fluorescent dye labeled probes by the 5' --> 3' endonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by a Sequence Detector System. The signal generated was directly proportional to the starting copy number of target molecules in the sample over 6 log concentrations and quantitative analysis of cDNA concentrations was performed in comparison to beta-actin or cytokine cDNA standards. mRNAs coding for beta-actin and TNF alpha were readily detectable in cDNAs prepared from the whole blood of eight healthy donors, while the other cytokines were expressed in lower amounts (IFN gamma, IL10) or were undetectable (IL2, IL4). The assay described is highly reproducible, requires no post PCR manipulation of the amplicons and permits the analysis of several hundred PCR reactions per day. Using this method it is possible to detect and quantify cytokine mRNA expression reliably in small amounts of previously frozen blood even after storage of samples for at least several months.

Characterization of early immunological responses in primary cultures of differentially activated human peripheral mononuclear cells.

Specific immune cell activation is a hallmark of infections and autoimmune disorders. Quantification of proliferative cell responses by (3)H-thymidine incorporation is a slow process and describes only one type of cellular reaction. We here investigated early immunological responses of purified human peripheral blood mononuclear cells to the direct stimulus alpha CD3 and antigen specific stimulation (human myelin basic protein (hMBP), tetanus toxoid, and influenza vaccine) and compared them to polyclonal LPS stimulation. Cytokine mRNA levels were quantified using real-time quantitative reverse transcriptase polymerase chain reaction (RT PCR) 4 h, 16 h, and 48 h after activation. Proliferation was measured 96 h after initiation of the cultures. Antigen specific responses were detected as early as 4 h after stimulation and followed different kinetics depending on the mode of activation. We demonstrated significant correlations of cytokine mRNA and protein expression for TNF alpha, IL10, and IFN gamma. Expression of IL2 mRNA at 16 h was correlated with proliferation indices at 96 h whereas IL4 mRNA levels were negatively correlated. Early cytokine mRNA expression in stimulated immune cells provides important functional data and is a powerful tool with which to study immunological reactions.