Unique strategies for therapeutic gene transfer in haemophilia A and haemophilia BWFH State-of-the-Art Session on Therapeutic Gene Transfer Buenos Aires, Argentina.

SUMMARY: Gene therapy of haemophilia has been initiated through a number of approaches including expression in muscle, liver and omental implanted fibroblasts, or i.v. injection of an expression construct under the control of a ubiquitous promoter. In all these approaches, the goal was to have factor VIII (FVIII) or factor IX (FIX) synthesized so that it restored the levels of the missing protein in blood. The three talks in this session are totally, or at least in part, directed at strategies that may be clinically effective even in the absence of correction of the missing plasma clotting factor, although the haematopoietic stem cell or blood outgrowth endothelial cell therapy could achieve plasma correction as well. Two of the approaches achieve localized coagulation factor expression without necessarily correcting the systemic defect--one is with synthesis of FVIII or FIX within the joint space and the other is with the local release of FVIII (or FIX) by platelets at the site of vascular injury. All of the three approaches have demonstrated efficacy in small animal models and are now the subject of larger animal studies. None has yet to progress to human trials.

Bleeding disorders: A common cause of menorrhagia in adolescents.

OBJECTIVE: To determine the frequency of underlying bleeding disorders in adolescents with menorrhagia. STUDY DESIGN: We retrospectively reviewed the charts of all girls, aged 10 to 19 years, who presented to our children's hospital for inpatient or outpatient evaluation of menorrhagia between January 1990 and November 1998. RESULTS: At presentation, 9 of the 71 girls (13%) had thrombocytopenia (platelet count <150,000/microL; range, 5000-106,000/microL). The most common causes for thrombocytopenia were immune thrombocytopenic purpura (n = 5) and myelosuppression caused by chemotherapy (n = 2). Of 14 girls who underwent a more detailed hemostatic evaluation, 8 were given a diagnosis of a hereditary coagulation disorder: 6 had platelet function defects and 2 had type 1 von Willebrand disease. Excessive menstrual bleeding commonly results in anemia. One half of the total group had anemia (hemoglobin <12.0 g/dL). Seven girls (10%) had potentially life-threatening anemia (hemoglobin <5.0 g/dL). CONCLUSIONS: Acquired and congenital bleeding disorders are common causes of menorrhagia in adolescent girls. Severe anemia is a frequent complication of menorrhagia. We recommend that adolescents without thrombocytopenia who present with menorrhagia receive a comprehensive hemostatic evaluation, including testing for von Willebrand disease and platelet function defects.

The epidemiology of myelopathy associated with human T-lymphotropic virus 1.

A progressive spastic myelopathy is one of the principal manifestations of the human T cell lymphotropic virus type 1. Recent research is reviewed on the geography and epidemiology of this specific form of tropical spastic paraparesis. First recognized in the Caribbean, Colombia and Japan, it is now also confirmed as a major neurological problem in areas of eastern Brazil, western equatorial Africa, Natal and Seychelles, with other scattered foci world-wide. Accurate surveys call for sophisticated techniques including polymerase chain reaction amplification. The only defined modes of spread are by breast feeding, sexual contact and blood cell infusion. The onset of neurological disease is associated with high antibody titres and a high rate of spontaneous T lymphocyte proliferation. Molecular analysis has revealed no nucleotide sequence variation in cases with or without myelopathy. In non-transfusion cases the clinical attack rate is low with a very long latent period, but there are unexplained regional differences and familial cases are an important exception. Current research is focused on possible local, ethnic, or genetic co-factors.

Diagnosis and treatment of homozygous protein C deficiency. Report of the Working Party on Homozygous Protein C Deficiency of the Subcommittee on Protein C and Protein S, International Committee on Thrombosis and Haemostasis.

This report summarizes the documented cases of homozygous protein C deficiency in the United States and Europe. Procedures for diagnosing and treating this disorder (both initially and over the long term) have been compiled by a working party on homozygous protein C deficiency of the Subcommittee on Protein C of the International Committee on Thrombosis and Haemostasis. Homozygous protein C deficiency is an autosomal recessive disorder that usually manifests itself by purpura fulminans and, less commonly, by massive large vein thrombosis; severe diffuse intravascular coagulation also develops in these infants, and there is evidence of intrauterine thrombosis. For confirmation of homozygous protein C deficiency in a neonate with purpura fulminans or massive venous thrombosis, the infant should have undetectable protein C activity and both parents should be heterozygous for protein C deficiency. At the onset of symptoms, the initial treatment should be plasma (8 to 12 ml/kg every 12 hours) until all lesions have healed. Two modalities for long-term treatment are accepted as useful in these children: oral anticoagulant therapy or protein C replacement (fresh frozen plasma or prothrombin complex concentrate). Liver transplantation has been performed in only one child, with success. Oral anticoagulation (vitamin K antagonists, maintaining the prothrombin time from one and one-half to two times control values or at the International Normalized Ratio of 2.5 to 4.4) is our recommendation of choice for long-term treatment. With appropriate care, these children are able to be free of coagulopathy and live relatively normal lives.

HTLV-III serology in hemophilia: relationship with immunologic abnormalities.

We investigated the relationship of the presence of antibodies to HTLV-III and immunologic abnormalities in patients with hemophilia. Serum antibodies to HTLV-III were analyzed by ELISA assay, immunoprecipitation of labeled cell extracts, and immunoprecipitation of purified HTLV-III p24. Thirty-four (61%) of the total group (n = 56) had antibody to HTLV-III; 34 (76%) of 45 patients given commercial factor VIII preparations were seropositive, compared with none of 11 patients treated exclusively with cryoprecipitate obtained from volunteer blood donors. Of patients who were seropositive for HTLV-III antibody, 94% had abnormal T4/T8 ratios, and 33% of those whose serum was antibody negative had abnormal T4/T8 ratios; five patients, each antibody positive, have lymphadenopathy syndrome. Sequential studies in a subset of patients indicate that there is a changing pattern of antibody production to HTLV-III antigens after seroconversion.

Severe homozygous protein C deficiency.

An infant with recurrent purpura fulminans in the first year of life was found to have severe homozygous deficiency of protein C (less than 1% of normal levels). The episodes of purpura fulminans were controlled by infusions of fresh frozen plasma containing protein C. The requirement of frequent plasma infusions, however, eventually resulted in several complications secondary to hyperproteinemia. Factor IX concentrates rich in protein C were then given to maintain adequate levels of the factor while minimizing the amount of extraneous proteins. The patient has remained asymptomatic and free of complications for greater than 10 months while receiving these concentrates every 48 hours.

Generalized lymphadenopathy and T cell abnormalities in hemophilia A.

Two patients with hemophilia A had generalized lymphadenopathy, lymphopenia, elevated IgG values, depressed T4 (helper) lymphocytes, elevated T8 (suppressor) lymphocytes, and abnormally low T4/T8 ratios. One of the patients, who also had hepatosplenomegaly, underwent cervical lymph node biopsy; the node contained 43% T8-lymphocytes, a marked elevation over the small fraction of T8 cells usually found in lymph nodes. These patients may have a form of the acquired immune deficiency syndrome described in male homosexuals, Haitians, intravenous drug abusers, and recently, in patients with hemophilia. We studied T cell phenotypes in 43 patients with hemophilia. Fourteen of 28 patients given commercial factor VIII concentrates had abnormal T4/T8 ratios; none of nine patients who used cryoprecipitate had abnormal values. T4 helper cells were significantly lower, T8 suppressor cells significantly elevated, and T4/T8 ratios significantly lower in the lyophilized concentrate users and in patients with hemophilia as a total group. The type of therapeutic factor VIII replacement may alter the risk of developing T4/T8 abnormalities or AIDS.

Observations on the vegetation of northeastern Mato Grosso II. Forests and soils of the Rio Suiá--Missu area.

The vegetation of the well drained soils along the Suiá--Missu road in the Serra do Roncador region of NE Mato Grosso is Evergreen Seasonal forest of Amazonian type. The area lies close to the meeting place of the Amazonian forest (the hylaea) and the cerrado (savanna) formation of Central Brazil. The structure of the forest is simple: the canopy is at about 18--23 m, and is exceeded by a few scattered emergents; no recognizable strata can be distinguished among the understorey trees and the shrub and herb layers are sparse. Table 1 lists the most important species and gives information on stratification and general distribution. Most of the species appear to have a hylaean centre of distribution but extend into other vegetation types. The forest differs from related communities which lie closer to the cerrado/forest boundary in its greater height and luxuriance, the presence of additional tall tree species, and the great reduction in abundance of a cerrado floristic element. A survey on the Xavantina--São Felix road allowed us to extend previous observations on the distance to which the cerrado tree Pterodon pubescens extends into the forest. The results obtained indicate a considerable extension of forest into cerrado during the life of an individual tree. A characteristic low forest occurs in the flood plain of the Rio Suiá--Missu while Swampy Gallery forests occur on permanently waterlogged soils around the headwaters of streams. The well drained soils of the Suiá--Missu forest are very uniform, deep latosols (oxisols) of very dystrophic nature with pH (in water) between 4.0 and 5.0 (see table 2, p. 203).

Clinical and pathological observations on Jamaican neuropathy: a report on 206 cases

The clinical features of 206 cases of a neuropathic syndrome in Jamaica are presented. The dominant feature is a spastic paraplegia. Approximately half of the cases have evidence of associated posterior column damage. In a minority optic atrophy, nerve deafness of selective anterior horn cell damage is found. The patients have been divided arbitrarily into two categories: 25 cases presenting predominantly with sensory ataxia, and in whom there is a high incidence of optic atrophy and eighth nerve deafness, with slight evidence of pyramidal tract damage. This group has a background of poor nutrition. (b) 181 cases presenting predominantly as a spastic myelopathy, and with a relative low incidence of optic atrophy and eight nerve deafness. The findings in ten necropsies from the spastic group are presented with their cases record. The histopathology is that of a chronic meningo-myelitis, with damage to the long tracts as the major lesion. Involvement of the brain, they grey matter of the cord and spinal nerve roots occurs to a lesser extent. An eleventh case is described with similar pathology, which clinically was thought to be an example of neurosyphilis. The relationship of this syndrome to other neuropathies is considered. The aetiology is discussed, with special reference to the possible role of syphilis and yaws in the spastic group. The majority of these cases have positive treponemal tests in the blood, but only 6 per cent have positive tests in the spinal fluid. The pathology has much in common with that of neurosyphilis, but lacks some of the features generally accepted as typical. The incidence is extremely high relative to other known forms of neurosyphilis in Jamaica. Other factors such as ingested toxins and vitamin deficiences may add to, or modify, the metabolic impairment. It is possible to envisage a spectrum of disease varying from a picture of non-inflammatory long tract degeneration to the active meningo-vascular reaction of florid adhesive arachnoiditis-a spectrum in which common causal factors could operate with variable degrees of intensity. This study emphasizes that our understanding of the pathogenesis and pathology of neurosyphilis in its many forms is far from complete (AU)

Observations on the cyanide content and toxicity of tropical pulses

The content of cyanogenetic glucosides in West Indian and other pulses was estimated by the release of hydrocyanic acid on hydrolysis. In the varieties of phaseolus lunatus (lima bean) examined, the CN content was under 20mg. per cent, and not of the order previously known to cause acute poisoning. Trace amounts were found in 5 other species, and a high content was found in a variety of vicia sativa seeds (common vetch). A comparison of hydrolytic procedures in ph. lunatus showed that whereas the cyanogenetic glucoside is stable on cooking the intact bean, neither human saliva nor dilute hydrochloric acid at 37oC was effective in releasing free HCN from beans crushed after cooking. Animal feeding tests of crushed uncooked beans showed that the toxicity of these varieties was unrelated to their CN content. Severe 'toxicity' of ph. vulgaris (kidney bean, red pea) in rats and guinea pigs was mainly, if not entirely, due to unpalatability, causing starvation. Palatability was much improved by cooking. Absorption and utilisation of other species were good when fed to rats, even at 50 per cent level. There was some evidence of pancreatic hypertrophy and of impaired absorption or utilization of vigna sp. (black-eye pea) and cajanus cajan (gungo pea) in guinea pigs. No neurological lesions were detected in rats in feeding tests of up to 6 month's duration (AU)