Prognostic significance of surfactant protein A, surfactant protein D, Clara cell protein 16, S100 protein, trefoil factor 3, and prostatic secretory protein 94 in idiopathic pulmonary fibrosis, sarcoidosis, and chronic pulmonary obstructive disease.

BACKGROUND: Identification of serum and bronchoalveolar lavage fluid (BALF) biomarkers may facilitate diagnosis and prognostication in various lung disorders. OBJECTIVE: Serum and BALF levels of surfactant protein A (SP-A), surfactant protein D (SP-D), Clara cell protein 16 (CC16), S100 protein, trefoil factor 3 (TFF3), and prostatic secretory protein 94 (PSP94) were evaluated in 94 consecutive patients (idiopathic pulmonary fibrosis (IPF; n=18), sarcoidosis (n=25), chronic obstructive pulmonary disease (COPD; n=51)), and in 155 healthy controls. METHODS: Biomarkers were measured at diagnosis and compared with disease characteristics. Both uniparametric and multiparametric analyses were used. RESULTS: Seven significant correlations were found: 1) BALF PSP94 level correlated with prognosis of sarcoidosis (P=0.035); 2) BALF SP-D level with pulmonary functions in IPF (P=0.032); 3) BALF SP-D and TFF3 with IPF mortality (P=0.049 and 0.017, respectively); 4) serum TFF3 level with COPD mortality (P=0.006,); 5) serum SP-A with pulmonary functions impairment in IPF (P=0.011); 6) serum SP-D level was associated with HRCT interstitial score in IPF (P=0.0346); and 7) serum SP-A was associated with staging of COPD according to spirometry (P<0.001). Moreover, our analysis showed that some biomarker levels differed significantly among the diseases: 1) BALF SP-D level differed between sarcoidosis and IPF; 2) serum SP-A level differed among IPF, sarcoidosis, COPD and was also different from healthy controls; 3) serum S100A6, S100A11 levels differed among IPF, sarcoidosis, COPD from healthy controls 4) serum SP-D, CC16, TFF-3 levels distinguished IPF patients from healthy controls; and 5) serum CC16, TFF3, PSP94 distinguished COPD patients from healthy controls. Our study shows that some of selected biomarkers should have prognostic value in the analysed lung disorders. On the other hand, these biomarkers do not appear to be unequivocally suitable for differential diagnosis of these disorders.

Acute hyperglycemia does not impair microvascular reactivity and endothelial function during hyperinsulinemic isoglycemic and hyperglycemic clamp in type 1 diabetic patients.

AIMS: The aim of this study was to evaluate the effect of acute glycemia increase on microvasculature and endothelium in Type 1 diabetes during hyperinsulinemic clamp. PATIENTS AND METHODS: Sixteen patients (51 ± 7 yrs) without complications were examined during iso- and hyperglycemic clamp (glucose increase 5.5 mmol·L(-1)). Insulin, lipid parameters, cell adhesion molecules and fibrinogen were analyzed. Microvascular reactivity (MVR) was measured by laser Doppler flowmetry. RESULTS: Maximum perfusion and the velocity of perfusion increase during PORH were higher in hyperglycemia compared to baseline (47 ± 16 versus 40 ± 16 PU, P < 0.01, and 10.4 ± 16.5 versus 2.6 ± 1.5 PU·s(-1), P < 0.05, resp.). Time to the maximum perfusion during TH was shorter and velocity of perfusion increase during TH higher at hyperglycemia compared to isoglycemic phase (69 ± 15 versus 77 ± 16 s, P < 0.05, and 1.4 ± 0.8 versus 1.2 ± 0.7 PU·s(-1), P < 0.05, resp.). An inverse relationship was found between insulinemia and the time to maximum perfusion during PORH (r = -0.70, P = 0.007). CONCLUSION: Acute glycemia did not impair microvascular reactivity in this clamp study in Type 1 diabetic patients. Our results suggest that insulin may play a significant role in the regulation of microvascular perfusion in patients with Type 1 diabetes through its vasodilation effect and can counteract the effect of acute glucose fluctuations.

Adiponectin, AFABP, and leptin in human breast milk during 12 months of lactation.

OBJECTIVES: Adiponectin, adipocyte fatty acid-binding protein (AFABP), and leptin have been shown to be present in human breast milk (BM). We determined intraindividual changes of BM levels of these proteins during 12 months of lactation. SUBJECTS AND METHODS: Proteins were measured using a high-sensitivity enzyme-linked immunosorbent assay method in 72 healthy mothers after delivery (day 0, D0) and after 1, 3, 6, and 12 months of lactation. RESULTS: Adiponectin levels in BM on D0 were 22.8 ± 0.8 (mean ± standard error of the mean), in 1 month (M1) 22.0 ± 0.6, in 3 months (M3) 20.5 ± 0.6, in 6 months (M6) 21.4 ± 0.8, and in 12 months (M12) 25.7 ± 1.4 ng/mL. AFABP levels were 12.3 ± 2.0, 6.2 ± 1.3, 1.3 ± 0.2, 2.5 ± 1.0, and 4.6 ± 1.9 ng/mL, respectively. Leptin levels were 0.3 ± 0.04, 0.2 ± 0.03, 0.1 ± 0.01, 0.1 ± 0.02, and 0.2 ± 0.04 ng/mL, respectively. We found significantly higher levels of adiponectin in M12 in comparison to M3 and M6 (P = 0.0026), higher levels of AFABP in D0 and M1 when compared with M3, M6, and M12 (P < 0.0001), and higher levels of leptin on D0 than in M1, M3, M6, and M12 (P < 0.0001). AFABP levels correlated negatively with infants' body weight in M1, but there was no correlation throughout the lactation period between body weight and other proteins. We found positive correlation between adiponectin, AFABP, and leptin throughout the lactation. CONCLUSIONS: All of the hormones were detectable in BM up to 12 months of lactation, with decreasing trend until M3 and subsequent increase till M12. We speculate that higher levels in M6 and M12 may be caused by longer intervals between breast-feeding due to the introduction of complementary food.

Influence of long-term thromboprophylaxis with low-molecular-weight heparin (enoxaparin) on changes of bone metabolism markers in pregnant women.

This is a first descriptive, retrospective, observational study aiming to evaluate the changes in bone turnover markers in pregnant women and to assess the effect of a long-term treatment with low-molecular-weight heparin (LMWH), specifically, enoxaparin. Study involved 50 pregnant Caucasian women with thrombophilia. The patients either received prophylactic enoxaparin once daily subcutaneously (N = 35) or were observed without treatment (N = 15). Concentrations of total serum alkaline phosphatase (total AP), bone alkaline phosphatase (bone AP), osteoprotegerin (OPG), and the receptor activator of nuclear factor κB ligand (RANKL) were measured at 15, 25, and 35 weeks of gestation. Total serum AP increased with gestational age. In the group treated with enoxaparin, the percentage of bone AP concentration was lower (P < .05) than in the control group. Serum OPG also increased with gestational age, but no significant difference was found between the groups with- and without treatment. Despite the OPG increased, RANKL did not change.

FXa inhibition and coagulation changes during DVT prophylaxis by enoxaparin over the course of a 15-day follow-up in septic patients.

The objective of our study was to examine the changes in coagulation parameters and inflammatory reaction over the course of 15 days in patients with severe sepsis. We tried to identify mechanisms by which sepsis-induced pathophysiological changes may influence the effectiveness of subcutaneously (SC) administered enoxaparin 40 mg once daily. A total of 16 patients (8 men, 8 women; age 35-83 years) meeting the inclusion criteria of severe sepsis were enrolled in this study. The follow-up was performed on days 1, 2, 3, 6, 9, 12, and 15 of hospitalization at the intensive care unit (ICU). Blood coagulation (activated partial thromboplastin time [aPTT], prothrombin time [PT], fibrinogen, antithrombin (AT), protein C [PC], D-dimer, fragment 1.2 [F1.2], factor Xa [FXa] inhibition) and inflammatory reactants (interleukin 6 [IL-6], C-reactive protein [CRP], orosomucoid, alpha-1-antitrypsin) were tested. The mean FXa inhibition was 0.17 (+ or - 0.17) IU/mL. The arbitrarily established range of FXa inhibition for prophylaxis, 0.2 to 0.4 IU/mL, was reached in 22 cases (20%), while in 74 cases (68%), it was below and in 13 cases (12%) above the aforementioned range. Factor Xa inhibition positively correlated with AT (r = .42; P < .001) and PC (r = .45; P < .001) activities. A negative correlation was found between the FXa inhibition and alpha-1-antitrypsin concentrations (r = -.33; P = .01) but only in the subgroup with alpha-1-antitrypsin concentrations > or = 2.2 g/L. We confirmed that in most patients with sepsis, the prophylaxis with enoxaparin did not lead to the required FXa inhibition. The inhibition of FXa by enoxaparin depends mainly on the AT and PC activities.

Older age and type of surgery predict the early inflammatory response to hip trauma mediated by interleukin-6 (IL-6).

Hip trauma and surgery are associated with systemic inflammatory reaction. However, little evidence exists about the role of IL-6. In order to assess the inflammatory response, we evaluated white blood cell (WBC) count, C-reactive protein (CRP) and IL-6 dynamics in sequential pre- and postsurgical samples collected from 125 elderly patients (mean age 78+/-9 years) undergoing osteosynthesis (OS) for extracapsular hip fractures (n=69), hemiarthroplasty (HA) or urgent total hip arthroplasty for intracapsular fractures (UA) (n=35), and elective total hip arthroplasty for osteoarthrosis (OA) (n=21). Both preoperative CRP and IL-6 levels were higher in patients with intracapsular fractures. IL-6 levels reached peak values immediately after the surgery, while CRP peak levels were reached 48 h after the surgery. The overall inflammatory reaction was more intense in HA patients compared to the other subgroups. Independent of each other, older age and the hip fracture type affected the IL-6 response, while the CRP response depended only on the type of surgery. The abrupt increase in IL-6 immediately after the procedure suggests its involvement in the early stages of the postoperative inflammatory reaction after hip surgery. This reaction is particularly pronounced in elderly patients receiving HA.

Favorable coagulation profile with fondaparinux after hip surgery in elderly patients.

Twenty-three patients with fondaparinux prophylaxis over 75 years of age who underwent hip fracture surgery were enrolled in the study. Fondaparinux sodium (2.5 mg) was administered subcutaneously 6 h postoperatively and then every 24 h for 28 days. Coagulation and inflammatory parameters were measured preoperatively, then 10 h, 2, 7, and 28 days postoperatively. Increased D-dimers, positive acute phase proteins, and IL-6, and decreased negative acute phase proteins were observed preoperatively (P < 0.05). Maximum values were reached 10 h postoperatively for IL-6 and D-dimer, and on postoperative days 2 and 7 for positive acute phase proteins (P < 0.05). Transferrin, prealbumin and antithrombin levels were lowest 10 h postoperatively and on postoperative day 2 (P < 0.05). Increased D-dimers, IL-6, and positive acute phase proteins, and decreased negative acute phase proteins persisted until postoperative day 28 (P < 0.05). Prothrombin fragments (F1 + 2) reached peak levels preoperatively and decreased gradually until postoperative day 28. Fondaparinux promoted the inhibition of thrombin generation, as documented by negative correlation between F1 + 2 and FXa inhibition (r = -0.46; P < 0.001). Fondaparinux-induced FXa inhibition increased gradually until postoperative day 28. This increase correlated positively with antithrombin activity (r = 0.4; P < 0.05). Fondaparinux prophylaxis counteracted pro-thrombogenic effect associated with hip fracture and subsequent surgery without severe bleeding complications.

Elevated inflammation markers in pheochromocytoma compared to other forms of hypertension.

OBJECTIVE: To investigate the effect of long-term catecholamine excess in pheochromocytoma on leukocyte and platelet count and on proteins of acute-phase response. METHODS: Fifteen subjects with pheochromocytoma, 16 with primary aldosteronism, 18 with essential hypertension and 17 healthy controls were studied. Sixteen subjects with pheochromocytoma were investigated after tumor removal. Leukocyte, neutrophil and platelet count, as well as C-reactive protein were measured in all subjects, while fibrinogen, alpha(1)-antitrypsin, alpha(2)-macroglobulin, orosomucoid, transferrin and prealbumin were only measured in subjects with pheochromocytoma, primary aldosteronism and essential hypertension. RESULTS: Subjects with pheochromocytoma showed significantly higher leukocyte [7.5 +/- 0.9 10(9)/l, p < 0.001 vs. primary aldosteronism (5.4 +/- 0.9 10(9)/l) and healthy controls (5 +/- 0.9 10(9)/l), p = 0.04 vs. essential hypertension (6.3 +/- 1.6 10(9)/l)], neutrophil (p < 0.001 vs. primary aldosteronism and healthy subjects) and platelet counts (p < 0.001 vs. primary aldosteronism; p = 0.01 vs. essential hypertension) compared to the other groups of subjects. Similar results were obtained for positive proteins of acute-phase response in subjects with pheochromocytoma [C-reactive protein: 0.62 +/- 0.52 mg/dl, p < 0.001 vs. healthy subjects (0.08 +/- 0.08 mg/dl), p = 0.001 vs. primary aldosteronism (0.17 +/- 0.19 mg/dl), p = 0.04 vs. essential hypertension (0.31 +/- 0.26 mg/dl); fibrinogen: p = 0.02 vs. primary aldosteronism; orosomucoid: p = 0.005 vs. primary aldosteronism; alpha(2)-macroglobulin: p = 0.009 vs. primary aldosteronism]. No significant differences were found in plasma levels of alpha(1)-antitrypsin, transferrin and prealbumin. Tumor removal led to a significant decrease in leukocyte (p = 0.004), neutrophil (p = 0.007) and platelet count (p = 0.003) and also to a significant decrease in acute-phase proteins (C-reactive protein: p = 0.03, fibrinogen: p = 0.008, alpha(1)-antitrypsin: p = 0.003, orosomucoid: p = 0.04). CONCLUSIONS: Chronic catecholamine excess in pheochromocytoma is accompanied by an increase in inflammation markers which was reversed by the tumor removal.

Isolation and biochemical characterization of two soluble iron(III) reductases from Paracoccus denitrificans.

Two soluble enzymes (FerA and FerB) catalyzing the reduction of a number of iron(III) complexes by NADH, were purified to near homogeneity from the aerobically grown iron-limited culture of Paracoccus denitrificans using a combination of anion-exchange chromatography (Sepharose Q), chromatofocusing (Mono P), and gel permeation chromatography (Superose 12). FerA is a monomer with a molecular mass of 19 kDa, whereas FerB exhibited a molecular mass of about 55 kDa and consists of probably two identical subunits. FerA can be classified as an NADH:flavin oxidoreductase with a sequential reaction mechanism. It requires the addition of FMN or riboflavin for activity on Fe(III) substrates. In these reactions, the apparent substrate specificity of FerA seems to stem exclusively from different chemical reactivities of Fe(III) compounds with the free reduced flavin produced by the enzyme. Observations on reducibility of Fe(III) chelated by vicinal dihydroxy ligands support the view that FerA takes part in releasing iron from the catechol type siderophores synthesized by P. denitrificans. Contrary to FerA, the purified FerB contains a noncovalently bound redox-active FAD coenzyme, can utilize NADPH in place of NADH, does not reduce free FMN at an appreciable rate, and gives a ping-pong type kinetic pattern with NADH and Fe(III)-nitrilotriacetate as substrates. FerB is able to reduce chromate, in agreement with the fact that its N-terminus bears a homology to the previously described chromate reductase from Pseudomonas putida. Besides this, it also readily reduces quinones like ubiquinone-0 (Q0) or unsubstituted p-benzoquinone.

Fine-tuned regulation by oxygen and nitric oxide of the activity of a semi-synthetic FNR-dependent promoter and expression of denitrification enzymes in Paracoccus denitrificans.

In Paracoccus denitrificans at least three fumarate and nitrate reductase regulator (FNR)-like proteins [FnrP, nitrite and nitric oxide reductases regulator (NNR) and NarR] control the expression of several genes necessary for denitrifying growth. To gain more insight into this regulation, beta-galactosidase activity from a plasmid carrying the lacZ gene fused to the Escherichia coli melR promoter with the consensus FNR-binding (FF) site was examined. Strains defective in the fnrP gene produced only very low levels of beta-galactosidase, indicating that FnrP is the principal activator of the FF promoter. Anoxic beta-galactosidase levels were much higher relative to those under oxic growth and were strongly dependent on the nitrogen electron acceptor used, maximal activity being promoted by N(2)O. Additions of nitrate or nitroprusside lowered beta-galactosidase expression resulting from an oxic to micro-oxic switch. These results suggest that the activity of FnrP is influenced not only by oxygen, but also by other factors, most notably by NO concentration. Observations of nitric oxide reductase (NOR) activity in a nitrite-reductase-deficient strain and in cells treated with haemoglobin provided evidence for dual regulation of the synthesis of this enzyme, partly independent of NO. Both regulatory modes were operative in the FnrP-deficient strain, but not in the NNR-deficient strain, suggesting involvement of the NNR protein. This conclusion was further substantiated by comparing the respective NOR promoter activities.

Detection, with a pH indicator, of bacterial mutants unable to denitrify.

In order to facilitate isolation of mutants with alterations in the denitrification pathway, a new screening procedure using phenol red incorporated into agar overlay has been defined. Alkalinization in the neighbourhood of denitrifying colonies respiring nitrate or nitrite gives rise to a red circular halo. Antimycin blocked these colour changes, which suggests their association with the periplasmic reduction of nitrite. Inhibition of nitrous oxide reductase by acetylene had no significant effect on alkalinization elicited by nitrate or nitrite. Several mutants negative by the phenol red staining test were generated by transposon Tn5 mutagenesis of Paracoccus denitrificans. All these mutants were defective in the activities of nitrite and nitric oxide reductases while the other denitrification activities were present at the wild-type level.