Decoupling of normal CD40/interleukin-4 immunoglobulin heavy chain switch signal leads to genomic instability in SGH-MM5 and RPMI 8226 multiple myeloma cell lines.

The processes mediating genomic instability and clonal evolution are obscure in multiple myeloma (MM). Acquisition of new chromosomal translocations into the switch region of the immunoglobulin heavy chain (IgH) gene (chromosome 14q32) in MM, often heralds transformation to more aggressive disease. Since the combined effects of CD40 plus interleukin-4 (IL-4) mediate IgH isotype class switch recombination (CSR), and this process involves DNA double strand break repair (DSBR), we hypothesized that CD40 and/or IL-4 activation of MM cells could induce abnormal DNA DSBR and lead to genomic instability and clonal evolution. In this study, we show that MM cell lines that are optimally triggered via CD40 and/or IL-4 demonstrate abnormal decoupling of IL-4 signal transduction from CD40. Specifically, CD40 alone was sufficient to trigger maximal growth of tumor cells. We further demonstrate that CD40 triggering induced both DNA DSBs as well as newly acquired karyotypic abnormalities in MM cell lines. Importantly, these observations were accompanied by induction of activation induced cytidine deaminase expression, but not gross apoptosis. These data support the role of abnormal CD40 signal transduction in mediating genomic instability, suggesting a role for the CD40 pathway and intermediates in myelomagenesis and clonal evolution in vivo.

Structural basis for the interaction of the free SH2 domain EAT-2 with SLAM receptors in hematopoietic cells.

The T and natural killer (NK) cell-specific gene SAP (SH2D1A) encodes a 'free SH2 domain' that binds a specific tyrosine motif in the cytoplasmic tail of SLAM (CD150) and related cell surface proteins. Mutations in SH2D1A cause the X-linked lymphoproliferative disease, a primary immunodeficiency. Here we report that a second gene encoding a free SH2 domain, EAT-2, is expressed in macrophages and B lympho cytes. The EAT-2 structure in complex with a phosphotyrosine peptide containing a sequence motif with Tyr281 of the cytoplasmic tail of CD150 is very similar to the structure of SH2D1A complexed with the same peptide. This explains the high affinity of EAT-2 for the pTyr motif in the cytoplasmic tail of CD150 but, unlike SH2D1A, EAT-2 does not bind to non-phosphorylated CD150. EAT-2 binds to the phosphorylated receptors CD84, CD150, CD229 and CD244, and acts as a natural inhibitor, which interferes with the recruitment of the tyrosine phosphatase SHP-2. We conclude that EAT-2 plays a role in controlling signal transduction through at least four receptors expressed on the surface of professional antigen-presenting cells.

CD150 is a member of a family of genes that encode glycoproteins on the surface of hematopoietic cells.

Human CD150 (SLAM) is a glycoprotein expressed on the surface of T, B, natural killer, and dendritic cells. The extracellular domain of CD150 is the receptor for measles virus and CD150 acts as a co-activator on T and B cells. We characterized the mouse and human CD150 genes, each of which comprises seven exons spanning approximately 32 kb. Mouse CD150 mRNA was detected in T cells and in most thymocyte subsets, except CD4-8- cells. Surprisingly, the CD4-8- thymocytes of CD3gammadeltanull mice, but not of Ragnull or severe combined immunodeficiency mice, expressed CD150. Whereas high levels of CD150 were found in Th1 cells, only small amounts were detectable in Th2 cells. CD150 expression was up-regulated upon in vitro activation of mouse T cells by anti-CD3. The complete mouse CD150 gene is highly homologous to its human orthologue in terms of nucleotide sequences and intron/exon organization. The human genomic sequences indicate that all isoforms detected so far have arisen from alternative splicing events. As judged by fluorescence in situ hybridization, mouse CD150 mapped to Chromosome (Chr) 1, band 1H2.2-2.3, and human CD150 was found on Chr 1q22. Human and mouse CD150 share sequence homologies with six other genes, five of which - CD84, CD229 (Ly-9), CD244 (2B4), CD48, and 19A - are localized in a 250-kb segment in close proximity to the human gene. Their location and their sequence similarities strongly suggest that the CD150 family of cell surface receptors arose via successive duplications of a common ancestral gene.

SAP controls T cell responses to virus and terminal differentiation of TH2 cells.

SH2D1A, which encodes signaling lymphocyte activation molecule (SLAM)-associated protein (SAP), is altered in patients with X-linked lymphoproliferative disease (XLP), a primary immunodeficiency. SAP-deficient mice infected with lymphocytic choriomeningitis virus had greatly increased numbers of CD8+ and CD4+ interferon-gamma-producing spleen and liver cells compared to wild-type mice. The immune responses of SAP-deficient mice to infection with Leishmania major together with in vitro studies showed that activated SAP-deficient T cells had an impaired ability to differentiate into T helper 2 cells. The aberrant immune responses in SAP-deficient mice show that SAP controls several distinct key T cell signal transduction pathways, which explains in part the complexity of the XLP phenotypes.

Generation of IL-2-dependent cytolytic T lymphocytes (CTLs) with altered TCR responses derived from antigen-dependent CTL clones.

Ag-specific CD8+ CTL clones require TCR stimulation to respond to IL-2 for growth. Because IL-2 may be produced in the vicinity of CD8+ CTLs when Ag is limiting at the end of an immune response, we have examined the effect of culturing viral-specific CTL clones in IL-2 in the absence of antigenic stimulation. Limiting dilution analysis revealed a high precursor frequency for CTL clones derived from IL-2 propagation (termed CTL-factor dependent (FD)) that are dependent upon exogenous IL-2 for growth and survival and no longer require TCR stimulation to proliferate. Culturing CTL-FDs with infected splenocytes presenting Ag and IL-2 did not revert the clones but did lead to a TCR-induced inhibition of proliferation. The derived CTL-FDs have lost the ability to kill via the perforin/granule exocytosis mechanism of killing, although they express similar levels of TCR, CD3epsilon, CD8alphabeta, CD45, and LFA-1 compared with the parental clones. The CTL-FDs retain Fas ligand/Fas-mediated cytotoxicity, and IFN-gamma production and regulate the expression of CD69 and IL-2Ralpha when triggered through the TCR. A parental CTL protected BALB/c mice from a lethal challenge of influenza virus, whereas a CTL-FD did not. These findings represent a novel regulatory function of IL-2 in vitro that, if functional in vivo, may serve to down-regulate cellular immune responses.

Ca2+ signaling modulates cytolytic T lymphocyte effector functions.

Cytolytic T cells use two mechanisms to kill virally infected cells, tumor cells, or other potentially autoreactive T cells in short-term in vitro assays. The perforin/granule exocytosis mechanism uses preformed cytolytic granules that are delivered to the target cell to induce apoptosis and eventual lysis. FasL/Fas (CD95 ligand/CD95)-mediated cytolysis requires de novo protein synthesis of FasL by the CTL and the presence of the death receptor Fas on the target cell to induce apoptosis. Using a CD8(+) CTL clone that kills via both the perforin/granule exocytosis and FasL/Fas mechanisms, and a clone that kills via the FasL/Fas mechanism only, we have examined the requirement of intra- and extracellular Ca2+ in TCR-triggered cytolytic effector function. These two clones, a panel of Ca2+ antagonists, and agonists were used to determine that a large biphasic increase in intracellular calcium concentration, characterized by release of Ca2+ from intracellular stores followed by a sustained influx of extracellular Ca2+, is required for perforin/granule exocytosis. Only the sustained influx of extracellular Ca2+ is required for FasL induction and killing. Thapsigargin, at low concentrations, induces this small but sustained increase in [Ca2+]i and selectively induces FasL/Fas-mediated cytolysis but not granule exocytosis. These results further define the role of Ca2+ in perforin and FasL/Fas killing and demonstrate that differential Ca2+ signaling can modulate T cell effector functions.

IL-2 induces Fas ligand/Fas (CD95L/CD95) cytotoxicity in CD8+ and CD4+ T lymphocyte clones.

IL-2 is a T cell growth factor that has pleiotropic functions in T cell differentiation, induction of lymphokine-activated killer cells, and regulation of immune responses. In studying TCR triggering of perforin or Fas ligand (FasL)/Fas (CD95 ligand/CD95) cytotoxicity in our influenza-specific T cell clones, we found that IL-2 can also induce FasL/Fas cytotoxicity. IL-2 induces FasL/Fas cytotoxicity in our CD8+ and CD4+ Th1 clones, but not in our CD4+ Th2 clones. IL-2 induction of cytolytic activity occurs when the CD8+ T cells are refractory to IL-2-induced proliferation. This killing is Ag independent, MHC unrestricted, and blocked by Fas.Fc fusion protein. IL-2 induces FasL/Fas cytotoxicity in a dose-dependent manner, but does not induce high levels of FasL expression as detected by flow cytometry. TCR triggered FasL/Fas cytotoxicity is detectable in CD8+ and Th1 clones by 3 h and peaks at 6 h; high levels of killing are maintained for at least 24 h. Similarly, IL-2 induces FasL/Fas killing in CD8+ and Th1 clones within 3 h of stimulation and maintains high levels for at least 24 h. TCR-triggered FasL/Fas killing is inhibited by emetine and cyclosporin A, whereas IL-2-induced FasL/Fas killing is inhibited by emetine, but not by cyclosporin A. These results demonstrate a second mechanism to induce FasL/Fas cytotoxicity in CD8+ and Th1 clones and may explain IL-2 induction of Ag-independent MHC-unrestricted lymphokine-activated killer cell activity.

[Endoscopic polypectomy of the stomach]. / Polipectomia endoscopica nello stomaco.

Until 1970 endoscopy of the digestive tract was the only diagnostic method available. After this date, the introduction of endoscopic polypectomy enabled patients affected by these lesions to be treated, and at the same time considerably reduced their length of stay in hospital. The paper reports the results of 346 gastric polypectomies performed in 187 patients from 1974 to the present.

[Laparoscopy in oncological pediatrics]. / La laparoscopia in pediatria oncologica.

Laparoscopy permits direct inspection of the peritoneal cavity. Its role is very important in oncology both in the adult and in the pediatric patient. From 1973 to 1985, 147 laparoscopies were performed on 137 patients, aged 2-16; indications were: definition of the nature of neoplastic disease in 8 cases, staging of known neoplasia in 132, restaging in 7. On Hodgkin's disease, splenic involvement in 8/113 cases was revealed by laparoscopy with biopsy. Histological examination was positive in 4 out of 22 cases of ovarian tumors. Morbidity was 1.2%. Although a laparotomy performed after laparoscopy has shown some false negatives, in most cases the clinical and instrumental follow-up has proved reliability of laparoscopic examination.

Esophageal carcinoma. Radiologic appearance of minimal lesions.

Twelve minimal lesions were found over a period of 18 months at the Istituto Nazionale Tumori of Milan at the end of the radiologic, endoscopic and histologic procedures. Eleven lesions were radiologically detected, and a radiologic diagnosis of malignancy was perspectively made in 10 of the identified lesions. The radiologic aspects of minimal lesions are described. Double contrast study of the esophagus allows excellent mucosal detail and good reproduction of lesions. Simplicity and minimal discomfort for the patient justify it as a first diagnostic step for detection of neoplastic pathology.

Endoscopic diagnosis of gastric lymphomas.

From January 1970 to June 1979, 180 gastroscopies were performed in patients affected by lymphoma (168 in patients with non-Hodgkin lymphoma and 12 in patients with Hodgkin's disease). A gastric localization was evidenced in 48 patients with non-Hodgkin lymphoma (15 primaries and 33 secondaries), and no lesions were found in patients with Hodgkin's disease. The gastroscopies were accompanied by histological samples in all the cases and by cytological samples in most of them. The examination allowed a histological classification in 96% of the cases. The endoscopic aspect most frequently observed (48%) was vegetation, which was often associated with multiple ulcerations. The most frequent site (67%) was the vertical portion of the stomach. Gastroscopy was also utilized in the follow-up of 22 of the 48 patients affected by gastric lymphoma: in two of these, gastric localizations missed at other examinations were found.

The effect of placement of cervical margins of class II amalgam restorations on plaque accumulation and gingival health.

The effects or restoring early proximal carious lesions were investigated in a series of thirty-six surfaces. In eighteen lesions the cervical margins of the restorations extended subgingivally, and in eighteen the margins were located supragingivally. Three months after restoration, plaque accumulation was not significantly different from pre-restoration levels. The degree of gingival inflammation showed a significant improvement after restoration where cervical margins were finished supragingivally (P less than 0.5). Where margins were finished subgingivally a highly significant deterioration in gingival health occurred (P less than 0.1).