Biochemical polymorphism and the 2,3-diphosphoglycerate in the sheep red blood cells

There was no significant difference in the level of 2,3-DPG in the red blood cells of sheep of different haemoglobin types (Hb A and Hb B) or potassium types (HK and LK). However, low glutathione (GSHL) sheep had significantly higher (p less than 0.01) level of 2,3-DPG in their red blood cells than high glutathione (GSHH) sheep. There was also significant effect of interactions between glutathione, haemoglobin and potassium types (p less than 0.05) and glutathione and haemoglobin types (p less than 0.01) on red cell 2,3-DPG levels.     

Haemolytic activity of Aeromonas species isolated in Libya

Twenty seven Aeromonas strains (5A. hydrophila, 8A. sobria and 14A. caviae) isolated from children with diarrhoea and 34 Aeromonas strains (9A. hydrophila, 7A. sobria an 18A. caviae) isolated from children without diarrhoea were tested from haemolysin production. The results obtained showed that haemolysin production using human, horse or sheep erythrocytes was significantly associated with A.hydrophila and A sobria but not with A.caviae, regardless of whether these strains were isolated from children with or without diarrhoea. Human or horse rather than sheep erythrocytes are recommended for use in the haemolysin assay.     

Interferon-gamma secretion defects in haemophilia A patients receiving highly purified plasma-derived or recombinant factor VIII

The outcome of developing immune responses is influenced by interactions among a large and complex network of secreted cytokines. T-cell secretion of interferon-gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha) and TNF-beta, or lymphotoxin contributes to the development of cell-mediated immunity, whereas secretion of interleukin (IL)-4, IL-5 and IL-6 contributes to development of humoral immunity. Humoral immunity to factor VIII (FVIII) develops in approximately 25% of severe haemophilia A patients. The aim of our research was to understand the underlying immune response to FVIII in patients with FVIII inhibitors. We report a defect in IFN-gamma secretion by peripheral blood mononuclear cells (PBMC) derived from haemophilia A patients, which was accompanied by a low level of mitogen-induced proliferation and a significant decrease in the percentage of natural killer (NK) cells. All of the observed defects were found in haemophilia A patients, both with and without FVIII inhibitors, who were free of viral infection and had been treated predominantly or exclusively with monoclonal antibody-purified or recombinant FVIII.     

Management of haemophilia in Sweden

The incidence of living haemophiliacs in Sweden (total population 8.1 millions) is about 1:15,000 males and about 1:30,000 of the entire population. The number of haemophiliacs born in Sweden in 5-year periods between 1931-1975 (June) has remained almost unchanged. The total number of haemophilia families in Sweden is 284 (77% haemophilia A, 23% haemophila B) with altogether 557 (436 with A and 121 with B) living haemophiliacs. Of the haemophilia A patients 40% have severe, 18% moderate, and 42% mild, haemophilia. The distribution of the haemophilia B patients is about the same. Inhibitors have been demonstrated in 8% of the patients with severe haemophilia A and in 10% of those with severe haemophilia B. There are 2 main Haemophilia Centres (Stockholm, Malm?) to which haemophiliacs from the whole of Sweden are admitted for diagnosis, follow-up and treatment for severe bleedings, joint defects and surgery. Minor bleedings are treated at local hospitals in cooperation with the Haemophilia Centres. The concentrates available for treatment in haemophilia A are human fraction I-0 (AHF-Kabi), cryoprecipitate, Antihaemophilic Factor (Hyland 4) and Kryobulin (Immuno, Wien). AHF-Kabi is the most commonly used preparation. The concentrates available for treatment in haemophilia B are Preconativ (Kabi) and Prothromplex (Immuno). Suffcient amounts of concentrates are available. In Sweden 3.2 million units of factor VIII and 1.0 millino units of factor IX are given per year. Treatment is free of charge. Only 5 patients receive domiciliary treatment, but since 1958 we in Sweden have practised prophylactic treatment of boys (4-18 years old) with severe haemophilia A. At about 5-10 days interval they receive AHF in amounts sufficient to raise the AHF level to 40-50%. This regimen has reduced severe haemophilia to moderate. The joint score is identical with that found in moderate haemophilia in the same age groups. For treatment of patients with haemophilia A and haemophilia B complicated by inhibitors we have used a large dose of antigen (factor VIII or factor IX) combined with cyclophosphamide. In most cases this treatment produced satisfactory haemostasis for 5 to 30 days and prevented the secondary antibody rise.     

Barium granuloma of the rectum

Every child with severe or moderate haemophilia A or B, born in Sweden during the period 1970-1990, was treated in the national haemophilia register, all 117 case records being surveyed for mode of delivery and perinatal complications. Of the 117 deliveries, 13 were by caesarean section and the remaining 104 vaginal. Of the 13 caesarean sections, 2 were performed because the woman was a haemophilia carrier, the remaining 11 (5 emergency, 6 elective) for other reasons. Neonatal complications were: subgaleal or cephalic haematoma (n = 12), intracranial haemorrhage (n = 4), umbilical bleeding (n = 4), haematuria (n = 1), retro-orbital bleeding (n = 1) and abnormal bleeding after surgery, injection or venepuncture (n = 28). Of the 12 infants with subgaleal/cephalic haematoma, 10 were delivered by vacuum extraction. Seven more infants were delivered by vacuum extraction and another 11 were born without abnormal bleedings after laborious (> 24 h) delivery. Of the 4 children with intracranial haemorrhage, all were sporadic cases of haemophilia, 1 was a premature birth by caesarean section in the 27th week, I was delivered by vacuum extraction and the remaining 2 vaginally. In these 4 cases there were no sequelae or only minor ones. We conclude that the risk of serious bleeding in conjunction with normal vaginal delivery is small, but that vacuum extraction should be avoided when delivering offspring of haemophilia carriers.     

Cross-reactivity to porcine factor VIII of factor VIII inhibitors in patients with haemophilia in Australia and New Zealand

BACKGROUND: Inhibitory antibodies which neutralise factor VIII develop in 10-20% of individuals with inherited haemophilia A and rarely as autoantibodies in normal individuals to cause acquired haemophilia. The antibodies are directed against human factor VIII but cross-react to varying degrees with porcine factor VIII. Porcine factor VIII can be used for treatment in individuals with low cross-reactivity. AIMS: To determine the cross-reactivity of factor VIII inhibitors between human factor VIII and porcine factor VIII, in a population of patients with inherited and acquired haemophilia A. Also, to determine whether patients with inherited haemophilia and inhibitors have a higher incidence of factor VIII gene inversion in intron 22. METHODS: Samples and data sheets from 43 patients with inherited and ten with acquired haemophilia were submitted from hospitals in Australia and New Zealand. Inhibitor levels to human and porcine factor VIII were measured by the Bethesda method in 39 with inherited and nine with acquired haemophilia A. RESULTS: Of 39 patients with inherited haemophilia A, cross-reactivity was 0% in 17 patients, 1-19% in six, 20-39% in 11 and 40-80% in five. In six of nine patients with acquired haemophilia cross-reactivity was < or = 7%. In inherited severe haemophilia A, the frequency of the intron 22 inversion was not greater in 37 study patients than in 28 patients without an inhibitor. CONCLUSIONS: Many patients in Australia and New Zealand with inhibitors to human factor VIII presently show a low or absent level of cross-reactivity to porcine factor VIII. These may respond to treatment with this concentrate at least in the short term. There remains a group of patients with high cross-reactivity who will respond only to recombinant factor VIIa or prothrombin complex concentrates.     

Abattoir survey of congenital reproductive abnormalities in ewes

A survey of abnormalities of the reproductive tract of female sheep was undertaken at two abattoirs in the south west of England over a period of 12 months. During the survey, 9970 reproductive tracts from cull ewes and 23,536 tracts from nulliparous sheep (prime lambs and hoggets) were examined. A total of 655 (6.57 per cent) ewes and 459 (1.95 per cent) nulliparous sheep had abnormalities of the reproductive tract. Of these, congenital abnormalities of the paramesonephric ducts accounted for 2.4 per cent of the ewes and 7.4 per cent of the nulliparous sheep, congenital abnormalities of the ovaries accounted for 2.6 per cent of the ewes and 7.4 per cent of the nulliparous sheep and cystic structures that were considered to have been of congenital origin accounted for 27.2 per cent of the ewes and 52.7 per cent of the nulliparous sheep. The most common lesion was paraovarian cysts (26.6 per cent of ewes and 39.0 per cent of nulliparous sheep), but few of these appeared to have affected the sheep's reproductive function. Several specific conditions were recorded, including some described for the first time in sheep. Uterus unicornis occurred in 20 sheep and other forms of segmental aplasia of parts of the paramesonephric ducts occurred in a further 13 animals. Uterus didelphys occurred in six sheep, and 11 animals were intersex. Intersex sheep had vestigial structures that were derived from the paramesonephric ducts, hypoplastic or masculinised gonads and some had masculinised external genitalia. Ovarian hypoplasia occurred in 34 sheep, and in a further 12 mainly nulliparous animals, the ovaries were fused. Sixty nulliparous animals and two ewes had hydatids of Morgagni.     

Postprandial plasma glucose, insulin, glucagon and triglyceride responses to a standard diet in normal subjects

A uterine pouch was prepared surgically in ewes before mating to study the production and chemical composition of uterine secretions during pregnancy. Pregnancy was established in 6 sheep and in the first 55 days uterine fluid was present in small amounts (less than 5 ml), whereas between 109 days post coitum and 3 days post partum the volume ranged from 90 to 775 ml. The chemical composition of uterine fluid in pregnancy differed from that of plasma especially in respect of its high concentration of total calcium (up to 83.5 mM) and prostaglandin (PG) F-2a (up to 1500 ng/ml). Progesterone was implicated as an important endocrine factor since uterine fluid (188 ml) with a high concentration of total calcium (53-5 mM) and PGF-2a(235ng/ml) was recovered from a non-pregnant sheep treated with progesterone for 115 days.     

Immunosuppressive treatment in haemophiliacs with inhibitors to factor VIII and factor IX

9 patients with severe haemophilia A and inhibitors (inhibitor levels between 0.1 to 5.8 U/ml) and 3 patients with severe haemophilia B and inhibitors (inhibitor levels between 0.1 to 11 U/ml) were treated on a total of 16 and 13 occasions, respectively, with a large dose of antigen (factor VIII or factor IX) and cyclophosphamide (10-15 mg/kg b.w. i.v. initially and then 2-3 mg/kg b.w. orally for 7-10 days) in connection with severe bleeding and surgery. All the patients had proved not to respond to treatment with factor VIII or factor IX concentrate alone, and all except one had shown strong secondary antibody increases. In 6 of the patients with haemophilia A the treatment (11 occasions) had a satisfactory haemostatic effect and even permitted neurosurgery without bleeding complications. The inhibitor level remained at zero for 5-10 days, after which it gradually began to return towards its original level. In these cases it was possible to give factor VIII in amounts which neutralised the inhibitor and afterwards raised the factor VIII initially to at least 50%. In the 3 patients with haemophilia B treatment (13 occasions) was successful except on one occasion, and surgery was performed without abnormal bleeding. The factor IX level was initially raised to at least 50% except in the one failure. The inhibitor level remained at zero for 12 days to 3 months, after which it gradually rose towards its original level. One patient was treated on 8 occasions.     

Polymorphism of the beta2-adrenoceptor and the response to long-term beta2-agonist therapy in asthma

DNA-based diagnosis of haemophilia A has previously been carried out by linkage analysis using two highly informative markers, Hind III RFLP and St14 VNTR, for affected Turkish families. In the present study the number and frequency of the microsatellite alleles at introns 13 and 22 in the factor VIII (FVIII) gene were analysed in order to increase the rate of informative females and accuracy of linkage analysis. Six alleles were observed at both loci. The two most frequent alleles of each locus were the same as the two common alleles found in Anglo-Americans. The comparison of heterozygosity of both microsatellite loci showed that the Turkish population is slightly less polymorphic than Anglo-Americans but more polymorphic than Chinese, Slavs and Uzbekians. The additional use of the two microsatellite repeat polymorphisms with the previously established informative markers has been accepted as the most effective strategy in DNA diagnosis by linkage analysis for the assessment of haemophilia A carriers and affected fetuses in the Turkish population. The modifications adopted in this study for the multiplex PCR analysis of the microsatellite repeat polymorphism eliminated the use of radioactivity and sequencing gels, reducing cost and labour.     

Revision consensus hemophilia: treatment and responsibility. Nederlandse Vereniging van Hemophilia Patients

Haemophilia is an X-linked clotting disease occurring in 1400 men in the Netherlands. As the result of factor VIII or IX deficiency, haemophilia patients suffer from severe bleedings, spontaneous or caused by trauma. Bleedings mostly occur in major joints and muscles. Repeated bleedings can cause disability. Treatment of haemophilia consists of replacement therapy with factor VIII or IX. The first aim of treatment is to prevent bleedings (prophylaxis). The second aim is to limit joint damage in case of bleeding by timely and adequate substitution therapy. In addition, physical therapy and sports are used to keep patients in shape. Until the introduction of recombinant factor VIII in 1992 only plasma derived factor VIII and IX products were available. Substitution therapy has caused various side effects, such as allergic reactions. Since the introduction of ultra-pure concentrates, allergic reactions have grown rare. Viral infections like HIV and hepatitis C, which were transmitted through blood products, have not occurred in Dutch haemophilia patients since the introduction of adequate viral inactivation steps. In case of development of antibodies against factor VIII or IX, therapy with factor VIII or IX products is insufficient. Antibody formation is more often seen in haemophilia A (25%) than in haemophilia B (2%). In most cases antibodies disappear during so-called immune tolerance induction. This therapy consists of regular infusion with factor VIII or IX. Once an inhibitor has disappeared, patients can be treated normally once more. As haemophilia is a rare and complex disease patients should be treated in specialized centres, preferably by a comprehensive care team. This is even more justified because treatment of haemophilia is costly. Over the last 20 years haemophilia treatment has improved much. This has resulted in a decrease of the number and of the duration of hospitalization, and a decrease in days lost at school or work. This has led to great improvement of the social life of haemophilia patients.     

Development of a factor VIII inhibitor in a newborn haemophiliac

A major problem in the treatment of haemophilia A is the development of inhibitors (antibodies) against factor VIII. We report the case of a newborn male with no family history of haemophilia who developed an intracerebral haemorrhage. On day 10 post-delivery severe haemophilia A was diagnosed and treatment with recombinant FVIII (rFVIII) concentrate was started. Seventy-two hours later the presence of inhibitors was suspected because high doses of rFVIII were required to maintain therapeutic FVIII plasma levels. Days after, the inhibitor was detected. The quick detection of the inhibitor in this newborn haemophiliac allowed us to start the immunotolerance early, without interruption in the administration of rFVIII.     

Haemophilia A and the blood transfusion service: a Scottish study

The demand for blood products containing factor VIII for treating patients with haemophilia A in south-east Scotland was reviewed. From 1961 to 1975 the demand for fresh frozen plasma (FFP), cryoprecipitate (CP), and antihaemophilic factor (AHF) increased by seven and a half times, while total donations increased by only a third. Patients with severe haemophilia A treated at the regional haemophilia centre used about 85% of the factor VIII issued in 1971-4, most of which was used on demand. A patient with severe haemophilia A on unlimited ondemand home treatment would need about 500 units of factor VII/kg body weight/year, and a regional haemophilia centre, treating moderate and mild cases as well as severe ones, would use 15000 units/patient/year. Altogether about 50 million units of factor VIII will be needed each year in the UK. Although cryoprecipitate is much harder to store and administer than AHF, its yield from plasma may be far greater and its cost far smaller. Unless the blood transfusion services receive increased amounts of money and reappraise their functions and operation, it seems likely that they will have to rely increasingly on commercial (and costly) sources for the major plasma fractions.     

Management of hemophilia in Spain

A single injection (100 mug i.m.) of Estrumate (I.C.I. 80996) was used to induce luteal regression on day 8 of the estrous cycle in 3 sheep. Progesterone levels in the utero-ovarian vein and femoral artery had fallen within 6 h to less than 50% of the concentrations seen before injection of the analogue. Luteolysis was not associated with endogenous production of PGF. The concentration of PGF in the uteroovarian vein began to increase 27-39 h after the administration of Estrumate, reaching a mean maximum concentration of 1455pg/ml 48 h after Estrumate. The mean concentration of PGF in the utero-ovarian vein between 36-69 h after Estrumate was significantly greater than during the 24 h before Estrumate (control period) or during the 0-30 h immediately after injection (both P less than 0.001). The maximum secretion of estradiol and the pre-ovulatory LH peak occurred during the period of elevated PGF concentrations in the utero-ovarian veins. The possible importance of endogenous PGF production at this time is discussed.     

Feasibility of prenatal diagnosis and carrier detection in South African haemophilia A patients

The feasibility of DNA diagnosis for haemophilia A was tested in South African patients and families by screening for the common inversion mutation in the factor VIII gene and for the intragenic microsatellite markers in introns 13 and 22. The allele frequencies at the two microsatellite loci were significantly different, with informativity being higher in the Negroid (100%) than the Caucasoid group (67%). In severely affected haemophiliacs the inversion was found in 43% (6/14) of Negroids but in only 32% (13/41) of Caucasoids. Presence of a second common unidentified mutation may account for the low frequency in the latter. Haplotype analysis shows a disproportionately high frequency of an (AC)20 intron 13-(AC)26 intron 22 inversion negative Caucasoid haemophilia chromosome, supporting a founder effect.     

Fitting contact lenses after excimer laser photorefractive keratectomy for myopia

The number and percentage of C-cells per unit area were investigated in 2-3-year-old sheep by an immunoperoxidase technique, using a digital-image analysis. C-cells were distributed throughout the thyroid lobes but were not present in either the isthmus or the superior and inferior poles of the thyroid. C-cells were more concentrated in the deep central region of the lobes and decreased gradually toward the periphery. There was a high correlation between the number and the percentage of C-cells per unit area of thyroid gland in sheep. Significant differences were not present between male and female sheep of 2-3 years of age.     

Food aversion conditioned in anesthetized sheep

We discovered that a food aversion could be conditioned in anesthetized sheep. Sheep were allowed to eat a familiar food (alfalfa-grain pellets) for 30 min, and 90 min later they were given either an intraruminal (IR) injection of water (C), an IR injection of LiCl (L), anesthesia followed by an IR injection of water (A), or anesthesia followed by an IR injection of LiCl (A+L). Induction of anesthesia was by an intravenous injection of pentobarbitone sodium, and maintenance of deep anesthesia was by halothane. Sheep were maintained in deep anesthesia for 2 h to ensure that the effects of LiCl on the acquisition of a food aversion, which occur within about 1 h, were completed before they awakened. When tested 5 days later, sheep that received LiCl (treatments L and A+L) consumed less alfalfa-grain pellets than sheep that did not receive LiCl (treatments C and A) (241 g vs. 306 g; p = 0.057). Intake of sheep that were anesthetized (treatments A and A+L) did not differ from that of sheep that were not anesthetized (treatments C and L) (295 g vs. 252 g; p = 0.183). Nor was there an interaction between LiCl and anesthesia (p = 0.423). Thus, we conclude that changes in preferences for foods caused by postingestive feedback occur automatically every time food is ingested (i.e., they are noncognitive), and the kind and amount of feedback is a function of the match between the food's chemical characteristics and its ability to meet the animal's current demands for nutrients.     

"Democracy was never intended for degenerates": Alberta''s flirtation with eugenics comes back to haunt it

BACKGROUND: Differences in the hepatitis C virus (HCV) genotype influence the severity of HCV related liver disease and response to interferon therapy. HCV infection is frequent in Australian haemophilia patients who have been exposed repeatedly to multiple HCV genotypes through non HCV virally inactivated clotting factor concentrates. The distribution of the various HCV genotypes in Australian haemophilia patients is unknown. AIM: To examine the HCV genotype distribution and clinical features of HCV associated liver disease in Australian haemophilia patients. METHODS: Forty patients with bleeding disorders who were known to be both HCV antibody and polymerase chain reaction (PCR) positive were evaluated by direct sequencing of the PCR products for the HCV genotype. RESULTS: Genotype 1 was found in 65% of patients (26/40), type 2 in 5% (2/40) and type 3 in 30% (12/40). No genotypes 4 to 6 were found. There was no association between the HCV genotype and the severity of haemophilia, alanine transaminase levels, or the presence of portal hypertension. Unlike European, Asian and American studies where the majority of type 1 infection is subclass 1b, in Australian haemophilia patients it is subclass 1a (73%-19/26) which may have a better prognosis and response to interferon. CONCLUSIONS: Despite patients with haemophilia being exposed to multiple HCV genotypes, it appears that there is no selection advantage of one genotype over another. Australian haemophilia patients with HCV have a different genotype distribution to that reported in other countries and care should be observed in interpreting non Australian studies concerning HCV.     

A patient with acquired haemophilia A and pemphigus

Haemophilia A is usually a genetic deficiency of coagulation factor VIII (F VIII). The development of antibodies against F VIII is a well known and frequent complication in the treatment of haemophilia A. Rarely, a F VIII inhibitor arises spontaneously, causing a condition which is known as acquired haemophilia A. We describe a patient with acquired haemophilia A and pemphigus, who presented with spontaneous haematomas of the extremities. Laboratory tests showed an activated partial thromboplastin time (aPTT) of 71 s (normal: 26-36 s), a F VIII concentration of 9% (normal: 60-140%), and a F VIII inhibitor-activity of 7.5 Bethesda Units/ml (B.U./ml, normal: 0). The haematomas disappeared within a few days and the laboratory tests normalized within 6 weeks, after administration of a booster of oral corticosteroids. One and a half years after the corticosteroids were stopped, both the clinical and the laboratory course of the patient has been uneventful. As far as we know, the combination of acquired haemophilia A and pemphigus has been reported in the literature only three times before. The diagnosis acquired haemophilia A should be considered in a patient presenting with a newly arisen haemorrhagic diathesis.