Long term effects of cigarette smoke extract or nicotine on nerve growth factor and its receptors in a bronchial epithelial cell line.

Long-term exposure to cigarette smoke induces severe injuries to respiratory system through several mechanisms, some of them are well defined, but many others are not yet elucidated. Beside its classical role in nervous system, we have previously shown that Nerve Growth Factor (NGF) and its receptors have a crucial role in airway inflammatory diseases, such as Chronic Obstructive Pulmonary Disease. To expand our knowledge about the relevance of NGF and its receptors in airway diseases induced by cigarette smoking, we exposed for 16 weeks the bronchial epithelial cell line BEAS-2B to sub-toxic concentrations of whole cigarette smoke extract or pure nicotine. Viability, cell cycle gene expression, cell morphology and migration ability were tested and compared to NGF release and gene expression. Modulation of its receptors TrKA and p75NTR was also analyzed. The present study shows that long term exposure of BEAS-2B cells to cigarette smoke extract or nicotine induces: (A) differences: in cell viability, in the expression of cell cycle-related genes, in NGF release and in gene expression of NGF and its receptors; (B) similarities: in morphology and migration ability. Taken together, our data provide new insights about the biological role of NGF and its receptors in airway diseases induced by long-term cigarette smoking and, finally, our data evidence the opportunity not to use nicotine lozenges or e-cigarettes as anti smoking replacement therapy in patients with a previous airway disease according to the ability of nicotine to increase the amount of the pro-inflammatory cytokine NGF into the bronchial environment.

Gene Expression and Localization of NGF and Its Cognate Receptors NTRK1 and NGFR in the Sex Organs of Male Rabbits.

Experiments were devised to characterize the expression of nerve growth factor, beta polypeptide (NGF), and its cognate receptors neurotrophic tyrosine kinase receptor type 1 (NTRK1) and nerve growth factor receptor (NGFR) in rabbit male sex organs, as well as the concentrations of NGF in both seminal and blood plasma of sexually mature male rabbits. Immunoreactivity and gene expression for NGF and cognate receptors were detected in testis, prostate gland and seminal vesicle. The highest levels of NGF and NTRK1 transcripts were found in the prostate, while intermediate expressions were found in the testis. NGFR transcripts were expressed at the same levels in both testis and prostate and were more abundant than in seminal vesicles. The widespread distribution of NGF in all prostate glandular cells, together with its relative high mRNA abundance, confirms that the prostate of rabbits is the main source of this neurotrophin. In conclusion, the present data suggest that the NGF system is involved in the testicular development and spermatogenesis of rabbits and that NGF may act as a potential ovulation-inducing factor being abundantly present in the seminal plasma.

[Identification of candidate genes and expression profiles, as doping biomarkers]. / Individuazione di geni candidati e profili d'espressione, come indicatori molecolari di assunzione di sostanze dopanti.

Administration of prohibited substances to enhance athletic performance represents an emerging medical, social, ethical and legal issue. Traditional controls are based on direct detection of substances or their catabolites. However out-of-competition doping may not be easily revealed by standard analytical methods. Alternative indirect control strategies are based on the evaluation of mid- and long-term effects of doping in tissues. Drug-induced long-lasting changes of gene expression may be taken as effective indicators of doping exposure. To validate this approach, we used real-time PCR to monitor the expression pattern of selected genes in human haematopoietic cells exposed to nandrolone, insulin-like growth factor I (IGF-I) or growth hormone (GH). Some candidate genes were found significantly and consistently modulated by treatments. Nandrolone up-regulated AR, ESR2 and PGR in K562 cells, and SRD5A1, PPARA and JAK2 in Jurkat cells; IGF-I up-regulated EPOR and PGR in HL60 cells, and SRD5A1 in Jurkat; GH up-regulated SRD5A1 and GHR in K562. GATA1 expression was down-regulated in IGF-1-treated HL60, ESR2 was down-regulated in nandrolone-treated Jurkat, and AR and PGR were down-regulated in GH-treated Jurkat. This pilot study shows the potential of molecular biology-based strategies in anti-doping controls.

Primary effusion lymphoma in HIV-infected patients with multicentric Castleman's disease.

Multicentric Castleman's disease (MCD) and primary effusion lymphoma (PEL) are two B-cell lymphoproliferative diseases associated with Kaposi's sarcoma-associated herpes virus/human herpesvirus-8 (KSHV/HHV-8). Although MCD is considered a prelymphoma state, it is not known whether a pathogenetic link exists between MCD and PEL. This paper reports the clinico-pathological features of four cases of PEL (two pericardial, one pleural, and one peritoneal) developing in the context of HIV-associated MCD. Effusions, lymph nodes, spleen, and additional tissues from three autopsies were examined for morphology/immunophenotype, search for HHV-8 DNA, and assessment of immunoglobulin heavy chain gene (IgH) configuration using polymerase chain reaction (PCR)-based techniques. MCD and PEL samples contained HHV-8 DNA. Clonal IgH rearrangements were detected only in PEL, whereas MCD tissues were polyclonal. Light-chain immunostaining confirmed B-cell clonality in PEL (two lambda, one kappa, one not tested) and polyclonality in MCD. The autopsies revealed different morphological variants of visceral KS and multi-organ atypical infiltrates exhibiting immunoblastic/plasmablastic features reminiscent of PEL morphology, with a restriction of lambda-positive cells. In two cases, using microdissection and IgH PCR analysis, multiple/discrete bands were found in the infiltrates, compatible with polyclonality/oligoclonality. The case showing an oligoclonal IgH ladder contained a rearrangement of identical junctional size to the PEL clone; however, further analysis with PEL-derived clonotypic primers and sequencing of PCR products showed no amplification and nucleotide diversity, respectively, indicating that the two B-cell populations examined were clonally unrelated. These data show that MCD and PEL may co-exist in HIV-infected patients, suggesting a relevant association between these two HHV-8-related disorders. Although a definite clonal relationship between MCD and PEL was not demonstrated, it is hypothesized that in some MCD cases, within expanded polyclonal B-cell populations secondary to HHV-8 infection, clonal expansions may occur that localize into a body cavity, i.e. PEL.

Human herpesvirus-8 in lymphomatous and nonlymphomatous body cavity effusions developing in Kaposi's sarcoma and multicentric Castleman's disease.

Human herpesvirus-8 (HHV-8) has been associated with Kaposi's sarcoma, multicentric Castleman's disease and primary effusion lymphoma. Kaposi's sarcoma and multicentric Castleman's disease patients may develop body cavity effusions that, unlike primary effusion lymphoma, are poorly characterized. To better define these effusions, pleural and peritoneal fluids derived from 12 human immunodeficiency virus-seropositive and one seronegative patients affected by Kaposi's sarcoma or multicentric Castleman's disease were analyzed by a combination of morphologic, immunophenotypic, and DNA analyses, including polymerase chain reaction amplification of HHV-8, Epstein-Barr virus, and immunoglobulin heavy-chain (IgH) gene sequences. In addition, HHV-8 serologic status was assessed by using an immunofluorescence assay. All patients were adult men with high antibody titers to HHV-8; 11 of the 13 patients were homosexual/bisexual. Effusions revealed monocyte/macrophage-rich infiltration (10 patients) or large-cell lymphoma with CD45(+)/non-T/non-B phenotype (three of 13 patients); polymerase chain reaction analysis showed the presence of HHV-8 sequences (nine of 13 patients), germline IgH (seven of 12 patients) or clonal IgH rearrangements (four of 12 patients), and rarely Epstein-Barr virus sequences (two of 12 patients). In the setting of HHV-8 infection, two effusion types may occur. One fulfills the criteria for HHV-8-positive PEL (lymphoma-morphology, HHV-8-DNA(+), IgH rearrangement). The other seems more reminiscent of an HHV-8-associated nonneoplastic process (monocyte-macrophage morphology, HHV-8-DNA(+/-), germline IgH). Interestingly, a single case of the latter effusion type harbored a B-cell monoclonal proliferation, which suggests the hypothesis that a prelymphomatous effusion may precede overt body cavity lymphoma.

Human herpes virus-8 associated primary effusion lymphoma of the pleural cavity in HIV-negative elderly men.

Human herpes virus-8 (HHV-8)-associated primary effusion lymphoma (PEL) is an unusual lymphoma confined to the body cavities, which primarily affects human immunodeficiency virus (HIV)-positive men at high risk for Kaposi's sarcoma (KS). We describe two HIV-negative elderly Italian men, who developed pleural HHV-8-positive PEL in association with other diseases (systemic hypertension, colonic carcinoma, chronic obstructive airways disease, dilated cardiomyopathy), but without KS. Thoracic computed tomography revealed unilateral pleural effusion and pleural thickening. Thoracentesis disclosed large lymphoma cells, with no T- or B-cell associated antigens, clonal rearrangement of the immunoglobulin heavy chain gene and the presence of HHV-8 but not Epstein-Barr virus deoxyribonucleic acid sequences. Our cases differ from most pleural effusion lymphomas, in that they are non-acquired immunodeficiency syndrome-related. This highlights the possible human herpes virus-8-associated primary effusion lymphoma risk among elderly human immunodeficiency virus-negative patients, particularly Italians, in whom human herpes virus-8 seroprevalence rates and incidence of classic Kaposi's sarcoma are high.

Kaposi's sarcoma following malignant mesothelioma.

We report the unusual occurrence of Kaposi's sarcoma following asbestos-related malignant mesothelioma, in a human deficiency virus (HIV)-negative Italian man. Seropositivity to human herpes virus 8 (HHV8) was documented at the time of mesothelioma diagnosis and preceded the onset of Kaposi' sarcoma with a time lapse of 13 months. HHV8 DNA was detected by polymerase chain reaction in lesional Kaposi's sarcoma but not within mesothelioma. By immunostaining, mesothelioma cells expressed interleukin-6 and platelet-derived growth factor, which are important for survival of Kaposi's sarcoma cells. Besides the possibility of a casual association, we hypothesize that mesothelioma-linked factors may have contributed to the development of Kaposi's sarcoma in the presence of HHV8 infection.

Absence of HHV-8 DNA sequences in malignant mesothelioma.

Human herpesvirus 8 (HHV-8) associated primary effusion lymphomas arise and grow in the body cavities as effusions, but it is not known whether the lining of body cavities and mesothelium derived malignancies are potential targets of HHV-8 infection. We examined a series of 13 diffuse malignant mesotheliomas and four mesothelial cell rich effusion samples of non-neoplastic aetiology from non-immunodepressed patients using the polymerase chain reaction to detect HHV-8 specific sequences. HHV-8 amplification products were absent in diffuse malignant mesotheliomas and in non-neoplastic effusions samples. These results suggest that HHV-8 has a selective tropism among body cavity based tumours, being confined to primary effusion lymphomas.

Early detection of relapse by prospective reverse transcriptase-polymerase chain reaction analysis of the PML/RARalpha fusion gene in patients with acute promyelocytic leukemia enrolled in the GIMEMA-AIEOP multicenter "AIDA" trial. GIMEMA-AIEOP Multicenter "AIDA" Trial.

Although the majority of patients with acute promyelocytic leukemia (APL) are potentially cured by treatments combining all-trans retinoic acid (ATRA) and chemotherapy (CHT), a sizable proportion (around 30%) will relapse during follow-up. Retrospective molecular monitoring studies using reverse transcriptase-polymerase chain reaction (RT-PCR) for the specific PML/RARalpha fusion gene, have shown that a positive test usually precedes the occurrence of hematologic relapse. Prospective RT-PCR analyses were performed since 1993 at diagnosis and at preestablished time intervals during follow-up in bone marrow (BM) samples of 163 patients with PML/RARalpha+ APL enrolled in the multicenter Gruppo Italiano Malattie Ematologiche Maligne dell' Adulto (GIMEMA) trial AIDA (All-trans retinoic acid plus Idarubicin). Treatment consisted of ATRA and idarubicin for induction followed by three polychemotherapy courses as consolidation. The sensitivity level of the RT-PCR assay for PML/RARalpha, as assessed by serial dilution experiments, was 10(-4). All patients were in hematologic remission and tested PCR- at the end of consolidation. Of 21 who converted to PCR-positive thereafter, 20 underwent hematologic relapse at a median time of 3 months (range, 1 to 14) from the first PCR+ result. Seventeen of these 21 (81%) PCR+ conversions were recorded within the first 6 months postconsolidation. Of 142 who tested persistently PCR- in >/=2 tests after consolidation, 8 had hematologic relapse and 134 remained in complete remission (CR) after a median follow-up of 18 months (range, 6 to 38) postconsolidation. Using a time-dependent Cox model, the relative risk of hematologic relapse of patients who converted to PCR+ was 31.8 (confidence limits 95%, 12.9 to 78.3). Our results indicate that conversion to PCR positivity for PML/RARalpha during remission is highly predictive of subsequent hematologic relapse and highlight the prognostic value of stringent molecular monitoring during the early postconsolidation phase in APL. As a result of the present study, salvage treatment in patients enrolled in the GIMEMA trial AIDA is now anticipated at the time of molecular relapse, defined as the conversion to PCR positivity in two successive BM samplings during follow-up.

Primary effusion lymphoma containing human herpesvirus 8 DNA in two AIDS patients with Kaposi's sarcoma.

BACKGROUND AND OBJECTIVE: Primary effusion lymphomas (PELs) containing Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8/HHV-8) DNA sequences represent a distinct but heterogeneous group of rare non-Hodgkin's lymphomas of null-cell phenotype/B-cell origin. We aimed to describe the clinicopathologic features of two human immunodeficiency virus (HIV)-related PELs occurring in homosexual men with Kaposi's sarcoma (KS). DESIGN AND METHODS: Thoracentesis was followed by morphologic plus immunophenotypic studies and molecular analysis of tumor cell DNA by means of combination of polymerase chain reaction and Southern blot analysis. RESULTS: Patients developed recurrent lymphomatous effusions lacking tissue involvement, in the context of severe immunodepression (CD4 count < 60/microL) and anti-retroviral therapy. The effusions disclosed an immunoblast-like population CD45/CD30+, but B-cell- and T-cell-associated antigen negative, showing clonal immunoglobulin heavy chain gene rearrangements and harbouring HHV-8 DNA sequences. One case contained Epstein-Barr virus genome with no evidence of c-myc, bcl-2 and bcl-6 gene alterations. Both patients had aggressive disease. INTERPRETATIONS AND CONCLUSIONS: These cases represent additional examples of PEL associated with HHV-8 and confirm that the group of HIV-positive homosexual men may be at highest risk for PEL.

Pathogenetic and clinical implications of Bcl-6 and Bcl-2 gene configuration in nodal diffuse large B-cell lymphomas.

Bcl-6 (LAZ-3) and Bcl-2 gene rearrangements have been respectively reported in 20-35 per cent and 10-25 per cent of diffuse large B-cell lymphomas (DLBCLs). Although these genetic lesions have been associated with different clinical outcomes (i.e., more favourable in Bcl-6 rearranged cases and poorer in Bcl-2 rearranged cases), their prognostic significance is still controversial. In the present study, we have investigated by Southern blot analysis the Bcl-6 and Bcl-2 gene configuration in a series of 80 lymph nodes involved by well-characterized DLBCLs, histologically defined according to the REAL and the updated Kiel classifications. The molecular findings have been correlated with the clinical features at presentation and with response to therapy. The majority of cases (57/80 = 71.2 per cent) had a centroblastic morphology. Bcl-6 rearrangements were detected in 23/80 cases (28.8 per cent), and were similarly associated with centroblastic (18/57 = 31.6 per cent) or immunoblastic (3/11 = 27.3 per cent) histotypes. In contrast, Bcl-2 was found to be rearranged in only three cases of centroblastic lymphoma (3.8 per cent). No significant differences were found between Bcl-6 rearranged and germline cases, as far as the clinical features at presentation are concerned. Forty-one patients, in whom the lymph node biopsy was performed at diagnosis, could be evaluated for response to treatment and clinical outcome. Most of these cases (30/41 = 73.2 per cent) were nodal DLBCL, without extranodal site involvement. Analysis of the clinical outcome showed no statistically significant differences between Bcl-6 rearranged and Bcl-6 germline cases (actuarial overall survival 50 per cent vs. 48 per cent, event-free survival 45 per cent vs. 46 per cent, at 4 years). These findings confirm that Bcl-6 rearrangements are the most frequent genetic lesion in DLBCL. The incidence of Bcl-2 involvement in our series is significantly lower than the figures reported in other studies, mainly from North American countries, probably reflecting heterogeneous patient selection and/or epidemiological variability. Finally, our results suggest that no relevant clinical differences are observed between Bcl-6 rearranged and Bcl-6 germline cases, when nodal DLBCLs are considered.

Improved rapid detection of the PML/RARalpha fusion gene in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a medical emergency which requires rapid diagnosis and tailored treatment. Detection of the PML/RARalpha fusion gene in APL blasts is critical to start promptly the specific therapy with all-trans retinoic acid (ATRA). APL lacking this genetic lesion have been reported as being ATRA resistant. Reverse-transcription polymerase chain reaction (RT-PCR) has been extensively used to detect the PML/RARalpha cDNA. The reported PML/RARalpha amplification techniques are laborious and time consuming, and include conventional RNA extraction, cDNA synthesis and a two-round (nested) PCR. We hereby describe a few variations of the commonly adopted RNA extraction and PML/RARalpha RT-PCR protocols which allow a molecular diagnosis of APL to be carried out in less than 5 h. Processing of small volumes of leukemic cell lysate (0.5 ml) in a microfuge allows extraction of good quality RNA in 1 h. After reverse transcription to obtain cDNA, a 'hot start' PCR procedure was adopted which enabled us to amplify clearly visible and specific products after a single (not nested) amplification round. The PML/RARalpha fusion gene was detected in the blasts of six consecutive APL at diagnosis, and an APL-tailored protocol including ATRA was started in each case within 6 h of admission. On repeated experiments, the assay proved highly specific and sensitive for the rapid detection of all PML/RARalpha transcript types. Our data should encourage the use of this rapid procedure for the diagnosis of both typical APL and, particularly, less typical cases awaiting urgent therapeutic intervention.

Effect of priming with diphtheria and tetanus toxoids combined with whole-cell pertussis vaccine or with acellular pertussis vaccine on the safety and immunogenicity of a booster dose of an acellular pertussis vaccine containing a genetically inactivated pertussis toxin in fifteen- to twenty-one-month-old children. Italian Multicenter Group for the Study of Recombinant Acellular Pertussis Vaccine.

OBJECTIVE: To evaluate the safety and the immunogenicity of a booster dose of recombinant acellular pertussis vaccine combined with diphtheria and tetanus toxoids (DTaP, Biocine SpA) in 15- to 21-month-old children primed in infancy with either whole-cell diphtheria-tetanus-pertussis (DTwP) vaccine or DTaP vaccine. DESIGN: Open-label second phase of a double-masked, controlled trail, with masked analysis of serum samples. PARTICIPANTS AND SETTING: Three hundred fifty children, 15 to 21 months of age, who had been primed at 2, 4, and 6 months of age with either three doses of DTaP vaccine (n = 173) or DTwP vaccine (n = 177). The children were enrolled in eight vaccination centers in Italy. INTERVENTIONS: All children received a booster dose of the DTaP vaccine and were examined for safety at 48 hours and at 7 days after vaccination. Serum samples for evaluation of immunogenicity were obtained from 196 (55%) of the 350 children. MAIN OUTCOME MEASURES: IgG antibodies to pertussis toxin (Ptox), filamentous hemagglutinin, 69-kilodalton protein, and tetanus toxoid were measured by enzyme-linked immunosorbent assay. Pertussis toxin-neutralizing antibodies were measured by the Chinese hamster ovary cell toxin neutralization assay. MAIN RESULTS: Adverse reactions to DTaP were infrequent, and there was no difference in the incidence of local or systemic reactions in children given DTaP as a fourth dose in comparison with a first dose. One month after the DTaP booster vaccination, both groups had 6- to 40-fold increases in serum antibody concentrations to all antigens tested; the concentrations against the three pertussis antigens were higher in the DTaP-primed children (p < 0.05). The antibody titers to diphtheria and tetanus toxoids were higher in the DTwP-primed group (p < 0.05), but both groups had protective titers. The geometric mean ratio of anti-Ptox neutralizing antibody per unit of IgG anti-Ptox antibody was higher in the DTaP-primed group (p < 0.001). CONCLUSIONS: There are quantitative and qualitative differences in booster responses to DTaP vaccine in young children, depending on whether they were given DTaP or DTwP as primary immunization. This DTaP vaccine is safe and highly immunogenic as a booster.

[Duodenal ulcer. Gastroscopic aspects before and after acupuncture treatment]. / Ulcera duodenale. Aspetti gastroscopici prima e dopo trattamento agopunturale.

4 cases of duodenal ulcer, diagnosed by gastroscopic examinations three of which were already treated medically with insufficient results, are presented. Acupuncture was carried out several times (about 5 to 7) during 1-2 months and gave rise to a complete disappearance of the clinical symptoms and complete healing of the former lesion was diagnosed through gastroscopy