E Di Marco

Transformation of NIH 3T3 cells by overexpression of the normal coding sequence of the rat neu gene.

While the normal human erbB-2 gene is potently transforming when overexpressed in NIH 3T3 cells, its rat homolog, the neu gene, seems to acquire transforming properties only upon alteration of its coding sequence. In this study, we compared the effects of different levels of expression of normal erbB-2 and neu in NIH 3T3 cells. Our results revealed that the normal rat neu gene acts as a potent oncogene when sufficiently overexpressed in NIH 3T3 cells.

Viral-load and B-lymphocyte monitoring of EBV reactivation after allogeneic hemopoietic SCT in children.

EBV-associated post transplant lymphoproliferative disease (EBV-PTLD) is a life-threatening complication that may occur after hemopoietic SCT. We prospectively screened 80 children on a weekly basis using nested quantitative PCR to evaluate EBV genome copies. EBV viral load <1000 copies per 105 PBMC was observed in 63% of transplants, whereas it was between 1000 and 9999 copies per 105 PBMC in 13%, and between 10 000 and 19 999 in 10%, with no significant increase in percentage of CD20+ lymphocytes. Viral load reached ⩾20 000 copies per 105 PBMC in 14% of patients, and rituximab was administered to 75% of them. None of the patients except one developed a lymphoproliferative disease. Our study found that only 13% of unrelated donor HSCT recipients had a very high risk of EBV-PTLD defined as ⩾20 000 geq per 105 PBMC associated with an increase in CD20+ lymphocyte. We suggest that rituximab could be administered in the presence of very high levels of EBV-DNA viral load or in the presence of mid levels of EBV-DNA viral load associated with an increase in the percentage of CD20+ lymphocytes. Through this approach, we significantly reduced the number of patients treated with rituximab, and consequently the acute and chronic adverse events related to this treatment.

Acute phase lipocalin Ex-FABP is involved in heart development and cell survival.

Ex‐FABP is an extracellular fatty acid binding protein, expressed during chicken embryo development in cartilage, muscle fibers, and blood granulocytes. Transfection of chondrocytes and myoblasts with anti‐sense Ex‐FABP cDNA results in inhibition of cell proliferation and apoptosis induction. Ex‐FABP expression is dramatically enhanced by inflammatory stimuli and in pathological conditions. In this paper, by in situ whole mount and immunohistochemistry analysis we show that, at early developmental stage, Ex‐FABP is diffuse in all tissues of chick embryos. Particularly high level of transcript and protein are expressed in the heart. During acute phase response (APR) induced by endotoxin LPS injection, a marked increase of Ex‐FABP mRNA was observed in embryos, highest Ex‐FABP expression being in heart and liver. To investigate in vivo the biological role of Ex‐FABP, we have directly microinjected chicken embryos with antibody against Ex‐FABP. Almost 70% of chicken embryos died and the target tissue was the heart. We detected in heart of the treated embryos a significant increase of apoptotic cells and high level of fatty acids. We propose that the accumulation of fatty acid, specific ligand of Ex‐FABP, in the cell microenvironment is responsible of heart cell death, and we suggest that Ex‐FABP may act as a survival protein by playing a role as scavenger for fatty acids.

Whole-genome sequencing as standard practice for the analysis of clonality in outbreaks of meticillin-resistant Staphylococcus aureus in a paediatric setting.

Meticillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of hospital-associated infections. This study investigated the potential use of whole-genome sequencing (WGS) for surveillance purposes by re-examining MRSA strains related to past outbreaks among hospitalized paediatric patients. WGS data ameliorated the genotypic profile previously obtained with Sanger sequencing and pulsed-field gel electrophoresis typing, and discriminated between strains that were related and unrelated to the outbreaks. This allowed strain clonality to be defined with a higher level of resolution than achieved previously. This study demonstrates the potential of WGS to trace hospital outbreaks, which may lead to WGS becoming standard practice in outbreak investigations.

Rare viral infections in children receiving hemopoietic stem cell transplant

Viral infections are a rare complication in autologous hemopoietic stem cell transplant (HSCT) recipients but represent a frequent cause of disease after allogeneic HSCT. In the last years, there has been an increase in the number of viral diseases observed in these patients. This fact may be at least partially due to an improvement in diagnostic facilities, but the increasing number of transplant procedures and the more severe immunosuppression may also have played an important role.

Severe cutaneous zygomycosis: evaluation of treatment with high doses of liposomal amphotericin B in a one-year old child.

We report a case of severe cutaneous infection by Absidia corymbifera and Syncephalastrum racemosum in a one-year old child treated successfully with surgical debridement and parenteral administration of liposomal amphotericin B. We underline the effectiveness of the multidisciplinary approach and the use of high doses of liposomal amphotericin B. Absidia corymbifera and Syncephalastrum racemosum, belonging to the class of zygomycetes, mucorales orders, are responsible for acute, necrotic fungal infections, characterized by angio-invasion, vascular thrombosis and tissue necrosis. Although healthy humans have a strong natural immunity to infection from zygomycetes, patients with underlying local or systemic predisposing factors can easier develop the disease and a high mortality is reported even when aggressive antifungal therapy and surgical debridement are used 1,2. The present report describes a case of cutaneous zygomycosis occurring in a pediatric patient affected by hyposplenia and previous pneumococcal purpura fulminans 3,4. Case report: In February, 2005 a one-year old child was hospitalized for septic shock and purpura fulminans. On admission, the patient was febrile and pale with hypotension, altered consciousness and diffuse hemorrhagic skin lesions extended in particular to the legs. Laboratory data included a total leukocyte count of 6,600/mm3 with 71% neutrophils, 22% lymphocytes; C-reactive protein was 9.36 mg/dl (n.v. <0.46 mg/dl). Besides the urinary Journal of Chemotherapy Vol. 19 n. 3 (347-349) 2007

Proliferative Activity of Human Solid Tumors Evaluated by Thymidine Labeling Index and Primer-Dependent α-DNA Polymerase

Proliferating cells, independent of their position in the cell cycle, contain a primer-dependent alpha-DNA polymerase (PDP); we have, therefore, tested the feasibility of the PDP assay in breast, ovarian, and head and neck tumors. Experimental results demonstrate that the PDP labeling index (PDP-LI) is constantly superior to the thymidine labeling index (TLI), which indicates the percentage of S phase cells. The PDP-LI/TLI ratios observed were 5.0 in breast cancer, 3.6 in ovarian cancer, and 2.5 in head and neck tumors. Higher PDP-LI scores have been obtained in metastatic breast tumor samples (PDP-LI = 8.8) compared to nonmetastatic tumors (PDP-LI = 4.9).

Genomic characterization of a paediatric MRSA outbreak by Next Generation Sequencing

INTRODUCTION Twelve strains of meticillin-resistant Staphylococcus aureus (MRSA) isolated during a suspected outbreak in a paediatric intensive care unit were analysed by whole-genome sequencing (WGS). AIM To define the clonality of MRSA strains to a high discriminative power, and to evaluate the presence of genetic determinants responsible for antibiotic resistance and virulence. RESULTS Ten out of 12 strains belonged to multi-locus sequence type ST2625, while the other two strains were ST8. Among the ST2625 strains, analysis based on 1126 genes showed that they were clonal, sharing more than 98.3% of allelic identities, and one strain was isolated from a healthcare worker. All ST2625 strains were characterized by the SCC-Mec cassette IVa, and resistoma analysis indicated correspondence between phenotypic and genotypic characteristics. The study of 63 genes associated with virulence was correlated with the pattern of clonality shown. CONCLUSION This analysis confirmed the occurrence of an outbreak. As such, standard infection control measures were strictly enforced, and this led to prompt termination of the outbreak.