Patient-reported outcomes of 182 adults with severe haemophilia in Germany comparing prophylactic vs. on-demand replacement therapy.

Clotting factor replacement therapy has a major impact on the quality of life in patients with haemophilia. To analyse and compare the outcomes of on-demand and prophylactic treatment regimens in child- and adulthood, a self-evaluation questionnaire was sent to 182 patients over 30 years of age with severe haemophilia A or B. Analysis of the questionnaire results revealed that most study participants had been treated on-demand in childhood, but that the majority of these patients subsequently switched to prophylaxis. However, of those patients who began with prophylaxis as children, the vast majority maintained prophylactic treatment as adults. Inhibitor development was reported significantly more frequently by patients who started with on-demand treatment than by those who started with prophylaxis. In the year prior to completing the questionnaire, adults with severe haemophilia who received prophylactic treatment reported a significantly lower incidence of bleeding as a result of more frequent factor consumption. These results are in close accordance with published prospective data. Although nearly all patients with severe haemophilia had joint pain due to bleeding, those who had always had prophylactic treatment reported superior outcomes in terms of the need for joint replacement, walking speed and distance, participation in school sports and further education. These data clearly underline the superiority of prophylactic treatment for the majority of individuals with severe haemophilia. The worst outcomes were found in those treated on-demand in childhood who later switched to prophylaxis. In contrast to most studies which have compared treatment regimens on the basis of data from healthcare professionals, this study reflects treatment outcomes from the patient's perspective.

Haemoassist--a hand-held electronic patient diary for haemophilia home care.

On-demand or prophylactic home-treatment is currently the treatment of choice for haemophilia patients. To allow physicians to monitor the amount of factor concentrates administered, the patients document each factor injection in a paper-diary. Nevertheless, because of the fact that most patients visit their physicians only two to four times a year, there could be considerable delay in detecting medication problems. The aim of this pilot study was to assess whether an electronic documentation tool could successfully replace traditional paper-diaries for haemophilia A patients and enable the physician to have a timely overview of the patient's treatment. An electronic, hand-held documentation tool, Haemoassist, was developed. In this study, patients using prophylaxis and on-demand therapies documented their factor consumption both electronically and on paper-diaries. Documentations were compared and descriptively evaluated. Patients also completed a survey to evaluate the feasibility and gather their opinions on the Haemoassist system. Ten patients from two haemophilia treatment centres in Germany submitted a total of 548 records via hand-held device during the observation period, from March 2006 to February 2007. Comparison of electronic and paper-based records showed differing responses among patients with some patients entering more electronic and some others more paper-based documentations. In the questionnaires on feasibility and usefulness of Haemoassist, three patients preferred the electronic tool, two patients wanted to continue using paper-based diaries, and one had no preference. The study shows that an electronic documentation system is feasible for haemophilia patients and provides the physician with the opportunity to more closely monitor patients. However, not all patients seem to be qualified for using an electronic tool, and the tool has to run reliably without major errors for ensuring reliability and acceptability. In the future, Haemoassist might support quality assurance in haemophilia treatment and improve guidance in the home-care setting.

Influence of diuresis on enzymuria.

Urinary excretion of renal brush border enzymes may serve as an early marker of renal injury. However, the distinction between physiological and pathological levels remains controversial, since enzymuria is affected by physiological parameters. To clarify the influence of diuresis, we investigated the urinary excretion of alanine-aminopeptidase (AAP; EC 3.4.11.2) as function of diuretic state. 17 healthy volunteers of both sexes were subjected to protocols with sudden or prolonged water load preceded and followed by a thirst period. Urinary excretion of AAP was measured using an enzyme kinetic assay. As expected AAP excretion increased with urine flow, the increments diminished yielding an overall excretion pattern that resembled saturation kinetics. This function is described by a mathematical model. This model assumes, that AAP is released in proximal tubules at a constant rate and reabsorbed or inactivated in the distal tubule and collecting duct. Non-linear fits of the model equation to our data allowed two parameters, chi and mu, to be defined. Chi describes the rate of AAP release independent of urinary flow, and mu the ratio of distal tubular reabsorption or inactivation. If a substrate is not reabsorbed at all, mu approximates zero. Since mu fitted for AAP differed significantly from zero, this indicates reabsorption or inactivation of AAP in the distal nephron. Therefore, our study supports the theory of flow-dependent reabsorption or inactivation of AAP in the distal nephron.

In hemophilia A and autoantibody inhibitor patients: the factor VIII A2 domain and light chain are most immunogenic.

Factor VIII (fVIII) is a protein cofactor essential for blood coagulation, and it binds in the factor Xase complex to factors IXa, X, and phospholipid. In about 30% of severe hemophilia A patients, treatment with fVIII leads to production of anti-fVIII antibodies. Anti-fVIII autoantibodies also rarely appear in normal individuals. Those antibodies that inactivate fVIII (inhibitors) prevent optimal fVIII therapy. Inhibitor epitopes were previously localized to the fVIII A2, A3, and C2 domains and to an acidic amino acid region between A1 and A2. Such anti-fVIII antibodies interfere with fVIII binding to components of the factor Xase complex and prevent blood coagulation. When total anti-fVIII titers were determined for each fVIII domain in 43 inhibitor plasmas by immunoprecipitation (IP) and inhibitor neutralization assays, the anti-light chain (LCh) antibody titer was highest, anti-A2 was intermediate, and anti-A1 and anti-B were low. The relative immunogenicity of the fVIII domains in hemophilic and autoantibody inhibitor patients was similar.

Low factor VIII recovery in haemophilia A patients without inhibitor titre is not due to the presence of anti-factor VIII antibodies undetectable by the Bethesda assay.

Measurements of factor VIII (FVIII) recovery in previously untreated patients with haemophilia A were done as part of the clinical trial of safety and efficacy of the recombinant FVIII, Recombinate. In 22 of 72 assessable patients, positive inhibitor titres > or = 0.6 Bethesda units mL-1 were detected by the Bethesda assay in one or more plasma samples, and the remaining 50 patients were negative at all timepoints. Of the latter group, 16 individuals without inhibitors unexpectedly had both normal (111) and low (52) recoveries during the study. We investigated the possibility that other antibodies not detectable in the Bethesda assay were responsible for the low recovery, by using a highly sensitive immunopreciptation (IP) assay for detection of all antiFVIII antibodies. Eight of the 16 patients with low and normal recoveries did indeed have antibodies detected by the IP assay, and the remaining eight were negative. Four antibody-positive individuals had insignificantly low titres, and the other four had modest to high titres. In the latter group, antibodies were found with similar frequencies and titre in plasmas from patients with low or normal recovery. Low recovery in haemophilia A patients without inhibitor titres must therefore be attributed to factors other than antiFVIII.

Nephrotoxic potential of antiinfective drugs as assessed by tissue-specific proteinuria of renal antigens.

In order to assess the nephrotoxic potential of antibiotics, various aminoglycosides and cephalosporins were tested for their potency to alter the excretion of tubular marker proteins (and brush border antigens) or to change the normal pattern of serumproteinuria as analyzed by SDS polyacrylamidgel gradient electrophoresis. After aminoglycosides, especially after gentamicin injection, a cumulative highly significant increase in the urinary output of marker proteins emerged (healthy volunteer model). In contrast, cephalosporins exhibited practically no nephrotoxic effect on proximal tubule cells. Excretion of tubular marker proteins was enhanced under combined administration of cephalosporins and aminoglycosides mainly due to the aminoglycoside component. There was no nephrotoxic synergy of both drugs. Image analysis of rat kidney sections after injection of aminoglycosides revealed that increased shedding of tubular membrane components under the toxic challenge is followed by rapid inductive repair processes (overshoot protein synthesis) of tubular cells. After a limited acute toxic damage tubular cells may recover within one week.

The natural history of the immune response to exogenous factor VIII in severe haemophilia A.

The development of inhibitory antibodies to factor VIII (fVIII) in severe haemophilia A patients is a serious therapeutic complication. Using a highly sensitive immunoprecipitation (IP) assay which measures all anti-fVIII antibodies, we have tested severe haemophilic plasmas from two clinical studies. Inhibitor titres in the range of 0.4 to 1 Bethesda units/ml (BU/ml) could not be verified by IP as being due to an immune response to fVIII in 35% of plasmas tested. Low fVIII recoveries were likewise correlated with the presence of antibodies in 29% of plasmas tested. However, 16% of plasmas without inhibitor titres had immune responses as measured by IP. The rapidity of antibody appearance did not allow their effective detection by IP before development of inhibitor titres. These results suggest that the IP assay can provide a valuable confirmation of anti-fVIII antibody production when the Bethesda assay is low or negative and where clinical observations suggest their presence, but they cannot be used reliably to detect early immune responses.

Carbon disulphide. III. Risk factors for coronary heart diseases in workers in the viscose industry.

To evaluate risk factors for coronary heart disease and factors which can influence the course of acute myocardial infarction in workers exposed to CS2 we performed a cross-sectional study of 247 workers in the viscose industry. The control group of 222 men from the same plant was comparable for age, social status and physical work. The CS2 exposure determined by personal air sampling ranged from < 0.2 ppm to 65.7 ppm (median: 4.0 ppm) and the duration of exposure ranged from 4 to 220 (median: 66) months. Using a multiple linear regression model we found neither higher blood pressure at rest or after exercise, nor hyperlipoproteinaemia in a higher degree, nor lower high-density lipoprotein (HDL) or lower apolipoprotein A-I levels, nor higher blood glucose values, nor indicators of direct cardiotoxic effects or signs of disturbances in blood coagulation in the exposed group in comparison to controls. Regarding the influence of chronic exposure on the investigated parameters, we found an inverse correlation of the cumulative exposure (mean CS2 exposure in the department multiplied by the duration of work in this department) with the HDL concentration. The HDL levels correlated with the duration but not with the intensity of exposure. In the same way the apolipoprotein A-I levels showed a negative association with the duration of exposure in the exposed group as well as in the control group. The HDL concentrations showed the same trend for the controls. It therefore seems that this finding is more likely due to confounding factors than to the CS2 exposure. As all subjects (exposed and controls) have done shift work, in some cases for a long time, this kind of work could be responsible for the negative relationship between the duration of employment as a shift worker and the apolipoprotein A-I and HDL levels. At the current air-borne levels no significant differences were found between the exposed persons and the controls in the distribution frequency for blood pressure values, lipoproteins, blood glucose, blood coagulation and indicators of direct cardiotoxic effects.

Screening of F.VIII:C antibodies by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (Elisa) method was developed in order to examine prevalence and titer of antibodies directed against the factor VIII coagulant protein (F.VIII:C) in hemophilia A and nonhemophilia A patients. Highly purified F.VIII:C was used as immunosorbent on microtiter plates with a peroxidase-conjugated goat anti human IgG antibody for F.VIII:C antibody detection. Results determined by Elisa were compared with measurements according to the Bethesda method. Initially 24 plasma samples containing an F.VIII:C inhibitory activity ranging from 0 to 7,700 Bethesda units (BU) were analysed. At plasma dilutions of 1:128 the optical density determined by our Elisa measurement and the corresponding BU showed a logarithmic correlation. The coefficient of correlation was r = 0.92 with a standard deviation of 0.002 from the regression curve. Plasma samples were analysed from 53 hemophilia A patients, from 21 nonhemophilia patients with acquired F.VIII:C antibodies and from 460 randomly selected nonhemophilia patients presenting for routine preoperative coagulation examination. F.VIII:C antibody-positive Elisa results and positive BU were found in 7 hemophilia A patients and the 2 patients with a history of acquired F.VIII:C antibodies. Positive Elisa results and negative BU were found in 1 hemophilia A patient and 25 out of 460 nonhemophilia A patients (5.43%) suggesting F.VIII:C antibodies without inhibitory potency on F.VIII:C in these cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the gene for apolipoprotein E5-Frankfurt (Gln81->Lys, Cys112->Arg) by polymerase chain reaction, restriction isotyping, and temperature gradient gel electrophoresis.

A new apolipoprotein (apo) E variant, apoE5-Frankfurt, was identified in a 43-year-old male with moderate hypercholesterolemia. On isoelectric focusing in an immobilized pH gradient, apoE5-Frankfurt migrated to a position more cathodic than apoE4 (Cys112->Arg). On sodium dodecyl sulfate-gel electrophoresis, its apparent molecular weight could not be distinguished from that of the three common apoE isoforms (E2, E3 and E4). Restriction isotyping with CfoI (HhaI) showed that apoE5-Frankfurt had arginine in positions 112 and 158 of the mature protein, suggesting that the mutation accounting for the additional positive charge had occurred in an epsilon 4 allele. The third and the fourth exon of the apoE gene were amplified using the polymerase chain reaction and analyzed by temperature gradient gel electrophoresis. This suggested that there were two mutations in the fourth exon of the mutant allele. Cloning and sequencing disclosed that, apart from the exchange of arginine for cysteine in position 112, a C to A substitution replaced glutamine (CAA) in position 81 by lysine (AAA).

[Assessment of drug nephrotoxicity by the excretion of tubule-specific membrane antigens and enzymes]. / Beurteilung der Nephrotoxizität von Pharmaka über die Ausscheidung tubulusspezifischer Membranantigene und Enzyme.

A possible tubulotoxicity of drugs can be judged with the help of the excretion of tubular membrane proteins in the urine. The brush border of the proximal tubular epithelia which react particularly sensitive to toxic influences contains surface antigens which easily release themselves from the membrane core membrane under pathological conditions and become provable in the urine by means of biochemical and immunological methods as signs of an early structural cell damage. Apart from these soluble membrane proteins which above all correspond to enzymes such as alanine aminopeptidase and gamma-glutamyl transpeptidase in severe lesions high molecular brush border fragments transformed to vesicles can appear. The clinical relevance of a pathological tissue proteinuria (histuria) of proteins of renal membranes is among others explained at the instance of the renal effects of cytostatics, antibiotics and x-ray contrast medias.