Circulating Klotho is linked to prognosis of acute intracerebral hemorrhage.

BACKGROUND: Klotho is an anti-aging protein and its increased plasma concentrations were related to good functional outcome of acute ischemic stroke. This study was designed to ascertain the prognostic significance of plasma Klotho in intracerebral hemorrhage. METHODS: Plasma Klotho concentrations in 96 intracerebral hemorrhage patients and 96 healthy controls were quantified. Poor prognosis was defined as modified Rankin scale scores >2 at 90 days. The association of plasma Klotho concentrations with stroke prognosis was assessed using regression model. RESULTS: Patients showed a substantially lower concentration of Klotho than healthy controls (P < .01). Klotho concentrations were highly correlated with National Institutes of Health Stroke Scale scores, Glasgow coma scale scores, intracerebral hemorrhage scores and hematoma volumes (r = -0.426, 0.382, -0.334 and - 0.432). Patients with the highest plasma Klotho concentration were less prone to have poor prognosis at 90 days compared with the lowest quartile (odds ratio, 0.092; 95% confidence interval, 0.015-0.562). Its optimal cutoff value for distinguishing patients at risk of poor prognosis was 345 pg/ml, which yielded a sensitivity value of 0.86 and a specificity value of 0.62. CONCLUSIONS: Decreased plasma Klotho concentrations were associated with increasing severity and poor prognosis significantly, indicating the prognostic role of plasma Klotho in intracerebral hemorrhage.

[Prokaryotic expression of human GITRaa27-165 and preparation of its polyclonal antibody].

AIM: To express and purify the extracellular region of human glucocorticoid-induced tumor necrosis factor (hGITR(aa27-165)), and to prepare and identify the polyclonal antibody (pAb) against this fusion protein. METHODS: The 429 bp DNA sequence of hGITR(aa27-165) was obtained from pGEM T-hGITR by PCR and then it was inserted into pQE30 plasmid to construct the prokaryotic expression plasmid pQE30-hGITR(aa27-165). pQE30-hGITR(aa27-165) was transformed into E.coli. The target fusion protein was expressed with the induction of Isopropyl beta-D-1-thiogalacto- pyranoside (IPTG) and purified by Ni(2+)-NTA affinity chromatography column. The antiserum against hGITR(aa27-165) was obtained from the rabbits immunized with hGITR(aa27-165). The titer of pAb was detected by ELISA and the specificity of pAb was identified by Western blot. RESULTS: The prokaryotic expression plasmid pQE30-GITR(aa27-165) was constructed successfully. The culture condition in which the target fusion protein was highly expressed was found out: the optimal concentration of IPTG was 0.5 mmol/L, the culture temperature was 30 degree centigrade and the culture time was 6 h. The GITR(aa27-165) fusion protein was effectively expressed in E.coli as inclusion body. Soluble protein was obtained through denaturation and refolding procedure. The fusion protein was purified by Profinity IMAC Ni-Charged Resin affinity column with above 90% purity. The anti-GITR(aa27-165) pAb was prepared by immunizing the rabbits with the purified target fusion protein. ELISA showed the titer of pAb was 1:1.6 x 10(5). Western blot analysis confirmed anti-GITR(aa27-165) pAb was of good specificity. CONCLUSION: The fusion protein hGITR(aa27-165) with high purity has been obtained and the anti-hGITR(aa27-165) pAb with high titer and good specificity has been prepared. Our study may be conductive to further research into the molecular mechanism of action between human GITR and GITRL.