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Biomedical subjects

Francesca Gualandi

Publications and source records attributed to Francesca Gualandi.

16 recordsLinked to original sources
PubMed2007

Mitochondrial dysfunction in the pathogenesis of Ullrich congenital muscular dystrophy and prospective therapy with cyclosporins.

Ullrich congenital muscular dystrophy is a severe genetically and clinically heterogeneous muscle disorder linked to collagen VI deficiency. The pathogenesis of the disease is unknown. To assess the potential role of mitochondrial dysfunction in the onset of muscle fiber death in this form of dystrophy, we studied biopsies and myoblast cultures obtained from patients with different genetic defects of collagen VI and variable clinical presentations of the disease. We identified a latent mitochondrial dysfunction in myoblasts from patients with Ullrich congenital muscular dystrophy that matched an increased occurrence of spontaneous apoptosis. Unlike those in myoblasts from healthy donors, mitochondria in cells from patients depolarized upon addition of oligomycin and displayed ultrastructural alterations that were worsened by treatment with oligomycin. The increased apoptosis, the ultrastructural defects, and the anomalous response to oligomycin could be normalized by Ca(2+) chelators, by plating cells on collagen VI, and by treatment with cyclosporin A or with the specific cyclophilin inhibitor methylAla(3)ethylVal(4)-cyclosporin, which does not affect calcineurin activity. Here we demonstrate that mitochondrial dysfunction plays an important role in muscle cell wasting in Ullrich congenital muscular dystrophy. This study represents an essential step toward a pharmacological therapy of Ullrich congenital muscular dystrophy with cyclosporin A and methylAla(3)ethylVal(4) cyclosporin.

Adult↗
PubMed2006

Boost of CD34+-selected peripheral blood cells without further conditioning in patients with poor graft function following allogeneic stem cell transplantation.

BACKGROUND AND OBJECTIVES: A proportion of patients develop poor graft function (PGF) following an allogeneic hemopoietic stem cell transplant (HSCT). It is uncertain whether a boost of donor marrow or blood cells is beneficial in terms of trilineage recovery and non-relapse-related mortality (NRM). DESIGN AND METHODS: The aim of this study was to compare outcomes in patients with PGF and full donor chimerism following an allogeneic HSCT who did or did not receive a boost of donor stem cells. The study included patients with primary PGF--i.e. those failing to achieve sustained graft function- and secondary PGF--i.e. those developing PGF after complete hematologic recovery. We studied 54 patients with PGF: 20 patients received no further donor cell infusion (group A), 14 received a boost of unmanipulated marrow or blood cells from the original donor, without further conditioning (group B), and 20 received donor cells after CD34 selection without conditioning (group C). The three groups were comparable for disease phase, patients' age, donor type, primary or secondary PGF, full donor chimerism and duration of PGF. RESULTS: Trilineage recovery was seen in 40%, 36% and 75% of the patients in, respectively, groups A, B and C (p=0.02). In multivariate Cox analysis trilineage recovery was more frequent in patients with secondary PGF (RR of complete recovery 2.82, p=0.01) and in patients receiving CD34+-selected cells (RR of complete recovery 3.0; p=0.007). There was no effect of donor type on hematologic recovery. The rate of NRM was 55%, 64%, 20% in groups A, B and C, respectively (p=0.06) and was highly correlated with trilineage recovery (RR 0.36, p<0.0001). PGF was the primary cause of death in 30%, 21% and 10% of the patients in the three groups, graft-versus-host disease (GVHD) in 5%, 36%, and 10%. INTERPRETATIONS AND CONCLUSIONS: In patients with poor graft function (a) a boost of CD34+-selected peripheral blood donor cells is associated with a high chance of trilineage recovery and a low risk of acute GVHD; (b) a boost of unmanipulated donor cells does not seem to offer a survival advantage over no infusion of cells; and (c) NRM is lower when using peripheral blood cells for the boost. These data may be useful when discussing second stem cell donations for patients with poor graft function.

Adolescent↗
PubMed2005

GJB2 mutations and degree of hearing loss: a multicenter study.

Hearing impairment (HI) affects 1 in 650 newborns, which makes it the most common congenital sensory impairment. Despite extraordinary genetic heterogeneity, mutations in one gene, GJB2, which encodes the connexin 26 protein and is involved in inner ear homeostasis, are found in up to 50% of patients with autosomal recessive nonsyndromic hearing loss. Because of the high frequency of GJB2 mutations, mutation analysis of this gene is widely available as a diagnostic test. In this study, we assessed the association between genotype and degree of hearing loss in persons with HI and biallelic GJB2 mutations. We performed cross-sectional analyses of GJB2 genotype and audiometric data from 1,531 persons, from 16 different countries, with autosomal recessive, mild-to-profound nonsyndromic HI. The median age of all participants was 8 years; 90% of persons were within the age range of 0-26 years. Of the 83 different mutations identified, 47 were classified as nontruncating, and 36 as truncating. A total of 153 different genotypes were found, of which 56 were homozygous truncating (T/T), 30 were homozygous nontruncating (NT/NT), and 67 were compound heterozygous truncating/nontruncating (T/NT). The degree of HI associated with biallelic truncating mutations was significantly more severe than the HI associated with biallelic nontruncating mutations (P<.0001). The HI of 48 different genotypes was less severe than that of 35delG homozygotes. Several common mutations (M34T, V37I, and L90P) were associated with mild-to-moderate HI (median 25-40 dB). Two genotypes--35delG/R143W (median 105 dB) and 35delG/dela(GJB6-D13S1830) (median 108 dB)--had significantly more-severe HI than that of 35delG homozygotes.

Adolescent↗
PubMed2005

Human mesenchymal stem cells modulate B-cell functions.

Human mesenchymal stem cells (hMSCs) suppress T-cell and dendritic-cell function and represent a promising strategy for cell therapy of autoimmune diseases. Nevertheless, no information is currently available on the effects of hMSCs on B cells, which may have a large impact on the clinical use of these cells. hMSCs isolated from the bone marrow and B cells purified from the peripheral blood of healthy donors were cocultured with different B-cell tropic stimuli. B-cell proliferation was inhibited by hMSCs through an arrest in the G0/G1 phase of the cell cycle and not through the induction of apoptosis. A major mechanism of B-cell suppression was hMSC production of soluble factors, as indicated by transwell experiments. hMSCs inhibited B-cell differentiation because IgM, IgG, and IgA production was significantly impaired. CXCR4, CXCR5, and CCR7 B-cell expression, as well as chemotaxis to CXCL12, the CXCR4 ligand, and CXCL13, the CXCR5 ligand, were significantly down-regulated by hMSCs, suggesting that these cells affect chemotactic properties of B cells. B-cell costimulatory molecule expression and cytokine production were unaffected by hMSCs. These results further support the potential therapeutic use of hMSCs in immune-mediated disorders, including those in which B cells play a major role.

B-Lymphocytes↗
PubMed2005

Genomic and transcription studies as diagnostic tools for a prenatal detection of X-linked dilated cardiomyopathy due to a dystrophin gene mutation.

X-linked dilated cardiomyopathy (XLDC) represents a form of dystrophinopathy with exclusive heart involvement. Here a prenatal diagnosis of this condition performed in a family with XLDC is described. In this family, the causative mutation was a pure intronic deletion, which induces the splicing of a novel, aberrant, and out-of-frame exon into the dystrophin transcript. The genetic test was performed by defining both the DNA (villous) and the RNA (amniocyte) configuration. The prenatal diagnosis determined that the fetus was female, and a carrier of the genomic deletion. RNA analysis on cultured amniocytes revealed the presence of an easily detectable dystrophin transcript, as well as the co-existence of both the wild-type and the abnormal splicing profile. Our analysis represents the first report of a prenatal diagnosis in XLDC and also indicates the feasibility of dystrophin mutation detection on RNA from amniocytes. This finding suggests that the dystrophin splicing pattern in amniocytes and skeletal muscle is similar, and that, therefore, this approach could be used in other prenatal dystrophin mutation detection, where abnormal RNA splicing is thought to play a role, or for specific cases in which no mutations have been identified in the coding regions.

Cardiomyopathy, Dilated↗
PubMed2005

Autologous stem cell transplantation as rescue therapy in malignant forms of multiple sclerosis.

Malignant forms of multiple sclerosis (MS) represent a limited group of very aggressive demyelinating diseases, which rapidly progress to severe disability leading often to life-threatening conditions. On these clinical entities, currently available therapies for MS are not very effective. Recently, it has been demonstrated that intense immunosuppression followed by autologous stem cell transplantation (ASCT) can affect the clinical course of individuals with severe MS and completely abrogate the inflammatory activity detected by magnetic resonance imaging (MRI). We report on the treatment with intense immune ablation followed by ASCT of three patients with malignant MS whose clinical course indicated a dramatically poor prognosis. This procedure succeeded in halting the rapidly worsening course of disease. The effect was long lasting, as demonstrated by a sustained efficacy over a two-year period in two subjects and 12 months in the third case. In addition, a striking effect on inflammation-related MRI findings was obtained. These results support a role for intense immunosuppression followed by ASCT as treatment in rapidly evolving malignant MS cases unresponsive to conventional therapies.

Adolescent↗
PubMed2004

Autologous HSCT for severe progressive multiple sclerosis in a multicenter trial: impact on disease activity and quality of life.

Hematopoietic stem cell transplantation (HSCT) has been proposed for the treatment of severe multiple sclerosis (MS). In a phase 2 multicenter study we selected 19 non-primary progressive MS patients showing high disease activity on the basis of both brain magnetic resonance imaging (MRI) and sustained clinical deterioration despite conventional treatments. After stem cell mobilization with cyclophosphamide (CY) and filgrastim, patients were conditioned with BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea), cytosine arabinoside, etoposide, and melphalan (BEAM) followed by antithymocyte globulin (ATG). Unmanipulated peripheral blood stem cells (PBSCs) were then infused. No maintenance treatment was administered with a median follow-up of 36 months (range, 12 to 72 months). All patients showed clinical stabilization or improvement; 3 subsequently deteriorated, 1 beyond the baseline. No MRI active lesions were detected after the HSCT except in 1 patient who showed a new lesion at 4.5 years. Infections were limited and restricted to 3 months after HSCT. Health-related quality of life was assessed through the 54-item MS quality of life (MSQOL-54) questionnaire, showing a statistically significant improvement in both composite scores and in most of the individual domains. HSCT is able to induce a prolonged clinical stabilization in severe progressive MS patients, resulting in both sustained treatment-free periods and quality of life improvement.

Adult↗
PubMed2004

In vivo study of an aberrant dystrophin exon inclusion in X-linked dilated cardiomyopathy.

We previously identified a dystrophin intron 11 rearrangement in one family with X-linked dilated cardiomyopathy, causing incorporation of an aberrant exon in a tissue-specific manner. In this study we analyzed the role of different intron 11 genomic regions in the regulation of splicing by using mini-genes based approach, in C2C12 (skeletal muscle) myoblasts and myotubes, H9C2 cardiomyocytes, and HeLa cells. We show that inclusion of the aberrant exon is favored in H9C2 and differentiated C2C12 myotubes. These data suggest that the aberrant exon undergoes a differentiation-specific splicing. Unexpectedly, length of intron has a favorable effect in inclusion of the aberrant exon in the cardiac cells, suggesting that cardiac cells might be more prone to steric hindrance of trans-acting factors, involved in the inclusion of the aberrant exon. Furthermore, the cultured cell system used can serve as a suitable model to study human alternative splicing.

Alternative Splicing↗
PubMed2004

Reducing transplant-related mortality after allogeneic hematopoietic stem cell transplantation.

BACKGROUND AND OBJECTIVES: Transplant-related mortality (TRM) following allogeneic hematopoietic stem cell transplantation (HSCT) has been reported to be related to disease stage, duratiion of disease and type of donor. Furthermore, the outcome of transplants performed in the 1990s appears to be better than that of transplants done in the previous decade. The aims of this study were to determine whether these relationships still hold and whether the outcome of transplants is continuing to improve. DESIGN AND METHODS: We analyzed 1180 consecutive patients with leukemia (n=979) or other hematologic malignancies (n=201) undergoing HSCT in 4 time periods: before 1990, 1991-1995, 1996-2000, and 2001-2002. Changes during these eras include increasing patient age, more unrelated transplants, more patients with advanced disease, different graft-versus-host disease (GvHD) prophylaxis, and different management of infections. RESULTS: The actuarial 2-year transplant-related mortality (TRM) differed significantly between the transplant eras (p<0.001) with a significant interaction with disease phase (p=0.018). In patients in first remission (n=585) TRM was 34%, 25%, 21% and 6% in the four transplant eras. The reduction in TRM was less evident in patients in second remission (n=284) (37%, 35%, 30%, 25%) and absent in relapsed patients (n=311) (TRM=45%, 41%, 29%, 51%). This is a consequence of reductions in GvHD, infections and multiorgan failure among patients in remission but not among those who relapse. The actuarial 2-year survival has improved significantly in patients in first remission (54%, 66%, 72%, 78%) but not in those in second remission (38%, 46%, 52%,45%), or relapsed patients (31%, 25%, 36%, 21%). INTERPRETATION AND CONCLUSIONS: In conclusion, TRM has been significantly reduced in first remission patients, suggesting an allograft should be considered in this phase, when appropriate, without delay. There has been no improvement in survival for patients beyond first remission, due to persisting high risk of infections and organ toxicity, a possible consequence of prolonged pre-transplant chemotherapy and neutropenia.

Actuarial Analysis↗
PubMed2003

Genomic definition of a pure intronic dystrophin deletion responsible for an XLDC splicing mutation: in vitro mimicking and antisense modulation of the splicing abnormality.

We characterised a dystrophin gene rearrangement in a previously described family with X-linked dilated cardiomyopathy and we demonstrated that it represents an 11 kb deletion occurring within intron 11. This unique deletion joined two physiologically distant intronic regions and brought adjacent two cryptic splice sites, generating a 159 bp sequence recognised as a novel alternative exon and spliced into the dystrophin transcript. Comparative analysis of the intronic region involved in the breakpoint revealed the presence of a LINE1 element (L1P_MA2), containing a 5' unconventional region (L1M1_5). This region provides the 5' cryptic splice site utilised by the novel exon, includes part of the region spliced into the dystrophin transcript and contains two short GA rich regions compatible with splicing motifs. We performed an in vitro splicing assay by using a minigene containing the patient minimal genomic rearrangement and we reproduced the inclusion of the novel alternative exon seen in the patient tissues. Antisense splicing modulation targeting the 3' cryptic splice site succeeded in restoring the canonical splicing. This represents a novel intronic mutational mechanism affecting the dystrophin gene and generating a splicing pathology. The definition of this mechanism might open perspectives in unravelling splicing regulatory motifs and their involvement in human genetic diseases.

Alternative Splicing↗
PubMed2003

Progress in understanding GJB2-linked deafness.

Mutations in the GJB2 gene (encoding for Connexin 26 protein) represent a leading cause of genetic hearing impairment. Extensive epidemiological and molecular studies have been reported, describing GJB2 mutations type, frequency and distribution. Moreover, several aspects of GJB2 mutations pathogenic effects have been elucidated taking advantage of in vitro and in vivo experimental approaches. Progress through reported studies is reviewed, highlighting recent major achievements in this field. Attention is focused on different unresolved questions regarding GJB2 deafness pathogenesis and genotype-phenotype relationships. Clarification of these important clues will significantly increase our understanding of the molecular basis of hearing loss and will improve the effectiveness of diagnosis and counselling of this frequent disease.

Connexin 26↗
PubMed2002

Transplant-related mortality and long-term graft function are significantly influenced by cell dose in patients undergoing allogeneic marrow transplantation.

We have studied the impact of cell dose on short- and long-term graft function and outcome in 905 patients undergoing an unmanipulated allogeneic bone marrow transplantation (BMT) from an HLA-identical sibling (n = 735), a one-antigen mismatched related donor (n = 35), or a matched unrelated donor (n = 135). Median number of nucleated cells infused was 3.4 x 10(8)/kg (25th percentile 2.4 x 10(8)/kg, 75th percentile 5 x 10(8)/kg). Patients were stratified according to cells infused in 3 groups: < or = 2.4 x 10(8)/kg (n = 247; low dose); >2.4 x 10(8)/kg and < or = 5 x 10(8)/kg (n = 452; intermediate dose); and >5 x 10(8)/kg (n = 206; high dose). Patients receiving high cell dose had significantly higher platelet counts on days +20, +50, +100, +180, and +365 after BMT (P <.01) and higher white blood cell counts on days +50, +100, and +180 after BMT (P <.01) as compared with other patients. The actuarial 5-year transplant-related mortality (TRM) was 41% versus 36% versus 28% (P =.01); overall survival was 45% versus 51% versus 56% (P =.0008); and disease-free survival was 41% versus 42% versus 48%, respectively, (P =.04) in patients receiving low, intermediate, or high cell dose. The cell dose effect was more pronounced in patients older than 30 years of age, with advanced disease, with chronic myeloid leukemia, and with alternative donors. In multivariate Cox analysis on TRM, cell dose was a significant predictor (P =.002; relative risk 0.6) together with donor type (P =.0001), year of transplantation (P =.0001), conditioning regimen (P =.02), and recipient age (P =.02). In conclusion, transplantation of high marrow cell dose is associated with reduced transplant mortality and improved survival and results in improved graft function both short and long term.

Adolescent↗
PubMed2002

Aspergillus galactomannan antigen in the cerebrospinal fluid of bone marrow transplant recipients with probable cerebral aspergillosis.

The Aspergillus galactomannan test was performed on cerebrospinal fluid and serum samples from 5 patients with probable cerebral aspergillosis and from 16 control patients. Cerebrospinal fluid galactomannan levels were significantly higher in aspergillosis patients, and most galactomannan was produced intrathecally. Comparison of serum galactomannan values in pulmonary and cerebral aspergillosis patients showed significant overlapping. Detection of Aspergillus galactomannan in cerebrospinal fluid may be diagnostic of cerebral aspergillosis.

Antigens, Fungal↗
PubMed2002

Treatment of acute graft versus host disease with low dose-alternate day anti-thymocyte globulin.

BACKGROUND AND OBJECTIVES: The efficacy of antithymocyte globulin (ATG) in the treatment of graft-versus-host disease (GvHD) is controversial. In the present study we report on the use of low dose ATG (thymoglobuline, Sangstat) and steroids in 28 patients with moderate to severe acute GvHD. DESIGN AND METHODS: Fifteen patients received ATG as first-line treatment within 14 days of the diagnosis of GvHD (median 8 days, range 4-13). Twelve patients received ATG as second-line therapy, more than 14 days after diagnosis (median 32 days, range 14 to 98). The proportion of patients with severe (grade III-IV) GvHD at the time of ATG therapy was 4/15 in the former group and 7/13 in the latter (p=0.1). RESULTS: On day 30 after ATG the overall proportion of responders was 80% in the group administered ATG early and 38% in those given it later (p=0.03). The overall actuarial 3-year transplant-related mortality was 40% vs 74% for the early vs late ATG groups (p=0.03); the actuarial 3-year survival was, respectively, 49% vs 23% (p=0.04). For patients with GvHD grade III-IV the actuarial 1-year TRM was 47% for those given ATG early, 87% for the late ATG group and 82% for a concurrent control group of 26 patients not treated with ATG. INTERPRETATION AND CONCLUSIONS: In conclusion, ATG may be considered for early treatment of acute GvHD, within a few days from the onset of the disease. A prospective trial has been started to test whether, in this setting, low dose ATG with steroids is superior to steroids alone.

Adult↗
PubMed2002

Investigating the mechanism of chromosomal deletion: characterization of 39 deletion breakpoints in introns 47 and 48 of the human dystrophin gene.

The region of the dystrophin gene containing introns 45-50 is characterized by a high rate of recombination events that give rise to large deletions causing dystrophinopathy. The nucleotide sequence of this intronic region has recently been released in GenBank. With the aim of further understanding the mechanism favoring the occurrence of these deletions, we have characterized the distribution of introns 47 and 48 deletion endpoints in 39 dystrophinopathy patients. In 14 of these patients we were able to sequence the break junction. On these sequences we were able to identify several intronic motifs that could predispose to DNA double-strand breaks. Our results, combined with other literature data, show that unequal homologous recombination is a very poorly represented event in the dystrophin gene, whereas junction features are suggestive of a model of recombination in which DNA double-strand breaks are incorrectly repaired by a nonhomologous end-joining mechanism. The correlation among recombination rate, deletion frequency, and percentage of repetitive elements is discussed.

Amino Acid Sequence↗