Therapeutic potential of vasopressin V2 receptor antagonist in a mouse model for autosomal dominant polycystic kidney disease: optimal timing and dosing of the drug.

BACKGROUND: The renoprotective effect of vasopressin V2 receptor antagonist (V2RA) is currently being tested in a clinical trial in early autosomal dominant polycystic kidney disease (ADPKD). If efficacious, this warrants life-long treatment with V2RA, however, with associated side effects as polydipsia and polyuria. We questioned whether we could reduce the side effects without influencing the renoprotective effect by starting the treatment later in the disease or by lowering drug dosage. METHODS: To investigate this, we administered V2RA OPC-31260 at a high (0.1%) and low (0.05%) dose to a tamoxifen-inducible kidney epithelium-specific Pkd1-deletion mouse model starting treatment at Day 21 (early) or 42 (advanced). After 3 and 6 weeks of treatment, we monitored physiologic and potential renoprotective effects. RESULTS: Initiation of V2RA treatment at advanced stage of the disease lacked renoprotective effects and had less pronounced physiologic effects than early initiation. After 3 weeks on a high dose, cyst ratio and kidney weight were reduced versus untreated controls (18 versus 25%, P = 0.05, and 0.33 versus 0.45 g, P = 0.03, respectively). After 6 weeks of treatment, however, this did not reach significance anymore, even at a high dose (cyst ratio 24 versus 27%, P = 0.12, and kidney weight 0.55 versus 0.66 g, P = 0.38). CONCLUSIONS: Our results suggest that intervention with V2RA should be instituted early in ADPKD and that it might be necessary to further increase the dosage of this drug later in the disease to decrease cyst growth.

Von Willebrand Disease type 2M "Vicenza" in Italian and German patients: identification of the first candidate mutation (G3864A; R1205H) in 8 families.

Von Willebrand disease type 2M "Vicenza" (VWD 2M V) is characterised by autosomal dominant inheritance, low von Willebrand factor (VWF) and the presence of "supranormal" multimers in plasma. This specific phenotype has been described in Italian and recently also in German patients. The molecular defect is linked to the VWF gene. However, no specific mutations have been identified until now. We analysed the complete coding region and adjacent intron sequences of the VWF gene in Italian families in comparison to German families with VWD 2M V by a PCR-based mutation screening, combined with SSC- and heteroduplex-analysis of exons 2 through 52, followed by direct sequencing. We identified the first heterozygous candidate mutation (G3864A; R1205H) in all affected members of the 7 Italian families and in 1 German patient but not in the unaffected family members nor on 100 chromosomes of normal subjects, suggesting a causal relationship between the mutation and the phenotype. Haplotype identity, with minor deviations in one Italian family, suggests a common but not very recent genetic origin of R1205H.

Prevalence of factor V Leiden in children with thrombo-embolism.

UNLABELLED: Hereditary resistance to the anticoagulatory action of activated protein C (APC resistance, APCR) was identified as a possible new thrombophilic factor in a high percentage (17%-60%) of young adults with thrombotic events. A single missense mutation (R506Q) due to a G/A transition (G1691A) in exon 10 of the factor V gene is regarded as the causative molecular defect, resulting in factor V Leiden which is correlated with APCR. Identification of this mutation by polymerase chain reaction-based methods is easy to perform and prevents pre-analytical and analytical errors in the coagulometric assay for APCR. Since the impact of this mutation in children with thrombo-embolic disease has not been determined to date, we initiated a multi centre prevalence study in two paediatric populations, with and without thrombo-embolic events. We compared 125 paediatric patients with thrombosis, divided into three different age groups (0 to < 0.5 years; > 0.5 to < 10 years; > 10 to < 18 years) with a normal population of 159 children. Although the mutation G1691A was found with an unexpectedly high prevalence of 12% in our normal controls, the prevalence was significantly higher in the age groups; 0 to < 0.5 years (26%) and > 10 to < 18 years (30%). In patients between > 0.5 and < 10 years the overall prevalence was similar to that of the control group (13%). However, in patients of this age with spontaneous thrombosis, G1691A was also a significant risk factor (5/17 approximately equal to 29%). Homozygosity for G1691A was detected in three patients but not in the control group. Including deficiencies of protein C, protein S, antithrombin, and the presence of anti-phospholipid antibodies, thrombosis was correlated with endogenous thrombophilic factors in 38/125 patients (30.4%). CONCLUSION: Our results emphasize the impact of factor V Leiden on thrombogenesis in children. However, the significance is age-dependent and may reflect the different physiology of haemostasis in the three age groups. The diagnostic workup of children with thrombosis should include tests for factor V Leiden. The correlation of factor V Leiden with the clinical course of thrombo-embolism in children is essential to establish rational guidelines for therapy and prophylaxis of APCR-related thrombosis which are not yet available.

Results of a screening for von Willebrand disease type 2N in patients with suspected haemophilia A or von Willebrand disease type 1.

A screening program for the detection of patients with von Willebrand disease type 2N (VWD 2N) was carried out in 177 unrelated patients previously diagnosed with haemophilia A and in 199 unrelated patients with VWD type 1 in comparison. By measuring the factor VIII (FVIII) binding capacity of von Willebrand factor (VWF), we detected 13 patients with VWD 2N within 8 unrelated families. The former diagnosis has been haemophilia A in 5 index patients, and VWD in the remaining 3. Included in this study were 14 patients with suspected haemophilia A, whose molecular analysis for mutations in the FVIII gene was unsuccessful. Five of them had VWD 2N. In all patients with the VWD 2N phenotype we were able to identify specific molecular defects in the corresponding DNA sequence of the FVIII binding domain of VWF. The most common defect was R91Q. Four patients from 4 families were homozygous for R91Q, six patients from 3 families were compound heterozygous for R91Q and a silent yet unidentified allele. The VWD 2N phenotype of father, daughter, and son in one family, was based on 2 different genotypes, compound heterozygosity for R91Q and VWD type 1 in the father and the daughter, and homozygosity for R91Q in the son. Two patients from one family were compound heterozygous for T28M and delta C2680-2685 in exon 18, and one patient was compound heterozygous for a novel candidate missense mutation in exon 18(E24K) and delta C2680-2685 in exon 18 which is regarded the most common defect causing severe VWD type 3. FVIII:C values of 0.07 and 0.14 IU/ml were observed in patients with the genotype T28M/delta C2680-2685 whereas in patients being homozygous or compound heterozygous for R91Q, FVIII:C was 0.19 +/- 0.071 IU/ml. In contrast to the other genotypes, E24K/delta C2680-2685 is correlated with a more severe haemophilic phenotype with a FVIII residual activity of only 0.01-0.02 IU/ml. This emphasizes the need for inclusion of the factor VIII binding assay in the diagnostic workup of suspected haemophilia A.

Defective dimerization of von Willebrand factor subunits due to a Cys-> Arg mutation in type IID von Willebrand disease.

The same heterozygous T -> C transition at nt 8567 of the von Willebrand factor (vWF) transcript was found in two unrelated patients with type III) von Willebrand disease, with no other apparent abnormality. In one family, both alleles were normal in the parents and one sister; thus, the mutation originated de novo in the proposita. The second patient also had asymptomatic parents who, however, were not available for study. The structural consequences of the identified mutation, resulting in the CyS2010 -> Arg substitution, were evaluated by expression of the vWF carboxyl-terminal domain containing residues 1366-2050. Insect cells infected with recombinant baculovirus expressing normal vWF sequence secreted a disulfide linked dimeric molecule with an apparent molecular mass of 150 kDa before reduction, yielding a single band of 80 kDa after disulfide bond reduction. In contrast, cells expressing the mutant fragment secreted a monomeric molecule of apparent molecular mass of 80 kDa, which remained unchanged after reduction. We conclude that CyS2010 is essential for normal dimerization of vWF subunits through disulfide bonding of carboxyl-terminal domains and that a heterozygous mutation in the corresponding codon is responsible for defective multimer formation in type III) von Willebrand disease.

Identification of a candidate missense mutation in a family with von Willebrand disease type IIC.

A screening project to identify candidate molecular defects causing von Willebrand disease type IIC (VWD IIC) in a German family was carried out using polymerase chain reaction (PCR) amplification of all 52 exons of the von Willebrand factor (VWF) gene, subsequent electrophoresis of single and double stranded DNA and direct sequencing of PCR products with aberrant electrophoretic patterns. Only one candidate mutation, G550R, caused by a G-->A transition, was detected in exon 14 of the pro-VWF gene sequence. This mutation was not found on 200 chromosomes of normal individuals. The propositus was homozygous for the mutation and for an extended intragenic haplotype, composed of eight polymorphic markers. Further family members were heterozygous for the mutation and were phenotypically normal or only mildly affected, in accordance with the recessive pattern of inheritance for VWD type IIC. The mutation could influence one of the presumed active centers for the suspected multimerizing enzymatic activity of pro-VWF localized in the D1 and D2 domain, which corresponds to exon 5 and exon 14 of the VWF gene.

Genetic heterogeneity of severe von Willebrand disease type III in the German population.

The genetic heterogeneity of severe von Willebrand disease (vWd) type III was estimated by analysing extended haplotypes of eleven intragenic restriction fragment length polymorphisms and one variable number of tandem repeat polymorphism in 32 patients from 28 families from Germany or of German origin. All patients were screened for gross deletions and for mutations at potential "hot spot" regions of the von Willebrand factor (vWf) gene. Disease-associated haplotypes were established in 24 families. Only a few, apparently unrelated families shared common haplotypes suggesting a considerable genetic heterogeneity in the German population of vWd type III patients. Defects causing vWd type III were identified on 14 out of 56 chromosomes (25%). Gross deletions were detected in two families. A complete homozygous deletion of the vWf gene was displayed in one patient. Another patient was compound heterozygous for a large deletion of at least 100 kb of the vWf gene with an additional, as yet unidentified, defect. One homozygous missense mutation was detected in exon 10, and two nonsense mutations were detected in exon 8 and exon 45 of the vWf gene, respectively. A frameshift mutation (delta C) in exon 18 was identified in five families and an additional frameshift mutation (delta G) was found in exon 28 in one family. It appears that delta C is the most common molecular defect in German patients with vWd type III. Its association with a number of different haplotypes suggests repeated de novo mutations at a mutation "hot spot". Evidence is presented that particular molecular defects causing vWd type III are associated with different patterns of inheritance, depending on their location within the vWf gene. Complete deletions of the gene and nonsense mutations in the pro-sequence are correlated with recessive inheritance, whereas frameshift and nonsense mutations in the gene sequence corresponding to the mature vWf subunit tend to be inherited in a dominant fashion.

Percent body fat in obese white females predicted by anthropometric measurements.

The percent body fat (PBF) and 15 anthropometric measurements were measured in 221 obese white females randomly assigned to validation and cross-validation groups. Two new anthropometric equations for the prediction of the percent of body fat were generated by multiple regression. Equation 1 includes the residual lung volume (RV) as a factor and had a correlation coefficient (r) of 0.85 and a standard error of the estimate (SEE) of 3.9%. Equation 2 does not include the RV and has an r of 0.82 and an SEE of 4.3%. Both equations were more precise than two previous widely used equations. In a subgroup of 37 subjects who underwent weight loss, equation 1 gave a more precise estimate of the change in PBF. We conclude that the new equations permit a better prediction of the PBF in obese white females.

Microbial growth in clinically used enteral delivery systems.

Potential contamination of enteral formulas has led to the development of policies limiting formula hangtimes. However, enteral administration bags can easily become contaminated during formula refilling. We prospectively studied enteral formula contamination when the hangtime of a prefilled 1000 ml pouch was compared with the standard 4-hour hangtime of a refilled enteral administration bag. Samples of formula collected from different locations along the enteral delivery system were cultured during 57 days of enteral hyperalimentation in 19 patients. The overall enteral formula contamination rate was 61%, where the greatest microbial growth occurred in reconstituted enteral formulas. The presence of microbial growth did not differ between canned formulas administered according to a 4-hour hangtime and the prefilled pouch. Greatest growth in all cases was at the distal tubing hub, where contamination during system manipulation or from the patient probably occurred. Use of prefilled enteral administration bags may delay formula contamination in the administration reservoir. A change in equipment design that would decrease the need to manipulate feeding sets or feeding tube connections should be further investigated.

[Swimming after myocardial infarct. Oxygen saturation, pulmonary arterial and capillary pressure in underwater immersion and during swimming after myocardial infarct]. / Schwimmbelastung nach Herzinfarkt. Sauerstoffsättigung, Pulmonalarteriendruck und Pulmonalkapillardruck bei subaqualer Immersion und während des Schwimmens nach abgelaufenem Herzinfarkt.

Pulmonary arterial (PA) and pulmonary capillary (PC) pressures and oxygen saturation were measured over a 5-minute period on immersion in water and during swimming in four symptom-free men (aged 52-58 years) who had sustained a transmural infarct 6-10 weeks previously. Measurements were made through an indwelling flow-guided percutaneously introduced catheter. The speed of swimming was 20-25 m/min. Static pressures on immersion up to the neck rose up to 23 mm Hg, on diving to five times as much above the resting recumbent value. Mean PC values after five minutes of swimming rose in all four subjects above those obtained after a 5-minute 100 W exercise: they were similar to the diastolic pressures. But the oxygen saturations on swimming were in all patients higher than after tread-wheel exercise. All patients felt swimming to be easier than the ergometric exercise. It is concluded that noninvasive methods, such as X-rays, ergometry and echocardiography, are not sufficient for estimating volume stress induced by immersion in water. Cardiac arrhythmias did not occur.

Enhancing enteral nutrition delivery: development of an enteral preparation facility.

As a result of prospective reimbursement, departments of clinical dietetics must define and maintain the nutrition services offered in the most cost-effective manner. The processing, preparation, and provision of quality enteral hyperalimentation to patients provide an example of a nutrition service in which cost-saving measures can be employed. Saint Vincent Charity Hospital and Health Center, Cleveland, decided to evaluate and increase the efficiency of tube-feeding preparation, delivery, inventory, and purchasing because of an increased demand for those services. The evaluation process resulted in the development of an enteral preparation facility, a specialty kitchen located within the foodservice department, specifically designed for cost-effective preparation and dispensation of all enteral formulas. The effort required physical space reallocation and personnel retraining. Implementation of the enteral preparation facility has resulted in improved quality of enteral nutrition services and has significantly aided nutrition support and cost-containment efforts at the hospital.

Microbial growth comparisons of five commercial parenteral lipid emulsions.

The ability of parenteral lipid emulsions to support microbial growth was compared using commercially available brands of lipid emulsion. Both 10 and 20% concentrations of soybean and safflower oil emulsions were used. Washed cultures of six gram-negative, three gram-positive, and one yeast, in concentrations of 1 x 10(4) to 2 x 10(4) colony-forming units/ml, were inoculated into lipid emulsion aliquots and stored at room temperature. There were than subcultured at 0, 6, 12, 24 and 48 hr. After 48 hr at 37 degrees C, growth was recorded as colony-forming units/ml. Normalized growth curves were expressed as mean +/- SEM. ANOVA demonstrated no difference in growth patterns due to the nature of the oil or its concentration. Gram-negative organisms multiplied faster when compared to gram-positive (p less than 0.05 at 12 hr, p less than 0.01 at 24 hr, and p less than 0.005 at 48 hr). Yeast grew as well as bacteria. The Center for Disease Control's recommendation of a 12-hr hang time for parenteral lipid emulsions should be observed until correlation of laboratory microbial growth patterns and clinical use are studied further.

Selenium deficiency with total parenteral nutrition: reversal of biochemical and functional abnormalities by selenium supplementation: a case report.

A patient with multiple intestinal fistulae maintained on total parenteral nutrition for 18 months developed low serum selenium. Erythrocyte glutathione peroxidase activity was 6% of normal. Erythrocytes were not able to metabolize H2O2 as well as those from controls, although the hexose monophosphate shunt itself was intact. Granulocytes from this patient had 15% of the erythrocyte glutathione peroxidase activity found in normals. Patient granulocytes were not able to metabolize H2O2 as well as controls, although the hexose monophosphate shunt was intact. Erythrocyte glutathione peroxidase-deficient granulocytes incubated with a respiratory burst stimulant, phorbol myristate acetate, had only 60% of the hexose monophosphate shunt activity present in control granulocytes. These abnormalities were reversed with selenium supplementation. Bacterial killing of Staphylococcus aureus 502A and cardiac function were not affected by selenium deficiency. Thus, selenium deficiency resulted in biochemical and functional abnormalities of erythrocytes and granulocytes. These abnormalities were reversed with selenium supplementation.

Alternate dietary lifestyles.

Various forms of vegetarian diets are discussed and evaluated for their nutritional adequacy. Health, philosophical, religious, ecological, and economic concerns are suggested as possible reasons for these alternate dietary lifestyles. Nutrients of specific concern ot the vegetarian are highlighted and suggestions given to help incorporate these in the diet, thereby avoiding marginal intakes. With judicious menu planning and careful thought to food selections, most vegetarian diets can supply excellent nutrition. Very restricted vegetarian diets or higher level macrobiotic diets may not be nutritionally complete, and individuals following these diets may benefit from special dietary counseling and dietary supplementation. Otherwise, these diets may place the adult as well as pregnant and lactating women, infants, and children at a nutritional risk. As vegetarian food habits are becoming more widespread, physicians and nutritionists must be knowledgeable about these alternate dietary lifestyles in order to counsel their patients appropriately, to understand the reasons for these eating habits, and to be supportive of the choice of diet.

Performance evaluation of a new nasogastric feeding tube.

A performance evaluation of a new polyurethane nasogastric feeding tube and stainless steel stylette (Nutriflex, No 8 French) was conducted at University Hospital. Thirty-eight tubes were successfully placed in 18 patients. Tubes remained in place less than 1-29 days and feedings were administered for a total of 196 patient days. Enteral formulas commonly available were administered via continuous drip. The tube use was surveyed for ease of insertion primarily; however, patency and comfort to the patient were felt to be positive with the use of this tube. With slight modifications the new feeding tube is a welcome addition to the expanding technology associated with enteral feedings.