Kurt Herkner

High levels of eosinophil cationic protein in wheezing infants predict the development of asthma

BACKGROUNDnIn association with respiratory tract infections, infants may have episodes of wheezing, which represent the onset of asthma in some of them. Activated eosinophils play a central part in asthmatic inflammation.nnnOBJECTIVEnWe investigated whether, in infants experiencing their first episode of wheezing, eosinophil activation is present and can predict the development of asthma.nnnMETHODSnIn a prospective trial, eosinophil activation was measured by eosinophil cationic protein (ECP) concentrations in serum from 33 nonatopic infants with their first episode of wheezing, 15 nonatopic infants with upper respiratory tract infection without wheezing, and 18 healthy nonatopic infants. One year later, the children were re-evaluated for a diagnosis of infantile asthma.nnnRESULTSnWheezing infants had higher median serum ECP levels (13.4 micrograms/L) than children with nonwheezy respiratory tract infection (7.6 micrograms/L, p < 0.005) or healthy subjects (7.1 micrograms/L, p < 0.005). In addition, wheezing infants (n = 13) with serum ECP concentrations greater than 20 micrograms/L were more likely to have asthma within 1 year than patients with ECP levels less than 20 micrograms/L (odds ratio = 12.4; confidence interval, 4.6-33.5).nnnCONCLUSIONnEosinophil activation measured by serum ECP is present in infants with their first episode of wheezing illness, especially in those infants in whom asthma subsequently develops within 1 year. These data may indicate a predictive value of serum ECP measurements in children with wheezing to identify those patients in whom infantile asthma is developing. These findings probably also indicate that serum ECP may be used to identify the children who need early antiinflammatory treatment.

Hematologic manifestations and impaired liver synthetic function during valproate monotherapy

In a prospective study 50 children with new onset epilepsy were investigated. Routine screening for complete blood count, serum protein, albumin, gamma-glutamyltransferase (gamma-GT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, and coagulation studies before, 3, 6 and 9 weeks after commencement of antiepileptic therapy with valproate were carried out. Serum B12 and folate levels were also determined in 29 patients. The aim of the study was to evaluate the effect of VPA on these laboratory findings. We found a significant reduction of red blood count and platelet count, whereas MCV showed a significant upward trend. Vitamin B12 levels were elevated after starting VPA therapy. We found no elevations of liver enzymes, but a significant transient reduction of ALT after 3 and 6 weeks and significantly reduced serum protein and albumin after 3, 6 and 9 weeks. Coagulation studies revealed a significant downward trend in serum fibrinogen and upward trend in thrombin time. The other parameters showed no significant changes after onset of VPA treatment. We think that reduced red blood cell and platelet counts, and elevated MCV indicate a direct toxic effect on a hematopoietic precursor or stem cell in patients treated with VPA. Furthermore, reduced protein, albumin and fibrinogen indicate an impaired liver synthetic function in asymptomatic children treated with VPA monotherapy.

Ischemic Conditioning Prevents Na,K-ATPase Dissociation from the Cytoskeletal Cellular Fraction after Repeat Renal Ischemia in Rats

Recent studies have suggested that heat shock proteins (HSPs) are involved in the restoration of the cytoskeletal anchorage of Na,K-ATPase after renal ischemia. To determine their role in ischemic conditioning, we investigated whether cytoskeletal Na,K-ATPase was stabilized during repeat ischemia concurrent with 25-kD and 70-kD HSPs induction. Anesthetized rats either underwent single unilateral renal ischemia or were conditioned with bilateral renal ischemia and, after 18 h of reflow, were then subjected to repeat unilateral renal ischemia. Renal cortex was harvested, and effects of single versus repeat ischemia were compared by Triton X-100 extraction, by immunohistochemistry, and by an in vitro assay of Na,K-ATPase association with isolated cytoskeletal fractions. In contrast to single ischemia, repeat ischemia did not result in increased Triton X-100 extractability of Na,K-ATPase. Levels of 25-kD and 70-kD HSPs were significantly induced by ischemic conditioning and redistributed into the cytoskeletal fraction after single and repeat ischemia. Immunohistochemistry also showed significant disruption of Na,K-ATPase within proximal tubules only after a single episode of ischemia, whereas repeat ischemia did not alter the pattern of restored Na,K-ATPase localization in conditioned renal cortex. The preserved association of Na,K-ATPase with the cytoskeletal fraction of conditioned renal cortex was effectively abolished in vitro by addition of antibodies against 25-kD or 70-kD HSP. These results suggest that 25-kD and 70-kD HSPs induced by ischemic conditioning stabilize the cytoskeletal anchorage of Na,K-ATPase during repeat renal ischemia.

Energy metabolism in graded perinatal asphyxia of the rat

Although information on energy metabolism during hypoxemic-ischemic states is abundant, data on perinatal asphyxia (PA) are limited. As results from hypoxia-ischemia cannot be directly extrapolated to PA, a clinical entity characterized by acidosis, hypoxemia and hypercapnia, we decided to use a rat model of graded PA during delivery. Cesarean section was performed at the 21st day of gestation and the pups, still in the uterus horns, were asphyxiated from 0 to 20 minutes. In this model survival decreases with the length of asphyxia. Early changes of energy-rich phosphates in brain, heart and kidney were determined by HPLC. ATP and phosphocreatine gradually decreased with the length of asphyxia, with highest ATP depletion rate occurring in the kidney. ATP: brain 1.39 +/- 0.71 (0 min) to 0.06 microM/g wwt (20 min); heart 4.73 +/- 0.34 (0 min) to 1.08 +/- 0.47 (20 min); kidney 1.62 +/- 0.11 (0 min) to 0.02 +/- 0.02 (20 min). Phosphocreatine: brain 1.65 +/- 0.68 (0 min) to 0.51 +/- 0.45 microM/g (20 min); heart 6.98 +/- 0.38 (0 min) to 6.17 +/- 1.07 (20 min); kidney 8.23 +/- 0.86 (0 min) to 3.76 +/- 0.54 (20 min). We present data on energy derangement in a rat model of PA, closely resembling the clinical situation, showing that energy depletion precedes cell damage and death.

Anticytokeratins are a potential source of false‐positive indirect immunofluorescence assays for C‐ANCA

Antibodies to neutrophil cytoplasmic antigens (ANCA) targeted toward granule enzymes have been recognized as a valuable diagnostic tool in the detection of Wegeners granulomatosis and systemic vasculitides. However, the most commonly used method of detection, the indirect immunofluorescence assay, is prone to false‐positive results due to antibodies of different pathological significance either targeted to, or cross‐reacting with, similarly distributed epitopes. Using double immunofluorescence, the present study demonstrates that anticytokeratin antibodies are able to produce false‐positive C‐ANCA immunofluorescence assays. In addition, a case of natural appearance of cytokeratin‐reactive antibodies causing a false‐positive “pseudo‐ANCA” staining pattern in a patient presenting with sepsis is reported. Since the expression of cytokeratins is almost exclusively confined to epithelial cells, the most plausible explanation for both phenomena is a crossreaction of anticytokeratin antibodies with granule associated epitopes. Due to the natural appearance of anticytokeratin antibodies in association with a variety of other pathologic entities, it is of crucial importance for the diagnostic significance of the C‐ANCA immunofluorescence assay to exclude anticytokeratin caused false‐positive results. It is shown that supplementary indirect immunofluorescence tests performed on cultured human epithelial cells readily distinguish anticytokeratin caused “pseudo‐ANCA” from true C‐ANCA. J. Clin. Lab. Anal. 12:54–59, 1998.

The effects of acid glycosaminoglycans on neonatal calvarian cultures — A role of keratan sulfate in morquio syndrome?

Morquio syndrome (mucopolysaccharidosis IV) presents with multiple bone dysplasia and is characterized by the inability to degrade keratan sulfate due to deficient N-acetylgalactosamine-6-sulfate sulfatase in Morquio A syndrome and deficient beta-D-galactosidase in Morquio B syndrome. The aim of our study was to investigate into the pathogenetic mechanism as it is not clear whether the accumulation of keratan sulfate is toxic for osteoblasts or inhibits osteoblast activity as e.g. bone resorption. The glycosaminoglycans keratan sulfate, heparan sulfate, dermatan sulfate, chondroitin-4,6-sulfate and hyaluronic acid were tested in rat neonatal calvarian cultures for their effects on bone resorption, osteoblast activity and toxicity. Bone resorption was evaluated by calcium release into the medium, osteoblast activity by the determination of alkaline phosphatase and toxicity by measuring lactate dehydrogenase in the culture media. Keratan sulfate had no effect on bone resorption but inhibited osteoblast activity at the low, nontoxic concentration of 10 ng per ml organ culture supernatant significantly (p<0.05). At a concentration of 100 ng per ml keratan sulfate revealed toxic effects as reflected by significantly (p<0.05) elevated lactate dehydrogenase activity. None of the other glycosaminoglycans inhibited osteoblast activities. Heparan sulfate showed at toxic levels (10 microg per ml supernatant) significantly increased bone resorption (p<0.05) accompanied by increased alkaline phosphatase activity. The specific keratan sulfate effects of inhibiting osteoblast activity and toxicity towards bone, which were never tested before, suggest a role for this glycosaminoglycan in the pathogenesis of bone dysplasia in Morquio syndrome.

Factors contributing to the occurrence and predictability of bronchial hyperresponsiveness to methacholine in children

Using a stepwise logistic regression analysis, we investigated clinical data, allergologic findings, spirometric data, and the cellular and humoral immune system in order to gain new insights into the role these parameters play in bronchial hyperresponsiveness to methacholine in children and to create a model for the prediction thereof. Bronchial hyperresponsiveness, which was found in 124 of 462 children (26.8%), was observed to have been influenced by an increased level of eosinophils, the positivity of the skin prick test for any of the allergens tested, a decreased baseline forced expiratory volume in 1 second (FEV1) (percent predicted), a decreased maximum expiratory flow at 50% expiration as a percent of forced vital capacity, and a decreased level of kappa-chain-assembled immunoglobulins. Logit analysis disclosed that the influence of all other parameters on the occurrence of bronchial hyperresponsiveness was of no further statistical significance. The degree of bronchial hyperresponsiveness (provocative dose causing a 20% fall in FEV1) showed a statistically significant correlation with the eosinophil count (Spearmans r = -0.198) and FEV1 (percent predicted) (Spearmans r= 0.203). Our findings suggest that allergic sensitization and eosinophilic reaction in children are major factors in contributing to the occurrence of bronchial hyperresponsiveness to methacholine.

Erythropoietin, erythropoesis and iron status in children after major surgical stress

The aim of our study was to evaluate bone marrow stimulation and bone marrow response to post-operative anaemia in children after open heart surgery. In 16 children (age 5.7±0.9 years, weight 20.1±3.2 kg) serum erythropoietin, haematocrit, reticulocyte count, ferritin, transferrin saturation and C-reactive protein were assessed perioperatively after cardiopumonary bypass for surgical repair of atrial septal defect. Erythropoietin increased seven fold from 14±6.2 (7–30) to 80±49 (20–171) mU/ml (P<0.05) and the reticulocyte count a 1.7-fold from 11.1±3.1 (6–19) to 18.4±5.9 (10–31) ‰ (P<0.05). Transferrin saturation was inversely correlated to C-reactive protein.ConclusionThese findings suggest adequate bone marrow stimulation but an inadequate bone marrow response during the immediate perioperative period, caused by inhibition of erythropoesis by acute postoperative inflammation in children after open heart surgery.

Creatine reduces collagen accumulation in the kidneys of diabetic db/db mice

In the present study, we tested the hypothesis whether creatine, a metabolite of arginine metabolism, shares the pharmacological activities of arginine reducing collagen accumulation in the diabetic kidney. Ten db/db mice were given, for 3 months, a solution containing a daily dosage of creatine of 50 mg/kg body weight. Eleven db/db mice served as controls. At the end of the 3-month study period, the mean N-carboxymethyllysine concentration in the untreated group was significantly higher than in the treated group (0.163 +/- 0.18 versus 0.096 +/- 0.017 nmol/mumol hydroxyproline, p < 0.001). Collagen accumulation was also significantly higher in the untreated than in the treated group (2.21 +/- 0.24 versus 1.68 +/- 0.22 mumol hydroxyproline/100 mg kidney weight, p < 0.001). We conclude that creatine led to a significant reduction in collagen type IV accumulation resembling arginine or aminoguanidine action. We do suggest that the guanidino group common to both compounds is able to block reactive carbonyls.

Influences of the Cellular and Humoral Immune System in Bronchoalveolar Lavage on Lung Function in Pulmonary Sarcoidosis

We investigated the changes in the cellular and humoral immune system in bronchoalveolar lavage (BAL) performed in 22 patients with pulmonary sarcoidosis and in 14 normal control subjects and their interactions with lung function parameters. Lymphocytosis, the increase in OKT4+ lymphocytes and OKT4+OKDR+ lymphocytes correlated with the increase in immunoglobulins, especially IgG, IgA and kappa chain assembled immunoglobulins. The transferrin levels obtained in BAL were found to be higher in patients with sarcoidosis, and they correlated with the cellular and, more closely, with other humoral findings. A negative correlation existed between the ventilatory parameters and the cell count and humoral findings. In addition, we found a negative correlation between the diffusing capacity for carbon monoxide and other cellular findings, which was most pronounced with reference to lymphocytes, OKT4+ lymphocytes and the OKT4+/OKT8+ ratio. These results underscore the role of OKT4+ lymphocytes, activated OKT4+OKDR+ lymphocytes and transferrin in the increase in immunoglobulins, mainly kappa chain isotypes. Because of the relationship between these changes and ventilatory parameters, and the diffusing capacity, the above results also reveal the clinical relevance of our findings.