Matthias Brandis

Plasma levels and gene expression of granulocyte colony-stimulating factor, tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-8, and soluble intercellular adhesion molecule-1 in neonatal early onset sepsis

Bacterial sepsis is still a leading cause of neonatal morbidity and mortality. Early onset sepsis in particular, presents with a different clinical course and involves other pathogens than sepsis later in life. In this study, plasma concentrations and mRNA expression of granulocyte colony-stimulating factor (G-CSF), tumor necrosis factor-α (TNF-α), IL-1β, IL-6, IL-8, and soluble intercellular adhesion molecule-1 (sICAM-1) of neonates with early onset sepsis were evaluated in cord blood and during the first days of life. Irrespective of prematurity, plasma levels of G-CSF, TNF-α, IL-1β, IL-6, and IL-8, but not sICAM-1, were excessively elevated in septic neonates when compared with both healthy infants and infants with clinically suspected but not confirmed sepsis. Compared with the corresponding maternal levels, neonatal cytokine cord plasma levels were likewise highly elevated, indicating the endogenous cytokine production by the neonate. With the exception of TNF-α, mRNA expression in blood cells from septic infants was, however, not more frequently detectable than in those from nonseptic patients. Cytokine levels decreased significantly within the first days of life, whereas levels of sICAM-1 and C-reactive protein increased during the same time period. In summary, in contrast to C-reactive protein and sICAM-1, cord blood plasma levels, but not the presence of mRNA, of G-CSF, TNF-α, IL-1β, IL-6, and IL-8 can predict neonatal early onset sepsis with a high sensitivity and specificity. Cell types other than blood cells are likely to contribute considerably to the high cytokine production in septic newborns.

Novel molecular variants of the Na-K-2Cl cotransporter gene are responsible for antenatal Bartter syndrome.

Antenatal Bartter syndrome is a variant of inherited renal-tubular disorders associated with hypokalemic alkalosis. This disorder typically presents as a life-threatening condition beginning in utero, with marked fetal polyuria that leads to polyhydramnios and premature delivery. Another hallmark of this variant is a marked hypercalciuria and, as a secondary consequence, the development of nephrocalcinosis and osteopenia. We have analyzed 15 probands belonging to 13 families and have performed SSCP analysis of the coding sequence and the exon-intron boundaries of the NKCC2 gene; and we report 14 novel mutations in patients with antenatal Bartter syndrome, as well as the identification of three isoforms of human NKCC2 that arise from alternative splicing.

Cholinergic ion secretion in human colon requires coactivation by cAMP

Cl-secretion in the colon can be activated by an increase of either intracellular Ca2+ or cAMP. In this study we examined a possible interdependence of the two second-messenger pathways in human colonic epithelium. When measured in a modified Ussing chamber, carbachol (CCH; 100 μmol/l, basolateral), via an increase in cytosolic Ca2+concentration ([Ca2+]i), activated a transient lumen-negative equivalent short-circuit current ( I sc) [change (Δ) in I sc = -79.4 ± 7.5 μA/cm2]. Previous studies indicated that intracellular Ca2+ directly acts on basolateral K+ channels, thus enhancing driving force for luminal Cl- exit. Increased intracellular cAMP (by basolateral addition of 100 μmol/l IBMX and 1 μmol/l forskolin) activated a sustained lumen-negative current (Δ I sc = -42.4 ± 7.2 μA/cm2) that was inhibited by basolateral trans-6-cyano-4-( N-ethylsulfonyl- N-methylamino)-3-hydroxy-2,2-dimethyl&2-chromane (10 μmol/l), a blocker of KvLQT1 channels. In the presence of elevated cAMP, the CCH-activated currents were augmented (Δ I sc = 167.7 ± 32.7 μA/cm2), suggesting cooperativity of the Ca2+- and cAMP-mediated responses. Inhibition of endogenous cAMP production by indomethacin (10 μmol/l) significantly reduced CCH-activated currents and even reversed the polarity in 70% of the experiments. The transient lumen-positive I sc was probably due to activation of apical K+channels because it was blocked by luminal Ba2+ (5 mmol/l) and tetraethylammonium (10 mmol/l). In the presence of indomethacin (10 μmol/l, basolateral), an increase of cAMP activated a sustained negative I sc. Under these conditions, CCH induced a large further increase in lumen-negative I sc(Δ I sc = -100.0 ± 21.0 μA/cm2). We conclude that CCH acting via [Ca2+]ican induce Cl- secretion only in the presence of cAMP, i.e., when luminal Cl- channels are already activated. The activation of a luminal and basolateral K+ conductance by CCH may be essential for transepithelial KCl secretion in human colon.

Modulation of Ca2+-activated Cl- secretion by basolateral K+ channels in human normal and cystic fibrosis airway epithelia.

Human airway epithelia express Ca2+-activated Cl− channels (CaCC) that are activated by extracellular nucleotides (ATP and UTP). CaCC is preserved and seems to be up-regulated in the airways of cystic fibrosis (CF) patients. In the present study, we examined the role of basolateral K+ channels in CaCC-mediated Cl− secretion in native nasal tissues from normal individuals and CF patients by measuring ion transport in perfused micro Ussing chambers. In the presence of amiloride, UTP-mediated peak secretory responses were increased in CF compared with normal nasal tissues. Activation of the cAMP pathway further increased CaCC-mediated secretion in CF but not in normal nasal mucosa. CaCC-dependent ion transport was inhibited by the chromanol 293B, an inhibitor of cAMP-activated hKvLQT1 K+ channels, and by clotrimazole, an inhibitor of Ca2+-activated hSK4 K+ channels. The K+ channel opener 1-ethyl-2-benzimidazolinone further increased CaCC-mediated Cl− secretion in normal and CF tissues. Expression of hSK4 as well as hCACC-2 and hCACC-3 but not hCACC-1 was demonstrated by reverse transcriptase PCR on native nasal tissues. We conclude that Ca2+-activated Cl− secretion in native human airway epithelia requires activation of Ca2+-dependent basolateral K+ channels (hSK4). Co-activation of hKvLQT1 improves CaCC-mediated Cl− secretion in native CF airway epithelia, and may have a therapeutic effect in the treatment of CF lung disease.

Identification of a New Gene Locus for Adolescent Nephronophthisis, on Chromosome 3q22 in a Large Venezuelan Pedigree

Nephronophthisis, an autosomal-recessive cystic kidney disease, is the most frequent monogenic cause for renal failure in childhood. Infantile and juvenile forms of nephronophthisis are known to originate from separate gene loci. We describe here a new disease form, adolescent nephronophthisis, that is clearly distinct by clinical and genetic findings. In a large, 340-member consanguineous Venezuelan kindred, clinical symptoms and renal pathology were evaluated. Onset of terminal renal failure was compared with that in a historical sample of juvenile nephronophthisis. Onset of terminal renal failure in adolescent nephronophthisis occurred significantly later (median age 19 years, quartile borders 16.0 and 25.0 years) than in juvenile nephronophthisis (median age 13.1 years, quartile borders 11.3 and 17.3 years; Wilcoxon test P=.0069). A total-genome scan of linkage analysis was conducted and evaluated by LOD score and total-genome haplotype analyses. A gene locus for adolescent nephronophthisis was localized to a region of homozygosity by descent, on chromosome 3q22, within a critical genetic interval of 2. 4 cM between flanking markers D3S1292 and D3S1238. The maximum LOD score for D3S1273 was 5.90 (maximum recombination fraction.035). This locus is different than that identified for juvenile nephronophthisis. These findings will have implications for diagnosis and genetic counseling in hereditary chronic renal failure and provide the basis for identification of the responsible gene.

Antibiotic resistance of urinary tract pathogens and rationale for empirical intravenous therapy.

AbstractEmpirical antibiotic treatment in urinary tract infection (UTI) in children must rely on surveillance data on the epidemiology and resistance patterns of common uropathogens. A retrospective analysis of bacteria isolated from children with UTI irrespective of underlying disease or pre-treatment was performed at the University Hospital of Freiburg, Germany, in 1997, and from 1999 to 2001. In the first study period, 261 positive urine samples and in the second period 684 positive samples were analyzed. Escherichia coli (57.2%) was the leading uropathogen followed by Enterococcus spp. (13.7%), Pseudomonas aeruginosa (7.0%), Proteus spp. (5.9%), Klebsiella spp. (4.7%), and Enterobacter/Citrobacter spp. (4.3%). Almost 50% of the E. coli isolates were resistant to ampicillin, but effectively no resistance against cephalosporins, aminogylcosides, ciprofloxacin, nitrofurantoin, and imipenem was observed. In Enterococcus spp. the resistance to ampicillin was about 15% and 40% to netilmicin, while none of the latter showed high-level aminoglycoside resistance. In P. aeruginosa, there was no resistance to aminoglycosides. No difference in resistance patterns between the two study periods was observed. We conclude that an empirical combination treatment of ampicillin and gentamicin, netilmicin, or tobramycin is appropriate in children with UTI independent of pre-treatment or underlying disease. This therapy should be clinically efficacious, well tolerated, and cost effective, and should prevent unnecessary development of antimicrobial resistance.

CFTR-mediated inhibition of epithelial Na+ conductance in human colon is defective in cystic fibrosis

Cystic fibrosis (CF) patients show characteristic defects in epithelial ion transport, such as failure in cAMP-dependent Cl-secretion. Because the cystic fibrosis transmembrane conductance regulator (CFTR) also functions as a downregulator of epithelial Na+ channels (ENaC), enhanced Na+ conductance was found in the airways of CF patients. Here, we examined whether enhanced epithelial Na+ conductance is also present in the colonic epithelium of CF patients and examined the underlying mechanisms. Thus transepithelial voltages were measured, and equivalent short-circuit currents ( I sc-eq) were determined by means of a novel type of Ussing chamber. Non-CF tissues demonstrated cAMP-dependent Cl- secretion that was absent in biopsies of CF patients. Correspondingly, Isc-eq was inhibited in non-CF but not in CF epithelia when synthesis of endogenous prostaglandins was blocked by indomethacin. In the presence of indomethacin, a larger portion of amiloride-sensitive Isc-eq was detected in CF tissues, suggesting enhanced ENaC conductance in colonic mucosa of CF patients. Increase of intracellular cAMP by forskolin and IBMX inhibited amiloride-sensitive ENaC currents in non-CF tissues but not in CF biopsies. Therefore, enhanced epithelial Na+ conductance is present in the CF colon and is probably due to missing downregulation by CFTR.

Mycophenolate mofetil in pediatric renal transplantation without induction therapy: results after 12 months of treatment. German Pediatric Renal Transplantation Study Group.

Background. Acute rejection episodes (ARE) of kidney transplants are considered as risk factor in the development of chronic rejection. In adult renal transplantation (RTx), ARE have been significantly reduced by mycophenolate mofetil (MMF) in combination with cyclosporin (CyA) and steroids (Pred). Reports of pediatric RTx on a maintenance immuno-suppression with MMF are restricted to patients (P) after antibody induction therapy. Methods. The efficacy and safety of MMF combined with CyA and Pred in pediatric RTx without induction therapy were evaluated in an open-labeled multicenter study. Results. From 10/1996 to 6/1999, 65 pediatric P (MMF group) were followed for at least 6 months, 58 of 65 for 12 months. These P were compared with 54 retrospectively analyzed pediatric P who were transplanted between 1990 and 1996 and had received CyA, Pred, and azathioprine for immunosuppression (historic AZA group). Within the first 6 months after RTx, 18 of 65 (MMF group) and 32 of 54 (historic AZA group) P showed clinical signs of acute rejection (P <0.01). Thereafter only one further P in the MMF group developed a first ARE. Graft loss due to rejection occurred in one MMF- and seven AZA-treated P (P <0.05). The creatinine-clearance 3 and 6 months after RTx was higher in the MMF group. Major adverse events (MMF group) included infections of the urinary and the upper respiratory tract, diarrhea, and leukopenia. Cytomegalovirus-infection occurred in 13 P and 2 P developed cytomegalovirus disease. One P developed PTLD 10 months after RTx and recovered after the reduction of immunosuppression. Conclusions. The combination of MMF, CyA, and Pred reduced ARE in pediatric RTx without incurring major side effects.

Cytokine Expression of Cord and Adult Blood Mononuclear Cells in Response to Streptococcus agalactiae

Neonatal bacterial sepsis is often characterized by a fulminant clinical course and highly elevated plasma levels of proinflammatory cytokines. To evaluate in vitro activation of the neonatal immune system by specific infectious stimuli, cord blood cells from healthy neonates were examined for expression of tumor necrosis factor-α (TNF-α), IL-1β, IL-6, and IL-8 in response to Streptococcus agalactiae (GBS), lipopolysaccharide (LPS), and lipoteichoic acid (LTA). Cytokine-expression was compared in mononuclear cells from cord and adult peripheral blood. TNF-α and IL-6 levels in the supernatant of cord blood cell cultures were significantly higher after stimulation with heat-killed GBS (107/mL) than with LPS (2 μg/mL) or LTA (2 μg/mL) (TNF-α: 2215 versus 267.5 versus 40 pg/mL, p = 0.001; IL-6: 9667 versus 4909 versus 919 pg/mL, p = 0.006). mRNA expression of TNF-α, IL-1β, IL-6, and IL-8 was equally pronounced after stimulation with either GBS, LPS, or LTA in cord or adult blood cells at various times. A MAb directed against the monocyte receptor molecule CD14 did not inhibit the release of cytokines in cord blood mononuclear cells after stimulation with GBS. In summary, activation of cord blood cells by infectious stimuli is comparable to the adult immune response in terms of expression of proinflammatory cytokines. GBS in particular proves to be a potent activator of the neonatal immune system when compared with LPS and LTA. CD14 seems not to be a crucial molecule for activation of cord blood cells by GBS.

Enterohemorrhagic Escherichia coli infections: following transmission routes.

Abstract Infections with enterohemorrhagic Escherichia coli (EHEC) are the major cause of hemolytic-uremic syndrome (HUS ), the most-common cause of acute renal failure in childhood. The mortality rate of HUS (0%–5% in most recent series and 10%–30% in individual reports) and residual chronic renal sequelae (in up to 50% of patients in long-term follow-up studies) emphasize the seriousness of HUS for public health. Several studies have described possible sources of EHEC infection. However, in the majority of cases the pathogen cannot be identified in food or animals and the routes of transmission remain unclear. In this review article the hypothesized routes of transmission are summarized. The medical data bases ”Medline” and ”Current contents” were screened for the years January 1966 through November 1998. The difficulties in following the chain of EHEC infection are discussed. A precise evaluation of the environmental aspects of the patient is a precondition for further analysis.