Glucose-6-phosphate dehydrogenase deficiency.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect, being present in more than 400 million people worldwide. The global distribution of this disorder is remarkably similar to that of malaria, lending support to the so-called malaria protection hypothesis. G6PD deficiency is an X-linked, hereditary genetic defect due to mutations in the G6PD gene, which cause functional variants with many biochemical and clinical phenotypes. About 140 mutations have been described: most are single base changes, leading to aminoacid substitutions. The most frequent clinical manifestations of G6PD deficiency are neonatal jaundice, and acute haemolytic anaemia, which is usually triggered by an exogenous agent. Some G6PD variants cause chronic haemolysis, leading to congenital non-spherocytic haemolytic anaemia. The most effective management of G6PD deficiency is to prevent haemolysis by avoiding oxidative stress. Screening programmes for the disorder are undertaken, depending on the prevalence of G6PD deficiency in a particular community.

Coagulation and splenectomy: an overview.

Venous thromboembolic events, such as pulmonary embolism, deep venous thrombosis, and portal vein thrombosis, have been observed in adult thalassemia patients, mainly in beta-thalassemia intermedia. The clinical findings are consistent with the observation of several alterations that indicate a state of activation of the hemostatic mechanisms in thalassemias. These alterations have usually been related to high platelet counts due to splenectomy and/or liver dysfunction. In a retrospective study of a large cohort of adults with thalassemia, we found a larger prevalence of venous thromboembolic events in transfusion-independent patients with thalassemia intermedia (29%) than in regularly transfused patients with thalassemia major (2%); moreover, the higher prevalence occurred particularly in splenectomized thalassemia intermedia patients. More recently, a multicenter study involving 56 tertiary referral centers in 7 countries was planned to assess the magnitude of thrombotic risk in thalassemia patients. The total number of patients who had thrombotic events was 146 (1.65%) out of 8860, with a prevalence of 0.9% in thalassemia major and 4% in thalassemia intermedia. The highest prevalence was confirmed in splenectomized patients. The observation that thrombotic events are more frequent in beta-thalassemia patients who are not receiving regular transfusions (thalassemia intermedia or thalassemia major patients in less developed countries with limited transfusion resources) or in thalassemic patients who have undergone splenectomy strongly supports the procoagulant activity of circulating damaged red blood cells.

Alcohol dehydrogenase: an autoantibody target in patients with alcoholic liver disease.

The link between alcohol consumption and liver disease is not direct and several factors including autoimmunity to hepatocyte components have been implicated. We have previously identified alcohol dehydrogenase (ADH) as an autoantigen in autoimmune liver disease and in a proportion of patients with alcoholic liver disease. The aim of the present study is to investigate the association between the presence of anti-ADH antibodies, alcohol consumption and severity of liver damage in alcoholic patients. The presence of antibodies to human ADH beta2 and horse ADH was investigated in 108 patients with documented history of alcohol consumption and alcohol related liver disease, 86 being active alcohol abusers and 22 on sustained alcohol withdrawal, 39 with non-alcohol related disease and 22 normal subjects. Antibodies to either ADH form were more frequently detected in active alcohol abusers (55/86, 64%) than in patients on sustained alcohol withdrawal longer than 6 months (1/8, 13 %, P < 0.005), HBV infection (2/8, 25 %, P=0.03), non-alcohol related disease (9/29, 23 %, P < 0.0001) and in normal controls (3/22, 14 %, P < 0.0001); were more frequent in patients with cirrhosis than in those with steatosis (26/34, 76 % vs 34/64, 53 %, P=0.02); and were associated with elevated levels of ALT (anti-ADH beta2, P < 0.05), immunoglobulin A (P < 0.05) and gamma-glutamyl transpeptidase (P=0.01). Anti-ADH antibody positive serum samples were able to inhibit the enzymatic activity of ADH. These findings suggest that anti-ADH antibodies may be triggered by alcohol consumption and act as a disease activity marker in alcoholic liver disease.

Characterization, regulation, and function of specific cell membrane receptors for insulin-like growth factor I on bone endothelial cells.

It is now widely accepted that insulin-like growth factor-I (IGF-I) has a local regulatory role in bone remodeling. IGF-I has also been demonstrated to regulate proliferation of bone-derived endothelial cells. Such studies suggest a role of IGF-I in skeletal angiogenesis. Using BBE cells, a bovine bone endothelial cell line, we characterized the kinetics and chemical properties of IGF-I receptors and examined the effect of IGF-I on bone endothelium migration. Two classes of binding sites with high affinity for IGF-I were detected by binding experiments on bone endothelial cells. Both competition analyses and cross-linking studies revealed the presence of type I IGF receptor in bone endothelial cells. Moreover, these cells produced and released authentic IGF-I into the medium, as evidenced by radioimmunoassay analyses of gel-filtered conditioned media. Both IGF-I binding capacity and release decreased either with increases in cell number or after treatment with 17 beta-estradiol (17 beta E2) and parathyroid hormone (PTH). Both hormones also inhibited chemotactic responses of bone endothelial cells to IGF-I. Taken together, these results strongly suggest that IGF-I, a growth factor that promotes the proliferation of various bone cell types, also induces growth and chemotactic responses in bone endothelium acting through the type I IGF receptor. This may be part of a generalized response of bone cells to IGF-I that facilitates cell migration.

Molecular analysis of the hydroxymethylbilane synthase (HMBS) gene in Italian patients with acute intermittent porphyria: report of four novel mutations.

Acute intermittent porphyria (AIP) is an autosomal disorder caused by molecular abnormalities in the hydroxymethylbilane synthase (HMBS) gene coding for the third enzyme in the heme biosynthetic pathway. So far, more than 160 different mutations responsible for AIP have been identified in this gene. We have now identified seven mutations in eight unrelated Italian patients with AIP: two splicing defects (IVS7+2T-->C, 612G-->T), three small deletions (308-309delTG, 730-731delCT, 182delA) and two missense mutations (134C-->A, 541C-->T). The splicing defects were responsible for activation of splicing cryptic sites respectively within intron 7 (15 bp insertion) and exon 10 (9 bp deletion). The small deletions resulted in frameshifts leading to the formation of premature stop codons. The 134C-->A and 541C-->T mutations caused the formation of stop codons likely to be responsible for drastic disruption of the HMBS structure (Ser45Ter, Gln181Ter). This is the first molecular study in AIP patients of Italian origin leading to the identification of four new mutations and three molecular defects that have already been described.

Seven novel point mutations in the uroporphyrinogen decarboxylase (UROD) gene in patients with familial porphyria cutanea tarda (f-PCT).

In this work, we describe seven novel molecular defects in the uroporphyrinogen decarboxylase gene responsible for familial porphyria cutanea tarda in Italian subjects with reduced erythrocyte URO-D activity. Four of these molecular abnormalities (R142Q, L161Q, S219F, P235S) are missense mutations, one (Q206X) is a nonsense mutation, one (IVS8-1 G>C) is a splicing defect causing the exon 9 deletion and one (1107 G>A) is located in the 3' untranslated region of UROD gene. All the amino acid substitutions fall in conserved regions in several organisms suggesting an important role in catalysis or in the protein structure stabilization. Three of these mutations have been detected in more than one subject. These results suggest a molecular heterogeneity at the UROD locus in Italian PCT patients although recurrent mutations have been identified.

Cyclic adenosine monophosphate stimulates insulin-like growth factor binding protein-4 and its messenger ribonucleic acid in a clonal endothelial cell line.

Endothelial cells regulate the passage of insulin-like growth factors (IGFs) or IGFs complexed to IGF-binding proteins (IGFBPs) from plasma to the extravascular space, and in addition respond to plasma and tissue IGFs. The IGFBPs are thought to determine the availability and localization of IGFs to tissues, and to inhibit or potentiate their biological activity. Because of the potential importance of the IGF system in endothelial cell pathophysiology, and because IGFBPs modulate IGF action, we have characterized the IGFBPs synthesized by a clonal endothelial cell line (BPE-1) established from bovine parathyroid microvessels. The only IGFBPs identified by ligand blotting in media conditioned by BPE-1 cells were N-glycosylated 28 kilodalton and non-N-glycosylated 24 kilodalton IGFBP-4 species. Both forms were immunoprecipitated by antibodies to human IGFBP-4. Northern blot hybridization of BPE-1 RNA identified messenger RNAs for IGFBP-4 and IGFBP-6, but not for IGFBP-1, 2, 3, or 5. Agents that increase cAMP including forskolin, (Bu)2cAMP, isobutyl-methylxanthine, and histamine increased IGFBP-4 and IGFBP-4 messenger RNA in BPE-1 cells 2- to 5-fold and 2- to 3-fold, respectively, similar to results previously reported in human osteosarcoma cells and fibroblasts. Increased IGFBP-4 was detected in BPE-1 media 6 h after forskolin addition and was maximal after 24 h. Maximal stimulation (6- to 9-fold) was observed with 1-30 microM forskolin. IGFBP-4 also was the predominant IGFBP synthesized by two other endothelial cell lines, a clonal cell line established from bovine bone microvessels, and a polyclonal cell line established from calf pulmonary artery. Further study is required to determine the role of endothelial cell IGFBP-4 on endothelium and adjacent cells, and on the transport of IGFs from plasma to specific subendothelial sites.

Epidermal growth factor receptors in human hyperplastic prostate tissue and their modulation by chronic treatment with a gonadotropin-releasing hormone analog.

We characterized the epidermal growth factor (EGF) receptor in the membrane fraction of prostatic tissue from men with benign prostatic hyperplasia (BPH). The maximum specific binding of [125I]EGF to the BPH membrane fraction was achieved after 30-min incubation at 35 C. Analysis of the binding data revealed two classes of binding sites, one of high affinity [Kd, 2.5 +/- 0.5 (+/- SE) x 10(-11) mol/L] and one of lower affinity (2.2 +/- 0.3 x 10(-9) mol/L). [125I]EGF binding was inhibited by excess EGF, but not by insulin, proinsulin, fibroblast growth factor, or insulin-like growth factors I and II. In prostatic tissue of men with BPH treated for 3 months with the GnRH agonist analog Goserelin (Zoladex, depot formulation), the binding capacities of both sites were significantly higher than those of BPH tissue from untreated men (P less than 0.001). These results demonstrate that prostatic tissue from men with BPH contains two classes of specific binding sites for EGF, and their levels are modulated by chronic GnRH agonists treatment.

Spermatic and peripheral plasma concentrations of testosterone and androstenedione in prepubertal boys.

Spermatic and peripheral plasma concentrations of testosterone (T) and androstenedione (A) have been measured in prepubertal boys affected by inguinal hernia (group I; n = 7) and unilateral undescended testis (group II; n = 18). Mean (+/- SE) spermatic T concentrations (47.7 +/- 14.8 ng/dl in group I; 36.3 +/- 3.4 ng/dl in group II) were significantly different from mean peripheral T concentrations (9.8 +/- 2.1 ng/dl in group I; 9.3 +/- 0.9 ng/dl in group II) in both groups (P less than 0.05 and P less than 0.0005, respectively). Mean spermatic A concentration (59.7 +/- 4.9 ng/dl) was significantly higher than mean peripheral A concentration (49.8 +/- 4.9 ng/dl) in group II (P less than 0.05) but not in group I. Mean spermatic and peripheral T and A values found in boys of group I were not significantly different from those found in group II. The mean spermatic/peripheral T ratio was higher (5.01 in group I; 4.42 in group II) than the corresponding mean spermatic/peripheral A ratio (1.27 in group I; 1.32 in group II) in both groups. Our data suggest that 1) although testicular T secretion is present in all prepubertal boys, A secretion is not constant and often negligible; 2) the contribution of testicular secretion to the circulating T is much more important than the contribution to the circulating A; 3) no significant differences were found between the testicular secretory pattern of prepubertal boys with inguinal hernia and unselected boys with unilateral undescended testis.

Dopamine D2 receptor gene expression and binding sites in adrenal medulla and pheochromocytoma.

To determine whether dopamine D2 receptors are present in normal and neoplastic chromaffin tissues, 10 pheochromocytomas and 5 human adrenal glands were studied. Dopamine D2 receptor messenger ribonucleic acid corresponding to a single band of approximately 2.5 kilobases was detected in both pheochromocytoma and human adrenal gland by Northern blot analysis. D2 receptor messenger ribonucleic acid levels determined by dot blot analysis were 3.1-fold lower in human adrenal medullas than in pheochromocytomas (P < 0.001). Simultaneous Scatchard analysis of [3H]spiperone binding experiments demonstrated the presence of two different binding sites in membrane preparations from bovine adrenal medullas [R1: Kd = 0.14 nmol/L; binding capacity (Bmax) = 6.2 fmol/mg protein: R2: Kd = 16 nmol/L; Bmax = 223 fmol/mg protein]. Similarly, two binding sites were present in membrane preparations from pheochromocytomas (R1: Kd = 0.39 nmol/L; R2: Kd = 61 nmol/L]. Binding capacities were greatly variable among pheochromocytomas (R1: Bmax = 12.0-372.5 fmol/mg protein; R2: Bmax = 1,000-11,586 fmol/mg protein). The relative potencies of different compounds to displace [3H]spiperone were spiperone > domperidone > (+)-butaclamol > quinpirole > SCH 23390 in bovine adrenal medulla, and spiperone >> domperidone > quinpirole > (+)-butaclamol > SCH 23390 in pheochromocytoma. We conclude that dopamine D2 receptors are synthesized in human adrenal medulla and pheochromocytoma tissues.

Three-month treatment with a long-acting gonadotropin-releasing hormone agonist of patients with benign prostatic hyperplasia: effects on tissue androgen concentration, 5 alpha-reductase activity and androgen receptor content.

The intraprostatic concentrations of testosterone (T) and dihydrotestosterone (DHT) have been measured in only a few men. We measured, in prostatic tissue obtained at surgery from seven men with benign prostatic hyperplasia, the effects of 3-month treatment with a long-acting GnRH agonist on 1) the intraprostatic concentrations of T, DHT, and 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol); 2) prostatic 5 alpha-reductase activity; and 3) the prostatic content of androgen receptors (AR). Plasma T, DHT, and 3 alpha-diol levels also were measured. Prostatic tissue samples obtained at surgery from a group of untreated men with benign prostatic hyperplasia also were studied. The mean DHT and 3 alpha-diol concentrations in the prostatic tissue of the treated men were about 10% of those in untreated men (n = 19; P less than 0.01 for DHT and P less than 0.05 for 3 alpha-diol), and the mean intraprostatic T concentration in the treated men was about 25% of that in the control group (0.10 greater than P greater than 0.05). The mean in vitro formation of DHT by the prostatic tissue of the treated men was about 50% lower (P less than 0.05) than that by prostatic tissue of the untreated men (n = 9). The mean cytosolic AR content in the prostatic tissue of the treated men was significantly higher (P less than 0.05), whereas the mean nuclear content of both salt-extractable and salt-resistant AR was significantly lower (P less than 0.05) than that in the prostatic tissue of the untreated men (n = 8). The mean plasma T levels in treated men decreased from 4.77 +/- 1.79 (SD) ng/mL (16.5 +/- 6.2 nmol/L) to 0.27 +/- 0.42 ng/mL (0.9 +/- 1.5 nmol/L) after 1 month of therapy and remained in the castrate range thereafter. We conclude that pharmacological castration resulting from 3-month treatment with a long-acting GnRH agonist decreases the intraprostatic T concentration to about one fourth and those of DHT and 3 alpha-diol to about one tenth of the levels in untreated men. Thus, GnRH agonist treatment may not completely abolish intraprostatic androgen concentrations in metastatic prostatic cancer patients. The decrease in prostatic 5 alpha-reductase activity as well as the decrease in nuclear receptors are probably secondary to the decrease in plasma T concentrations.

Epidermal growth factor, epidermal growth factor receptor, and transforming growth factor-alpha in human hyperplastic prostate tissue: expression and cellular localization.

It is widely accepted that polypeptide growth factors are involved in the growth and development of normal and neoplastic human prostate. It has been previously reported that epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) receptors are present in the human hyperplastic prostate tissue (BPH). To add information on the mechanism of action of EGF and transforming growth factor-alpha (TGF alpha), a peptide correlated to EGF, and the EGF receptor (EGF-R) in the human prostate, we studied the expression and cellular localization of messenger ribonucleic acid (RNA) encoding EGF, EGF-R, and TGF alpha in BPH tissue. Reverse transcriptase-PCR of total RNA extracted from BPH tissues documented the presence of specific transcripts for EGF, EGF-R, and TGF alpha. In situ hybridization with specific RNA probes synthesized from the respective complementary DNA demonstrated that EGF, EGF-R, and TGF alpha messenger RNAs were mainly localized in the epithelial cells. Immunprecipitation and Western blot analysis showed that BPH tissue contained the corresponding proteins, EGF and TGF alpha. Our findings provide additional support for the idea that EGF and TGF alpha may be considered specialized symbols in the language of cell-cell interactions and for the hypothesis that in the human prostate they seem to act in an autocrine fashion.

Insulin-like growth factor-I (IGF-I) and its binding protein IGFBP-4 in human prostatic hyperplastic tissue: gene expression and its cellular localization.

It has been previously reported that 1) type I insulin-like growth factor (IGF) receptors are present in the human prostatic tissue; 2) IGF-I receptors are mainly localized in the epithelial cells; 3) IGF-I is a mitogen for prostatic epithelial cells in culture; and 4) IGF-binding proteins (IGFBPs) are released by these cells in the conditioned medium. To add information on the mechanism of IGF-I action in the human prostate, we studied the expression and cellular localization of mRNA encoding IGF-I and IGFBP-4 in human prostatic hyperplastic (BPH) tissue. Northern analysis of total RNA extracted from BPH tissues with cDNA probes containing the entire coding regions for IGF-I and IGFBP-4 documented the presence of multiple IGF-I mRNA transcripts with lengths of 7.5, 1.7, 1.3, and 1.1 kilobases and a single 2.1-kilobase transcript of IGFBP-4 mRNA. In situ hybridization with the cDNA probes used for Northern analysis and with cRNA probes synthesized from the respective cDNA demonstrated that IGF-I mRNA was only localized in the stromal cells, whereas IGFBP-4 mRNA was predominantly expressed by epithelial cells. In addition, immunoreactive IGF-I was measured in BPH tissue extracts after acidification and reverse phase chromatography. The mean (+/- SD) IGF-I content of six BPH tissues was 28.1 +/- 4.0 ng/g tissue. Our results suggest that in the human prostate, the locally secreted IGF-I exerts its principal biological effects with a paracrine mode of action and demonstrate that IGFBP-4 is mainly expressed by IGF-I target cells.

Insulin-like growth factor-I receptors in human hyperplastic prostate tissue: characterization, tissue localization, and their modulation by chronic treatment with a gonadotropin-releasing hormone analog.

Insulin-like growth factor I (IGF-I) receptors were characterized in membranes obtained from prostate tissue of patients affected by benign prostatic hyperplasia (BPH) before and after treatment with a GnRH agonist analog. Binding of [125I]IGF-I to membranes obtained from untreated patients was specific and time and temperature dependent. Analysis of the binding data yielded two classes of binding sites, one of high affinity (Kd, 10(-11) mol/L) and one of lower affinity (Kd, 10(-9) mol/L). BPH membrane preparations were affinity-cross linked to labeled IGF-I, and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis by autoradiography revealed one labeled protein with an apparent Mr = 300K under nonreducing conditions and two labeled protein with Mr = 270K and Mr = 130K under reducing conditions. Excess unlabeled IGF-II reduce both of them, whereas the same excess of IGF-I completely abolished them. In membrane preparations of prostatic tissues from patients affected by BPH and treated for 2 months with a GnRH agonist analog, the binding capacities of both binding sites were significantly higher than those of BPH tissue from untreated patients, whereas binding affinities were unchanged. The IGF-I receptor in BPH prostate tissue of untreated patients was mainly localized in the basal layer of the epithelium, as demonstrated by immunohistochemical staining, whereas in the tissue from treated patients positive staining was found also in the glandular epithelium. These results demonstrate that: 1) specific binding sites for IGF-I are present in prostatic tissue from patients with BPH, 2) androgen deprivation increases their binding capacities and seems to modify their epithelial localization.

Insulin-like growth factor-I receptors in nonfunctioning thyroid nodules.

We have recently demonstrated the production of insulin-like growth factor-I (IGF-I) as well as the presence of type I IGF receptors in human thyroid cells in primary culture. The role of IGF-I in the control of thyroid cell growth has been well established. In order to investigate the involvement of IGF-I in abnormal thyroid growth, the density of IGF-I receptors in solitary, cold, micro- and macro-follicular thyroid adenomas, and in extranodular histological normal tissue was studied. Forty-three euthyroid patients with isolated cold nodules were selected for the study. In 30 patients the presence of IGF-I receptors was evaluated by using quantitative immunohistochemistry; in 10 patients by using radioligand binding studies, and in 3 patients by using affinity labeling. Cross-linking and binding studies clearly demonstrated the presence of a homogeneous class of binding sites for type I IGF receptors. Furthermore, radioligand studies did not show any significant differences in receptor density between the 2 types of thyroidal tissues. Conversely, the computerized analysis of 900 fields of nodular and normal thyroid tissues immunostained with the monoclonal antibody alpha-IR3, strongly indicated that higher concentrations of IGF-I receptors were present in the epithelial cells of non-functioning thyroid nodules than in the adjacent extranodular thyroid tissues. These studies strongly suggest that the same type I IGF receptor is present in thyroid follicular adenomas as in histological normal thyroid tissue removed from the same patient. The higher concentration of IGF-I receptors as documented by immunostaining in the adenomas suggests that IGF-I may contribute to the abnormal growth of the neoplasms.

Effect of heparin on proliferation and signalling in BC3H-1 muscle cells. Evidence for specific binding sites.

We studied binding and growth inhibitory properties of different glycosaminoglycans in growing and differentiated BC3H-1 muscle cells. Heparin (10 micrograms/ml) and heparan sulfate (10 micrograms/ml) significantly inhibited DNA synthesis in growing and differentiated cells, as monitored by [3H]thymidine incorporation. Binding of heparin to BC3H-1 cells was specific and time-dependent. Heparan sulfate was the only glycosaminoglycan able to displace [3H]heparin (IC50, 3.2 x 10(-7) M), although it was 10-fold less effective than heparin itself (IC50, 3.6 x 10(-8) M). Scatchard analysis revealed the existence of high-affinity heparin binding sites (Kd, 5 x 10(-8) M). Furthermore, heparin inhibited serum-induced stimulation of inositol lipid turnover. Taken together, these results indicate that heparin inhibits BC3H-1 cell growth by interacting with the cell surface, possibly disrupting the flow of growth factor-related mitogenic signalling.

Characterization and function of the receptor for IGF-I in human preosteoclastic cells.

Using a coculture system, we have recently demonstrated that insulin-like growth factor I (IGF-I) is a mediator of preosteoclastic cell migration toward bone-derived endothelial cells. To better characterize the mechanisms of IGF-I action on preosteoclastic cells we evaluated the expression of type I IGFs receptor in the human leukemic cell line, FLG 29.1, which differentiates toward the osteoclastic phenotype following phorbol ester (TPA) treatment. Scatchard analysis of 125I-labeled IGF-I to FLG 29.1 cells revealed the presence of a single high affinity binding site in both untreated and TPA-treated cells with a similar Kd value (0.3 +/- 0.2 nmol/L and 0.4 +/- 0.1 nmol/L, respectively). In untreated cells, IGF-I binding capacity (1.43 +/- 0.41 fmol/10(6) cells) was significantly (p < 0.05) lower than in TPA-treated cells (2.62 +/- 0.87 fmol/10(6) cells). Competition analyses and crosslinking studies revealed the presence of type I IGF receptor both in untreated and TPA-treated cells. Northern analysis demonstrated that mRNA for IGF-I receptor was expressed by both untreated and TPA-treated FLG 29.1 cells. In addition, FLG 29.1 cells released in the conditioned medium IGFBP-2 and IGFBP-4, whose expression was increased by TPA treatment as demonstrated by ligand and immunoblot analyses. The previous observations of chemotactic action of IGF-I on FLG 29.1 cells was confirmed by ultrastructural observations. Indeed, these cells revealed a marked migratory activity in response to nanomolar concentrations of IGF-I. In addition, the IGF-I receptor alpha IR-3 antiserum inhibited the IGF-I-induced FLG 29.1 cell's migratory activity. These findings clearly show that type IIGF receptor is expressed by osteoclast precursors and that IGF-I induces migration of these through the binding to type I IGF receptors. Binding proteins expressed by osteoclast precursors may play an autocrine role in modulating the IGF-I bioeffects.

A modified rosette inhibition test in renal allotransplantation.

The rosette inhibition test, with a modification of the technique which enables highly accurate marking of T lymphocytes, has been employed in the followup of 55 renal transplant patients. The minimal inhibitory concentration of antilymphocyte globulin (that is, that concentration of antilymphocyte globulin causing 25% inhibition of rosette formation) was higher than 1:16,000 in 63 (97%) of 65 separate determinations made during acute rejection episodes, and lower than 1:16,000 in 377 (92%) of 410 determinations after which no clinical evidence of rejection developed. The results presented in this paper indicate that this modified test is a useful tool either to predict the occurrence of or to confirm the diagnosis of rejection.

Combined use of DNA probes in first-trimester prenatal diagnosis of hemophilia A.

First-trimester prenatal diagnoses of hemophilia A were heretofore obtained by using either intragenic factor VIII markers or linked extragenic polymorphic markers. Postulating that the combined use of all the available intragenic and extragenic markers can render such diagnoses more frequently feasible and more reliable, we carried out ten first-trimester prenatal diagnoses in male fetuses at risk for hemophilia A by DNA analysis of chorionic villus employing in combination the intragenic Bcl I polymorphism and the St 14 (DXS 52) or DX 13 (DXS 15) extragenic probes. A diagnosis of hemophilia was obtained in three fetuses, with a diagnosis of normal fetus obtained in the remaining seven. Seven diagnoses are confirmed by factor VIII assays carried out at the time of abortion, in the mid-trimester or at birth. A factor VIII probe recognizing Bcl I polymorphism was useful in 4 of 6 diagnoses; St 14, in 5 of 6; and DX 13 in 3 of 5. In two cases, St 14 was the only useful probe for diagnosis. Even though no recombination between extragenic probes and factor VIII gene was detected in this study, when only extragenic markers were informative we advised diagnostic confirmation on fetal plasma obtained by fetoscopy. Hence, first-trimester prenatal diagnosis of hemophilia A is feasible for the great majority of fetuses at risk through combined use of all the available intragenic and extragenic probes, providing key family members are available.

Binding and growth-inhibitory effect of heparin and oligo-heparin (2kDa) in Balb/c 3T3 cells: lack of effect on PDGF- or serum-induced inositol lipid turnover.

1. The ability of heparins (bovine heparin sm 1026, Av. mol. wt. 36.9 kDa and bovine heparin EP 756, Av. mol. wt. 12.9 kDa) and heparin fractions of different molecular weights (low molecular weight heparin, LMW 2123/OP, Av. mol. wt. 4.5 kDa and oligo-heparin, Av. mol. wt. 2 kDa) to inhibit the proliferation and signalling of Balb/c 3T3 fibroblasts was investigated. 2. Heparin and heparin fractions of 4.5 and 2 kDa significantly inhibited DNA synthesis as monitored by [2H]-thymidine incorporation. 3. 3H-labelled heparin fractions of 4.5 and 2 kDa were prepared by gel-chromatography fractionation on Sephadex G-75 of an 3H-labelled commercial heparin after treatment with heparinase. 4. The binding of unfractionated and oligo-heparin of 2 kDa to Balb/c 3T3 fibroblasts was studied; we determined the specificity of heparin and oligo-heparin binding to the cells by means of displacement of bound 3H-labelled compound in response to increasing concentrations of unlabelled compounds. Scatchard analysis of binding data obtained using [3H]-heparin as ligand revealed the presence of a single class of high affinity binding sites (Kd = 28 nM) for heparin. Scatchard analysis of binding data obtained using [3H]-oligo-heparin as ligand revealed the presence of a single class of low affinity binding sites (Kd = 3.2 microM) for oligo-heparin. 5. In addition heparin displaced [3H]-oligo-heparin at a concentration of approximately 100 fold of the Kd determined in displacement studies. Furthermore, oligo-heparin significantly displaced [3H]-heparin at a concentration of approximately 10 fold of the Kd determined by displacement studies. 6. Both heparin and oligo-heparin exert their inhibitory effects on Balb/c 3T3 DNA synthesis stimulated by PDGF or serum. However these molecules did not affect the inositol lipid turnover triggered by PDGF at a concentration which did not produce maximal response. The increase of inositol phosphate metabolism produced by 20% serum was also unaffected by heparin. This concentration of serum elicited a response comparable to that induced by a submaximal concentration of PDGF.