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.

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.

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.

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.

Immunocytochemistry of the olfactory marker protein.

The olfactory marker protein has been localized, by means of immunohistochemical techniques in the primary olfactory neurons of mice. The olfactory marker protein is not present in the staminal cells of the olfactory neuroepithelium, and the protein may be regarded as indicative of the functional stage of the neurons. Our data indicate that the olfactory marker protein is present in the synaptic terminals of the olfactory neurons at the level of the olfactory bulb glomeruli. The postsynaptic profiles of both mitral and periglomerular cells are negative.

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).

Experimental studies on the olfactory marker protein. III. The olfactory marker protein in the olfactory neuroepithelium lacking connections with the forebrain.

Total unilateral bulbectomy induces degeneration of the mature olfactory neurons and disappearance of the olfactory marker protein from the primary sensory pathway. Owing to the presence of a neurogenetic matrix in the neuroepithelium, reconstitution of a new population of neuronal elements occurs. In this experiment, connections of the regrown olfactory axons with the spared forebrain are barred by the formation of scar tissue. In spite of the absence of a target, new neurons differentiate and produce olfactory marker protein.

Experimental studies on the olfactory marker protein. II. Appearance of the olfactory marker protein during differentiation of the olfactory sensory neurons of mouse: an immunohistochemical and autoradiographic study.

The time interval between the incorporation of [3H]thymidine and the appearance of olfactory marker protein (OMP) in autoradiographically labeled neurons which have differentiated from stem cells, has been determined by autoradiographic and immunohistochemical techniques. The first [3H]thymidine-labeled, OMP-containing elements have been observed 7 days after administration of the radioactive thymidine. This result allows some speculation on the potential function of the olfactory marker protein.

Experimental studies on the olfactory marker protein. I. Presence of the olfactory marker protein in tufted and mitral cells.

Partial, unilateral olfactory nerve section was performed in mice, and the behavior of the olfactory marker protein (OMP) studied, after this experimental manipulation, with the peroxidase--antiperoxidase method. The protein, which in normal mice is present only in mature olfactory sensory neurons, after unilateral lesion of the fila olfactoria was observed in mitral and tufted cells of both olfactory bulbs. Positive elements, present at 5 days postoperative, increased in number up to 30 days and some could still be detected at 60 days. The functional implications of this finding are briefly discussed.

Reinnervation of the olfactory bulb after section of the olfactory nerve in monkey (Saimiri sciureus).

Section of the fila olfactoria in squirrel monkey, a non-human primate, induces rapid degeneration of the sensory axon terminals in the olfactory bulb glomeruli. A population of axons, from newly formed sensory neurons in the olfactory neuroepithelium, regrow, passes the lamina cribrosa and, upon reaching the olfactory bulb, reinnervates the glomeruli. A new set of synaptic contacts is reformed between the sensory terminals and the post-synaptic dendritic processes of the glomeruli. Our observations indicate that this portion of the CNS of a non-human primate can be reinnervated after deafferentiation, and that active synaptogenesis occurs.

Neurogenesis of sensory neurons in the primate olfactory system after section of the fila olfactoria.

Axotomy of the olfactory sensory neurons in the adult primate squirrel monkey induces retrograde degeneration of the perikarya in the nasal neuroepithelium. The process of neuronal degeneration is rapid and by the 10th day the olfactory neuroepithelium is deprived of all mature neurons. Basal cells, supporting cells and the Bowman's glands are unaffected by the surgical procedure. The degeneration of the neurons is followed by intense mitotic activity of the basal cells of the neuroepithelium. At 30 days survival several young, mature neurons are present again in the neuroepithelium. At 60--90 survival days the neuroepithelium reacquires a population of neurons similar to controls. The persistence of neurogenesis and the replacement of experimentally degenerated neurons in an adult, non-human primate is briefly discussed.

Denervation in the primary olfactory pathway of mice. IV. Biochemical and morphological evidence for neuronal replacement following nerve section.

Unilateral olfactory nerve section was performed in the mouse. Three biochemical markers of the olfactory chemoreceptor neurons: carnosine, carnosine synthetase activity and the olfactory marker protein, were measured in the olfactory bulb and epithelium. Parallel observations were made by light microscopy as well as at the ultrastructural level. The specific biochemical markers decrease rapidly in both bulb and epithelium and reach a minimum by the end of the first week after surgery. They then slowly return to 80% of control values by one month. Carnosinase activity in epithelium was essentially unaffected. These biochemical observations coincide temporally with the onset of degenerative changes seen morphologically, in both the bulb and epithelium. The degenerative changes persist for up to two weeks in the bulb and for about one week in the epithelium. At this time basal cell division and differentiation begins in the epithelium with subsequent regrowth of olfactory axons into the glomerular layer of the olfactory bulb with ther reappearance of olfactory axon terminals. The temporal coincidence of these biochemical and morphological observations suggests they are manifestations of the same process, and is consistent with the idea that the olfactory chemoreceptor neurons are perhaps unique in being able to be replaced from undifferentiated stem cells.

Intraocular transplants of olfactory neuroepithelium in rat.

Olfactory neuroepithelium of neonatal rat pups has been transplanted in the anterior chamber of the eye of adult rats. Structural and ultrastructural observations at 5, 10, 30, 50, 90 and 120 days show that mature neurons degenerate rapidly in the expiant (before 5 days) while the basal elements proliferate and produce a new population of young neurons between 1 and 10 days. At longer survivals (30-120 days) it is seen that the neurons acquire morphological maturity, and positivity to the olfactory marker protein, as demonstrated by immunohistochemical methods. Our observations show that new neurons can be generated in the transplanted neuroepithelium and that their generation and maturation occurs in the absence of connections with a brain target.

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.

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.