Carole Hennequin

Inhibitory effect of bikunin on calcium oxalate crystallization in vitro and urinary bikunin decrease in renal stone formers

Abstract Two proteins of 17 and 24 kDa, respectively, which were immunologically related to bikunin, were purified from urine of healthy men, using in the last step a trypsin CNBr-sepharose affinity column. These proteins strongly inhibited calcium oxalate (CaOx) crystallization in two in vitro models. In the first model, the presence of 8 μg/ml protein in a medium containing 0.76 mM CaCl2 (with 45Ca) and 0.76 mM ammonium oxalate inhibited the crystallization process by 80%, as estimated by supernatant radioactivity after 60 min of incubation. A similar inhibition was observed in the second turbidimetric model, where the CaOx crystallization kinetics were followed for 10 min at 620 nm in a medium containing 4 mM CaCl2 and 0.5 mM Na2Ox. These proteins were used as standard protein for the development of an enzyme-linked immunosorbent assay (ELISA) in urine. Mean (± SEM) urinary bikunin concentration in 18 healthy subjects was 5.01 ± 0.91 μg/ml. This was a concentration range of strong inhibitory activity in vitro. Bikunin values were nearly 50% lower (2.54 ± 0.42 μg/ml, P=0.007) in 31 CaOx renal stone formers (having weddelite crystals in their first morning urine) than in the healthy volunteers. A correlation was found between urinary bikunin and alpha-1 microglobulin concentrations in the control group (y=0.73x + 1.09, r2=0.8) while no such correlation existed in the lithiasis group. In conclusion, bikunin exerts a strong inhibitory action of CaOx crystallization in vitro. Its involvement in urinary CaOx crystallization of stone formers is highly probable, based on the significant decrease in its urinary concentration in the majority of stone formers studied.

Can procalcitonin measurement help the diagnosis of osteomyelitis and septic arthritis? A prospective trial

ObjectivesProcalcitonin (PCT) is an accurate marker for differentiating bacterial infection from non-infective causes of inflammation or viral infection. However, there is only one study in children which tested procalcitonin as a diagnostic aid in skeletal infections. With this study we sought to evaluate the sensitivity, specificity and predictive values of procalcitonin for identifying bone and joint infection in children evaluated in the emergency department for non traumatic decreased active motion of a skeletal segment.MethodsPatients aged 1 month to 14 years were prospectively included in the emergency department when suspected for osteomyelitis or septic arthritis. Procalcitonin levels, C reactiv protein, white blood cell count were measured and bacteriological samples were collected before initiation of antibiotic treatment. Patients were assigned to 3 groups according to the degree of suspected infection: group 1 confirmed infection, group 2 presumed infection and group 3 non infected patients.ResultsThree hundred thirty nine patients were included (118 girls and 221 boys). Group 1 comprised 8 patients (2 had PCT levels > 0.5 ng/ml). Two had osteomyelitis and 6 septic arthritis. Forty children were incuded in group 2 (4 had PCT levels > 0.5 ng/ml). Eighteen had presumed osteomyelitis and 22 presumed septic arthritis. Group 3 comprised 291 children (9 PCT levels > 0.5 ng/ml) who recovered without antibiotic treatment. The specificity of the PCT as a marker of bacterial infection (comparing Group 1 and Group 3) was 96.9% [95% CI, 94.2-98.6], the sensitivity 25% [95% CI, 3.2-65.1], the positive predictive value (PPV) 18.2% [95% CI, 2.3-51.8] and the negative predictive value (NPV) 97.9% [95% CI, 95.5-99.2].ConclusionPCT is not a good screening test for identifying skeletal infection in children. Larger studies are needed to evaluate still more the place of PCT measurements in the diagnosis of osteomyelitis and septic arthritis.

Apoptotic pathways involved in U937 cells exposed to LDL oxidized by hypochlorous acid.

Oxidized low-density lipoproteins (oxLDL) play a critical role in atherogenesis. One oxidative pathway of LDL involves myeloperoxidase, which catalyzes the production of hypochlorous acid (HOCl) in monocytes. We investigated the apoptotic mechanism induced by oxLDL, generated by HOCl treatment of native LDL, in human monocytic U937 cell line. The involvement of the mitochondrial apoptotic pathway was analyzed in Bcl-2-overexpressing clones, generated from U937 cells. HOCl-oxLDL induced in U937 cells (i) a marked caspase-dependent increase of apoptosis, (ii) a loss of mitochondrial membrane potential, (iii) a specific activation of caspase-2, -3, -8, and -9, and (iv) a similar degree of apoptosis in presence or absence of anti-Fas and anti-TNF-R1 antibodies. Moreover, the degree of HOCl-oxLDL-induced caspase-3 and -8 activation, and apoptosis was significantly reduced in U937/Bcl-2 cells, with no activation of caspase-9. By contrast, Cu-oxLDL-mediated apoptosis in U937 cells involved exclusively the mitochondrial pathway. In conclusion, the mechanism of HOCl-oxLDL-induced apoptosis in monocytic U937 cells involves the two pathways of apical caspase activation: (i) death receptor-mediated caspase-8 and (ii) mitochondria-mediated caspase-9. This converges in the activation of executing caspases, including caspase-3, and apoptosis. The interference of Bcl-2 overexpression with HOCl-oxLDL-induced apoptosis suggests the importance of mitochondrial involvement in this apoptotic mechanism.

A stable animal model of diet-induced calcium oxalate crystalluria.

Abstract Twenty male Wistar rats, weighing 150 g, were placed in metabolic cages on a 30% sucrose diet for 7 days, before allocation to two groups: a control group (n = 5) and a lactose group (n = 15). They received respectively a 30% sucrose diet or a 30% lactose diet for 8 weeks, each containing 0.67% calcium and 0.38% phosphorus. After 4 (T1) and 8 (T2) weeks, the serum calcium (Ca) and citrate levels were significantly (P < 0.01) higher in rats fed the lactose diet. Serum alkaline phosphatase activity was increased in the lactose group (P < 0.01) at T1 and T2. The lactose-rich diet induced an increase in urinary Ca excretion at T1 and T2; citrate excretion was only enhanced at T2 (P < 0.001). No difference between the two groups was observed in urinary oxalate (Ox) excretion or creatinine clearance. Crystalluria analysis revealed a marked number (>300/mm3 at T1 and T2) of calcium oxalate dihydrate crystals (COD) in rats fed the lactose-rich diet, whereas no COD crystals were observed in sucrose-fed control rats at any time point. The formation of COD crystals in lactose-fed rats was related to an increase in calcium oxalate (CaOx) product (pCaOx), which was respectively 12.6 vs 3.9 at T1 and 10.5 vs 1.8 at T2, and an increase in CaOx ratio (Ca/Ox), which was 99.1 vs 7.5 and 67.5 vs 18.5 at T1 and T2, respectively. The high pCaOx and Ca/Ox ratios in the lactose group were due to hypercalciuria, in agreement with the number and the type of crystals. The present experimental model confirms that the ingestion of a 30% lactose diet increases urinary Ca excretion without changing urinary Ox excretion and shows for the first time that it induces a stable and marked crystalluria composed of COD. Such a non-nephrotoxic and stable model is of interest for the study of CaOx crystal formation secondary to hypercalciuria, and thus afterwards eventually for CaOx nephrolithiasis.