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.

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.

Isolation of kidney brush border gamma-glutamyl transpeptidase from urine by specific antibody gel chromatography.

The IgG fraction of antiserum directed against gamma-glutamyl transpeptidase (gammaGTP, EC 2.3.2.2) isolated from human kidney brush border membranes after limited proteolysis, was covalently bound to cyanogen bromide-activated Sepharose. With this antibody-gel, gammaGTP present in the urine of patients as a result of tubular damage was immunospecifically prepared by affinity chromatography. The enzyme isolated from the urine samples gave a complete cross-reaction wiht gammaGTP artificially cleaved off from brush border fragments. Since labelled anti-gammaGTP sera gave a specific immunofluorescence only of the luminal portion of cortical tubule, the use of immunosorption chromatography appears to be an important approach for the isolation of urinary kidney tissue antigens of a defined origin.

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.

[Quantitative enzymatic and enzyme histochemical analysis in detecting inductive and nephrotoxic effects of various antibiotics]. / Quantitative enzymatische und enzymhistochemische Analysen zum Nachweis induktiver und nephrotoxischer Wirkungen verschiedener Antibiotika.

In order to evaluate quantitatively changes of kidney tubular membrane and lysosomal proteins following administration of aminoglycosides and cephalosporins enzymatic and enzyme histochemical investigations were performed. Alterations of kidney enzyme concentrations of the proximal tubule were considered as a parameter indicating nephrotoxic and inductive effects of antibiotics. Following administration of aminoglycosides to volunteers significantly (Wilcoxon test, 2P less than 0.05) increased enzymuria was found as compared to untreated control persons. Under experimental conditions inductive effects were analysed following treatment with aminoglycosides, especially gentamicin. In contrary to these findings cephalosporins were administered without effects an the proximal tubule. Once daily administration of aminoglycosides were observed to be less nephrotoxic than twice daily injections to volunteers. Quantitative evaluation of tubular specific proteins appears to be a tool in monitoring the degree of alterations (enzyme induction, -liberation, -excretion) caused by antibiotics.

Excretion of kidney brush border antigens as a quantitative indicator of tubular damage.

The excretion of antigens and enzymes derived from the brush border region was studied in patients with kidney diseases, after kidney transplantation, during administration of potential nephrotoxic drugs, before and after operations etc. The main portion of membrane constituents was excreted in the urine at an increased rate, compared to healthy persons, and was identical with glycoproteins artificially released from the brush border membrane surface. Antisera against brush border antigens, which had been isolated from urine by affinity chromatography, were used to localise the origin of urinary kidney tissue-proteins applying immunofluorescence microscopy.

Effect of a proteinase inhibitor, aprotinin, on brush border membrane associated aminopeptidase of human kidney cortex.

In order to study the influence of the basic proteinase inhibitor aprotinin (Trasylol) on renal tubular alanine-aminopeptidase (E.C.3.6.11-), a brush border fraction was prepared from human kidney cortex by differential centrifugation. Incubation of brush border fragments, rich in membrane bound alanine-aminopeptidase, resulted in an activation of the enzyme. However, aminopeptidase, which was cleaved off from the membranes by limited proteolysis and subsequently purified by Concanavalin A-affinity chromatography exhibited activation by low but inhibition by higher concentration of aprotinin. Thus inhibition was associated with presence of the solubilized form of the brush border enzyme. On the other hand, as was shown by quantitative electroimmunoassay, formation of antigen-antibody complexes between soluble renal aminopeptidase and its specific rabbit antibody was not changed by the basic polypeptide.

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.

[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.

Quantitative enzymatic and immunologic computer-assisted histophotometry of human kidney tissue following neoplastic and other clinically significant alterations.

Renal tissue sections from 178 patients, whose kidneys were either normal or altered by various conditions such as hydronephrosis, interstitial nephropathies, chronic graft rejection, renal cancer etc., were investigated by computer-assisted histophotometry. We used enzyme histochemical and immunologic methods to measure kidneys suffering from various urological diseases quantitatively. Through this procedure, we were able to obtain information that allowed us to determine the degree of alteration in the metabolic state of tubular epithelial cells. The tissue activities of the following enzymes of the proximal tubule were investigated: alanine aminopeptidase (AAP), alkaline phosphatase (AP) and maltase (Ma) as membrane-bound markers, and beta-glucuronidase (beta-Gl) as a lysosomal marker. In addition, AAP and gamma-glutamyltranspeptidase (GGTP) were measured by immunofluorescent microscopy after having added specific anti-enzyme antibodies to the tissue sections. Compared to normal kidneys, quantitative enzyme histograms of diseased kidneys revealed a significant decrease in marker protein concentration of the tubule. The decline in tissue enzyme activities of AP, AAP, Ma and beta-Gl was accompanied by a significant decrease of enzyme concentrations as measured by the immuno histological method. This was especially true in cases with kidney cancer and in kidney tissues adjacent to infiltration adenocarcinoma. Morphological analyses of alterations were generally improved by enzymatic and/or immunologic histophotometry.

Biochemical, immunological and ultrastructural studies on brush-border membranes of human kidney.

Brush border (BB) membranes, isolated from human kidney cortex by density gradient centrifugation, revealed a distinct pattern of structural proteins as could be shown by bio- and immunochemical studies. Marker enzymes such as gamma-glutamyltranspeptidase (GGTP) and alanine-aminopeptidase (AAP) were characterized as extrinsic; alkaline phosphatase (AP) was characterized as an integral constituent of the BB membrane. The surface of the BB membranes exhibited numerous 5 nm particles bound through a linear component to the peripheral BB matrix (negative staining). Increase of AAP and GGTP (30%) activity in the supernatant after proteolytic treatment of BB fragments paralleled selective release of these constituents from the membranes. The surface components were found to be part of BB concanavalin A and wheat germ agglutinin receptor sites. Labelled antisera directed against surface glycoprotein fractions gave a specific immuno fluorescence staining of only the luminal plasma-membrane from the proximal tubule epithelia.

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).

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)