Possible donor-recipient bartonellosis transmission in a pediatric liver transplant.

Bartonella henselae is the causative agent of cat-scratch disease and other disorders, including hepatosplenic granulomatosis. This infection has only rarely been reported after solid organ transplantation, where it can mimic the more common post-transplant lymphoproliferative disease. Here we present a case of asymptomatic B. henselae hepatic and lymph nodal granulomatosis in a pediatric patient who had received orthotopic liver transplant 2 months before; we hypothesize that the causative agent was transmitted from the donor. This infection developed early in the post-transplant period; the disease involved only the graft liver and the regional lymph nodes, and the patient did not have a cat or any history of contact, scratches, or bites by a cat. In our patient this infection resolved successfully with a combination of 2 associated antibiotics and reduction of immunosuppressive therapy.

Cyclotron production of 64Cu by deuteron irradiation of 64Zn.

The short-lived (12.7h half-life) (64)Cu radioisotope is both a beta(+) and a beta(-) emitter. This property makes (64)Cu a promising candidate for novel medical applications, since it can be used simultaneously for therapeutic application of radiolabelled biomolecules and for diagnosis with PET. Following previous work on (64)Cu production by deuteron irradiation of natural zinc, we report here the production of this radioisotope by deuteron irradiation of enriched (64)Zn. In addition, yields of other radioisotopes such as (61)Cu, (67)Cu, (65)Zn, (69m)Zn, (66)Ga and (67)Ga, which were co-produced in this process, were also measured. The evaporation code ALICE-91 and the transport code SRIM 2003 were used to determine the excitation functions and the stopping power, respectively. All the nuclear reactions yielding the above-mentioned radioisotopes were taken into account in the calculations both for the natural and enriched Zn targets. The experimental and calculated yields were shown to be in reasonable agreement. The work was carried out at the Scanditronix MC-40 Cyclotron of the Institute for Health and Consumer Protection of the Joint Research Centre of the European Commission (Ispra site, Italy). The irradiations were carried out with 19.5 MeV deuterons, the maximum deuteron energy obtainable with the MC-40 cyclotron.

Optimisation study of alpha-cyclotron production of At-211/Po-211g for high-LET metabolic radiotherapy purposes.

The production of no-carrier-added (NCA) alpha-emitter (211)At/(211g)Po radionuclides for high-LET targeted radiotherapy and immunoradiotherapy, through the (209)Bi(alpha,2n) reaction, together with the required wet radiochemistry and radioanalytical quality controls carried out at LASA is described, through dedicated irradiation experiments at the MC-40 cyclotron of JRC-Ispra. The amount of both the gamma-emitter (210)At and its long half-lived alpha-emitting daughter (210)Po is optimised and minimised by appropriate choice of energy and energy loss of alpha particle beam. The measured excitation functions for production of the main radioisotopic impurity (210)At-->(210)Po are compared with theoretical predictions from model calculations performed at ENEA.

The additionally glycosylated variant of human sex hormone-binding globulin (SHBG) is linked to estrogen-dependence of breast cancer.

Sex Hormone-Binding Globulin (SHBG), the plasma carrier for androgens and estradiol, inhibits the estradiol-induced proliferation of breast cancer cells through its membrane receptor, cAMP, and PKA. In addition, the SHBG membrane receptor is preferentially expressed in estrogen-dependent (ER+/PR+) breast cancers which are also characterized by a lower proliferative rate than tumors negative for the SHBG receptor. A variant SHBG with a point mutation in exon 8, causing an aminoacid substitution (Asp 327-->Asn) and thus, the introduction of an additional N-glycosylation site, has been reported. In this work, the distribution of the SHBG variant was studied in 255 breast cancer patients, 32 benign mammary disease patients, and 120 healthy women. The presence of the SHBG mutation was evaluated with PCR amplification of SHBG exon 8 and Hinf I restriction fragment length polymorphism (RFLP) procedure. This technique allowed us to identify 54 SHBG variants (53 W/v and 1 v/v) in breast cancer patients (21.2%), 5 variants (4 W/v and 1 v/v) in benign mammary disease patients (15.6%), and 14 variants (W/v) in the control group (11.6%). The results of PCR and RFLP were confirmed both by nucleotide sequence of SHBG exon 8 and western blot of the plasma SHBG. No differences in the mean plasma level of the protein were observed in the three populations. The frequency of the SHBG variant was significantly higher in ER+/PR+ tumors and in tumors diagnosed in patients over 50 years of age than in the control group. This observation suggests the existence of a close link between the estrogen-dependence of breast cancer and the additionally glycosylated SHBG, further supporting a critical role of the protein in the neoplasm.

An alternatively spliced form of CD79b gene may account for altered B-cell receptor expression in B-chronic lymphocytic leukemia.

Several functional anomalies of B-chronic lymphocytic leukemia (B-CLL) cells may be explained by abnormalities of the B-cell receptor (BCR), a multimeric complex formed by the sIg homodimer and the noncovalently bound heterodimer Igalpha/Igbeta (CD79a/CD79b). Because the expression of the extracellular Ig-like domain of CD79b has been reported to be absent in the cells of most CLL cases, we have investigated the molecular mechanisms that may account for this defect. Peripheral blood lymphocytes (PBL) from 50 patients and two cell lines (MEC1, MEC2) obtained from the PBL of one of them were studied. MEC1, MEC2, and 75% of CLL cases did not express detectable levels of the extracellular Ig-like domain of CD79b, which was nevertheless present in greater than 80% CD19(+) cells from normal donors. In healthy subjects the expression of CD79b was equally distributed in CD5(+) and CD5(-) B-cell subsets. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD79b RNA from all patients and from MEC1 and MEC2 cell lines consistently yielded two fragments of different size (709 bp and 397 bp). The 709-bp band corresponds to CD79b entire transcript; the 397-bp band corresponds to an alternatively spliced form lacking exon 3 that encodes the extracellular Ig-like domain. Both fragments were also visible in normal PBL. The expression of the 397-bp fragment was increased in normal activated B cells, while no difference was seen between CD5(+) and CD5(-) B cells. To obtain a more accurate estimate of the relative proportions of the two spliced forms, a radioactive PCR was performed in 13 normal and 22 B-CLL samples and the results analyzed using a digital imager. The mean value of the CD79b to the CD79b internally deleted ratio was 0.64 +/- 0.20 SD in normal donors and 0.44 +/- 0.27 SD in B-CLL (P =.01). Direct sequencing of 397-bp RT-PCR products and of genomic DNA corresponding to exon 3 from MEC1, MEC2, their parental cells, and five fresh B-CLL samples did not show any causal mutation. Single-strand conformation polymorphism analysis of exon 3 performed in 18 additional B-CLL cases showed a single abnormal shift corresponding to a TGT --> TGC polymorphic change at amino acid 122. We propose a role for the alternative splicing of CD79b gene in causing the reduced expression of BCR on the surface of B-CLL cells. As normal B cells also present this variant, the mechanism of CD79b posttranscriptional regulation might reflect the activation stage of the normal B cell from which B-CLL derives.

MEC1 and MEC2: two new cell lines derived from B-chronic lymphocytic leukaemia in prolymphocytoid transformation.

We report the establishment and characterization of two cell lines, MEC1 and MEC2, that grew spontaneously on two subsequent occasions from the peripheral blood (PB) of a patient with B-chronic lymphocytic leukemia (B-CLL) in prolymphocytoid transformation. The patient was EBV-seropositive, his leukemic cells were EBNA negative, but the spontaneously grown cell lines are EBNA-2 positive. In liquid culture MEC1 cells grow adherent to the vessel wall and as tiny clumps; MEC2 cells do not adhere and form large clumps. The doubling time of MEC1 is 40h and of MEC2 is 31h. Both cell lines express the same light (kappa) and heavy chains (mu, delta) as the fresh parental B-CLL cells at the same high intensity, share the expression of mature B cell markers (CD19, CD20, CD21, CD22), differ in the expression of CD23 and FMC7, are CD11a+, CD18+, CD44+, CD49d+, CD54+ and express at high levels both CD80 and CD86. CD5 is negative on MEC1 cells (as on the vast majority of parental cells) and it has been lost by MEC2 cells after several months of culture. The cells have a complex karyotype. The tumour origin of MEC1 and MEC2 has been demonstrated by Southern blot analysis of the IgH loci and by Ig gene DNA sequencing. They use the VH4 Ig family and have not undergone somatic mutations (94.8% homology with germline Ig gene 4-59). Cytofluorographic analysis and RT-PCR reveal that MEC1 and MEC2 overexpress Bcl-2 together with Bax, express large amounts of Bcl-xL and trace amounts of Bcl-xS.

Fludarabine ability to down-regulate Bcl-2 gene product in CD5+ leukaemic B cells: in vitro/in vivo correlations.

CD5+ B-chronic lymphocytic leukaemia (B-CLL) and mantle cell lymphoma (MCL) in leukaemic phase are characterized by defects in cell death induction that primarily involves the Bcl-2 family of genes. Fludarabine (9-beta-D-arabinofuranosyl-2-fluoradenine, F-ara-A) is a potent inducer of apoptosis in CLL cells. This study aimed to determine whether F-ara-A-induced apoptosis might be related to Bcl-2 modifications and to evaluate in vitro/in vivo correlations. Peripheral blood lymphocytes from eight B-CLL and four leukaemic MCL were cultured in the presence of different concentrations of F-ara-A +/- methylprednisolone (MP). F-ara-A down-regulated the expression of Bcl-2 in 5/12 cases. mRNA down-regulation was maximal at 48 h; protein down-regulation was prominent after 48 h. Both events were dose-dependent. The amount of apoptosis was significantly higher in the samples treated with F-ara-A than in those exposed to MP alone. In the seven remaining cases, no Bcl-2 down-regulation was observed after exposure to F-ara-A and the degree of F-ara-A-induced apoptosis overlapped that induced by MP. The in vivo outcome after treatment with three to six courses of F-ara-A was evaluable in 10 patients: 4/5 cases, whose cells had shown in vitro Bcl-2 down-regulation and prominent apoptosis after exposure to F-ara-A, had a complete response (CR) and a partial response (PR) was observed in the remaining patient. Of the five patients whose cells had shown no in vitro Bcl-2 modulation after exposure to F-ara-A, two had a PR, but the other three did not show any in vivo clinical response.

In leukaemic CD5+ B cells the expression of BCL-2 gene family is shifted toward protection from apoptosis.

This study further investigated the mechanisms that control apoptosis in leukaemic CD5+ B cells, and focused on the Bcl-2 gene family. The pattern of expression of Bcl-2, Bcl-xL, Bcl-xS and Bax genes, selected because of their interrelated role in the control of apoptosis, was analysed in a series of CD5+ B-cell chronic lymphoid leukaemias. Cells from 34 patients with chronic lymphoid leukaemia of B-cell type (23 B-chronic lymphocytic leukaemia (B-CLL) and 11 mantle cell lymphoma (MCL) in leukaemic phase) were investigated. High levels of Bcl-2 mRNA were observed by Northern blot and high levels of Bcl-2 protein were detected by cytofluorograph analysis with a specific monoclonal antibody (MAb) in all cases. Strong Bax expression was detected by RT-PCR in 20/23 B-CLL cases; Bax was also observed in 8/11 MCL in leukaemic phase with variable degree of intensity. In both B-CLL and MCL samples the presence of Bax protein was confirmed by cytofluorograph analysis. RT-PCR detected high levels of Bcl-xL in 16/23 B-CLL and in 8/11 MCL in leukaemic phase, whereas Bcl-xS was detectable in low to trace amounts respectively in 13/23 B-CLL and in 6/11 MCL in leukaemic phase. According to the functional role of Bcl-2, Bcl-xL, Bcl-xS and Bax, these data indicate that the pattern of Bcl-2 family genes expression in leukaemic CD5+ B cells is skewed toward prevention of apoptosis and may thus favour the relentless accumulation of CD5+ leukaemic B cells.

[Hypertrophic obstructive cardiomyopathy in a patient with Turner syndrome]. / Cardiomiopatia ipertrofica ostruttiva in una paziente con sindrome di Turner.

A case of hypertrophic obstructive cardiomyopathy in a patient with Turner syndrome is reported. The most frequently associated cardiac anomalies are coarctation of the aorta and bicuspid aortic valve. Hypertrophic cardiomyopathy has never been reported in this syndrome but is frequent in Noonan syndrome. In these two conditions the phenotype may be indistinguishable but the cariotype is different: normal in Noonan and 45X in Turner syndrome. Our patient had the typical somatic features, and the cariotype was 45X in all examined cells. A familial form of hypertrophic cardiomyopathy was excluded by the normal clinical examination of other members of the family. The presence of hypertrophic cardiomyopathy also in Turner syndrome and the recent localization on the long arm of the chromosome 12 of the gene for Noonan syndrome might postulate a common pathogenesis of the two syndromes.

Expression of GATA-1 mRNA in human myeloid leukemic cells.

Expression of the transcription factor GATA-1, which regulates several erythroid specific genes and possibly also some megakaryocytic genes, has been previously detected in normal erythroblasts, megakaryocytes, and basophils, and in some myeloid cell lines. It has been suggested that GATA-1 may be first expressed in a common progenitor and then further activated during erythroid-megakaryocytic and basophilic differentiation and repressed during myeloid maturation. We investigated GATA-1 mRNA expression in highly purified leukemic blasts representing different lineages and stages of myeloid differentiation and in a recently established leukemic cell line, GF-D8, which exhibits morphological, cytochemical and immunophenotypic characteristics of early myeloid progenitor cells. We found GATA-1 expression in five of five myeloid and in one megakaryocytic blast crisis of CML, in four of six cases of myelomonocytic leukemias (M4 according to FAB classification), in one case of erythroleukemia (M6), whereas lymphoid blast crisis of CML and all other FAB groups were completely negative. In addition, a low level of GATA-1 mRNA was also expressed by the GF-D8 cell line. These data further support the hypothesis that GATA-1 expression may occur not only in erythroid and megakaryocytic progenitors, but also in early myeloid progenitors, and then be further regulated during lineage-specific maturation.

The nature of the B lymphocyte in B-chronic lymphocytic leukemia.

We have analyzed phenotypic, functional, and molecular properties of B-chronic lymphocytic leukemia (B-CLL) cells as compared to normal B cell differentiation stages and/or subsets. The possibility that the target B cell population transformed by the I primary oncogenic event(s) belongs to the normal CD5+ B cell subset from B mantle zone of secondary follicles is highly likely on phenotypic grounds. Though the genes responsible for the primary oncogenic event are presently unknown, a number of functional and molecular findings indicate that the end-product of their transforming activity is a cell frozen in the G0 phase of the cell cycle. This cell has several abnormalities that prevent an appropriate mitogenic response and presents a pattern of apoptosis-related gene expression that hinders apoptotic death. Pivotal to this apoptosis-escaping capacity is the expression of Bcl-2. We suggest that the increased expression of Bcl-2 together with an asynchronism between the expression of Bcl-2, c-myc, and APO1/Fas gene products shift the cellular balance away from apoptosis thereby helping the progressive accumulation in G0 of malignant CD5+ B cells.

Molecular characterization and functional studies on Hb Kempsey, beta 99 (G-1) Asp----Asn, a high-oxygen affinity variant.

BACKGROUND: Hb Kempsey (beta 99 Asp----Asn) is a high-oxygen affinity hemoglobin, never before reported in Italy, associated with secondary erythrocytosis. It has been found in the heterozygous state in two subjects from the same family originating from the Verona area in Northern Italy. METHODS: The abnormal hemoglobin was studied both at the protein and at the DNA level. The amino acid substitution was identified by fingerprinting and amino acid analysis. The nucleotide replacement was investigated by means of polymerase chain reaction (PCR) of the beta gene and direct sequencing. Oxygen affinity and other functional parameters were assessed on whole blood and on the separate hemoglobin fractions. RESULTS: These studies allowed us to establish the molecular substitution GAT (Asp)----AAT (Asn) at codon 99. Functional studies revealed a left-shifted and biphasic dissociation curve of the proposita, with a very low p50. The two carriers of this hemoglobinopathy have different degrees of polyglobulia, since iron deficiency in one of them reduces total and abnormal Hb. CONCLUSIONS: The compensatory mechanisms for tissue hypoxia are discussed with the conclusion that erythrocytosis has to be preserved in these patients to maintain adequate tissue oxygenation.

Wild-type and e antigen-minus hepatitis B viruses and course of chronic hepatitis.

Using an oligonucleotide hybridization assay, we studied the clinical implication of wild-type hepatitis B virus (HBV) and a HBV mutant that is unable to secrete hepatitis B e antigen (HBeAg) because of a translational defect due to a stop codon in the pre-C region in 106 hepatitis B surface antigen-positive patients with chronic hepatitis B. Wild-type HBV was detected in 31 of 42 (73.8%) HBeAg-positive patients, whereas a mixed viral population was present in 10 (23.8%). Significant differences in the severity and outcome of liver disease were not observed in the two groups of patients. However, the emergence of HBeAg-minus HBV in wild-type HBV carriers was associated with an exacerbation of liver disease and was followed by the presence of antibodies against HBeAg (anti-HBe) in serum in 50% of the cases. In 61 of 64 (95.3%) anti-HBe-positive patients, HBeAg-minus HBV was the predominant virus: HBeAg-minus HBV was detected in 42 patients (65.6%), whereas both wild-type and HBeAg-minus HBV were present in 19 (29.7%). HBeAg-minus HBV was associated with a course of hepatitis characterized by flare-ups of liver cell necrosis interspersed with periods of asymptomatic HBV carriage (P less than 0.01). These data support the hypothesis that genetic heterogeneity of HBV significantly influences the course and outcome of chronic hepatitis B. Wild-type HBV secreting HBeAg induces immunologic tolerance and causes chronic infection. HBeAg-minus HBV might be unable to induce chronic infection without the helper function of wild-type HBV, but it appears to be more pathogenic. Once chronic infection is established, HBeAg-minus HBV variants may prevail and displace wild-type virus.

Screening of beta-thalassemia mutations by PCR and ASO analysis in an Italian population of mixed geographic origin.

We used the polymerase chain reaction (PCR) and allele-specific-oligonucleotide hybridization (ASO) or restriction enzyme analysis (RE) to investigate the molecular defect in 100 Italian subjects heterozygous for beta-thalassemia, members of 50 couples at risk for the disease: 93 out of the 100 alleles studied were identified after investigating 9 known mutations. The mutation was identified in both members of the couple in 43/50 cases; in the remaining 7 couples the defect was identified only in one subject. The PCR-ASO or-RE method is a suitable, though still complex, approach to prenatal diagnosis of beta-thalassemia in a population with heterogeneous molecular defects.