Cytokine analysis

The supernatant from the centrifugation can be stored for cytokine analysis by enzyme-linked immunosorbent assay (if desired). 

A number of commercial antibody-based microarrays for multiplexed cytokines analysis are now available (Beckman Coulter BD Biosciences Panom-ics Pierce S S Zyomyx and others). Cytokines are essentially biomarkers of cell injury, inflammation, and apoptosis. They are released by cells in culture in response to drug action (Turtinen et al., 2004) or are elevated in serum in various disease states. Moreover, numerous cytokines are involved in cellular response and many serve as dual effectors (Asao and Fu, 2000). As a result, anticytokine microarrays are being evaluated in drug discovery for off-target toxicity testing to replace standard ELISA plate formats. 

Collect all the supernatants and aliquot them individually (50 pL each). Freeze at -80 °C until use for cytokine analysis see Subheading 3.11) (tee Note 23). 

Dupont NC, Wang K, Wadhwa PD, Culhane IF, Nelson EL. Vahdation and comparison of luminex multiplex cytokine analysis kits with ELISA determinations of a panel of nine cytokines in clinical sample culture supernatants. J Reprod Immunol 2005 66 175-191. 

A prospective clinical and serological evaluation of cytokine analysis in patients receiving botulinum toxin for movement disorders prospectively studied 117 patients (females 67.5% mean age 59.74 12.39years) treated with botulinum toxin in a total of 143 treatment cycles [23 j. While no patient reported any flu-like symptoms (FLS) at baseline, the symptom complex was subsequently reported in 19 patients (16.3%) and in 20 (14%) treatment cycles, with at least one systemic symptom reported in 49 (42%) patients in 59 (41.3%) treatment cycles. FLS and at least one symptom were reported more frequently by women (p = 0.006 and p=0.049, respectively) and by younger patients (55.6 vs 61.7years (p = 0.022)). Although the symptoms were usually considered mild, they were rated as moderate to serious after 18 (12%)cycles. Interleukin (IL)-1 3, IL-8, GRO-a, eotaxin, MCP-1 and 2, RANTES, TARCs and inducible protein 10 (IPIO) showed increased levels at 7-10 days after the botulinum toxin injections but only IPIO showed significantly increased levels in patients with FLS 69 versus 3pg/mL (p=0.007). FLS and other systemic symptoms occur after about 14% severity of FLS. 

DNA was isolated from the gel block using the DNA extraction kit (Cytokine, Russia). DNA added to the tube containing the reagents for PCR (DNA-Technology, Russia) and amplified in 35 cycles (30s 93°C, 30s 59°C and 30s at 72°C). For subsequent analysis 5 pi of the product was analyzed by gel electrophoresis as described above. 

Wetzel MA, Steele AD, Eisenstein TK, Adler MW, Henderson EE, Rogers TJ (2000) Mu-opioid induction of monocyte chemoattractant protein-1, RANTES, and IFN-gamma-inducible protein-10 expression in human peripheral blood mononuclear cells. J Immunol 165 6519-6524 Widmer U, Manogue KR, Cerami A, Sherry B (1993) Genomic cloning and promoter analysis of macrophage inflammatory protein (MIP)-2, MIP-1 alpha, and MIP-1 beta, members of the chemokine superfamily of proinflammatory cytokines. J Immunol 150 4996-5012 Ye RD (2001) Regulation of nuclear factor kappaB activation by G-protein-coupled receptors. [Review] [136 refs]. J Leukoc Biol 70 839-848... 

The chemoreceptive mechanisms in amphibia are undoubtedly worthy of further analysis, not only for their own sake, but to provide clues as to the origination of advanced chemosignal systems. As noted above, a pheromonal signal from the mental gland acts as a courtship/ receptivity inducer. The plethodontid receptivity factor (PRF) (Chap. 3) despite its size (22 kD), seems to have been converted from its internal role as an inter-cellular cytokine, to an inter-individual coordinator of reproductive activity (Rollmann et al., 1999). Endocrine or... 

Broxmeyer HE, Sherry B, Cooper S, et al. Comparative analysis of the human macrophage inflammatory protein family of cytokines (chemokines) on proliferation of human myeloid progenitor cells. Interacting effects involving suppression, synergistic suppression, and blocking of suppression. J Immunol 1993 150(8 Pt... 

Bacterial products such as lipopolysaccharides (endotoxins) and cytokines (IL-2) are able to activate the contact system in vitro and in vivo (D9, H4, H7, M41). Immediately after severe trauma or after surgical intervention and particularly during sepsis, a reduction of plasma contact system proteins has been found (C10, K1, N9). Gel filtration studies of plasma demonstrated that plasma PK after activation becomes complexed with a2-M and Cl-Inh (W4). These complexes are rapidly eliminated from the circulation in vivo. In experimental studies in which pulmonary insufficiency was induced in dogs, a significant reduction of plasma kallikrein inhibitors was observed together with reduced HMK. Analysis of the relation be-... 

Chomej, P., et al., Differential analysis of pleural effusions by fuzzy logic based analysis of cytokines, Respir. Med., 98, 308, 2004. 

Garside, P., Bunce, C., Tomlinson, R.C., Nichols, B.L. and Mowat, A.M. (1993) Analysis of enteropathy induced by tumour necrosis factor alpha. Cytokine 5, 24-30. 

Fig. 11.4 Dose-response effect of HU on ET-1 peptide release (a) from TrHBM EC cells incubated with various concentrations of HU during 48 h in the presence (A) and absence ( ) of cytokines (TNFaand IFNy at 100 U mL 1). Under the same conditions, quantitative mRNA analysis was also performed and the residual percentage of expression, in the presence (b) and absence (c) of pro-inflammatory cytokines is given.

Initial classification of some cytokines was also undertaken on the basis of the specific biological activity by which the cytokine was first discovered (e.g. TNF exhibited cytotoxic effects on some cancer cell lines CSFs promoted the growth in vitro of various leukocytes in clumps or colonies). This, too, proved an unsatisfactory classification mechanism, as it was subsequently shown that most cytokines display a range of biological activities (e.g. the major biological function of TNF is believed to be as a regulator of both the immune and inflammatory response). More recently, primary sequence analysis of cytokines coupled to determination of secondary and tertiary structure reveal that most cytokines can be grouped into one of six families (Table 8.2). 

Analysis of cytokine genes established the exact evolutionary relationships between these molecules. 

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