Transcranial color Doppler in stroke-free adult patients with sickle cell disease.

The threshold velocity ≥200 cm/s at transcranial Doppler (TCD) evaluation is a useful cut-off for preventing the stroke (STOP trial) in pediatric patients with sickle cell disease (SCD), term including different types of sickle genotypes. Scanty data are available for adult SCD patients. We compared intracranial blood flow velocities between adult SCD patients and controls using transcranial color Doppler (TCCD), measuring the peak of systolic velocity (PSV) with the insonation angle correction and the pulsatility index (PI), an indicator of endothelial elasticity. Fifty-three adult SCD patients (aged >18 years) were enrolled (15 sickle cell anemia, 26 sickle cell thalassemia, and 12 HbS/HbC). None of the patients presented neurological signs. PSVs in middle cerebral artery (MCA) were higher in SCD patients than in controls (p = 0.001). In sickle cell anemia patients, PSVs were higher when compared to HbS/ßThal (p < 0.0060) and HbS/HbC patients (p < 0.0139). PI was within the lower range of normality in SCD patients compared to controls. Moreover, MCA-PSV was higher with lower Hb levels and higher HbS%; PI did not change with variation of Hb levels and HbS%.PSV and PI in SCD adult patients could be a relevant index to indicate the abnormal cerebral blood flow and to detect the sickle endothelial damage, in order to prevent cerebrovascular accidents.

Bone quality in beta-thalassemia intermedia: relationships with bone quantity and endocrine and hematologic variables.

We report the first evaluation of bone quality in 70 thalassemia intermedia (TI) patients (37 males, 33 females, age 41 ± 12 years). Thirty-three patients (47%) had been transfused, 34 (49%) had been splenectomized, 39 (56%) were on iron chelation therapy, and 11 (16%) were on hydroxyurea. Mean hemoglobin was 9.2 ± 1.5 g/dl, median ferritin 537 ng/dl (range 14-4893), and mean liver iron concentration 7.6 ± 6.4 mg Fe/g dw. Fifteen patients (21%) had endocrinopathies, and 29 (41%) had vitamin D deficiency. Bone quantity (bone mineral density, BMD) and bone quality (trabecular bone score, TBS) were evaluated by densitometry. In 53/70 patients (76%), osteopathy was found (osteoporosis in 26/53, osteopenia in 27/53). BMD values were higher in the never-transfused patients and in the not-chelated group. A highly significant correlation was found between splenectomy and BMD at all the sites, with lower values in the splenectomized patients. TBS values were significantly lower in TI patients than in 65 non-thalassemic controls (1.22 vs 1.36, p < 0.01), mainly in those splenectomized and in the transfused and chelated groups (p < 0.01). TBS did not correlate with liver iron concentration values. Our data disclose the major role of non-invasive bone quality evaluation in TI patients, especially those with the worst health state, to obtain a comprehensive assessment of fracture risk. Splenectomy seems to play a major part in bone complications.

X-chromosomal inactivation directly influences the phenotypic manifestation of X-linked protoporphyria.

X-linked protoporphyria (XLP), a rare erythropoietic porphyria, results from terminal exon gain-of-function mutations in the ALAS2 gene causing increased ALAS2 activity and markedly increased erythrocyte protoporphyrin levels. Patients present with severe cutaneous photosensitivity and may develop liver dysfunction. XLP was originally reported as X-linked dominant with 100% penetrance in males and females. We characterized 11 heterozygous females from six unrelated XLP families and show markedly varying phenotypic and biochemical heterogeneity, reflecting the degree of X-chromosomal inactivation of the mutant gene. ALAS2 sequencing identified the specific mutation and confirmed heterozygosity among the females. Clinical history, plasma and erythrocyte protoporphyrin levels were determined. Methylation assays of the androgen receptor and zinc-finger MYM type 3 short tandem repeat polymorphisms estimated each heterozygotes X-chromosomal inactivation pattern. Heterozygotes with equal or increased skewing, favoring expression of the wild-type allele had no clinical symptoms and only slightly increased erythrocyte protoporphyrin concentrations and/or frequency of protoporphyrin-containing peripheral blood fluorocytes. When the wild-type allele was preferentially inactivated, heterozygous females manifested the disease phenotype and had both higher erythrocyte protoporphyrin levels and circulating fluorocytes. These findings confirm that the previous dominant classification of XLP is inappropriate and genetically misleading, as the disorder is more appropriately designated XLP.

Beta-thalassaemia intermedia: evaluation of endocrine and bone complications.

OBJECTIVE: Data about endocrine and bone disease in nontransfusion-dependent thalassaemia (NTDT) is scanty. The aim of our study was to evaluate these complications in ß-TI adult patients. METHODS: We studied retrospectively 70 ß-TI patients with mean followup of 20 years. Data recorded included age, gender, haemoglobin and ferritin levels, biochemical and endocrine tests, liver iron concentration (LIC) from T2*, transfusion regimen, iron chelation, hydroxyurea, splenectomy, and bone mineralization by dual X-ray absorptiometry. RESULTS: Thirty-seven (53%) males and 33 (47%) females were studied, with mean age 41 ± 12 years, mean haemoglobin 9.2 ± 1.5 g/dL, median ferritin 537 (range 14-4893), and mean LIC 7.6 ± 6.4 mg Fe/g dw. Thirty-three patients (47%) had been transfused, occasionally (24/33; 73%) or regularly (9/33; 27%); 37/70 (53%) had never been transfused; 34/70 patients had been splenectomized (49%); 39 (56%) were on chelation therapy; and 11 (16%) were on hydroxyurea. Endocrinopathies were found in 15 patients (21%): 10 hypothyroidism, 3 hypogonadism, 2 impaired glucose tolerance (IGT), and one diabetes. Bone disease was observed in 53/70 (76%) patients, osteoporosis in 26/53 (49%), and osteopenia in 27/53 (51%). DISCUSSION AND CONCLUSIONS: Bone disease was found in most patients in our study, while endocrinopathies were highly uncommon, especially hypogonadism. We speculate that low iron burden may protect against endocrinopathy development.

Heparin induced thrombocytopenia: pathogenetic, clinical, diagnostic and therapeutic aspects.

Heparin induced thrombocytopenia (HIT) in addition to bleeding complications are the most serious and dangerous side effects of heparin treatment. HIT remains the most common antibody-mediated, drug-induced thrombocytopenic disorder and a leading cause of morbidity and mortality. Two types of HIT are described: Type I is a transitory, slight and asymptomatic reduction of platelet count occurring during 1-2 days of therapy. HIT type II, which has an immunologic origin, is characterized by a thrombocytopenia that generally onset after the fifth day of therapy. Despite thrombocytopenia, haemorrhagic complications are very rare and HIT type II is characterized by thromboembolic complications consisting in venous and arterial thrombosis. The aim of this paper is to review new aspects of epidemiology, pathophysiology, clinical features, diagnosis and therapy of HIT type II. There is increasing evidence that platelet factor 4 (PF4) displaced from endothelial cells, heparan sulphate or directly from the platelets, binds to heparin molecule to form an immunogenic complex. The anti-heparin/PF4 IgG immune-complexes activates platelets through binding with the Fcgamma RIIa (CD32) receptor inducing endothelial lesions with thrombocytopenia and thrombosis. Cytokines are generated during this process and inflammation could play an additional role in the pathogenesis of thromboembolic manifestations. The onset of HIT type II is independent from dosage, schedule, and route of administration of heparin. A platelet count must be carried out prior to heparin therapy. Starting from the fourth day, platelet count must be carried out daily or every two days for at least 20 days of any heparin therapy regardless of the route of the drug administration. Patients undergoing orthopaedic or cardiac surgery are at higher risk for HIT type II. The diagnosis of HIT type II should be formulated on basis of clinical criteria and confirmed by in vitro demonstration of heparin-dependent antibodies detected by functional and antigen methods. However, the introduction of sensitive ELISA tests to measure anti-heparin/PF4 antibodies has showed the immuno-conversion in an higher number of patients treated with heparin such as the incidence of anti-heparin/PF4 exceeds the incidence of the disease. If HIT type II is likely, heparin must be immediately discontinued, even in absence of certain diagnosis of HIT type II, and an alternative anticoagulant therapy must be started followed by oral dicumaroids, preferably after resolution of thrombocytopenia. Further studies are required in order to elucidate the pathogenetic mechanism of thrombosis and its relation with inflammation; on the other hand large clinical trials are needed to confirm the best therapeutic strategies for HIT Type II.

Membrane-bound iron contributes to oxidative damage of beta-thalassaemia intermedia erythrocytes.

Iron-dependent oxidative reactions in beta-thalassaemic erythrocyte membranes are involved in premature cell removal and anaemia. We studied 22 beta-thalassaemia intermedia patients (i) to assess if membrane iron accumulation influences the oxidative damage to thalassaemic cells, and (ii) to see whether the mechanisms of haemoglobin destabilization described in vitro have indicators in circulating erythrocytes. Serum non-transferrin-bound iron as potentially toxic iron for erythrocytes was also evaluated. Membrane-bound free iron significantly correlated to bound haemichromes, suggesting a causal relation, but was poorly related to serum non-transferrin iron, which seems to contribute little to damage from outside the cells. The spleen played an important role in the removal of cells with more membrane iron.

Oral isobutyramide therapy in patients with thalassemia intermedia: results of a phase II open study.

A pilot phase II open study on 12 patients with thalassemia intermedia (7 men, 5 women; age 31 +/- 2.0 years SE) treated with oral isobutyramide, a derivative of butyric acid (150 mg/kg body wt/day), was performed in order to evaluate the effect of this compound in stimulating hemoglobin F (HbF) production. No patient underwent blood transfusion in the 1-year time frame prior to the study. Nine patients were splenectomized. Safety was monitored by clinical and laboratory tests. Efficacy was assessed in terms of the non-alpha/alpha globin chain biosynthetic ratio and the percentage increase of HbF. The study design consisted of a screening phase, a treatment phase of 28 days, and a posttreatment follow-up of 28 days. All patients completed the study. Compliance to treatment was 100%. No drug-related adverse event was recorded. We observed little or no increase in the non-alpha/alpha ratio in the majority of patients. Six patients showed a percentage increase of HbF at the end of treatment and in 5 of those 6 further increases at the end of the follow-up period were observed. The change in percentage of HbF over time was close to significance both in the treatment period (P = 0. 06) and in the follow-up period (P = 0.08). These results indicate that butyrate derivatives can stimulate fetal hemoglobin in patients with intermediate thalassemia. Testing of the effects of different schedules of administration of isobutyramide will be required in order to determine the optimal use of this compound in the treatment of the beta-thalassemia syndromes.

Metabolic indicators of oxidative stress correlate with haemichrome attachment to membrane, band 3 aggregation and erythrophagocytosis in beta-thalassaemia intermedia.

Haematological data, genotype, transfusion requirements, metabolic indicators of oxidative stress (flux via hexose-monophosphate shunt (HMPS); steady state level of GSH and GSSG, NADPH and NADP; activity of anti-oxidant enzymes), parameters of membrane damage (aggregated band 3; membrane-bound haemichromes, autologous immunoglobulins (Igs) and C3 complement fragments) and erythrophagocytosis were measured in erythrocytes (RBC) of 15 beta-thalassaemia intermedia patients (nine splenectomized) with low, if any, transfusion requirements. Patients presented increased aggregated band 3, bound haemichromes, Igs and C3 complement fragments, and increased erythrophagocytosis. Bound haemichromes strongly correlated with aggregated band 3. Anti-band 3 Igs were predominantly associated with aggregated band 3. Erythrophagocytosis positively correlated with aggregated band 3, haemichromes and Igs, suggesting the involvement of haemichrome-induced band 3 aggregation in phagocytic removal of beta-thalassaemic RBC. Splenectomized patients showed higher degrees of membrane damage and phagocytosis, significantly higher numbers of circulating RBC precursors, and tendentially higher numbers of reticulocytes. Basal flux via HMPS was increased twofold, but HMPS stimulation by methylene blue was decreased, as was the glucose flux via HMPS. GSH was remarkably decreased, whereas NADPH was increased. Except for unchanged catalase and glutathione reductase, anti-oxidant enzymes had increased activity. Negative correlation between HMPS stimulation by methylene blue and bound haemichromes indicated that the ability to enhance HMPS may counteract haemichrome precipitation and limit consequent membrane damage leading to erythrophagocytosis.

Butyrate trials.

The aims of this study were to ascertain tolerability, safety and efficacy of oral isobutyramide (150 mg/kg bw/day) in stimulating fetal hemoglobin production in twelve thalassemia intermedia patients. Patients were treated for 28 days and followed for a further 28 days. Efficacy was monitored by non-alpha/alpha globin chain ratio and percentage of HbF. Five patients experienced increases of non-alpha/alpha ratio ranging between 5.3 and 100% at the end of treatment. Five patients show an increase of HbF ranging between 4.4 and 26%. Their HbF% continues to increase during follow-up period. The analysis of variance for HbF showed a time effect close to significance both in treatment period (p = 0.06) and in follow-up period (p = 0.08). Moreover, to evaluate a possible erythropoietic modification, serum Erythropoietin (sEpo) and serum Transferrin Receptor (sTfR) were evaluated. Serum Epo and sTfR levels were significantly increased during treatment (p < 0.05 vs baseline).

Plasmablastic lymphoma of the stomach. A case report.

We report the case of a 53 year-old woman with a gastric tumor showing morphological, phenotypical and molecular features of a plasmablastic lymphoma, a recently recognized subtype of diffuse large B-cell lymphoma. The tumor was composed of plasmablast-like cells, lacked CD45 and B-cell associated antigens, expressed the plasma cell-associated antigen CD38, and showed clonally rearranged IgH genes in the absence of bcl-2 and bcl-6 genes rearrangement.

The effect of dietary magnesium supplementation on the cellular abnormalities of erythrocytes in patients with beta thalassemia intermedia.

BACKGROUND AND OBJECTIVE: Reduced serum or erythrocyte Mg have been reported in human beta thalassemia. These deficiencies may play a role in the cellular abnormalities characteristic of this disorder. We have therefore studied the effect of dietary Mg supplementation in patients with beta thalassemia intermedia in order to establish whether it improves the abnormalities of thalassemic erythrocytes. DESIGN AND METHODS: Plasma and erythrocyte Mg were determined in 11 patients with b thalassemia intermedia, not requiring chronic transfusion therapy, and in 17 normal controls. Inclusion criteria included normal renal and liver function and performance status of 70% or greater. Seven patients were enrolled for the Mg supplementation study, after the appropriate informed consent was obtained. They were given a starting dose of 0.6 mEq/kg/day of magnesium pidolate, divided into two oral daily doses, for four weeks. In a 70-kg subject, a daily Mg dose of 42 mEq corresponds to 504 mg of Mg, with the daily Mg intake of normal subjects being 418 +/- 120 mg for males and 343 +/- 94 mg for females. After 28 days of treatment, five of the patients continued the protocol with a daily dosage increased to 1.2 mEq magnesium pidolate/kg/day, divided into two oral administrations, for an additional four weeks. RESULTS: In patients with untransfused beta thalassemia intermedia we found reduced erythrocyte Mg (in mmol/kg Hb, 6.12 +/- 1.5, n = 11 vs. 8.69 +/- 0.89, n = 17, respectively, p < 0.0001) and normal serum Mg. In the seven patients given oral Mg supplements, at Mg dosages of 0.6 mEq/kg/day we observed significant increases in erythrocyte Mg, and significant improvement in some of the characteristic abnormalities of beta that erythrocytes (increased Na-K pump, KCl cotransport, cell dehydration, increased osmotic resistance). These changes were maintained in the 5 patients who were treated with 1.2 mEq of Mg/kg/day. Follow-up studies showed a return to baseline conditions. There were no signs of Mg toxicity, with the only side effect being diarrhea, which was generally mild, but led to discontinuation for one patient after the first four weeks. INTERPRETATION AND CONCLUSIONS: These data indicate that dietary Mg supplementation improves some of the characteristic cellular function abnormalities of b thalassemia intermedia. The possible therapeutic value of this strategy should be further tested in these patients.

Low-grade MALT lymphoma involving multiple mucosal sites and bone marrow.

Mucosa-associated lymphoid tissue (MALT) lymphomas are indolent neoplasms which tend to remain localized for a long time before spreading. We describe here the case of a 36-year-old woman with a low-grade MALT lymphoma involving the lung, stomach, lingual tonsil, and bone marrow at the time of diagnosis. The clonal origin of the pulmonary and bone marrow neoplastic infiltrates was assessed by means of gene rearrangement analysis. All of the involved sites were infiltrated by centrocyte- and monocytoid-like cells expressing the B-cell-associated antigens CD19 and CD20 and showed IgM lambda chain restriction; no CD5, CD10, or CD43 expression was detectable. As the patient had a history of recurrent bronchitis, and computed tomography performed 3 years before the lymphoma diagnosis had already revealed a lesion of the left lung, we conclude that the present case probably represents a pulmonary low-grade MALT lymphoma characterized by an early and unusual involvement of different mucosal sites and bone marrow.

Serum erythropoietin and circulating transferrin receptor in thalassemia intermedia patients with heterogeneous genotypes.

BACKGROUND: Thalassemia intermedia patients usually do not require blood transfusions; however, all show variable degrees of erythropoietic marrow expansion to compensate for more or less marked anemia, and this represents the major cause of complications in untransfused individuals. MATERIALS AND METHODS: To assess the degree of erythropoietic expansion in thalassemia intermedia, serum erythropoietin (sEpo) and serum transferrin receptor (sTfr) were determined in thirty Italian patient's characterized by their beta-globin genotype. RESULTS: Six patients showed inappropriately low sEpo levels (O/P ratio < 0.85). Even excluding these cases, no clear relationship was observed between Hb levels and sEpo or sTfr. Two groups of patients were compared: the first with low HbF (< 40%) that included the majority of beta(+) genotypes, and the second with high HbF (> 40%) that contained a prevalence of beta(0) genotypes. Hb levels were similar in the two groups: 8.09 +/- 1.15 g/dL in low HbF and 8.82 +/- 1.28 g/dL in high HbF patients. Mean sEpo was 112 +/- 78.02 mU/mL (O/P ratio = 0.98 +/- 0.22) in the first and 246.62 +/- 184.30 mU/mL (O/P ratio = 1.25 +/- 0.30) in the second group, with a statistically significant difference, as expected, because of HbF oxygen hyperaffinity. No significant difference in sTfr levels was observed, indicating a comparable erythropoietic response in the two groups. CONCLUSIONS: The relationships between anemia, HbF and total erythropoiesis in thalassemia are more complex than expected. Further studies of subjects with high HbF and benign conditions, such as HPFH, could be of help in clarifying this point, to the aim of safely increasing HbF in thalassemia intermedia.

[Transplantation of embryonic brain tissue into newborn rats after unilateral bulbectomy]. / Transplantácie embryonálneho mozgového tkaniva neonatálnym krysám po unilaterárnej bulbektómii.

Embryonic tissue (E14-E16) of the olfactory bulb and substantia nigra resp. was transplanted to neonatal rats into cavities formed by partial or total unilateral bulbectomy. After 40 days of survival the animals were perfused with Bouin's fluid and processed for histological examination. Complete series of 10 microns horizontal or sagittal sections were stained with Gill haematoxylin and Bodian's silver method. To demonstrate the olfactory protein immunohistochemical staining with peroxidase-antiperoxidase was used. After bulbectomy degeneration of the axons of the olfactory cells occur, which subsequently regenerate, penetrate into the transplant and form typical glomerular structures in the latter. By immunohistochemical staining in both types of transplants olfactory structures were reliably differentiated from non-olfactory elements.

Neuronal changes in the forebrain of mice following penetration by regenerating olfactory axons.

Following total, unilateral bulbectomy in neonatal mice, the olfactory sensory axons regrow from a reconstituted population of sensory neurons, cross the lamina cribrosa, and invade the spared forebrain that has leaned forward toward the anteroventral wall of the cranial cavity. The sensory axons invade several regions of the spared forebrain, at times penetrating deeply into the brain parenchyma. These axons terminate in characteristic globose structures resembling the glomeruli of the olfactory bulb. However, they can be distinguished from the latter by the absence of periglomerular cells. These ectopic glomerular structures are formed by the commingling of the olfactory axon terminals and the dendrites of brain neurons that lie in their proximity. Previously we have established that synaptic contacts occur between the sensory axon terminals and the dendrites of the brain neurons. Our present study describes large neurons, resembling mitral cells, that expand their dendrites into the intracerebral glomeruli. These neurons are recognized by virtue of their relatively large diameter, their selective stainability with silver methods, and the unorthodox arrangement of their dendrites in comparison with the neurons of the region. Their appearance is contingent upon the presence of ectopic glomeruli. The possibility is discussed that the large argyrophilic neurons may be derived from developing neuronal elements of the brain.

Neonatally bulbectomized rats with new olfactory-neocortical connections are anosmic.

Rats with one olfactory bulb removed when neonates and the second bulb removed when adults were tested on tone-light discrimination and odor detection tasks. On the neonatally operated side reconstituted olfactory receptor cell axons penetrated the frontal neocortex or portions of the anterior olfactory nucleus, and formed glomerular-like structures. On the adult operated side there was extensive scar formation which prevented in-growing sensory axons from contacting the brain. All experimental animals acquired the tone-light discrimination but failed to show any evidence of odor detection. These results indicate that reconstituted olfactory projections which terminate in the frontal neocortex or anterior olfactory nucleus do not support olfaction.