[Anemia and hemoglobin diseases in patients with migration background]. / Anämien und Hämoglobinkrankheiten bei Patienten mit Migrationshintergrund.

Among the German population with migration background there are probably 150 000-200 000 carriers of thalassemia (α und ß) and sickle cell disease, respectively, who have no or little symptoms. Compared to neighboring countries the number of sickle cell (1000-1500) and thalassemia patients (500-600) in Germany is rather low. This may explain the fact that hemoglobin diseases are not yet considered a public health problem in Germany. With optimal care 85-90 % of children with sickle cell disease and 100 % of children with thalassemia reach adulthood. In order to increase awareness for patients with hemoglobin diseases we discuss the most pertinent disease manifestations of adult patients and point out possibilities to obtain information. Specialists in regional centers should be addressed for acute management problems. Up to now it is difficult for many adult sickle cell and thalassemia patients to find a physician well enough informed and experienced to take over the care of their complex disease. Many adult patients are still taken care of by pediatricians. Urgently needed are reference centers with experience in management of hemoglobin diseases who are qualified for training hematologists and who can assure the transition of these patients from pediatrics to adult medical care.

[Prevention of infections and thromboses after splenectomy or because of functional loss of the spleen]. / Prävention von Infektionen und Thrombosen nach Splenektomie oder bei Funktionsverlust der Milz.

Overwhelming Post-Splenectomy Infection (OPSI or PSS), most frequently caused by encapsulated Gram-positive pathogens, is a complication after splenectomy. Reasons for splenectomy include trauma, or malignant and non-malignant hematologic diseases. OPSI-inducing bacteria are mainly Streptococcus pneumoniae and less frequently Haemophilus influenzae, Neisseria meningitides and Gram-negative bacilli. There exist very efficient--albeit often neglected--strategies, how to prevent infections in patients after splenectomy. These include vaccination, prophylactic antibiotics (always for 3 years during childhood and adolescence) and prompt antibiotic treatment, if an infection is suspected. Patients need to know the nature and likelihood of PSS and they should seek immediate medical attention if they become ill or febrile. Each patient should carry at all times a letter or card documenting the splenectomy. With these measures and precautions, the PSS-risk can be significantly reduced or at best be completely avoided.

Mode of splenectomy and immunogenicity of meningococcal vaccination in patients with hereditary spherocytosis.

BACKGROUND: Splenectomy predisposes patients to invasive disease from pneumococci, meningococci, and Haemophilus influenzae; immunization is mandatory. However, data on the impact of the splenectomy on vaccine immunogenicity are scarce. METHODS: A total of 41 children with hereditary spherocytosis (aged 5.8-14.4 years) had complete (16) or near-total (25) splenectomy. All received one dose of monovalent meningococcal C conjugate vaccine (MCV-C) and, 2 months later, a tetravalent meningococcal polysaccharide vaccine (MPV-ACWY). Serum bactericidal activity and antibodies against serogroups A and C were determined before and after they received MCV-C, and 4 weeks after they received MPV-ACWY. RESULTS: Before vaccination, only four of the 16 children who had a complete splenectomy were protected against serogroup A, compared with 15 of the 25 who had near-total splenectomy (P < 0.050), with the latter responding to immunization with significantly higher serogroup A serum bactericidal activity: geometric mean (95 per cent confidence interval) 1625.5 (49.9 to 3201.1) versus 980.6 (2.00 to 6204.1) (P < 0.050). All patients achieved putative protective serum bactericidal activity titres (at least 8) against serogroup C. CONCLUSION: Near-total splenectomy provides a favourable immunological basis for natural and vaccine-induced protection against meningococcal serogroup A and C infections. Sequential meningococcal vaccination is immunogenic in patients splenectomized for hereditary spherocytosis.

Favorable response of pediatric stem cell recipients to human protein C concentrate substitution for veno-occlusive disease.

Plasminogen activator inhibitor 1 is known to be elevated in patients with hepatic VOD after intensive chemotherapy. To re-establish endogenous fibrinolysis and to inhibit thrombin formation, we used non-APC (zymogen) to normalize PAI-1 levels. As a consequence of thrombin formation inhibition and the consecutive inhibition of the coagulation cascade, this treatment is expected to reduce the elevated D-dimer level. Six pediatric stem cell recipients with moderate or severe VOD after busulfan or total body irradiation conditioning regimen are reported here who were therapy-refractory to defibrotide or rt-PA therapy. All patients had low levels of PC activity (16-39%). The administration of PC (60-240 IU/kg) led to a rapid and sustained rise in PC activity (target level >80%) with near normalization of prothrombin and partial thromboplastin time in all patients. Elevated PAI-1 levels declined. Five of the six patients showed a good clinical response with prompt resolution of clinical, sonographic, and laboratory signs of hepatic blood flow obstruction, while one patient with severe VOD, as well as concomitant liver GVHD and CMV disease, had a slow but detectable response to PC therapy. All patients survived.

Monitoring the Cry1Ab susceptibility of European corn borer in Germany.

The European corn borer, Ostrinia nubilalis (Hübner), is one of the most important insect pests in corn, Zea mays L. Transgenic corn cultivars expressing Bacillus thuringiensis (Bt) toxin provide a promising crop protection strategy against European corn borer; however, management is needed to avoid resistance development of the target pest species. The aim of this work was to establish the baseline susceptibility of different European corn borer populations in Germany to be able to forecast a possible development of resistance at an early stage. To standardize test procedures for future resistance management, the efficiency of Cry1Ab toxins from different suppliers and different production was assessed. Furthermore, two different test methods, surface treatment and the incorporation method, were compared with regard to their practicability and efficiency. Neither method provided significant differences in the baseline susceptibility of populations from different German regions. Overall, the data suggested little differentiation among German populations in terms of their susceptibility to Bt toxin and their genetic background. Future monitoring could therefore use a single European corn borer population as a representative for southwestern Germany. However, toxins from different suppliers and different production batches produced a vast range of LC50 values. Changes because of different toxin batches may be mistaken as a change in baseline susceptibility or even as the start of a resistance development. Thus, it is important throughout insect resistance management that the same toxin batches will be available for baseline susceptibility bioassays and for future tests.

[Red cell membrane defects]. / Erythrozytäre Membrankrankheiten.

Genetic defects of red cell membrane proteins constitute in Europe the most common cause of congenital hemolytic anemias. During the past decennium the defects and the pathogenetic mechanisms have been eludicated. Relatively simple hematologic tests allow for differentiation into groups with different severity of the disease. This allows prognostic assessment and careful risk-benefit evaluation for splenectomy.

A phase II trial of liposomal busulphan as an intravenous myeloablative agent prior to stem cell transplantation: 500 mg/m(2) as a optimal total dose for conditioning.

We conducted a phase I/II trial, to evaluate the efficacy and safety of an intravenous liposomal formulation of busulphan (LBu) as a myeloablative agent for stem cell transplantation (SCT). The liposomal busulphan was administered as a 3 h infusion twice daily over 4 consecutive days. Six adults received 1.6-2 mg/kg/dose and 18 children received 1.8-3 mg/kg/dose. Pharmacokinetic parameters were studied after the first and the last dose of busulphan. No significant difference in clearance, AUC, elimination half-lives or distribution volume between the first and the last dose was found in either groups. A significantly (P < 0.005) higher clearance was observed in children after the first and the last dose (3.61 and 3.79 ml/min/kg, respectively) compared to adults (2.40 and 2.33 ml/min/kg, respectively). The elimination half-lives after the first and the last dose were significantly (P < 0.005) shorter in children (2.59 and 2.72 h, respectively) compared to adults (3.35 and 3.61 h, respectively). Clearance correlated significantly with age. However, no significant correlation with age was observed when clearance was adjusted to the body surface area. Two cases of VOD following a total dose of 24 mg/kg were observed. Six patients experienced mucositis. No other organ toxicity was observed. We conclude that intravenous liposomal busulphan pharmacokinetics is age dependent. A dosage schedule based on body surface area should be used especially in young children to reduce the age-dependent difference in kinetics. An intravenous liposomal dose of busulphan of 500 mg/m(2) is suggested to reach a similar systemic exposure and myeloablative effect in both children and adults. Moreover, the novel liposomal form of busulphan showed a favorable toxicity profile and seems safe as a part of the high-dose therapy prior to SCT.

Effect of cellulose on the digestibility of high starch versus high fat diets in dogs.

The effects of cellulose added in three levels (7, 15 and 20% crude fibre in dry matter) to three different basal diets (a high fat diet, a high starch diet with raw starch and a high starch diet with cooked starch) on apparent digestibility were investigated in eight adult dogs. Cellulose had little effect on the apparent digestibility of fat. In the high fat diet there was no significant decrease, not even at the highest cellulose level (98.3% compared with 98.1% in the basal high fat diet). In the cooked starch diets, fat digestibility decreased from 95.1% in the basal cooked starch diet to 93.8% at the highest cellulose level. In the raw starch diets, digestibility did not decrease with increasing cellulose levels. The apparent digestibility of crude protein was considerably decreased by cellulose in all diets. Starch also decreased protein digestibility and the effects of cellulose and starch appeared to be additive (high fat diet decrease of protein digestibility from 86.7 to 83.5%, cooked starch from 81.6 to 78.6%, raw starch from 79.0 to 70.8%, basal diets to highest cellulose levels, respectively). The apparent digestibility of nitrogen-free extract decreased from 93.9% in the basal cooked starch diet to 84.5% at the highest cellulose level. The figures for the raw starch diets were similar (decrease from 93.4 to 85.9%). Cellulose decreased the apparent digestibility of energy in all diets. This decrease was more marked in the high starch diets (cooked starch decrease from 89.1 to 69.6%, raw starch from 88.9 to 70.2%) than in the high fat diet (decrease from 90.1 to 76.1%). An evaluation of previous data showed that in general fibre has a higher impact on the apparent digestibility of energy in high carbohydrate diets than in low carbohydrate diets. The apparent digestibility of potassium, sodium and chloride was impaired by cellulose. The apparent digestibility of these minerals tended to be lower in the high starch diets, especially in the raw starch diet, and the effect of cellulose was usually more marked in those diets.

Amphiphile-induced vesiculation in aged hereditary spherocytosis erythrocytes indicates normal membrane stability properties under non-starving conditions.

Aged HS erythrocytes with a defined primary defect in band 3 protein or ankyrin were incubated with amphiphiles (detergents) at sublytic concentrations (37 C, 60 min) or glucose-starved (37 C, 24 h). In line with previous studies, the release of AChE (exovesicles) from HS erythrocytes during glucose-starvation was significantly higher (11%) compared to that from control erythrocytes (1%). Control and HS cells responded, however, similarly to amphiphile-treatment (non-starving conditions). Amphiphiles induced similar types of shape alterations and a similar amount of AChE release (14-15%). Furthermore, the size and shape of amphiphile-induced exo- and endovesicles released from control and HS erythrocytes were similar. The results suggest that the stability properties of the membrane are not seriously disturbed in aged HS erythrocytes under non-starving conditions.

[Antiinfectious prophylaxis in asplenia]. / Infektionsprophylaxe bei Asplenie.

Asplenia in childhood may be congenital (e.g. Ivemark-syndrome) or acquired (functional hyposplenism in sickle cell disease; after splenectomy or bone marrow transplantation). Hereditary spherocytosis is the most common indication for splenectomy in childhood. Virtually every patient without spleen has a significantly increased risk of severe postsplenectomy infection (mostly caused by Streptococcus pneumoniae). Therefore, vaccinations against pneumococci, haemophilus influenzae and, under certain circumstances, meningococci are recommended. In addition a continuous prophylaxis with antibiotics should be performed for at least three years (or even longer depending on the disease) after splenectomy followed by lifelong interventional application of broad spectrum antibiotics in case of any unclear infection or high fever. This prophylaxis must be started as early as four months of age in sickle cell disease. In future the use of penicillin may be hampered by the growing resistance of pneumococci. Due to this fact the indication for splenectomy in childhood should be restricted to patients with hematologic disease (spherocytosis and other hemolytic anemias, chronic ITP etc.) and moderate to severe symptoms. It is unclear whether partial splenectomy for spherocytosis (and other hemolytic anemias) is an alternative regarding both longlasting reduction of hemolysis and prevention of severe infection. After trauma every effort should be undertaken to preserve a splenic remnant.

Molecular analysis of the fibrinogen gene cluster in 16 patients with congenital afibrinogenemia: novel truncating mutations in the FGA and FGG genes.

Congenital afibrinogenemia is an autosomal recessive disorder characterized by the complete absence of detectable fibrinogen. We previously identified the first causative mutations for this disease in a non-consanguineous Swiss family. These were homozygous deletions of approximately 11 kb of the fibrinogen alpha chain gene (FGA). Our subsequent study revealed that the majority of cases were attributable to truncating mutations in FGA, with the most common mutation affecting the donor splice site in FGA intron 4 (IVS4+1 G-->T). Here, we report 13 further unrelated patients with mutations in FGA, confirming the relative importance of this gene compared with FGG and FGB in the molecular aetiology of afibrinogenemia. Three other patients were homozygous for mutations in FGG. Eight novel mutations were identified: five in FGA and three in FGG. Sufficient mutation data is now available to permit an effective strategy for the genetic diagnosis of congenital afibrinogenemia.

Clinical and molecular evaluation of non-dominant hereditary spherocytosis.

About 75% of hereditary spherocytosis (HS) patients have the autosomal dominant form of the disease, whereas both parents of the remaining HS patients are clinically and haematologically normal. These patients could have either the autosomal recessive form of the disease or a de novo mutation. We studied 80 randomly chosen, Italian HS children with normal parents. They had different clinical phenotypes (16 mild, 40 moderate, 16 moderately severe and eight severe). These patients were screened for the occurrence of ankyrin or beta-spectrin de novo mutations. To search for ankyrin de novo mutations affecting mRNA accumulation, we studied a (AC)(n) microsatellite located in the non-coding sequence of the last exon of the ankyrin gene, and four different exonic polymorphisms in the beta-spectrin gene were utilized for the detection of de novo mutations influencing beta-spectrin mRNA stability. They were also screened for the presence of alpha-spectrin(LEPRA) as well as for the mutation -108T-->C in the ankyrin promoter, two variants previously found in some cases of genuinely recessive HS. Twenty-five patients showed ankyrin de novo mutations and 10 HS subjects had beta-spectrin de novo mutations. Furthermore, we found five patients to be heterozygous for alpha-spectrin(LEPRA) and one heterozygous for the mutation in the ankyrin promoter. Therefore, a molecular diagnosis was achieved in about 50% of the cases. Our data demonstrate that, among HS patients with normal parents, de novo dominant mutants are six times more common than recessive mutations. These results should be considered in view of the genetic counselling of a normal couple with a HS child.

Uroguanylin treatment suppresses polyp formation in the Apc(Min/+) mouse and induces apoptosis in human colon adenocarcinoma cells via cyclic GMP.

The enteric peptides, guanylin and uroguanylin, are local regulators of intestinal secretion by activation of receptor-guanylate cyclase (R-GC) signaling molecules that produce cyclic GMP (cGMP) and stimulate the cystic fibrosis transmembrane conductance regulator-dependent secretion of Cl- and HCO3-. Our experiments demonstrate that mRNA transcripts for guanylin and uroguanylin are markedly reduced in colon polyps and adenocarcinomas. In contrast, a specific uroguanylin-R-GC, R-GCC, is expressed in polyps and adenocarcinomas at levels comparable with normal colon mucosa. Activation of R-GCC by uroguanylin in vitro inhibits the proliferation of T84 colon cells and elicits profound apoptosis in human colon cancer cells, T84. Therefore, down-regulation of gene expression and loss of the peptides may interfere with renewal and/or removal of the epithelial cells resulting in the formation of polyps, which can progress to malignant cancers of the colon and rectum. Oral replacement therapy with human uroguanylin was used to evaluate its effects on the formation of intestinal polyps in the Min/+ mouse model for colorectal cancer. Uroguanylin significantly reduces the number of polyps found in the intestine of Min/+ mice by approximately 50% of control. Our findings suggest that uroguanylin and guanylin regulate the turnover of epithelial cells within the intestinal mucosa via activation of a cGMP signaling mechanism that elicits apoptosis of target enterocytes. The intestinal R-GC signaling molecules for guanylin regulatory peptides are promising targets for prevention and/or therapeutic treatment of intestinal polyps and cancers by oral administration of human uroguanylin.

Characteristic features of the genotype and phenotype of hereditary spherocytosis in the Japanese population.

Hereditary spherocytosis (HS) is the most common hemolytic anemia of congenital origin in the Japanese population. Among 844 cases of 520 kindred with congenital red cell membrane disorders studied at the Kawasaki Medical School in the last 25 years (1975-1999), 407 cases (48.2%) of 215 kindred had HS. Among the recent 60 kindred with HS, autosomal dominant (AD) transmission was proven in 19. The remaining 41 non-AD HS included 1) homozygous patients with autosomal recessive inheritance, 2) HS patients with de novo gene mutations, and 3) mild HS with AD inheritance. The extent of clinical severity in the non-AD HS cases was nearly identical to that in the AD cases. The incidence of membrane protein abnormalities in our 60 Japanese HS kindred was unique: there were lower ankyrin deficiencies (7%), moderate band 3 deficiencies (20%), and much higher protein 4.2 deficiencies (45%), with 28% of unknown etiology. The incidence of membrane protein deficiencies corresponded to that determined by gene analyses; i.e., mutations mostly in band 3 and/or in protein 4.2 genes and fewer ankyrin gene mutations. In the band 3 gene, 11 mutations pathognomonic for HS were identified (3 frameshift and 8 missense mutations). There were 5 mutations of the protein 4.2 gene (3 missense mutations, 1 nonsense mutation, and 1 splicing mutation) pathognomonic for HS. On the other hand, 2 missense mutations were detected in the ankyrin gene in this study. The genetic abnormalities in our HS patients correlated well with the phenotypic ultrastructural abnormalities of red cell membranes in situ. Ankyrin mutations (ankyrin Marburg and ankyrin Stuttgart with frameshift mutations) were associated mostly with a disrupted cytoskeletal network, and band 3 mutations (band 3 Kagoshima with frameshift mutation) typically demonstrated anomalies of intramembrane particles (IMPs). Protein 4.2 mutations (homozygotes of protein 4.2 Nippon) with complete protein 4.2 deficiency showed abnormalities of both the cytoskeletal network and IMPs.

[Diabetes mellitus type I, celiac disease and Imerslund-Gräsbeck syndrome: only an incidental combination of rare diseases?]. / Diabetes mellitus Typ I, Zöliakie und Imerslund-Gräsbeck-Syndrom: nur eine ungewöhnliche Kombination seltener Erkrankungen?

We describe the clinical course of a girl with onset of type I diabetes mellitus at the age of 3 years. At the age of 10, coeliac disease and shortly thereafter a vitamin B12 deficiency anemia (Imerslund-Gräsbeck-syndrome) was diagnosed. Her younger sister also suffered from Imerslund-Gräsbeck-syndrome when she was 11 year old. This unusual, so far not described association of rare diseases suggests a common autoimmune etiology.

Increased urinary excretion of uroguanylin in patients with congestive heart failure.

Uroguanylin is a small-molecular-weight peptide that activates membrane-bound receptor-guanylate cyclases in the intestine, kidney, and other epithelia. Uroguanylin has been shown to participate in the regulation of salt and water homeostasis in mammals via cGMP-mediated processes, bearing a distinct similarity to the action of the atriopeptins, which play a defined role in natriuresis and act as prognostic indicators of severe congestive heart failure (CHF). The objectives of this study were to measure the urinary levels of uroguanylin and the circulating plasma levels of atrial natriuretic peptide (ANP) in healthy individuals (n = 53) and patients with CHF (n = 16). Urinary excretion of uroguanylin was assessed by a cGMP accumulation bioassay employing human T84 intestinal cells. In individuals without CHF, the concentration of uroguanylin bioactivity was 1.31 +/- 0.27 nmol cGMP/ml urine and 1.73 +/- 0.25 micromol cGMP/24-h urine collection. The urinary bioactivity of uroguanylin in males (1.74 +/- 0.55 nmol cGMP/ml urine; n = 27) tended to be higher than the excretion levels in females (0.94 +/- 0.16 nmol cGMP/ml urine; n = 26) over a 24-h period but did not achieve statistical significance. Both male and female groups showed 24-h temporal diurnal variations with the highest uroguanylin levels observed between the hours of 8:00 AM and 2:00 PM. The circulating level of ANP was 12.1 +/- 1.6 pg/ml plasma and did not significantly vary with respect to male/female population or diurnal variation. In patients with CHF, the concentration of plasma ANP and urinary uroguanylin bioactivity increased substantially (7.5-fold and 70-fold, respectively, both P

Congenital deficiency of vitamin K dependent coagulation factors in two families presents as a genetic defect of the vitamin K-epoxide-reductase-complex.

Hereditary combined deficiency of the vitamin K dependent coagulation factors is a rare bleeding disorder. To date, only eleven families have been reported in the literature. The phenotype varies considerably with respect to bleeding tendency, response to vitamin K substitution and the presence of skeletal abnormalities, suggesting genetic heterogeneity. In only two of the reported families the cause of the disease has been elucidated as either a defect in the gamma-carboxylase enzyme (1) or in a protein of the vitamin K 2,3-epoxide reductase (VKOR) complex (2). Here we present a detailed phenotypic description of two new families with an autosomal recessive deficiency of all vitamin K dependent coagulation factors. In both families offspring had experienced severe or even fatal perinatal intracerebral haemorrhage. The affected children exhibit a mild deficiency of the vitamin K dependent coagulation factors that could be completely corrected by oral substitution of vitamin K. Sequencing and haplotype analysis excluded a defect within the gamma-carboxylase gene. The finding of highly increased amounts of vitamin K epoxide in all affected members of both families indicated a defect in a protein of the VKOR-multienzyme-complex. Further genetic analysis of such families will provide the basis for a more detailed understanding of the structure-function relation of the enzymes involved in vitamin K metabolism.

Frequency of very late fatal sepsis after splenectomy for hereditary spherocytosis: impact of insufficient antibody response to pneumococcal infection.

Very late sepsis in splenectomized patients with hereditary spherocytosis has been seen rarely up to now; the frequency and the immunodeficiency causing it are largely unknown. Within the past 7 years we have learned of four cases of sepsis or meningitis (three fatal) in adult patients with hereditary spherocytosis who had been splenectomized years earlier. The estimated frequency of very late postsplenectomy infections is 0.69 cases of sepsis or meningitis in 1000 patient-years (0.46 deaths in 1000 patient-years). Pneumococci were proven in two patients. The surviving patient showed low antibody titers against pneumococcal serotypes even after pneumococcal meningitis and subsequent vaccination. There have been several reports of an insufficient response to pneumococcal vaccination in patients with severe infections. We recommend determination of pneumococcal antibody titers after immunization in every splenectomized patient: Nonresponders to vaccination may be at high risk for overwhelming postsplenectomy infection. Our data demonstrate that there is a lifelong risk for severe postsplenectomy infections and therefore the lasting need for immediate antibiotic therapy in any case with sudden onset of high fever.

Identification of new prognosis factors from the clinical and epidemiologic analysis of a registry of 229 Diamond-Blackfan anemia patients. DBA group of Société d'Hématologie et d'Immunologie Pédiatrique (SHIP), Gesellshaft für Pädiatrische Onkologie und Hämatologie (GPOH), and the European Society for Pediatric Hematology and Immunology (ESPHI).

Diamond-Blackfan anemia (DBA) is a constitutional disease characterized by a specific maturation defect in cells of erythroid lineage. We have assembled a registry of 229 DBA patients, which includes 151 patients from France, 70 from Germany, and eight from other countries. Presence of malformations was significantly and independently associated with familial history of DBA, short stature at presentation (before any steroid therapy), and absence of hypotrophy at birth. Two hundred twenty-two patients were available for long-term follow-up analysis (median, 111.5 mo). Of these individuals, 62.6% initially responded to steroid therapy. Initial steroid responsiveness was found significantly and independently associated with older age at presentation, familial history of DBA, and a normal platelet count at the time of diagnosis. Severe evolution of the disease (transfusion dependence or death) was significantly and independently associated with a younger age at presentation and with a history of premature birth. In contrast, patients with a familial history of the disease experienced a better outcome. Outcome analysis revealed the benefit of reassessing steroid responsiveness during the course of the disease for initially nonresponsive patients. Bone marrow transplantation was successful in 11/13 cases; HLA typing of probands and siblings should be performed early if patients are transfusion dependent, and cord blood should be preserved. Incidence of DBA (assessed for France over a 13-y period) is 7.3 cases per million live births without effect of seasonality on incidence of the disease or on malformative status. Similarly, no parental imprinting effect or anticipation phenomenon could be documented in families with dominant inheritance.