[A case report of pyridoxine-responsive homocystinuria]. / Slucaj piridoksinresponzivne homocistinurije.

INTRODUCTION: Homocystinuria is a rare, congenital, autosomal-recessive, metabolic disease biochemically characterized by homocysteinemia and homocystinuria and by multi-system clinical disorders. It is a biochemical abnormality of methionine metabolism caused either by transulfuration pathway disorders or by disorders of homocysteine remethilation into methionine and as such it can be a result of numerous specific and different genetic lesions. CASE REPORT: Homocystinuria is most commonly caused by deficiency of cystationine beta synthetase enzyme which catalyses the synthesis of cystathionine from homocysteine and serine in the methinione pathway. This results in accumulation of homocysteine and methionine in plasma and leads to excretion of excessive urinary homocysteine. Depending on specific property of the mutant enzyme molecule some patients respond to very high doses of pyridoxine with decreases of methionine and homocystine and some not. Pyridoxine responsiveness is based on the presence of small residual activity of cystathionine beta synthetase which is not present in nonresponsive patients. Homocystinuria due to cystathionine beta-synthase (CBS) deficiency is characterized by numerous different clinical abnormalities, but changes in four organ systems are dominant (eye, skeletal, central nervous and vascular system). CASE DESCRIPTION: Six years ago a six year-old boy was admitted to the hospital with vision problems. Ophthalmologic examination revealed a lens dislocation and because of many stigmates the child was sent to endocrinologist. The child had a marfanoid stature, with pectus carinatum and genu valgum, ataxic gait with motoric discoordination, muscle tone which ranged from hypotonia to hypertonia of extrapvramidal type and low intellectual abilities. A simple cyanide-nitroprusside test of patient's urine was positive suggesting homocystinuria. The diagnosis was established after plasma and urine amino acid analysis by HPLC. Concentration of homocystine and methionine were 52 mumol/l and 57 mumol/l in plasma and 249 mumol/du and 55 mumol/du in urine, respectively. After four months of treatment with pyridoxine there were not any significant changes in plasma homocysteine and methionine, but at the same time decrease in urine of these two amino acids were more than 2.5 times higher. This confirms that the patient has a pyridoxine-responsive type of homocystinuria and the dose was increased to 60 mg/day and 600 mg/day. This results in further decline of homocysteine and methionine in plasma and urine which persists up to now (for six years).

Effect of serum from thrombocytopenic patients on platelet count in blood of mice.

Estimation of the effect of sera obtained from patients suffering from several forms of megakaryocytic (immune) thrombocytopenia (19), and those with acute non-lymphoblastic leukemia (13), on the platelet count in the peripheral blood of mice was carried out. Several groups of mice were injected intravenously with 0.2 ml of patients' sera, and the platelet count was followed up for 10 days. A marked and highly significant decrease of platelet count in recipient mice was established even 6 hours after the application of both groups of patients' sera. In the control group of mice receiving pooled serum of healthy persons this decrease was not registered. It can be presumed with great probability that the mechanism of development of thrombocytopenia in both groups of patients is very similar: there is an extremely increased destruction of platelets in the peripheral blood in cases with immune thrombocytopenia by antibodies, and in cases with acute leukemias by antibodies and/or other cytotoxic substances. In further investigations the influence of thrombopoietin on this phenomenon will be tested.

[Clinical use of thrombopoietin (c-Mpl Ligand)]. / Klinicka upotreba trombopoetina (c-Mpl LIGANDA).

INTRODUCTION: Results in clinical use of thrombopoietin have been published later than of other hematopoietic growth factors, because until recently the research was the least understood aspect of blood cell development. Reasons for this time gap were numerous, from inconvenient methods for measurement of thrombopoietin activity, to difficulties of its chemical purification. It is claimed recently that the understanding of platelet production has been profoundly advanced by the recombinant-gene synthesis and characterization of c-Mpl ligand (Megakaryocyte Growth and Development Factor), a substance which strongly enhances the proliferation of megakaryocytic line and the production of platelets. In this paper, some historical facts and biology of thrombopoietin are briefly discussed as well as the recent of the clinical use of thrombopoietin. THE HISTORY OF RESEARCH AND PRODUCTION OF THROMBOPOIETIN: The concept that the platelet production is underlying humoral regulation was first promoted by a group of Hungarian authors and they also named that humoral regulator--thrombopoietin. Further research followed in several countries including our own, and the initial studies proved that the serum of thrombocytopenic animals induced proliferation and maturation of megakaryocytic cell line and thrombocytosis in the peripheral blood of recipient animals. Later, when in vitro techniques were developed, it was shown that this humoral regulator has also a colony stimulating activity on megakaryocytic precursors. During the following two decades, studies of megakarycytopoiesis supported the hypothesis that two types of factors are involved in platelet production: early acting--megakaryocyte colony stimulating factors (Meg-CSF), and late acting--megakaryocyte potentiators, first of all thrombopoietin (TPO). However, extensive attempts on the purification of substances that either stimulate megakaryocyte development or augment platelet production failed to yield a homogeneous protein adequate for protein sequencing and cDNA cloning, the usual route which led to the production of other hematopoietic growth factors. Furthermore, a large number of other cytokines were described that possessed activity in various assays of megakaryocyte development. In spite of great number of accumulated data, it seemed in early 1990s that the production of a distinct, clinically useful lineage specific thrombopoietin will not be soon possible to achieve. The breakthrough occurred in 1994, when four groups of investigators published simultaneously their successful results on production of c-Mpl ligand, a substance which specifically binds to the Mpl receptors on megakaryocytes and has a very potent thrombocytopoietic effect. This production is based on genetic engineering and two companies (Kirin and Amgen) are already able to produce recombinant human thrombopoietin in large amounts, for clinical use. Although this substance is not commercially available yet, it passed the preclinical and clinical trials whose results are presented here. RESULTS OF THE PRECLINICAL TRIALS OF RECOMBINANT THROMBOPOIETIN: The chemical structure of human recombinant thrombopoietin (rTPO) is well defined, it is a glycoprotein consisting of 353 amino acids and molecular weight of 30 kD. The biologic actions of this molecule are in vitro: stimulation of megakaryocyte colony forming, endoreduplication of chromosomes and megakaryocyte maturation, and in vivo: increase of the number of progenitors and of megakaryocytes in the bone marrow, and an extensive elevation of platelet count in the peripheral blood 4-7 days after its application. Also, in synergism with other pluripotent cytokines, it can stimulate the proliferation of other progenitors including CD34+ stem cells. Based on these data it is considered that c-Mpl ligand is the main physiological humoral regulator of thrombocytopoiesis, having the biological actions both of MegCSF and TPO.

[Homocystinuria--biochemical, clinical and genetic aspects]. / Homocistinurija--biohemijski, klinicki i genetski aspekti.

Homocystinuria is a rare autosomal recessive disease characterized by homocystinuria and multisystemic clinical disorders. The term denotes a biochemical abnormality of methionine metabolism caused both by transsulfuration pathway disorders and remethylation of homocysteine into methionine, and as such it can be a result of numerous specific and different genetic lesions. Homocystinuria is most commonly caused by deficiency of cystathionine beta-synthase (CBS) activity (EC 4.2.1.22). In this lesion, depending on specific characteristics of mutant enzyme molecules, in regard to existence of residual activity, responsive and nonresponsive homocystinuria can be differed regarding clinical response to high doses of pyridoxine. Although there are numerous different clinical abnormalities, changes on four organ systems are dominant. The most common symptoms of homocystinuria include lens dislocation, vascular disorders, skeletal abnormalities and mental retardation. Laboratory findings are the first diagnostic procedure, while determination of enzymatic activity is a direct parameter for making diagnosis. Prenatal diagnosis and early detection are extremely important for the course and prognosis of the disease as they enable application of currently available therapy as soon as possible. The presently available therapy can, only in such cases, prevent occurrence of serious clinical symptoms, prevent their advancement to some extent or improve reversible clinical manifestations.

[Clinical use of granulocyte colony stimulating-factors (GM-CSF and G-CSF]. / Klinicka upotreba faktora stimulacije granulocitnih kolonija (GM-CSF i G-CSF).

Humoral regulation of granulocytopoiesis was proven 30 years ago by discovery of factors which stimulate production of granulocyte colonies (CSF-colony stimulating factors), while clinical utilization of recombinant human colony stimulating factors (rhGM-CSF and rh-CSF) is present for the last 10 years. On the basis of this, today we have a lot of experience in regard to indications, modes and results of their clinical utilization. Clinical utilization of CSF is divided into three fields: treatment of the neutropenic syndrome, utilization in oncologic patients and in bone marrow transplantation. The best results have been achieved in neutropenic syndrome therapy, both chronic (congenital, cyclic and idiopathic neutropenia, aplastic anemia and myelodysplastic syndrome) and acute (granulocytopenia drug induced, postirradiation neutropenia). In oncologic patients it is used to eliminate neutropenia which occurs after cytostatic therapy with standard doses or high dose cytostatic therapy with better effects on the tumor, but with myeloablation. Granulocyte colony-stimulating factor is also used for stimulating fast granulocytopoiesis in autologous or allogeneic bone marrow transplantation, in unsatisfactory transplants especially. In recent years it has been used for mobilization of progenitor CD34+ cells in the donor in order to perform their transplantation, especially in treatment of chronic myeloid leukemia. At our institution G-CSF has been successfully used in 17 patients.

[Clinical use of erythropoietin]. / Klinicka upotreba eritropoetina.

Humoral regulation of erythropoiesis has been known for 100 years, while clinical utilization of recombinant human erythropoietin (rhEPO) only for two decades. It can be said that there is much experience in regard to indications, models and results of its clinical utilization. According to the standpoint of secretion of erythropoietin, anemias can be divided into those where secretion is increased and satisfactory, those where it is increased but not satisfactory and into those where it is not increased or it is even decreased. Anemias of the first group are not an indication for rhEPO utilization, the second group is relative and the third group absolute indication for its utilization. The best results are achieved with absolute indications and it is anemia in chronic renal insufficiency and nonphysiologic anemia of premature babies. Good results can be expected, but not predicted in relative indications, such as anemias in chronic infections, anemias in malignant diseases, myelodysplastic syndrome, aplastic anemia and other secondary anemias. Utilization of rhEPO is useful also in certain states without anemia, especially in transfusiology.

[Clinical use of hematopoietic growth factors--general principles]. / Klinicka upotreba hematopoeznih faktora rasta--opsti principi.

Biology of haematopoietic growth factors in the process of haematopoiesis is well known, but their clinical utilization started with production of recombinant preparations. Today only preparations of Erythropoietin, GM-CSF and G-CSF are commercially at disposal. Absolute indications for utilization of haematopoietic growth factors are states caused by decreased production of certain classes of blood cells as a consequence of shortage of a growth factor necessary for production of a certain class. As these states are very rare, relative indications spread to other states characterized by a decreased number of blood cells or necessity for stimulation of haematopoiesis due to any other reason. This paper contains results of clinical researches only for those growth factors which are not commercially utilized.: M-CSF, Interleukin 3, PIXY321, SCF, Interleukin 6, Interleukin 11, Interleukin 1, Interleukin 2 and Thrombopoietin. Our institution utilizes only the preparations of Erythropoietin (Eprex) and G-CSF (Neuprogen) in 38 patients.

[The effect of blood from patients with idiopathic thrombocytopenia and thrombopoietin on the thrombocyte count in the blood of mice]. / Uticaj seruma osobe obolele od idiopatske trombocitopenije i trombopoetina na broj trombocita u krvi misa.

We examined effects of serum belonging to persons suffering from chronic autoimmune thrombocytopenia on the number of thrombocytes in the peripheral blood of mice. The serum, 0.2 ml, was given to groups of mice intravenously and then the number of thrombocytes was examined during 14 days. A significant decrease of thrombocytes was established in mice receivers during the period of 6 hours to 12 days (it was maximal from the 4th to the 6th day). The same serum was given to two groups of mice and then one of them was also given thrombopoietin preparation. The number of thrombocytes was established 4 and 6 days later and in the group where only serum was given there was a significant decrease of thrombocytes. In the group where thrombopoietin was given apart from the serum, the number of thrombocytes remained normal. On the basis of these findings it has been suggested that, using such an experimental model, a biological in vivo test could be made to demonstrate antibodies in autoimmune thrombocytopenias and to establish that thrombopoietin could be a useful drug in cases of chronic autoimmune thrombocytopenia.

[Blood of patients with acute leukemia increases the leukocyte count in the peripheral blood of mice]. / Serum bolesnika obolelih od akutne leukemije povecava broj leukocita u perifernoj krvi miseva.

Serum of 23 patients suffering from acute myeloid leukemia were given to groups of six mice (s.c. 0.6 ml); 3 and 5 days later the total number of leucocytes, granulocytes and lymphocytes was determined in the peripheral blood of mice. Serums of all patients caused significant increase of leucocytes in mice 3 and/or 5 days after the serum's application. In most cases the increase was caused by increased number of granulocytes and lymphocytes, and in 6 cases only by increase of granulocytes or only lymphocytes. This increase can also be thought of as a stimulation of leukocytopoiesis by increased concentration of hematopoetic growth factors in blood of those suffering from acute leukemia.