Marta Miorin

Chronic lymphocytic leukemia B cells contain anomalous Lyn tyrosine kinase, a putative contribution to defective apoptosis

B cell chronic lymphocytic leukemia (B-CLL) is a neoplastic disorder characterized by accumulation of B lymphocytes due to uncontrolled growth and resistance to apoptosis. Analysis of B cells freshly isolated from 40 patients with chronic lymphocytic leukemia demonstrated that the Src kinase Lyn, the switch molecule that couples the B cell receptor to downstream signaling, displays anomalous properties. Lyn is remarkably overexpressed at the protein level in leukemic cells as compared with normal B lymphocytes, with a substantial aliquot of the kinase anomalously present in the cytosol. Whereas in normal B lymphocytes Lyn activation is dependent on B cell-receptor stimulation, in resting malignant cells, the constitutive activity of the kinase accounts for high basal protein tyrosine phosphorylation and low responsiveness to IgM ligation. Addition of the Lyn inhibitors PP2 and SU6656 to leukemic cell cultures restores cell apoptosis, and treatment of malignant cells with drugs that induce cell apoptosis decreases both activity and amount of the tyrosine kinase. These findings suggest a direct correlation between high basal Lyn activity and defects in the induction of apoptosis in leukemic cells. They also support a critical role for Lyn in B-CLL pathogenesis and identify this tyrosine kinase as a potential therapeutic target.

Cardiac and respiratory involvement in advanced stage Duchenne muscular dystrophy

This study aimed to describe myocardial involvement, respiratory impairment and pulmonary blood flow abnormalities in advanced-stage Duchenne muscular dystrophy (DMD). Twenty-one wheelchair-bound patients, aged from 10 to 24 yr, underwent electrocardiographic and echocardiographic examination, conventional spirometry, diurnal arterial blood gas analysis, and nocturnal polysomnography (SaO2 monitoring). Diagnosis was confirmed by neurological examination, dystrophin analysis at protein and DNA level. Patients were classified into two groups: group A normoxemic (14 cases) and group B with nocturnal hypoxemia (seven cases). Group A was further split into two subgroups, one without, and one with, left ventricular dilation (A1 = nine patients, end diastolic volume (EDV) = 51 ml m-2, ejection fraction (EF) = 56 per cent; A2 = five patients, EDV = 112 ml m-2, EF = 32 per cent; P < 0.05). Left ventricular regional wall motion abnormalities were found in 55, 40, and 43 per cent of groups A1, A2, and B patients respectively. Analysis of pulsed Doppler pulmonary data highlighted a significant reduction in corrected time to peak velocity in group B patients, when compared with control, A1, and A2 groups respectively. In group A, we observed a direct correlation between ejection fraction and corrected time-to-peak velocity. Two patterns of cardiac involvement may be recognized in advanced-stage DMD: left ventricular wall motion abnormalities and dilated cardiomyopathy. Doppler data which could suggest pulmonary hypertension may be observed in patients with dilated cardiomyopathy, and in patients with nocturnal hypoxemia. Therefore, in the management of advanced-stage DMD, a careful diagnosis of the heart-lung relationship should be performed, and both conventional treatment of heart failure and ventilatory therapy are necessary to improve the quality of life and survival in these patients.

Genetic epidemiology of congenital muscular dystrophy in a sample from north-east Italy

Congenital muscular dystrophy (CMD) is a heterogenous disease with autosomic recessive transmission. In an epidemiological study in four provinces of Veneto (region of 2586830 inhabitants in north-east Italy), the recorded incidence rate for the period 1979–1993 was 4.65 x 10−5; the prevalence rate in the year 1993 was 6.8 × 10−6. The incidence and the prevalence rates that we have obtained during the course of our investigation represent the first estimates for CMD in Europe and show that this myopathy is among the most frequent neuromuscular diseases with autosomic recessive transmission.

Facioscapulohumeral muscular dystrophy: epidemiological and molecular study in a north-east Italian population sample

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disease associated with a partial deletion on chromosome 4q35. Few relevant investigations have been reported on its epidemiology and were essentially based on clinical diagnosis, having been performed before recognition of the molecular mutation. We report an epidemiological survey on FSHD patients, in which the diagnosis was obtained by combined clinical and molecular evaluation. The survey concerned the north‐east Italian province of Padova, an area of 871,190 inhabitants (1 January 2004). We identified 40 patients affected by FSHD based on clinical diagnosis. In 33 of them, the EcoRI fragment size in the 4q35 region ranged from 14 to 35 kb. Four other patients belonging to the same family harbored a 38‐kb fragment. In these four cases, the relationship between the borderline deletion with the mild FSHD phenotype was corroborated by additional haplotype reconstruction and segregation analysis. Interestingly, the same mild facial‐sparing clinical pattern was apparent only in one other patient with an EcoRI fragment of 32 kb, suggesting that this unusual FSHD phenotype may be due to very small 4q35 deletions. On the whole, estimating a prevalence rate of 44 × 10−6, our survey confirmed FSHD as one of the most frequent neuromuscular disorders in Western populations.

Multiple myeloma plasma cells show different chemokine receptor profiles at sites of disease activity

Chemokines and their receptors play a pivotal role in the regulation of B‐lymphocyte trafficking. This study was aimed at investigating the pattern of chemokine receptor expression, including CCR1 to CCR3, CCR5 to CCR7, CXCR1 to CXCR5, and the migration ability of multiple myeloma (MM) plasma cells (PC). PC were recovered from the bone marrow (BM) of 29 MM patients, extramedullary sites of 10 patients and the BM of five controls. Flow cytometry analysis showed that the receptors mainly expressed on malignant BM PC were represented by CXCR4 (70% of patients), CCR1 (25%), CCR2 (25%), CCR5 (17%) and CXCR3 (20%), while other receptors were commonly lacking. The analysis performed on extramedullary (peripheral blood and pleural effusion) malignant PC demonstrated that the most represented receptors were CXCR4 (100%), CCR2 (66%) and CXCR1 (60%). The migratory capability of malignant PC at resting conditions identified three groups of patients with different migration (low, intermediate and high). As CXCR4 was the relevant chemokine receptor expressed by MM PC, its ligand CXCL12 induced their migration. These data suggest that malignant PC from MM display different chemokine receptor profiles and that CXCR4 is fully functional and might play a role in the spreading of the disease.

Epithelial CXCR3-B regulates chemokines bioavailability in normal, but not in Sjögren's syndrome, salivary glands

Expression of CXCR3-targeting chemokines have been demonstrated in several diseases, suggesting a critical role for CXCR3 in recruiting activated T cells to sites of immune-mediated inflammation. Sjögren’s syndrome (SS) is an autoimmune disease characterized by a mononuclear cell infiltrate of activated T cells around the duct in the salivary gland. Analysis of minor salivary gland biopsy specimens from 20 healthy subjects and 18 patients with primary SS demonstrated that CXCR3, in particular, the B form of this receptor, is constitutively expressed by human salivary gland epithelial cells. Salivary gland epithelial cell cultures demonstrated that CXCR3 participate in removing relevant amount of agonists from the supernatant of exposed cells without mediating calcium flux or chemotaxis while retaining the ability to undergo internalization. Although in normal salivary gland epithelial cells, CXCR3 behaves as a chemokine-scavenging receptor, its role in SS cells is functionally impaired. The impairment of this scavenging function might favor chemotaxis, leading to heightened immigration of CXCR3-positive T lymphocytes. These findings suggest that epithelial CXCR3 may be involved in postsecretion regulation of chemokine bioavailability. They also support a critical role for CXCR3 in the pathogenesis of SS and identify its agonists as potential therapeutic targets.

Expression and role of CCR6/CCL20 chemokine axis in pulmonary sarcoidosis

We have shown previously that the chemokine receptors CXCR3 and CXCR6 are coexpressed by Th1 cells infiltrating the lung and the granuloma of patients with sarcoidosis. In this study, we evaluated the role of CCL20/CCR6 interaction in the pathogenesis of acute and chronic pulmonary sarcoidosis. By flow cytometry and molecular analyses, we have demonstrated that Th1 cells isolated from the bronchoalveolar lavage (BAL) of patients with sarcoidosis and T cell alveolitis are equipped with CCR6. Furthermore, CCR6+ T cells coexpressed the chemokine receptors CXCR3 and CXCR6. Immunohistochemical analysis of lung specimens has shown that CCR6+ T cells infiltrate lung interstitium and surround the central core of the granuloma. It is interesting that CCR6 was never detected on the alveolar macrophage (AM) surface, and it is observed in the cytoplasm of AMs from patients with sarcoidosis and alveolitis. The CCR6 ligand CCL20 was expressed by macrophages, multinucleated giant cells, and epithelioid cells infiltrating the granuloma. Furthermore, detectable levels of CCL20 protein are seen in the BAL fluid components of patients with active sarcoidosis, and sarcoid AMs release the CCR6 ligand in vitro. From a functional point of view, sarcoid Th1 cells were able to respond to CXCL10, CXCL16, and CCL20 in migratory assays. In vitro kinetic studies demonstrated that CCR6 is induced rapidly by IL‐2, IL‐18, and IFN‐γ. In conclusion, T cells expressing CCR6, CXCR3, and CXCR6 act coordinately with respective ligands and Th1 inflammatory cytokines in the alveolitic/granuloma phases of the disease.

Lack of expression of inhibitory KIR3DL1 receptor in patients with natural killer cell-type lymphoproliferative disease of granular lymphocytes

Background Natural killer cell-type lymphoproliferative disease of granular lymphocytes is a disorder characterized by chronic proliferation of CD3−CD16+ granular lymphocytes. By flow cytometry analysis, we previously demonstrated a dysregulation in killer immunoglobulin-like receptor (KIR) expression in natural killer cells from patients with this lymphoproliferative disease, the activating KIR receptors being mostly expressed. We also found that patients with natural killer cell-type lymphoproliferative disease of granular lymphocytes usually had KIR genotypes characterized by multiple activating KIR genes. Design and Methods We investigated the mRNA levels of the KIR3DL1 inhibitory and the related KIR3DS1 activating receptors in 15 patients with natural killer cell-type lymphoproliferative disease of granular lymphocytes and in ten controls. These genes are usually expressed when present in the genome of the Caucasian population. Results We demonstrated the complete lack of KIR3DL1 expression in most of the patients analyzed, with the receptor being expressed in 13% of patients compared to in 90% of controls (P<0.01). Interestingly, studies of the methylation patterns of KIR3DL1 promoter showed a significantly higher methylation status (0.76 ± 0.12 SD) in patients than in healthy subjects (0.49±0.10 SD, P<0.01). The levels of expression of DNA methyl transferases, which are the enzymes responsible for DNA methylation, did not differ between patients and controls. Conclusions In this study we showed, for the first time, a consistent down-regulation of the inhibitory KIR3DL1 signal due to marked methylation of its promoter, thus suggesting that together with the increased expression of activating receptors, the lack of the inhibitory signal could also play a role in the pathogenesis of natural killer cell-type lymphoproliferative disease of granular lymphocytes.

Prevalent cardiac involvement in dystrophin Becker type mutation.

Myocardial involvement is frequently present in Xp21-linked muscular dystrophy, due to a lack of dystrophin in cardiac fibres. We describe a 41-yr-old man affected by dilated cardiomyopathy with sporadic episodes of myoglobinuria induced by effort and increased levels of serum creatine kinase. Very mild signs of skeletal myopathy were clinically evident. His mother was affected by an indefinite cardiopathy and suddenly died when she was 36 yr old. Muscle biopsy of the patient showed a dystrophic process. Dystrophin analysis together with a genetic DMD locus study led us to diagnose Becker type muscular dystrophy, with truncated dystrophin and a gene deletion extending from exon 45 to 48. Prevalent cardiac involvement in a Becker type mutation of the dystrophin gene further confirms clinical variability of dystrophinopathies.

A study on duplications of the dystrophin gene : evidence of a geographical difference in the distribution of breakpoints by intron

Starting from a group of 265 Italian patients affected with Duchenne or Becker muscular dystrophy a screening for duplications in the dystrophin gene was performed on 112 cases in which no deletions had previously been detected. The 21 intragenic duplications detected account for 7.9% of the total. Among these, one duplication including exons from 3 to 43 is the largest reported so far. Data from this study were combined with those from the literature and breakpoint distribution by intron was analysed. In general breakpoints occur mostly in the proximal third of the gene, in particular in intron 7. However, both the frequency of duplications and the distribution of breakpoints by intron are different in the Japanese sample compared with the other groups of patients. The role of geographical differentiation of intron sequences by genetic drift and of transposon-like sequences in explaining these differences is discussed.