Sepsis-Induced Acute Lung Injury (ALI) Is Milder in Diabetic Rats and Correlates with Impaired NFkB Activation

Acute lung injury (ALI) develops in response to a direct insult to the lung or secondarily to a systemic inflammatory response, such as sepsis. There is clinical evidence that the incidence and severity of ALI induced by direct insult are lower in diabetics. In the present study we investigated whether the same occurs in ALI secondarily to sepsis and the molecular mechanisms involved. Diabetes was induced in male Wistar rats by alloxan and sepsis by caecal ligation and puncture surgery (CLP). Six hours later, the lungs were examined for oedema and cell infiltration in bronchoalveolar lavage. Alveolar macrophages (AMs) were cultured in vitro for analysis of I kappa B and p65 subunit of NF kappa B phosphorylation and MyD88 and SOCS-1 mRNA. Diabetic rats were more susceptible to sepsis than non-diabetics. In non-diabetic rats, the lung presented oedema, leukocyte infiltration and increased COX2 expression. In diabetic rats these inflammatory events were significantly less intense. To understand why diabetic rats despite being more susceptible to sepsis develop milder ALI, we examined the NF kappa B activation in AMs of animals with sepsis. Whereas in non-diabetic rats the phosphorylation of I kappa B and p65 subunit occurred after 6 h of sepsis induction, this did not occur in diabetics. Moreover, in AMs from diabetic rats the expression of MyD88 mRNA was lower and that of SOCS-1 mRNA was increased compared with AMs from non-diabetic rats. These results show that ALI secondary to sepsis is milder in diabetic rats and this correlates with impaired activation of NF kappa B, increased SOCS-1 and decreased MyD88 mRNA.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)National Institutes of HealthNational Institutes of Health [NIH R00HL103777-03

Expression of human protein S100A7 (psoriasin), preparation of antibody and application to human larynx squamous cell carcinoma

Background\ud Up-regulation of S100A7 (Psoriasin), a small calcium-binding protein, is associated with the development of several types of carcinomas, but its function and possibility to serve as a diagnostic or prognostic marker have not been fully defined. In order to prepare antibodies to the protein for immunohistochemical studies we produced the recombinant S100A7 protein in E. coli. mRNA extracted from human tracheal tumor tissue which was amplified by RT-PCR to provide the region coding for the S100A7 gene. The amplified fragment was cloned in the vector pCR2.1-TOPO and sub-cloned in the expression vector pAE. The protein rS100A7 (His-tag) was expressed in E. coli BL21::DE3, purified by affinity chromatography on an Ni-NTA column, recovered in the 2.0 to 3.5 mg/mL range in culture medium, and used to produce a rabbit polyclonal antibody anti-rS100A7 protein. The profile of this polyclonal antibody was evaluated in a tissue microarray.\ud \ud \ud Results\ud The rS100A7 (His-tag) protein was homogeneous by SDS-PAGE and mass spectrometry and was used to produce an anti-recombinant S100A7 (His-tag) rabbit serum (polyclonal antibody anti-rS100A7). The molecular weight of rS100A7 (His-tag) protein determined by linear MALDI-TOF-MS was 12,655.91 Da. The theoretical mass calculated for the nonapeptide attached to the amino terminus is 12,653.26 Da (delta 2.65 Da). Immunostaining with the polyclonal anti-rS100A7 protein generated showed reactivity with little or no background staining in head and neck squamous cell carcinoma cells, detecting S100A7 both in nucleus and cytoplasm. Lower levels of S100A7 were detected in non-neoplastic tissue.\ud \ud \ud Conclusions\ud The polyclonal anti-rS100A7 antibody generated here yielded a good signal-to-noise contrast and should be useful for immunohistochemical detection of S100A7 protein. Its potential use for other epithelial lesions besides human larynx squamous cell carcinoma and non-neoplastic larynx should be explored in future.FAPESP doctoral fellowship n°. 05/50781-2CTC/CEPID/FAPESP [grant n. 1998/14247-6

Effects of isoflavones on the coagulation and fibrinolytic system of postmenopausal women

Objective: We evaluated the effects of soy isoflavone supplementation on hemostasis in healthy postmenopausal women. Methods: In this double-blinded, placebo-controlled study, 47 postmenopausal women 47-66 y of age received 40 mg of soy isoflavone (n = 25) or 40 mg of casein placebo (n = 22) once a day for 6 mo. Levels of factors VII and X. fibrinogen, thrombin-antithrombin complex, prothrombin fragments I plus 2, antithrombin, protein C, total and free protein S, plasminogen, plasminogen activator inhibitor-1, and D-dimers were measured at baseline and 6 mo. Urinary isoflavone concentrations (genistein and daidzein) were measured as a marker of compliance and absorption using high-performance liquid chromatography. Baseline characteristics were compared by unpaired Student`s t test. Within-group changes and comparison between the isoflavone and casein placebo groups were determined by a mixed effects model. Results: The levels of hemostatic variables did not change significantly throughout the study in the isoflavone group; however, the isoflavone group showed a statistically significant reduction in plasma concentration of prothrombin fragments I plus 2; both groups showed a statistically significant reduction in antithrombin, protein C, and free protein S levels. A significant increase in D-dimers was observed only in the isoflavone group. Plasminogen activator inhibitor-l levels increased significantly in the placebo group. However, these changes were not statistically different between groups. Conclusion: The results of the present study do not support a biologically significant estrogenic effect of soy isoflavone on coagulation and fibrinolysis in postmenopausal women. However, further research will be necessary to definitively assess the safety and efficacy of isoflavone. (D 2008 Elsevier Inc. All rights reserved

Inflammatory cell infiltration in BAL fluid and lung COX2 expression at 6h after CLP.

<p>Diabetic and non-diabetic rats were submitted to CLP or SHAM (false surgery) and after 6h bronchoalveolar lavage (BALF) was performed. (<b>A</b>) mononuclear and PMN cell were counted in haematoxilin-eosin stained cytospin preparations of BALF cells after total cell count was performed under light microscopy. (<b>B</b>) Expression of COX2 protein in lung homogenates six hours after CLP analysed by immunoblotting using antibodies to COX-2 and quantified by densitometric analysis of the immunoblot bands. Density values of bands were normalized to the total β-actin present in each lane and expressed as a percentage of control. n = 5/group, data are presented as mean ± SEM; *, P<0.05.</p

NFkB activation in alveolar macrophages 6h after CLP.

<p>Alveolar macrophages <b>(</b>AM) were obtained by lung lavage six hours after CLP and allowed to adhere in culture plates for 1 h. Total mRNA or total protein was extracted from AMs. (<b>A</b>) 20 µg of total protein analysed by immunoblotting using antibodies to phosphorylated – IκBα and β-actin.The bands were quantified by densitometric analysis. Density values of bands were normalized to the total β-actin present in each lane and expressed as percentage of control. (<b>B</b>) 50 µg of total protein analysed by immunoblotting using antibodies to phosphorylated – p65 and β-actin. (<b>C</b>) cDNA was synthesized from total mRNA extracted and the expression of SOCS-1. (<b>D</b>) MyD88 mRNA was analysed by RT-PCR. mRNA expression levels were calculated by the comparative Ct method and normalized to GAPDH levels with non-diabetic CLP given an arbitrary value of one. n = 5/group, data were presented as mean ± SEM.* P<0.05; ***, P<0.001.</p

Blood glucose levels (mg/dL) in diabetic^* and non-diabetic rats and effect of sepsis induced by CLP^**.

*<p>Alloxan (42 mg/Kg) was given i.v. and glucose levels were determinate 10 days later.</p>**<p>CLP – Colon Ligation and Puncture (20 G needle –12 punctures).</p

Lung oedema at 6 h after CLP.

<p>Diabetic and non-diabetic rats were submitted to CLP and after 6 h the lungs were removed and processed. (<b>A</b>) Photomicrographs of peribronchovascular axis in lung stained with haematoxylin-eosin; ‘<b>B</b>’ stands for bronchiole and ‘<b>V</b>’ for venule. Note the presence of oedema around the venule (leakage area marked in black bars). Photographs were taken at an original magnification of 200x. (<b>B</b>) Quantification of perivascular oedema by light microscopy with an integrating eyepiece with a coherent system consisting of a grid with 100 points and 50 lines (known length).The number of points falling on areas of perivascular oedema and the number of intercepts between the lines of the integrating eyepiece and the basal membrane of the vessels were counted. The oedema index was calculated as follows: number of points^1/2/number of intercepts. Ten random non-coincident microscopic fields containing a bronchus and a venule were evaluated for each group, n = 5 per group. (<b>C</b>) Evaluation of lung oedema by total protein content in the BAL after 6 hours of CLP or sham-operated, n = 5/group and scale bar  = 50 µm. Data are presented as mean ± SEM. ***P<0.001.</p

Sequence of primer pairs used in semi-quantitative real-time PCR.

<p>Sequence of primer pairs used in semi-quantitative real-time PCR.</p

Inflammatory cell infiltration in lung at 6h after CLP.

<p>Diabetic and non-diabetic rats were submitted to CLP and after 6h the lungs were removed and processed. (<b>A</b>) Photomicrographs of lung parenchyma stained with haematoxylin-eosin. (<b>B</b>) Mononuclear and polymorphonuclear cell index was determined in the parenchyma. The cell index quantification was performed with an integrating eyepiece with a coherent system consisting of a grid with 100 points and 50 lines (known length); cells were evaluated at x1,000 magnification. Points falling on mononuclear or PMN cells were counted and divided by the total number of points falling on tissue areas in each microscopic field. Ten random non-coincident microscopic fields were evaluated for each group, n = 5/group and scale bar  = 50 µm. Data are presented as mean ± SEM. * P<0.05; ***P<0.001.</p