Biomedical subjects
Publications and source records attributed to A Aiello.
Three sulfated alkene and alkanes-(R)-2,6-dimethylheptyl sulfate (1), 6-methylheptyl sulfate (2a), and (E)-5-octenyl sulfate (3a)-with cytotoxic activity in vitro, have been isolated from the Mediterranean ascidian Halocynthia papillosa. The structures of the new compounds 2a and 3a have been elucidated by spectroscopic analysis.
A new N-methylpyridinium alkaloid, with an interesting antiproliferative activity in vitro, has been isolated from the Mediterranean tunicate Microcosmus vulgaris. Its structure has been elucidated by spectroscopic analysis, including extensive 2D NMR experiments.
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A 44-year-old woman with a 12-year history of Sjögren's syndrome (SS) developed a low-grade mucosa-associated lymphoid tissue (MALT) lymphoma in the parotid gland. Two years later, she presented with generalized lymphadenopathy and hepatosplenomegaly and a follicular lymphoma was diagnosed. To investigate the relationship of the two histologically distinct lymphomas, we re-examined their histology and immunophenotype and studied the lymphomatous tissue from the parotid, cervical lymph node, and spleen using molecular genetic methods. Histologic and immunophenotypic studies confirmed the previous diagnoses and also identified a previously unnoticed focus of follicular lymphoma in the second parotid gland biopsy. Polymerase chain reaction (PCR) amplification of the rearranged Ig heavy-chain gene showed the same sized dominant product in the MALT lymphoma and the follicular lymphoma. Similarly, PCR analysis of the t(14:18) translocation yielded an identical sized band from both MALT and follicular lymphoma. Cloning and sequencing of the Ig PCR products showed an identical CDR3 sequence from each lesion, indicating a common clonal lineage. The follicular lymphoma of the parotid gland lymph node and the follicular lymphoma of the spleen showed an identical mutation signature to that of the salivary gland MALT lymphoma. We propose that follicular lymphoma in the parotid gland lymph node may have resulted from colonization of lymphoid follicles by MALT lymphoma cells, following which the tumor cells were induced to express a follicular lymphoma phenotype, due to Bcl-2 overexpression caused by t(14;18), leading to a change in clinical behavior resulting in rapid widespread dissemination of disease. These observations suggest that the distinct phenotypes of low-grade B-cell lymphomas may be the consequence of interplay between genetic and local microenvironmental factors.
A distinct pathologic entity (ALK+ lymphoma) that is characterized by expression of the anaplastic lymphoma kinase (ALK) protein has recently emerged within the heterogeneous group of CD30(+) anaplastic large-cell lymphomas. Information on clinical findings and treatment outcome of ALK+ lymphoma is still limited, and no data are available concerning the value of the International Prognostic Index when applied to this homogeneous disease entity. To clarify these issues, a recently developed monoclonal antibody ALKc (directed against the cytoplasmic portion of ALK) was used to detect expression of the ALK protein in paraffin-embedded biopsies from 96 primary, systemic T/null anaplastic large-cell lymphomas, and the ALK staining pattern was correlated with morphological features, clinical findings, risk factors (as defined by the International Prognostic Index), and outcome in 78 patients (53 ALK+ and 25 ALK-). Strong cytoplasmic and/or nuclear ALK positivity was detected in 58 of 96 ALCL cases (60.4%), and it was associated with a morphological spectrum (common type, 82.7%; giant cell, 3.5%; lymphohistiocytic, 8. 6%; and small cell, 5.2%) that reflected the ratio of large anaplastic elements (usually showing cytoplasmic and nuclear ALK positivity) to small neoplastic cells (usually characterized by nucleus-restricted ALK expression). Clinically, ALK+ lymphoma mostly occurred in children and young adults (mean age, 22.01 +/- 10.87 years) with a male predominance (male/female [M/F] ratio, 3.0) that was particularly striking in the second-third decades of life (M/F ratio, 6.5) and usually presented as an aggressive, stage III-IV disease, frequently associated with systemic symptoms (75%) and extranodal involvement (60%), especially skin (21%), bone (17%), and soft tissues (17%). As compared with ALK+ lymphoma, ALK- cases occurred in older individuals (mean age, 43.33 +/- 16.15 years) and showed a lower M/F ratio (0.9) as well as lower incidence of stage III-IV disease and extranodal involvement at presentation. Overall survival of ALK+ lymphoma was far better than that of ALK- anaplastic large-cell lymphoma (71% +/- 6% v 15% +/- 11%, respectively). However, within the good prognostic category of ALK+ lymphoma, survival was 94% +/- 5% for the low/low intermediate risk group (age-adjusted International Prognostic Index, 0 to 1) and 41% +/- 12% for the high/high intermediate risk group (age-adjusted International Prognostic Index, >/=2). Multivariate analysis identified ALK expression and the International Prognostic Index as independent variables that were able to predict survival among T/null primary, systemic anaplastic large-cell lymphoma. Thus, we suggest that such parameters should be taken into consideration for the design of future clinical trials.
Somatic hypermutation is the most critical mechanism underlying the diversification of Ig genes. Although mutation occurs specifically in B cells during the germinal center reaction, it remains a matter of debate whether the mutation machinery also targets non-Ig genes. We have studied mutations in the 5' noncoding region of the Bcl6 gene in different subtypes of lymphomas. We found frequent hypermutation in follicular lymphoma (25 of 59 = 42%) (germinal center cell origin) and mucosa-associated lymphoid tissue (MALT) lymphoma (19 of 45 = 42%) (postgerminal center), but only occasionally in mantle cell lymphoma (1 of 21 = 4.8%) (pregerminal center). Most mutations were outside the motifs potentially important for transcription, suggesting they were not important in lymphomagenesis but may, like Ig mutation, represent an inherent feature of the lymphoma precursor cells. Therefore, we investigated their normal cell counterparts microdissected from a reactive tonsil. Bcl6 mutation was found in 13 of 24 (54%) clones from the germinal centre but only in 1 of 24 (4%) clones from the naive B cells of the mantle zone. The frequency, distribution, and nature of these mutations were similar to those resulting from the Ig hypermutation process. The results show unequivocal evidence of non-Ig gene hypermutation in germinal center B cells and provide fresh insights into the process of hypermutation and lymphomagenesis.
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AIMS: We evaluated polymerase chain reaction (PCR) amplification of specific immunoglobulin heavy chain (IgH) gene rearrangements as a means of demonstrating monoclonality during follow-up of conservatively treated gastric MALT lymphoma, and compared the reproducibility of PCR on sequential frozen and paraffin-embedded endoscopic biopsies. We established an association between clonality detected by PCR and the histological observations. METHODS AND RESULTS: Sixty-nine pairs of sequential frozen and paraffin-embedded endoscopic biopsies from 21 conservatively treated patients were graded according to the Wotherspoon-Isaacson histological scoring system, which provides a measure of diagnostic confidence on a scale 0-5. PCR amplification of the IgH gene was performed using FR3/JH and FR2/JH primers. 68/69 paired samples (98.5%) showed identical mono- or polyclonal PCR amplification patterns. Forty-seven out of 48 pairs of samples sharing similar histological features produced identical amplification patterns in both fresh and paraffin-embedded tissues. In comparison with the histological grading, monoclonality was detected in 64.2% and 41.6% of samples scored 5 and 4, respectively. Conversely, among 64 samples scored 0-3, a monoclonal pattern was observed only in two samples, one of which was from a patient who relapsed 9 months later. CONCLUSIONS: PCR-based clonality analysis by demonstration of specific IgH gene rearrangement can be easily and reliably performed on both frozen and paraffin-embedded endoscopic biopsies. In conjunction with histological observation, this method can be used as a complementary tool to monitor MALT lymphoma regression during conservative treatment.
In a recent paper (Lampariello: Cytometry 15:294-301, 1994), we proposed a method for the automated evaluation of the percentage of positive cells from flow cytometric immunofluorescence histograms. The method is based on a suitable mathematical representation of the control histogram, which is used to identify the negative cell distribution in the test histogram. In this paper we present an improvement of the previous method, where we assume that the positive cell distribution in the test can also be modeled making use of an empirical distribution of the same kind as employed for modeling the control. The parameters of this distribution are estimated directly from the test. In this way, a mathematical representation of the whole test distribution is calculated without having to set up a purely positive control. In order to evaluate the accuracy of the method in the determination of the positive percentage, we carried out a set of measurements of double-labeled and suitably treated cells, mixed in different ratios with control cells, and from each sample we obtained histograms with overlapped and well-separated positive and negative distributions. These last histograms allow us to determine the actual positive percentages and thus to evaluate the performance of the analysis method applied to the histograms with overlapped distributions.
Follicular lymphomas are thought to arise from the follicle center B cells and are characterized by follicular structures that recapitulate many features of normal secondary lymphoid follicles. The neoplastic B cells of follicular lymphoma reside not only in follicles but also in the interfollicular zone in which they form a diffuse infiltrate. We have investigated the frequency, extent, and biological characteristics of this interfollicular component in 30 cases of follicular lymphoma. An interfollicular B-cell infiltrate of variable extent (minimal, moderate, or prominent) was present in all cases. Morphologically interfollicular neoplastic B cells were small centrocyte-like cells with lower grade cytology and lower proliferation fraction compared with the neoplastic follicles. The neoplastic phenotype of these cells (CD20+, light chain restricted) was confirmed in 18 cases. Clonal identity between the follicular and interfollicular components was shown in five cases using microdissection and PCR amplification of immunoglobulin heavy chain genes. Analysis of Ig heavy chain gene sequences showed identical variants of tumor subclones in both follicular and interfollicular compartments, indicating active tumor cell traffic between the two. In six cases in which frozen tissue was available, the immunophenotype of follicular and interfollicular tumor cells were compared using immunohistochemistry. Activation markers such as CD10, CD38, and CD95 and T-cell costimulatory molecules CD80 and CD86, which were expressed by neoplastic follicles, were either downregulated or absent in the interfollicular component in most of the cases. The low-grade cytological features, low proliferation fraction, and downregulation of activation markers in the interfollicular neoplastic B cells suggests that these are resting cells analogous to memory B cells of normal lymphoid tissues. The presence of such a resting tumor cell subpopulation in the majority of follicular lymphomas may partly account for the remarkable resistance to therapy of this disease.
Mutation of the bax gene has been reported previously in lymphoid cell lines. In vitro experiments have shown that alterations in promoter and coding sequences of the gene abolish its apoptosis initiation function, which is considered crucial for tumour development. To assess bax gene mutations in lymphomagenesis, polymerase chain reaction-based single strand conformation polymorphism analysis (PCR-SSCP) and direct sequencing were used to detect altered sequences in the promoter region and all the six exons and their flanking sequences of the gene. Nodal and extranodal B-cell lymphomas (n = 112) including follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, splenic marginal zone B-cell lymphoma and low- and high-grade mucosa-associated lymphoid tissue (MALT) lymphomas were studied. Sequence alterations were found in 11 cases. Nine also showed the same altered sequences in corresponding non-tumour control tissue samples, indicating polymorphism. In the remaining two cases, sequence alterations (in exons 3 and 6) which altered the bax open reading frame were observed only in tumour tissues, indicating tumour-specific point mutation. These results suggest that inhibition of apoptosis through bax gene mutations is unlikely to be a common event in B-cell lymphoma, at least in the major types of nodal and extranodal B-cell lymphomas.
A study carried out on primary school population in Campobasso district permitted us to evaluate the goitre prevalence and to effect advertising campaign about use of iodate-salt. In six months of 1997 we effected a screening of 7041 children aged 6-11 years. The project provided a clinical stage (goitre observation by means of inspection and palpation, auxological data picking) and an informative stage (divulgation of informative material, conferences with the teaching staff, television publicity). We attributed a score to the thyroid volume according to WHO classification. Among the 7041 children, 2093 (29.7%) presented goitre of 1A degree and 84 (1.2%) a goitre 1B. Our results showed in Molise a thyroid pathology prevalence higher to the threshold (10%) over that we can define a population affected with endemic goitre. From that the importance of screening programmes in the whole region and the promotion of interventions of iodine-prophylaxis.
The radiation response was investigated in two lymphoblastoid cell lines (LBC) derived from families with heterozygous germ-line missense mutations of p53 at codon 282 (LBC282) and 286 (LBC286), and compared to cells with wt/wt p53(LBC-N). By gel retardation assays, we show that p53-containing nuclear extracts from irradiated LBC282 and LBC286 markedly differ in their ability to bind to a p53 DNA consensus sequence, the former generating a shifted band whose intensity is 30-40% that of LBC-N, the latter generating an almost undetectable band. Unlike LBC286, which fail to arrest in G1 after irradiation, LBC282 have an apparently normal G1/S checkpoint, as they arrest in G1, like LBC-N. While in LBC-N, accumulation of p53 and transactivation of p21WAF1 increase rapidly and markedly by 3 h after exposure to gamma-radiation, in LBC286 there is only a modest accumulation of p53 and a significantly delayed and quantitatively reduced transactivation of p21WAF1. Instead, in LBC282 while p53 levels rise little after irradiation, p21WAF1 levels increase rapidly and significantly as in normal LBC. Apoptotic cells present 48 h after irradiation account for 32% in LBC-N, 8-9% in LBC282 and 5-7% in LBC286, while the dose of gamma-radiation required for killing 50% of cells (LD50) is 400 rads, 1190 rads and 3190 rads, respectively, hence indicating that the heterozygous mutations of p53 at codon 282 affects radioresistance and survival, but not the G1/S cell cycle control. In all LBC tested, radiation-induced apoptosis occurs in all phases of the cell cycle and appears not to directly involve changes in the levels of the apoptosis-associated proteins bcl-2, bax and mcl-1. Both basal as well as radiation-induced p53 and p21WAF1 proteins are detected by Western blotting of FACS-purified G1, S and G2/M fractions from the three cell lines. p34CDC2-Tyr15, the inactive form of p34CDC2 kinase phosphorylated on Tyr15, is found in S and G2/M fractions, but not in G1. However, 24 h after irradiation, its levels in these fractions diminish appreciably in LBC-N but not in the radioresistant LBC286 and LBC282. Concomitantly, p34CDC2 histone H1 kinase activity increases in the former, but not in the latter cell lines, hence suggesting a role for this protein in radiation-induced cell death. Altogether, this study shows that, in cells harbouring heterozygous mutations of p53, the G1 checkpoint is not necessarily disrupted, and this may be related to the endogenous p53 heterocomplexes having lost or not the capacity to bind DNA (and therefore transactivate target genes). Radiation-induced cell death is not cell cycle phase specific, does not involve the regulation of bcl-2, bax or mcl-1, but is associated with changes in the phosphorylation state and activation of p34CDC2 kinase.
The cancer chemopreventive synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) possesses antiproliferative and apoptotic activity at pharmacological doses. In this study we show that addition of antioxidants to HL-60 cells cultured in the presence of 3 microM HPR, markedly suppresses the apoptopic effect of the retinoid and significantly prolongs cell survival (48-96 h). We also show, by the use of the oxidation-sensitive probe 2',7'-dichlorofluorescin diacetate (DCF-DA) and in combination with flow cytometric and spectrofluorimetric analysis, that treatment of cells with 3 microM HPR results in an immediate and sustained production of intracellular free radicals, most likely hydroperoxides. Interestingly, the formation of these HPR-induced free radicals is effectively blocked by the water soluble antioxidants L-ascorbic acid and N-acetyl-L-cysteine. Neither 3-15 microM N-(4-methoxyphenyl) retinamide (MPR), the structurally similar but biologically inert analog of HPR, nor 3 microM doses of the retinoids all-trans retinoic acid, 9-cis-retinoic acid, TTNPB and SR11237 induce intracellular free radicals, thus indicating that the specificity of this phenomenon is restricted to HPR. Altogether, we provide the first direct evidence that HPR stimulates the generation of intracellular free radicals, which appear to have a causative role in the induction of apoptosis in vitro. Our findings raise the possibility that the therapeutic efficacy of HPR may, at least in part, depend on these apoptosis-inducing oxidative phenomena.
In order to improve the cytomorphologic diagnosis of malignant lymphoma on lymph node fine-needle aspiration (FNA), and to make a confident discrimination between low-grade follicular non-Hodgkin's lymphoma (NHL) and lymphoid hyperplasia, polymerase chain reaction (PCR) analysis was performed of the Ig CDR3 region and BCL2 breakpoint region in 25 nonselected cases of malignant lymphoma (17 NHL and 8 Hodgkin's disease [HD]) with histologic control, and 22 cases of lymph nodal hyperplasia with histologic and/or clinical control. Among lymphomas, IgH monoclonality was detected in 7 (77%) of 9 NHLs and BCL2 rearrangement in 3 (17.6%) of 17 NHLs, all of which were follicular centroblastic-centrocytic (FCBCC). Three BCL2/JH negative FCBCC cases were monoclonal for CDR3. Neither IgH monoclonality nor BCL2 rearrangement were found in HD. Among cytologically diagnosed lymphoid hyperplasias, one IgH polyclonal case was considered false-negative, being histologically diagnosed as lymphoplasmacytic NHL on the subsequent excisional biopsy. Another 4 cases (2 BCL2 rearranged and 2 monoclonal for IgH) were considered false-positive on the basis of histologic features or clinical control. These data indicate that the combined PCR analysis of IgH and BCL2 rearrangements can confirm a cytologic diagnosis of lymphoma in FNAs while, due to the occurrence of both false-positive and false-negative results, it is of limited value in the distinction between follicular lymphoma and lymphoid hyperplasia without morphologic or clinical support.
The A-myb gene encodes a transcription factor that is related both functionally and structurally to the v-myb oncogene. Following our observations that A-myb is expressed in a restricted subset of normal mature human B lymphocytes, with the phenotype CD38+, CD39-, slgM-, we have now investigated the pattern of A-myb expression in neoplastic B cells representating the whole spectrum of B-cell differentiation and compared it to that of c-myb and B-myb. In a panel of 32 B-cell lines, A-myb was very strongly expressed in most Burkitt's lymphoma (BL) cell lines, but weak or negative in 2 pre-B acute lymphoblastic leukemia (ALL), 4 non-Hodgkin's lymphoma (NHL), 6 Epstein-Barr virus-immortalized lymphoblastoid cell lines, and 6 myeloma lines. Protein expression paralleled that of the RNA. We have also investigated A-myb expression in 49 fresh cases of B leukemias. Among 24 ALL, 6 were of the null and 11 of the common type and all these were negative for A-myb expression; on the other hand, all 7 B-ALL cases (slg+), as well as one fresh BL case with bone marrow infiltration, expressed A-myb. A-myb was undetectable in 4 prolymphocytic leukemias (PLL) but was strongly expressed in 5/20 (25%) of chronic lymphocytic leukemia (CLL) samples. In the latter A-myb did not correlate with phenotype or clinical stage. Finally, we have studied the progression of one case of CLL into Richter's syndrome and have found that the Richter's cells expressed about 25-fold less A-myb RNA than the CLL cells from the same patient. The pattern of c-myb and B-myb was clearly distinct from that of A-myb. C-myb and B-myb were expressed in all neoplastic groups, except in CLL cells. Thus, A-myb expression, unlike that of c-myb and B-myb, is restricted to a subset of B-cell neoplasias (in particular BL and slg+B-ALL) representative of a specific stage of B-cell differentiation. This expression may in part reflect expression of A-myb by the normal germinal center B cells that are the normal counterpart of these transformed B cells. The data presented strongly support a role for this transcription factor in B-cell differentiation and perhaps in B-cell transformation in some neoplasias.
The cancer chemopreventive retinoid N-(4-hydroxyphenyl)-all-trans retinamide (HPR) was recently shown by us to have antiproliferative and apoptotic effects on human leukemic cell lines, including those unresponsive to all-trans retinoic acid (ATRA). We have now characterized further the process of HPR-induced cell death. We report that inhibitors of RNA transcription and of protein synthesis, activators of protein kinase C (PKC), inhibitors of tyrosine kinases, Zn++, and the antioxidants acetylcysteine, ascorbic acid, alpha-tocopherol, and deferoxamine suppressed HPR-induced apoptosis. HL60 cells induced toward monocytic differentiation by 1,25 dihydroxyvitamin-D3 [1,25(OH)2D3], but not those induced toward the granulocytic differentiation by ATRA, showed reduced responses to HPR. The transport of HPR by cells with different sensitivity to the retinoid, however, was similar, even after treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), which induces unresponsiveness to HPR. The expression of the apoptosis-related genes bcl-2, p53, and c-myc was examined to determine their role in HPR-triggered cell death. The levels of bcl-2 mRNA were markedly diminished by 24 hours of HPR treatment in all cell lines except in the relatively HPR-insensitive line K422. However, probably because of its long half-life, bcl-2 protein levels were either unchanged or only slightly decreased. Downregulation of p53 mRNA was also observed within 24 hours of HPR exposure in NB4 but not K422 cells, but no changes in the amount of p53 protein were found. Suppression of c-myc transcription was observed in all cells except K422. The protective role of bcl-2 on cell death by HPR was investigated in HL60 as well as 697 pre-B leukemia and Jurkat T-acute lymphocytic leukemia (T-ALL) cells constitutively expressing high levels of bcl-2 proteins due to gene transfer manipulation. Compared with control cells, the onset of apoptosis in these cells with deregulated bcl-2 production was delayed by at least 24 hours. These findings establish that cell death by HPR requires RNA transcription and protein synthesis and is regulated by the activation of PKC. Although changes in bcl-2, p53, and c-myc expression are found in cells treated with HPR, the time-course of these events suggests that HPR-triggered apoptosis is not directly controlled by these genes. Finally, while ectopic overexpression of bcl-2 does not protect cells from death by HPR, it markedly delays its onset.(ABSTRACT TRUNCATED AT 400 WORDS)
The aim of our work was to study apoptosis as a possible mechanism of CD4+ lymphocyte depletion in AIDS patients and to test whether IL-12 could limit this phenomenon. As an in vitro model, we used a human IL-2-dependent Th1 clone from an uninfected individual. We found that CD4 cross-linking, obtained either by mouse anti-CD4 mAb plus goat anti-mouse or by recombinant gp120 plus anti-gp120 mAb, followed by activation with immobilized anti-CD3 or anti-TCR mAb, induced apoptosis at early times (15-25% apoptotic cells at 4 hr), whereas IL-2 deprivation required longer times (20-40 hr) to induce apoptosis. Both CD4 cross-linking and IL-2 deprivation-induced apoptosis appeared to be PTK-dependent and were inhibited by either IL-2 or IL-12. Our data suggest that in vivo CD4/gp120 interactions could directly prime the apoptosis of Th1 lymphocytes and that IL-2 and IL-12 could be used to prevent this phenomenon.