Prospective multicentre study on azole resistance in Aspergillus isolates from surveillance cultures in haematological patients in Italy.

OBJECTIVES: This study was conducted to assess the prevalence of azole resistance in Aspergillus isolates from patients with haematological malignancies or who were undergoing haematopoietic stem cell transplantation and to identify the molecular mechanism of resistance. METHODS: In this 28-month prospective study involving 18 Italian centres, Aspergillus isolates from surveillance cultures were collected and screened for azole resistance, and mutations in the cyp51A gene were identified. Resistant isolates were genotyped by microsatellite analysis, and the allelic profiles were compared with those of resistant environmental and clinical isolates from the same geographical area that had been previously genotyped. RESULTS: There were 292 Aspergillus isolates collected from 228 patients. The isolates belonged mainly to the section Fumigati (45.9%), Nigri (20.9%), Flavi (16.8%) and Terrei (4.8%). Three isolates showed itraconazole resistance: Aspergillus fumigatus sensu stricto, Aspergillus lentulus (section Fumigati) and Aspergillus awamori (section Nigri). The itraconazole resistance rates were 1% and 1.48% considering all Aspergillus spp. isolates and the Aspergillus section Fumigati, respectively. The prevalence of azole resistance among all the patients was 1.3%. Among patients harbouring A. fumigatus sensu stricto isolates, the resistance rate was 0.79%. The A. fumigatus isolate, with the TR34/L98H mutation, was genotypically distant from the environmental and clinical strains previously genotyped. CONCLUSIONS: In this study, the Aspergillus azole resistance rate was 1% (3/292). In addition to A. fumigatus sensu stricto, A. lentulus and A. awamori azole-resistant isolates were identified. Therefore, it is important have a correct identification at the species level to address a rapid therapy better, quickly understand the shift towards cryptic species and have an updated knowledge of the local epidemiology.

Treatment of refractory chronic GVHD with rituximab: a GITMO study.

The anti-CD20 chimaeric monoclonal antibody Rituximab has recently been shown to induce significant clinical response in a proportion of patients with refractory chronic graft-versus-host disease (cGVHD). We now report 38 patients, median age 48 years (22-61), receiving Rituximab for refractory cGVHD, assessed for clinical response and survival. Median duration of cGVHD before Rituximab was 23 months (range 2-116), the median number of failed treatment lines was 3 (range 1 to > or =6) and the median follow-up after Rituximab was 11 months (1-88). Overall response rate was 65%: skin 17/20 (63%), mouth 10/21 (48%), eyes 6/14 (43%), liver 3/12 (25%), lung 3/8 (37.5%), joints 4/5, gut 3/4, thrombocytopaenia 2/3, vagina 0/2, pure red cell aplasia 0/1 and, myasthenia gravis 1/1. During the study period 8/38 died: causes of death were cGVHD progression (n=3), disease relapse (n=1), infection (n=3), sudden death (n=1). The actuarial 2 year survival is currently 76%. We confirm that Rituximab is effective in over 50% of patients with refractory cGVHD and may have a beneficial impact on survival.

Morphologic changes leading to bronchiolitis obliterans in a patient with delayed non-infectious lung disease after allogeneic bone marrow transplantation.

A 37-year-old man developed delayed non-infectious lung disease after undergoing bone marrow transplantation (BMT) for acute myeloid leukaemia. Over a 15-month period, the progression of morphologic changes from cellular interstitial pneumonia to bronchiolitis obliterans organizing pneumonia and cicatricial bronchiolitis obliterans was documented. Pulmonary function tests, high-resolution CT, bronchoalveolar lavage, lung biopsy and extensive microbiological studies were used as diagnostic tools either at onset and during the follow-up. This represents the first reported case in which a model--supported by longitudinal biopsy results--for the evolution of histologic lesions toward bronchiolitis obliterans after BMT is suggested; therapeutic implications are discussed.

Reduced incidence of GVHD without increase in relapse with low-dose rabbit ATG in the preparative regimen for unrelated bone marrow transplants in CML.

SUMMARY: Antithymocyte globulin (ATG) treatment prevents graft failure and results in a low incidence of GVHD, but an increased risk of relapse could be expected as a consequence of reduced GVHD. From September 1995 to June 2001, 28 consecutive chronic myeloid leukemia (CML) patients underwent unrelated bone marrow transplants: 21 were in chronic phase (CP) and seven in advanced phase (AP). Median age was 35.5 years (range 20-50). HLA typing was based on high-resolution molecular techniques; in eight cases there were one or more allele mismatches. The preparative regimen consisted of TBI, EDX 120 mg/kg and rabbit ATG 15 mg/kg. All patients engrafted and no rejection occurred. Acute GVHD grade III-IV occurred in six patients (21%). Chronic GVHD occurred in 10 (40%) and it was extensive in one. Four out of seven patients transplanted in AP had a hematological relapse. Of 21 in CP, there was one cytogenetic and one molecular relapse: these two patients are now in complete remission with imatinib mesylate. With a median follow-up of 45.7 months, the 5-year survival is 76.2% for those transplanted in CP. These data demonstrate that transplants performed in CP, with low-dose ATG, are associated with a good outcome, low incidence of GVHD and no increase of relapse.

Different immune reconstitution in multiple myeloma, chronic myeloid leukemia and acute myeloid leukemia patients after allogeneic transplantation of peripheral blood stem cells.

In this study we compared the lymphocyte reconstitution in 13 multiple myeloma (MM), nine acute myeloid leukemia (AML) and 10 chronic myeloid leukemia (CML) patients after allogeneic G-CSF-mobilized PBSC transplantation from HLA-identical siblings. Conditioning regimens included standard total body irradiation + cyclophosphamide (CY), or busulphan + CY, whereas VP-16 was added in patients with advanced disease. Overall comparable numbers of mononuclear cells, CD34+ cells and CD3+ T cells were infused in each group. A significantly higher CD3+ T cell number was observed in MM and AML than in CML patients 1 month after transplant. However, MM patients showed a faster and better recovery of CD4+ T cells than both AML and CML patients at 3 months (P = 0.01 and P = 0.01, respectively) and 12 months (P = 0.01 vs AML, while P = NS vs CML) after transplant, and had a CD4:CD8 ratio > 1 with a median CD4+ T cell value > 400/microl 1 year after transplant. Development of acute graft-versus-host disease (GVHD) did not affect CD4:CD8 ratios but patients who experienced acute GVHD > grade I had lower CD4+ and CD8+ T cell numbers at all time points. However, after excluding patients with GVHD > grade I, MM patients still showed a significantly higher CD4+ T cell value than patients with myeloproliferative diseases 1 year after transplant. These findings suggest that although allogeneic PBSC transplantation induces rapid immune reconstitution, different kinetics may occur among patients with hematological malignancies. In particular, the rapid reconstitution of CD4+ T cells in MM patients may contribute to the low transplant-related mortality achieved in this disease.

Use of anti-BDCA-2 antibody for detection of dendritic cells type-2 (DC2) in allogeneic hematopoietic stem cell transplantation.

TH2-inducing dendritic cells (DC2) are commonly identified as negative for lineage markers and positive for HLA-DR and CD123 expression. More recently, normal blood DC2 were shown also to be positive for BDCA-2 and BDCA-4 antigens. The aim of this study was to evaluate whether BDCA-2 expression on DC2 is impaired in patients undergoing an allogeneic hematopoietic stem cell transplantation (HSCT) and in healthy donors treated with G-CSF for HSC mobilization. Flow cytometry assays for DC2 detection using either a triple staining with anti-HLA-DR PerCP, anti-Lin(+) anti-CD34 FITC and anti-CD123 PE monoclonal antibodies (mAbs), or a double staining with anti-HLA-DR PE and anti-BDCA-2 FITC mAbs were compared in blood samples from patients who underwent an allogeneic HSCT (n = 30) or from healthy donors before (n = 11) and after (n = 8) G-CSF mobilization, as well as in healthy donors' leukapheresis products (n = 12) or bone marrow (n = 4). Staining of BDCA-2(+) cells with other markers such as anti-CD38, anti-CD54 and anti-CD58 were also performed. Median values of CD123(+) DC2 and BDCA-2(+) DC2 were not statistically different in the blood of patients previously treated with chemotherapy, nor in the blood or bone marrow of heathy donors. Also, a 5 day G-CSF treatment did not affect BDCA-2 or adhesion molecule expression on healthy donors' blood DC2 significantly. A correlation between all the results (n = 65) obtained with the two assays was demonstrated in a linear regression curve (r = 0.914) (P = 0.00001). BDCA-2 is a marker highly specific for DC2 that is not downregulated by chemotherapy or G-CSF treatment. Therefore, the anti-BDCA-2 mAb can be efficiently combined with other mAbs and used in studies addressing the role of DC2 in the allogeneic HSCT setting.

Discrepancy between serological complete remission and concomitant new bone lytic lesions after infusion of escalating low doses of donor lymphocytes in multiple myeloma: a case report.

A graft-versus-myeloma effect has been previously induced by infusing high numbers of donor lymphocytes after allogeneic stem cell transplantation in relapsed/refractory multiple myeloma (MM) patients. A 43-year-old patient with MM refractory to standard chemotherapy and autologous transplantation received an allogeneic HLA-matched T cell-depleted marrow transplant from his sister after conditioning with single dose total-body irradiation, melphalan and cyclophosphamide. Twenty-four months after transplant neither a significant reduction of serum M protein nor evidence of acute or chronic graft-versus-host disease (GVHD) were observed. The patient was then treated with four escalating low doses of donor lymphocyte infusions (DLI) (0.1, 1.0, 5.0 and 5.0 x 106 CD3+ T cells/kg, respectively) over a 13 month period. Following the second infusion a mild liver acute GVHD and a partial, but transient, response occurred. After the last DLI the patient achieved a complete remission and developed extensive chronic GVHD. However, concomitant with the disappearance of clonal plasma cells from the marrow and of serum M protein, two new bone lytic lesions appeared requiring treatment with radiotherapy. In conclusion, escalating low doses of DLI may be effective in MM and may prevent severe acute but not chronic GVHD. However, the efficacy of DLI in extramedullary MM lesions is still unclear.

Alloantigen presenting capacity, T cell alloreactivity and NK function of G-CSF-mobilized peripheral blood cells.

In this study we addressed whether the proportion and the function of antigen presenting cells (APC), T and NK lymphocytes are modified in the apheresis product of six healthy donors who received a stem cell mobilizing treatment with glycosylated G-CSF at 10 microg/kg/day x 5 days s.c. Flow cytometry analysis showed comparable percentages of HLA-DR+, CD19+, CD86+, CD80+ and CD1a+ cells in preG-CSF-peripheral blood mononuclear cells (preG-PBMC) and after mobilization in G-PBMC, whereas the proportion of CD14+ monocytes significantly increased in G-PBMC (3+/-1% vs 17+/-8%, P = 0.003). Analysis of lymphocyte subsets in preG-PBMC and G-PBMC showed similar proportions of CD3+, CD4+, CD8+ and CD28+ T cells, but a significantly lower percentage of CD16+ (11+/-7% vs 4+/-1%, P=0.01), CD56+ (15+/-6% vs 5+/-2%, P= 0.008), CD57+ (16+/-9% vs 5+/-2%, P=0.04), CD25+ (19+/-2% vs 9+/-6%, p=0.009) and CD122+ (5+/-2% vs 2+/-1%, P = 0.05) cells in G-PBMC. Unfractionated preG-PBMC and G-PBMC were irradiated and tested in primary mixed leukocyte culture (MLC) with two HLA-incompatible responders and induced efficient alloresponses in four of six cases, whereas G-PBMC stimulated poorly in the remaining two cases. Also, in allo-MLC with irradiated G-PBMC we detected lower amounts of IFN-gamma (P = 0.04) and of IL-2 (P = 0.06) than in allo-MLC with preG-PBMC. Furthermore, freshly isolated preG-PBMC and G-PBMC from each donor exerted comparable allogeneic responses to HLA-incompatible irradiated mononuclear cells in all cases. However, G-PBMC showed no NK activity against K562 target cells at any effector:target ratio tested. These data suggest that normal G-PBMC may prevent Thl alloresponses, maintain efficient alloreactivity to HLA mismatched antigens and have impaired NK activity.

Delayed non-infectious lung disease in allogeneic bone marrow transplant recipients.

BACKGROUND AND AIM OF THE WORK: The studies on late-onset non-infectious respiratory complications after allogeneic bone marrow transplantation (allo-BMT) have been mainly focused on bronchiolitis obliterans to date. The aim of this work was to analyze the incidence, clinico-pathologic characteristics and outcome of the entire spectrum of entities falling into the group of delayed non-infectious lung disease (DLD). METHODS: Retrospective chart review was carried out of 112 patients who underwent allo-BMT for hematologic malignancies between April 1995 and November 1998 at a single Institution. The categorization of the pulmonary disease was made by analyzing clinical data, bronchoalveolar lavage (BAL), high-resolution computed tomography (HRCT) and histology when possible. RESULTS: DLD occurred in 10 (10%) out of 97 recipients who survived at least 100 days following allo-BMT and was defined as bronchiolitis obliterans (BO; 4 cases), acute lung injury (ALI; 1 case) and subacute cellular interstitial pneumonia (SCIP; 5 cases). The BAL-profile was characterized by a marked increase of the neutrophil percentage in BO cases and of the lymphocyte (predominantly CD8+) percentage in parenchymal DLDs (SCIP, ALI). HRCT proved to be helpful to correctly identify BO cases, whereas histology was always needed to better define DLD presenting with an interstitial and/or alveolar pattern. The predominant airway involvement as well as the acute-onset of a respiratory illness with histological evidence of diffuse alveolar damage were associated with a worse prognosis because of a poor response to the immunosuppressive treatment. CONCLUSIONS: DLDs represent a group of entities heterogeneous in regard to variables such as onset and clinical behaviour (acute, subacute or chronic), predominant pattern of lung involvement (airway or parenchymal), response to treatment. Although immunopathologic mechanisms related to c-GVHD probably have a relevant pathogenic importance in this setting, the possible role of associated events (eg, drug toxicity and infections) at least in priming the lung damage need to be better clarified for its therapeutical implications.

Effects of defibrotide after oral and parenteral administration in patients with peripheral obliterative arterial disease (POAD).

Defibrotide (D) a polidesoxyribonucleotidic derivative provided with fibrinolytic and antithrombotic activity has already proven effective when administered by parenteral route in patients with peripheral obliterative arterial disease (POAD). Bioavailability studies gave evidence that the drug is absorbed by 50-70% when administered orally. Thus, aim of this trial was to evaluate whether the drug might exert similar clinical and biological effects after oral/parenteral dosing in a 2:1 ratio. This was a randomized cross-over study including 17 out patients with POAD (Leriche stage II). D was administered by oral (400 mg b.i.d.) and intramuscular route (200 mg b.i.d.), both treatments lasting 15 days. In basal conditions and at the end of both treatments the following evaluations were made: (1) absolute walking distance (tread mill); (2) Doppler ultrasonographic examination (Winsor index); (3) strain-gauge plethysmography (rest flow and peak flow). In addition in the same occasions plasma samples were collected for the assessment of plasminogen (chromogenic assay) and fibrinolytic activity (fibrin plates). Defibrotide administration was followed by a significant increase in walking distance both after oral and parenteral administration [basal conditions (IRL): 232.7 +/- 23.0 meters; oral: 273.1 +/- 28.1 m; i.m.: 277.9 +/- 26.8 m, p less than 0.01 - (IRA) basal conditions: 380.1 +/- 25.6; oral: 437.1 +/- 31.5 m; i.m.: 442.5 +/- 34.0 m, p less than 0.01] and by a significant increase in peak flow (basal conditions: 9.66 +/- 1.04; oral: 10.90 +/- 0.90; i.m.: 11.12 +/- 0.98, p less than 0.05), while Winsor index and rest flow were unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Doppler flowmetry and the diagnosis of breast lumps.

The aim of the study was to ascertain the reliability of a Doppler signal in the diagnosis of breast cancer. Computerized blood flow analysis was performed on 117 patients who presented with a breast lump at the Division of Diagnostic Oncology of the Istituto Nazionale Tumori of Milan. The values of systolic peak and diastolic frequency were evaluated in relation to the histologic diagnosis. Ten patients had a spontaneous regression of the lump and were excluded from the study. The number of evaluable cases with histologic confirmation was 107: 69 carcinomas and 38 benign lesions. Systolic peak values for the 69 carcinomas ranged from 1500 to 7400 Hz, with a mean value of 3243.4 Hz; diastolic frequency ranged from 200 to 3700 Hz, with mean value of 1413.9 Hz. No diagnostic signals were found in 4 breast cancers (false negatives). Twenty-three of 38 benign lesions (60.5%) and 65 of 69 malignant nodes (94.2%) were correctly diagnosed. The Doppler signal with computerized spectral analysis in addition to more specific ultrasonographic parameters could be considered a useful tool in the diagnosis of breast cancer.

[Increase in erythrocyte and plasma viscosity in patients with atherosclerotic vasculopathy of different sites]. / Aumento della viscosità eritrocitaria e plasmatica in pazienti affetti da vasculopatia aterosclerotica a diversa localizzazione.

Peripheral atherosclerotic arteriopathies are characterised by increased blood viscosity and, according to some, by a reduction in red blood cell deformability. Red blood cell viscosity was studied in 40 atherosclerosic patients (14 carotid and 26 aortoiliacofemoral lesions). All patients were subjected to the standard diagnostic examination (Doppler velocimetry, etc.). Fifty healthy non-smokers of the same sex and 20 heavy smokers were studied as controls. The viscosity of the whole blood, and plasma and blood fibrinogen were assayed. In studying erythrocyte viscosity, cells poor in leucocytes and platelets suspended in a saline phosphate buffer were employed. The atherosclerotic patients presented significantly higher mean levels of erythrocyte viscosity (p less than 0.01) and the increase in plasma viscosity and blood fibrinogen was also statistically significant. The viscosity of the whole blood was also increased in these patients (p less than 0.001). The increase in blood viscosity in subjects with peripheral arteriopathies therefore appears to be attributable to the plasma and to rheological alterations in the red blood cells.

[Effect of calciparine in the mid-term treatment of peripheral arteriopathy of the lower extremities]. / Effetto della calciparina nel trattamento a medio termine dell'arteriopatia periferica degli arti inferiori.

The authors examine a series of 20 patients suffering from AOCP (?) at Fontaine's 2nd stage treated with calciparine s.c. in a single daily dose of 12500 IU for three months. The parameters of clinical assessment of claudicatio intermittens show statistically significant increases of ILR and ILA which were not correlated to marked variations of the Windsor index. The extremely positive results in terms of the clinical trend of the disease are encouraging with a view to a more extensive application of this therapeutic approach in peripheral arterial disease of the lower limbs.

[Kinetics of hepatic enzymes in anorexia nervosa]. / Cinetica degli enzimi epatici nell'anoressia nervosa.

Nervous anorexia in advanced stage causes a generalized deterioration of organs' and apparatuses' functions. In particular, well-known are disorders of both CNS and PNS, changes of the haematic crasis and worsening of the metabolic-endocrine axis. Less well-known, however, is the compromission of function. Analysis of the data obtained in this trial points out a statistically significant correlation between the loss of weight and the variation of level of some liver enzymes (SGPT, SGOT, LDH). These indexes of hepatic necrosis in our opinion, can therefore, be regarded as sound markers not only for a more careful evaluation of the clinical evolution of the anorexic patient, but also for a better monitoring of the effectiveness of diet therapy.

Alemtuzumab plus cyclosporine treatment of the autoimmune hemolytic anemia in an adult bowel transplant.

An adult male underwent a bowel transplant for tufting enteropathy, receiving alemtuzumab, tacrolimus, and steroids as immunosuppressants. Five years later, he developed an autoimmune hemolytic anemia (AIHA), anti-IgG positive, with reduced reticulocyte count, leukopenia, and thrombocytopenia with antiplatelet antibodies. After an unsuccessful initial treatment with high dose steroids, reduction in tacrolimus dose, and intravenous immunoglobulin (IVIG), a bone marrow biopsy revealed absence of erythroid maturation with precursor hyperplasia. The patient was switched to sirolimus and received four doses of rituximab plus two courses of plasmapheresis, which decreased his transfusion requirements. After a febrile episode one month later, the AIHA relapsed with corresponding decreases in platelet and leukocyte count: cyclosporine A (CsA) was started with a second course of rituximab and IVIG without response, even though repeat bone marrow biopsy did not reveal morphology correlated to an acquired pure red cell aplasia (APRCA). Considering the similarity in his clinical and laboratory findings to APRCA, alemtuzumab was added (three doses over a week) with CsA followed by steroids. The patient was eventually discharged transfusion-independent, with increasing hemoglobin (Hb) levels and normal platelet and leukocyte count. One year later he is still disease-free with functioning graft.

Intensification of GVHD prophylaxis with low-dose ATG-F before allogeneic PBSC transplantation from HLA-identical siblings in adult patients with hematological malignancies: results from a retrospective analysis.

Several studies have shown that chronic GVHD (cGVHD) is more frequent in patients receiving transplants from PBSC than in those receiving BM. In the setting of PBSC-unrelated transplants, the addition of anti-T-cell globulin (ATG) has shown a significant decrease in incidence/severity of cGVHD, without an increase in relapses or infections. However, no prospective data are yet available in the sibling setting. We retrospectively analyzed the effects of intensification of standard GVHD prophylaxis (CsA+MTX) by the addition of low-dose ATG in 245 patients receiving a transplant from HLA-identical sibling. From 1996 to 2001, patients received PBSC as the preferred source (group 2), and then ATG was added before transplant (group 3) because of a high cGVHD rate. Patients receiving BM in the same time period were analyzed as a control group (group 1). The incidence of grade III-IV acute GVHD and cGVHD was not significantly different in the three groups, but extensive cGVHD was highest in group 2 (38%) compared with group 3 (21%) or group 1 (28%; P=0.03). OS, TRM and time to relapse/progression were similar in the three groups. Our analysis shows that adding ATG to PBSC sibling allogeneic transplants can lower cGVHD, without an increase of relapse. Further prospective studies are needed to confirm these findings.