Technical note: Automatic evaluation of infrared thermal images by computerized active shape modeling of bovine udders challenged with Escherichia coli.

Mastitis causes substantial economic losses and animal suffering in the dairy industry. The trend toward larger herd sizes complicates the monitoring of udder health in individual animals. Infrared thermography has successfully been used for early mastitis detection. However, manual thermogram analysis is time consuming and requires a skilled examiner, and automated image processing has not been tested. The aim of this study was to determine whether automatic evaluation of thermograms showed results comparable to those of manual evaluation of thermograms. Five healthy cows underwent an intramammary challenge with Escherichia coli to induce clinical mastitis. Multiple udder thermograms were taken every 2 h for 24 h before and after the challenge, resulting in 4,143 images in total. All images were evaluated using image recognition software (automatically) and a polygon tool (manually) to calculate the average and maximum surface temperatures. Because of the slightly different regions of interest, temperatures ascertained from the thermograms using the automatic method were consistently lower than those ascertained using the manual method. However, average udder surface temperatures evaluated using both methods were strongly correlated (r = 0.98 in the left hindquarter, and r = 0.99 in the right hindquarter) and showed maximum temperature peaks at the same time, 13 and 15 h after intramammary challenge. In the receiver operating characteristic analysis, both methods provided good results for sensitivity and specificity in detecting clinical E. coli-induced mastitis at different threshold values. For automatically evaluated maximum right hindquarter temperature, sensitivity was 93.75% and specificity was 94.96%, and for manually evaluated maximum right hindquarter temperature, sensitivity was 93.75% and specificity was 96.40%. Thus, automatic thermogram evaluation is a promising tool for automated mastitis detection.

Analysis of high resolution transmission electron microscope images of crystalline-amorphous interfaces.

For the analysis of images of homogeneous crystalline-amorphous interfaces we propose to average them along the interface obtaining the averaged interface image or the averaged intensity profile. Due to averaging, contrast components with the periodicity of the crystalline area of the image are extracted. Thus, the contrast features originating from the random overlap of the projected potentials of atoms in the amorphous layer are suppressed. It is shown that averaged images can be simulated by the multi-slice method using the novel approach to model the near interfacial amorphous structure by its mean atomic density distribution in front of the crystalline boundary. The crystalline structure is represented by its known atomic positions. We apply the proposed method to the investigation of the near interfacial short-range order in the c-Si/ a-Ge crystalline-amorphous interface.

Eight novel mutations and consequences on mRNA and protein level in pyruvate kinase-deficient patients with nonspherocytic hemolytic anemia.

Pyruvate kinase (PK) deficiency (PKD) is an autosomal recessive disorder with the typical manifestation of nonspherocytic hemolytic anemia. We analyzed the mutant enzymes of 10 unrelated patients with PKD, whose symptoms ranged from a mild, chronic hemolytic anemia to a severe anemia, by sequence analysis for the presence of alterations in the PKLR gene. In all cases the patients were shown to be compound heterozygous. Eight novel mutations were identified: 458T-->C (Ile153Thr), 656T-->C (Ile219Thr), 877G-->A (Asp293Asn), 991G-->A (Asp331Asn), 1055C-->A (Ala352Asp), 1483G-->A (Ala495Thr), 1649A-->T (Asp550Val), and 183-184ins16bp. This 16 bp duplication produces a frameshift and subsequent stop codon resulting in a drastically reduced mRNA level, and probably in an unstable gene product. Surprisingly, the existence of M2-type PK could be demonstrated in the patient's red blood cells. The study of different polymorphic sites revealed, with one exception, a strict linkage of the 1705C, 1738T, IVS5(+51)T, T(10) polymorphisms and the presence of 14 ATT repeats in intron 11. Our analyses show the consequences of a distorted structure on enzyme function and we discuss the correlations between the mutations identified and the parameters indicative for enzyme function.

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.

Application of RHD and RHCE genotyping for correct blood group determination in chronically transfused patients.

BACKGROUND: In chronically transfused patients, conventional blood group typing may be impossible because of mixed-field agglutination. STUDY DESIGN AND METHODS: In 27 patients with congenital anemia and lifelong transfusion history, genotyping for D, RHD, and RHCE was performed with polymerase chain reactions. These results were compared with the blood group typing results documented in the medical record. RESULTS: Two of 27 cases had been typed D-negative by serologic tests and D-positive by genotyping. In 20 patients, the CDE formula had been determined serologically according to the medical record; 4 of these patients were Cc by serologic tests and C/C by genotyping. One patient typed ee by serologic tests, and genotyping revealed heterozygosity (E/e). CONCLUSION: In patients with a lifelong transfusion history, serologic blood group determination may be impossible, and pretransfusion test results are not always available or reliable. In whites, Rh-matched transfusions are possible with genotyping. The genetic background of the RH genes has to be elucidated in other ethnic groups, such as in black patients with sickle cell disease, before genotyping can be applied without restriction.

Identification of a novel promoter mutation in the human pyruvate kinase (PK) LR gene of a patient with severe haemolytic anaemia.

Using direct sequencing we analysed the pyruvate kinase (PK) LR gene of a patient with severe haemolytic anaemia due to PK deficiency. A novel promoter mutation -249delA relative to the translation initiation site and the common 1529A mutation in exon 11 of the gene could be identified. Reverse transcription (RT)-PCR analysis combined with restriction digestion revealed that the -249delA mutation leads to a reduction in the amount of mRNA produced from this allele to about 6% of normal. We assume that both mutations would account for the PK deficiency in the compound heterozygous patient.

Cryohemolysis test as a diagnostic tool for hereditary spherocytosis.

The cryohemolysis test has been proposed as a new method of identifying hereditary spherocytosis. The purpose of the present study was to analyze the sensitivity and specificity of this method in comparison to the measurement of osmotic fragility. The examination included 61 patients suffering from hereditary spherocytosis and 58 patients with other hemolytic and nonhemolytic anemias. Hereditary spherocytosis patients showed significantly higher cryohemolysis values (median 29.7%, range 12.3-50.2%) than both normal subjects (median 3%, range 0.5-27%) and all other anemic patients excepting those with immune hemolytic anemia (median 4%, range 0.5-10.1%). Analysis of immune hemolytic anemia revealed broadly scattered values ranging from 1.4% to 53.5% (median 8.6%). Taking 15% as the threshold value, the sensitivity and specificity of the cryohemolysis test for hereditary spherocytosis were 95% and 96%, respectively. It is concluded that the simple-to-perform cryohemolysis test is quite comparable to the estimation of red cell osmotic fragility and therefore very useful as a diagnostic measure of hereditary spherocytosis.

Molecular basis of neurological dysfunction coupled with haemolytic anaemia in human glucose-6-phosphate isomerase (GPI) deficiency.

Glucose-6-phosphate isomerase (GPI) deficiency, an autosomal recessive genetic disorder with the typical manifestation of nonspherocytic haemolytic anaemia, can be associated in some cases with neurological impairment. GPI has been found to be identical to neuroleukin (NLK), which has neurotrophic and lymphokine properties. To focus on the possible effects of GPI mutations on the central nervous system through an effect on neuroleukin activity, we analysed DNA isolated from two patients with severe GPI deficiency, one of them with additional neurological deficits, and their families. The neurologically affected patient (GPI Homburg) is compound heterozygous for a 59 A-->C (H20P) and a 1016 T-->C (L339P) exchange. Owing to the insertion of proline, the H20P and L339P mutations are likely to affect the folding and activity of the enzyme. In the second family studied, the two affected siblings showed no neurological symptoms. The identified mutations are 1166 A-->G (H389R) and 1549 C-->G (L517V), which are located at the subunit interface. We propose that mutations that lead to incorrect folding destroy both catalytic (GPI) and neurotrophic (NLK) activities, thereby leading to the observed clinical symptoms (GPI Homburg). Those alterations at the active site, however, that allow correct folding retain the neurotrophic properties of the molecule (GPI Calden).

Hemoglobin F in myelodysplastic syndrome.

Reactivation of fetal hemoglobin synthesis in adulthood can be seen in hematological disorders affecting the erythropoietic system. The objective of the present study was to evaluate the incidence and prognostic significance of increased hemoglobin F in patients with myelodysplastic syndrome. Hemoglobin F concentrations and Ggamma/Ggamma + A gamma-globin chain ratios were determined in 26 patients with primary myelodysplastic syndrome. Median age of the patients was 65 years; all FAB subtypes were included. Increased hemoglobin F concentration of up to 20% of total hemoglobin (normal: below 2%) was seen in 16 patients; ten patients had normal values. There was a significant relation between hemoglobin F concentration and the course of disease, e.g., 12 of the 16 patients with elevated hemoglobin F survived at least 1 year after the examination, in contrast to only three of the ten patients with normal hemoglobin F (p < 0.025). All of six patients with hemoglobin F above 5% survived at least 1 year. There was no significant difference in the hemoglobin F concentration between patients with and without cytogenetic anomalies. The Ggamma/Ggamma + A gamma-globin chain ratio was slightly elevated in all patients, with a weak correlation to the degree of hemoglobin F elevation. The values were not of additional prognostic significance. The data of the present study suggest that the hemoglobin F concentration may be a prognostic parameter in myelodysplastic syndrome; increased hemoglobin F concentration may indicate a better prognosis.

Hyperphagia in children with craniopharyngioma is associated with hyperleptinaemia and a failure in the downregulation of appetite.

Patients with craniopharyngioma frequently suffer from severe obesity. Leptin induces an inhibition of appetite via hypothalamic receptors. This study was undertaken to investigate whether a relationship exists between serum leptin levels and pituitary/hypothalamic lesions in craniopharyngioma patients. Serum leptin levels were evaluated by RIA in 14 patients (age 7-21 years; 7 females, 7 males) after they had undergone neurosurgical treatment for craniopharyngioma. Normal controls had a positive correlation between leptin levels and body mass index (BMI) with higher levels in the females than in the males. Significantly elevated leptin levels with respect to BMI were found in 11 craniopharyngioma patients who had been affected by a suprasellar tumour, whereas 3 patients with an intrasellar tumour had lower, almost normal serum leptin levels. Our data suggest that craniopharyngioma patients develop hypothalamic obesity because their hypothalamic structures are insensitive to endogenous leptin. The elevated serum leptin concentrations found only in patients with a suprasellar tumour may be explained by a disturbed feedback mechanism from the hypothalamic leptin receptors to the adipose tissue.

Short stature and failure of pubertal development in thalassaemia major: evidence for hypothalamic neurosecretory dysfunction of growth hormone secretion and defective pituitary gonadotropin secretion.

UNLABELLED: In patients with beta-thalassaemia major, frequent blood transfusions combined with desferrioxamine chelation therapy lead to an improved rate of survival. Endocrine disorders related to secondary haemosiderosis such as short stature, delayed puberty and hypogonadism are major problems in both adolescent and adult patients. A total of 32 patients with beta-thalassaemia major undergoing treatment at the Children's Hospital, University of Göttingen were examined. Fourteen of these were short in stature. Growth hormone (GH) secretion was investigated in 13 patients exhibiting either a short stature or reduced growth rate. The stimulated GH secretion of 10 patients in this subgroup lay within the normal range. Studies of their spontaneous GH secretion during the night revealed that these patients had a markedly reduced mean GH and reduced amplitudes in their GH peaks. Low insulin-like growth factor (IGF)-I levels were seen in the growth-retarded thalassaemic patients. Eight were subjected to an IGF generation test and showed a strong increase in both IGF-I and insulin-like growth factor binding protein (IGFBP)-3 levels indicating intact IGF-I generation by the liver. Hypogonadotropic hypogonadism was found to be present in both the male and female patients with impaired sexual development. After priming with LH-releasing hormone (GnRH) per pump in 2 female and 5 male patients, no change in either their serum oestradiol or testosterone levels or in LH/FSH response to GnRH was observed suggesting that they were suffering from a severe pituitary gonadotropin insufficiency. Three male patients at the age of puberty but exhibiting short stature. low GH, low IGF-I and hypogonadism received low dose long-acting testosterone. After 3 12 months of therapy there was a marked growth spurt, higher nocturnal GH levels and an increase in both IGF-I and IGFBP-3. CONCLUSION: Reduced GH secretion and low IGF-I in thalassaemic patients are related to a neurosecretory dysfunction due to iron overload rather than to liver damage. Hypogonadotropic hypogonadism is caused by the selective loss of pituitary gonadotropin function. In patients with both GH deficiency and hypogonadism, low dose sexual steroid treatment should be considered either as an alternative or an additional treatment before starting GH therapy.

Glucose phosphate isomerase deficiency: biochemical and molecular genetic studies on the enzyme variants of two patients with severe haemolytic anaemia.

UNLABELLED: Biochemical and molecular genetic studies were performed on the enzyme variants of two patients compound heterozygous for glucose phosphate isomerase (GPI) deficiency, both suffering from severe haemolytic anaemia. The enzymes of case 1 (GPI 'Zwickau') and case 2 (GPI 'Nordhorn' [25]), revealed reduced GPI activity and remarkable thermolability. Glucose-6-phosphate (Gluc-6-P) concentration was elevated 2.3 times in case 1 and 3.8 times in case 2. Sequencing the patients' GPI genes showed four different point mutations, two of them involving highly conserved amino acids. The c1039 C-->T substitution, found in the gene of GPI 'Zwickau', has been described recently [30] and causes an Arg 347-->Cys substitution close to the putative catalytic site. The second mutation in this case is a novel c1538 G-->A substitution causing a Trp-->stop mutation at position 513 apparently resulting in premature RNA degradation thus resulting either in a complete lack of protein or a protein which does not show GPI activity. In the gene of GPI 'Nordhorn' a c1028 A-->G mutation was discovered, also previously described [1, 9] causing a Gln 343-->Trp substitution. The second mutation was a novel splice site mutation at the border of intron 15 to exon 16: IVS15-(-2) A-->C which leads to an aberrant splicing of exon 16, thus resulting either in a truncated and most likely inactive enzyme or in no protein at all. CONCLUSION: Biochemical and molecular genetic studies performed with the enzyme variants GPI 'Zwickau' and GPI 'Nordhorn' showed that in both cases the simultaneous occurrence of a single amino acid substitution affecting the active site, together with a nonsense mutation leading to the loss of major parts of the enzyme probably explains the severe clinical course of the disease.

Pitfalls in monitoring tacrolimus (FK 506).

Tacrolimus (FK 506) is a new, potent immunosuppressive drug for primary and rescue therapy in liver and kidney transplantation. Therapeutic drug monitoring is essential for this drug because of its narrow therapeutic window. Blood levels are monitored routinely by enzyme linked immunoassay (ELISA) or by microparticle enzyme immunoassay (MEIA). In a 13-year-old recipient of a liver transplant who had poor hepatic function during the first postoperative week, the authors observed unusually high tacrolimus blood concentrations using either the ELISA (26.6 to 49.0 microg/l) or MEIA (58.5 to 64.5 microg/l). Parent drug levels measured in the same blood samples by high-performance liquid chromatography/mass spectrometry (HPLC/MS) were up to 10-fold lower (5.1 to 9.0 microg/l). The discrepancies between the immunoassay and HPLC/MS results could not be attributed to any of the known metabolites of tacrolimus.

Ankyrin-1 mutations are a major cause of dominant and recessive hereditary spherocytosis.

Hereditary spherocytosis (HS) is the most common inherited haemolytic anaemia in Northern Europeans. The primary molecular defects reside in the red blood cell (RBC) membrane, particularly in proteins that link the membrane skeleton to the overlying lipid bilayer and its integral membrane constituents. Ankyrin-1 is the predominant linker molecule. It attaches spectrin, the major skeletal protein, to the cytoplasmic domain of band 3, the RBC anion exchanger. Two-thirds of patients with HS have combined spectrin and ankyrin-1 deficiency; deficiency of band 3 occurs in about 15 to 20% (ref.1). These data suggest that ankyrin-1 or band 3 defects may be common in HS. To test this we screened all 42 coding exons plus the 5' untranslated/promoter region of ankyrin-1 and the 19 coding exons of band 3 in 46 HS families. Twelve ankyrin-1 mutations and five band 3 mutations were identified. Missense mutations and a mutation in the putative ankyrin-1 promoter were common in recessive HS. In contrast, ankyrin-1 and band 3 frameshift and nonsense null mutations prevailed in dominant HS. Increased accumulation of the normal protein product partially compensated for the ankyrin-1 or band 3 defects in some of these null mutations. Our findings indicate that ankyrin-1 mutations are a major cause of dominant and recessive HS (approximately 35 to 65%), that band 3 mutations are less common (approximately 15 to 25%), and that the severity of HS is modified by factors other than the primary gene defect.

[Diagnosis of pyruvate kinase deficiency in the presence of an elevated reticulocyte count]. / Diagnostik des Pyruvatkinase-Mangels bei erhöhter Retikulozytenzahl.

HISTORY AND CLINICAL FINDINGS: Three members of one family (mother, now aged 44 years; daughter, now aged 23 years, and son, now aged 19 years) have had chronic haemolytic anaemia since their infancy. All three have had several blood transfusions a year because of haemoglobin (Hb) concentrations as low as 5 g/dl due to intermittent increases in haemolysis. Despite an unclear diagnosis all three had splenectomies: their spleens had been enlarged to about 5-7 cm below the rib margin. After splenectomy their haemoglobin concentration rose to 9-12 g/dl and the previously raised reticulocyte count of 150-250/1000 rose further to 400-700/1000. None of the patients had symptoms other than a slight decrease in exercise tolerance. They were admitted to hospital for further evaluation. Physical examination was unremarkable except for slight jaundice and well healed splenectomy scar. TESTS: All three patients had normochromic haemolytic anaemia (Hb 10-12 g/dl), bilirubin 2-3 mg/dl, lactate dehydrogenase activity 400-600 U/l). The reticulocyte count was raised to 420-690/1000. In all of them pyruvate kinase (PK) activity was normal or slightly increased if related to Hb concentration. Reticulocytes from healthy persons were isolated by density-gradient centrifugation so that samples could be prepared with different numbers of reticulocytes: PK activity was found to be dependent on the sample's reticulocyte count. In the three patients PK deficiency could, therefore, only be diagnosed by taking into account the raised reticulocyte count. CONCLUSION: As reticulocytes have a higher PK activity than erythrocytes the reticulocyte count must be taken into account when assessing the significance of a given value of this enzyme's activity.

Triosephosphate isomerase deficiency: biochemical and molecular genetic analysis for prenatal diagnosis.

Inherited deficiency of the glycolytic enzyme triosephosphate isomerase leads to a multisystem disorder characterized by progressive neuromuscular dysfunction, chronic nonspherocytic hemolytic anemia and increased susceptibility to severe infections. Most patients die within the first 6 years. We examined a family with severe triosephosphate isomerase deficiency. The 1-year-old index patient suffered from hemolytic anemia, neuromuscular impairment and pneumonias, with the necessity of intermitten mechanical ventilation. Triosephosphate isomerase activity in erythrocytes was reduced to about 20% of normal. Heat stability of the enzyme was strongly reduced; concentration of the physiological substrate, dihydroxyacetone phosphate was increased 20-fold due to the metabolic block. Direct sequencing of the triosephosphate isomerase gene revealed homozygosity for the formerly described GAG-->GAC-mutation changing 104 Glu-->Asp. During a 2nd pregnancy we examined a cord blood sample obtained in the 19th gestational week. The biochemical data on enzyme activity, heat stability of the enzyme and concentration of dihydroxyacetone phosphate were in the normal range. The molecular genetic analysis confirmed the presence of the normal triosephosphate isomerase alleles. Pregnancy was continued, resulting in the delivery of an unaffected, healthy newborn.

Mutations in the R-type pyruvate kinase gene and altered enzyme kinetic properties in patients with hemolytic anemia due to pyruvate kinase deficiency.

The biochemical properties of erythrocyte pyruvate kinase (PK) together with mutations found in the coding sequence of the R-PK gene in five patients with severe hemolytic anemia due to PK deficiency are described. The enzyme variants were designated PK 'Mosul' (homozygote), PK 'Bukarest', PK 'Hamburg', PK 'Köln', and PK 'Essen' (compound heterozygote). PK 'Mosul' showed normal positive cooperative substrate binding, PK 'Bukarest' exhibited non-cooperative behavior, and PK 'Hamburg' and PK 'Köln' displayed mixed cooperativity, whereas PK 'Essen' was negative cooperative. PK 'Mosul' was found to be homozygous for the mutation 1151 ACG to ATG, resulting in an amino acid substitution 384 Thr to Met. In one allele of PK 'Bukarest' a single nucleotide substitution GAG-TAG was found at nucleotide 721, causing a change of 241 Glu to a chain termination codon (PK 'Bukarest'). Additionally, in the second allele of this patient a point mutation at position 1594 (CGG-TGG) occurs, changing 532 Arg to Trp (PK 'Bukarest'). Direct sequencing showed the heterozygosity of the patient's mother (PK 'Bukarest'/normal) at position 721 and of the patient's father (PK 'Bukarest'/normal) at position 1594. A point mutation at position 1529 (CGA-CAA), causing an amino acid substitution 510 Arg-Gln, was identified in PK 'Hamburg' and PK 'Köln'. The second mutation in these variants was not detected. In PK 'Essen' no mutation in the coding sequence was found at all. Screening for the mutation at position 1529 in further compound heterozygote patients and in normal subjects of Western European origin showed that this exchange is a common mutation responsible for PK deficiency in this population.

[Mortality of premature infants 1980-1990: analysis of data from the Göttingen perinatal center]. / Mortalität unreifer Neugeborener von 1980-1990: Analyse aus dem Perinatalzentrum Göttingen.

We analyzed retrospectively (1980-1990) the causes of death and by using a logistic regression model the perinatal and neonatal risk factors influencing the mortality in preterm infants < 34 weeks of gestation (n = 1132). When comparing the interval from 1980-1986 to 1987-1990 we observed a decreasing mortality in infants < or = 1000 g from 57% to 19% as well as in the preterm infants > 1000 g from 8.3% to 3.0% (p < 0.001). The causes of death changed considerably. During 1980-1986 fifty-two (8.2%) out of the 632 preterm infants and during 1987-1990 only seven (1.3%) out of the 600 preterm infants died in the course of a severe respiratory distress syndrome or intracranial hemorrhages. From 1980 to 1986 21% (n = 10) and from 1987 to 1990 77% (n = 10) of the neonatal deaths in preterm infants > 1000 g were attributed to lethal malformations. In those infants without lethal malformations (n = 1109) we performed a logistic regression analysis. 87 (7.8%) of these neonates died. The risk of dying was significantly higher in infants born before 1987, in male newborns and in infants suffered from a severe respiratory distress syndrome III-IV or septicemia (p < 0.0001). An increasing gestational age of one week resulted in a lowered risk of mortality (odds ratio 0.59, p < 0.0001). Adjusted for these basic variables the mortality risk was also significantly higher for birth weights < or = 1000 g, low Apgar scores, peripartal acidosis, hypothermia and intracranial hemorrhages. An intrauterine growth retardation < 10. percentile resulted in a lower mortality risk.