DNase I mediates internucleosomal DNA degradation in human cells undergoing drug-induced apoptosis.

Internucleosomal DNA fragmentation following the activation of endonucleases is the common end point of apoptosis. DNase I, a Ca(2+) / Mg(2+)-dependent endonuclease ubiquitously expressed in mammalian tissues, is believed to play a role in this process. To analyze the in vivo function of this enzyme in human cells, we have generated a cell line with targeted disruption of the DNase I gene, as well as several stable cell lines which overexpress the DNase I gene. Inactivation of the human DNase I gene was obtained in the Jurkat T cell clone JA3, characterized by high susceptibility to apoptotic cell death induced by pharmacological stimuli. JA3 cells, after disruption of the DNase I gene, became resistant to apoptotic stimuli. DNase I was overexpressed in the human cell lines JA3, K562 (erythroleukemia), M 14 (melanoma) and CEM (T cell lymphoma). Remarkably, stable overexpression of DNase I gene resulted in accelerated apoptosis in JA3 cells and induced apoptosis in K562, CEM and M14 cell lines, which are otherwise resistant to internucleosomal DNA degradation following pharmacological stimuli. Our study provides the first in vivo evidence that DNase I mediates internucleosomal DNA degradation in human cells undergoing drug-induced apoptosis.

Natural killer lymphocytes: "null cells" no more.

Natural Killer (NK) lymphocytes were initially described as potent effector cells that, unlike T lymphocytes, were able to kill targets in the absence of a priori stimulation and without specific recognition mechanisms. Over the past ten years however, it has been clearly demonstrated that NK cell function is regulated by a number of surface receptors that bind specific ligands expressed by target cells. Some of these receptors display inhibitory functions and recognize MHC class I molecules expressed by normal autologous cells that, as a consequence, are spared from indiscriminate NK-mediated killing. Other receptors are involved in NK cell activation against allogeneic cells or cells that, upon viral infection or tumor transformation, down-regulate MHC Class I expression. Altogether these data provide important advances toward the understanding of the complexity of the molecular mechanisms that regulate NK-mediated functions.

Involvement of natural cytotoxicity receptors in human natural killer cell-mediated lysis of neuroblastoma and glioblastoma cell lines.

The surface receptors involved in natural killer (NK) cell triggering during the process of target cell lysis have been at least in part identified. These are members of a novel family of receptors that has been termed natural cytotoxicity receptors (NCR). The first three members of this emerging group of receptors are the NKp46, NKp44 and NKp30 molecules that all belong to the immunoglobulin superfamily. Blocking of these receptors inhibits NK-mediated cytotoxicity against a wide variety of tumor target cells. In the present study, we show that these NCR are also involved in NK-mediated killing of tumor cells of neural origin. Glioblastoma and neuroblastoma target cells were efficiently killed by all NK clones analyzed since little protection from NK lysis was mediated by HLA class I molecules. Blocking of one or another NCR inhibited cytotoxicity; however, optimal inhibition was only observed when the three receptors were blocked simultaneously. A sharp difference in cytotoxicity against neural tumors was demonstrated between NCR(bright) and NCR(dull) NK clones, further supporting the notion that NCR play a critical role in the induction of cytotoxicity against tumor target cells of different histotype. Finally, our data also indicate that CD16 does not function as a triggering receptor involved in lysis of neural tumors since no difference in cytotoxicity could be substantiated between CD16(+) and CD16(-) NK clones and no correlation could be detected between the NCR(bright)/NCR(dull) phenotype and CD16 expression.

Glycine-rich cell wall proteins act as specific antigen targets in autoimmune and food allergic disorders.

Our objective was to investigate the presence of a B and T cell immune response directed against the glycine-rich cell wall protein (GRP) in patients with different autoimmune disorders and with food allergy. GRP is an ubiquitous food protein that has high homology with cytokeratins and other self proteins [Epstein-Barr virus nuclear antigen-1 (EBNA-I), heterogeneous nuclear ribonucleoprotein, fibrillar collagen] which are common targets in autoimmune disorders. A peptide (GGYGDGGAHGGGYGG) derived from GRP was used to screen human sera in direct and competitive ELISA assay. Anti-GRP-specific IgG were analyzed for their ability to cross-react with autoantigens. The intracellular cytokine profiles of the peptide-specific T cell clones obtained from representative patients have been studied. BALB/c mice were immunized with the peptide coupled to the carrier protein keyhole limpet hemocyanin (KLH). Serum IgG antibodies directed against the GRP peptide were detected in several autoimmune disorders and in food allergic patients, and were able to cross-react with autoantigens including keratin, collagen and EBNA-I. Twenty-five T cell clones showed a specific proliferative response to the GRP peptide and were of the T(h)0 phenotype. Eight of the 10 BALB/c mice immunized with the peptide coupled to KLH developed an autoimmune response. Our data suggest that phylogenetically highly conserved epitopes in plants, viruses and humans may be responsible for an autoimmune response in susceptible individuals. They also indicate that the antigen spreading of a particular sequence among apparently divergent proteins may participate to initiate or amplify an immune response.

The human natural cytotoxicity receptors (NCR) that induce HLA class I-independent NK cell triggering.

The cytolytic activity mediated by human natural killer (NK) cells is the result of a balance between signals delivered by inhibitory and activating receptors. The inhibitory receptors are represented by different families of HLA-specific receptors characterized by immunoreceptor tyrosine-based inhibiting motif(ITIM) sequences in their cytoplasmic portion. The function and the specificity of the inhibitory receptors imply the existence of triggering receptors specific for non-HLA ligands that are responsible for the induction of the cytolytic activity against HLA class I-deficient target cells. These receptors have remained elusive until recently when three distinct NK-specific molecules, termed natural cytotoxicity receptors (NCR), were identified and cloned. The different members of this novel family of receptors play a complementary role in the recognition and lysis of target cells. The NCR family is composed by a heterogeneous group of molecules belonging to the Ig superfamily that associate to different immunoreceptor tyrosine-based activating motif (ITAM)-containing signal transducing polypeptides.

Human natural killer cell activating receptors.

Natural killer (NK) cells were poorly characterized until 10 years ago and few molecules expressed on their cell surface were known. Now the situation has changed dramatically, since a plethora of receptors characterized by opposite functions have been functionally and molecularly defined. NK cells express clonally distributed inhibitory receptors specific for different groups of HLA class I alleles, thus protecting normal cells from NK-mediated lysis. On the contrary, various activating receptors are involved in triggering of NK-mediated natural cytotoxicity. Their engagement induces human NK cells to kill target cells that are either HLA class I-negative or -deficient. Here a brief description of the activating receptors and coreceptor and of their ligand(s) is given.

Triggering receptors involved in natural killer cell-mediated cytotoxicity against choriocarcinoma cell lines.

The lack of classical HLA-class I molecules on trophoblast is necessary to prevent allorecognition by maternal CTL, but may induce activation of NK cells. A protective role against NK cells equipped of suitable inhibitory receptors has been proposed for nonclassical HLA-class I molecules including HLA-E and HLA-G. In the present study we show that the NK-mediated killing of two choriocarcinoma cell lines, JAR and JEG3, is induced upon engagement of natural cytotoxicity receptors (NCR) with their specific ligands. In particular, we show that NKp44, a triggering receptor expressed at the NK cell surface only after in vitro culture in the presence of IL-2, plays a central role in triggering NK cytotoxicity against trophoblast cells. Also NKp46 appear to contribute to this function by cooperating with NKp44. On the other hand, other triggering receptors such as NKp30, 2B4, and NKG2D are not involved in killing of choriocarcinoma. Our findings suggest that resistance of trophoblast to NK-mediated cytotoxicity is the result of insufficient activating interactions between the various triggering NK receptors and their target cell ligands. On the other hand, the interaction of nonclassical HLA class I molecules with inhibitory NK receptors appears to play only a marginal role in regulating the susceptibility of choriocarcinoma to NK mediated cytotoxicity.

Molecular and functional characterization of IRp60, a member of the immunoglobulin superfamily that functions as an inhibitory receptor in human NK cells.

In this study we describe the functional and molecular characterization of IRp60 (inhibitory receptor protein 60), an inhibitory receptor expressed on all human NK cells. The IRp60 molecule has been identified by the generation of three novel monoclonal antibodies (mAb). Cross-linking of IRp60 by specific mAb strongly inhibits the spontaneous cytotoxicity of NK cells as well as the NK-mediated cytolytic activity induced via different non-HLA-specific or HLA-specific activating receptors. IRp60 is a 60-kDa glycoprotein that, upon sodium pervanadate treatment, becomes tyrosine phosphorylated and associates with the SH2-containing phosphatases SHP-1 and SHP-2. The IRp60 gene is located on human chromosome 17 and encodes a molecule belonging to the immunoglobulin (Ig) superfamily characterized by a single V-type Ig-like domain in the extracellular portion. The cytoplasmic tail contains three classical immunoreceptor tyrosine-based inhibitory motifs. Southern blot analysis revealed cross-hybridization with monkey and mouse genomic DNA, thus suggesting that IRp60 may be conserved among different species. Moreover, based on the use of different anti-IRp60 mAb, we could identify two IRp60 allelic variants. Since IRp60 is also expressed by other cell types, including T cell subsets, monocytes and granulocytes, it may play a more general role in the negative regulation of different leukocyte populations.

Natural killer cell-mediated recognition of human trophoblast.

Large numbers of natural killer cells are present within the maternal decidua close to the extravillous trophoblast cells. Most of these natural killer cells express high levels of inhibitory NK receptors (KIR) specific for HLA class I molecules. Since trophoblast cells only express HLA-C and HLA-G, the KIR expressed by decidual NK cells can only recognize these HLA class I molecules in order to avoid NK-mediated rejection of fetal tissues. We show that HLA-C recognition by decidual NK cells can be mediated by p58, LIR-1 and (indirectly) by CD94/NKG2A receptors. On the other hand, HLA-G recognition is not only mediated by LIR-1 and (indirectly) by CD94/NKG2A but also by a newly identified receptor termed p49. The p49 receptor, unlike the other KIR, appears to be selectively expressed by decidual NK cells.

Chronic parvovirus B19 infection induces the production of anti-virus antibodies with autoantigen binding properties.

Human parvovirus B19 infection in adults shows some clinical features similar to those found in autoimmune connective tissue diseases. To better clarify the relationship between viral infection and autoimmunity, we have evaluated the ability of anti-parvovirus antibodies to specifically recognize autoantigens in ten patients with chronic symmetric arthritis resembling rheumatoid arthritis or with recurrent episodes of arthritis and cutaneous manifestations and persistence of specific IgM antibodies against B19 parvovirus. We synthetized a 24-amino acid immunodominant peptide corresponding to a part of the virus protein 1 and virus protein 2 overlapping region. The peptide has been used to test patients' sera at different time points with an enzyme-linked immunosorbent assay (ELISA) and to purify anti-virus antibodies by affinity chromatography on a peptide-Sepharose column. Eluted immunoglobulins recognized the B19 peptide in both direct and competitive ELISA. Affinity-purified anti-parvovirus antibodies were then tested on a panel of autoantigens including human keratin, collagen type II, thyreoglobulin, single-strand (ss)DNA, cardiolipin and ribonucleoprotein antigen Sm. Eluted antibodies specifically recognized keratin, collagen type II, ssDNA and cardiolipin. Autoantibody activity was not detected in the immunoglobulin fraction after complete removal of anti-peptide antibodies and in antibodies eluted from normal donors. Epstein-Barr virus-transformed cell clones obtained from two subjects produced antibodies which simultaneously recognize the viral peptide and several autoantigens. To further confirm the role of the virus in inducing an autoantibody response, eight BALB/c mice were immunized with the viral peptide coupled to a carrier protein. Autoantibody activity against keratin, collagen II, cardiolipin and ssDNA was detected in six of the eight mice which developed a strong anti-virus response. Together, these data indicate that B19 parvovirus may be linked to the induction of an autoimmune response.

Function and specificity of human natural killer cell receptors.

In humans, natural killer lymphocytes express HLA class I-specific inhibitory receptors belonging to at least two different molecular families. The first is represented by members of the Ig superfamily that are involved in the recognition of different groups of HLA class I alleles, and the second is represented by a molecular complex formed by CD94 and NKG2A that displays a broad specificity for various class I molecules including the 'non-classical' HLA-G molecules. In addition to the inhibitory receptors, a series of activating receptors has been identified. Some display the same specificities as the corresponding inhibiting receptors and can be viewed as HLA class I-specific activating receptors. Another group of activating receptors appear to be involved in the cytolytic activity against HLA-'negative' target cells. These receptors are clearly non-MHC specific and, under physiological conditions, their function is suppressed by the HLA class I-specific inhibitory receptors.

[Bone metastasis of leiomyosarcoma. Apropos of a case]. / Métastase osseuse d'un leiomyosarcome. A propos d'un cas.

INTRODUCTION: Bone leiomyosarcoma is a rare tumor, whether it may be primary or secondary. The authors report on the case of a woman, aged 67, admitted in January 1992 complaining of pain in the left hip and the upper end of the femur. CASE REPORT: In 1985 the patient underwent surgical excision of a soft tissue tumor in the right thigh, histologically diagnosed as a benign fibrous tumor. This lesion recurred locally four times and repeated excisions were performed throughout the years, always with a histological diagnosis of a benign lesion. On admission to hospital, the physical examination as well as laboratory data and plain roentgenograms were unremarkable. Both tomography and MRI showed a lesion in the upper end of the left femur. An isotopic bone scan showed marked increased uptake in the left hip extending to the femoral diaphysis. An open biopsy was performed for histology, immunohistochemistry and electron microscopy. A diagnosis of metastatic leiomyosarcoma was made. The retrospective histological examination of specimens of the soft tissue tumor excised in 1985 showed the same immunohistochemical features of the contralateral leiomyosarcoma. On this basis, one stage resection of the left hip and the upper end of the femur was performed and a Kotz modular prosthesis was inserted. Postoperative healing was achieved without any complications and the function of the operated limb was satisfactory. Three months after the operation pulmonary lesions were noted on chest radiographs and CT scan. The patient died two years after the first admission for widespread metastasis. DISCUSSION: In the reported case, the bony metastasis appeared to be the presenting finding of the soft tissue tumor of the contralateral thigh. This presentation is rare in previously published series. The misdiagnosis of the primary tumor had caused local recurrences, and an increased malignity occurred. According to the literature, a soft tissue leiomyosarcoma can be easily confused with other spindle cell lesions. Therefore an accurate histological and ultrastructural diagnosis is necessary for adequate surgical treatment.

Novel surface molecules involved in human NK cell activation and triggering of the lytic machinery.

Three new monoclonal antibodies (MAbs) termed 7A6, PP35 and A6/143 were isolated after mouse immunization with CD3- CD16+ NK clones. The screening procedure was based on the ability of MAbs to trigger cytolytic activity of the immunizing clones in a re-directed killing assay against the P815 murine mastocytoma cell line. The 7A6 MAb reacts with 58 kDa surface molecules that appear to belong to the same molecular family defined by the previously described NK-sub-set-specific GL183 and EB6 MAbs. However, unlike from these MAbs, the 7A6 MAb reacted with (and activated) all CD3- NK lymphocytes, independent of their sub-set assignment (based on the expression or lack of expression of EB6, GL183 and CD16). The PP35 MAb reacted with a 70 kDa surface molecule expressed on all CD3- NK cells, as well as on TCR gamma/delta + cells and on a small sub-set of TCR alpha/beta + CD8+ lymphocytes. The PP35 MAb induced activation of essentially all NK cells, although clonal analysis revealed quantitative differences in the magnitude of the cytolytic responses elicited in different clones. Finally, the A6/143 MAb reacted with a molecule of 115 kDa expressed by all human PBL. Similarly to 7A6 and PP35 MAbs, the A6/143 MAb activated all sub-sets of cloned NK cells.

Morphologic and functional characterization of human peripheral blood T cells expressing the T cell receptor gamma/delta.

The morphologic and functional characteristics of cells freshly isolated from human peripheral blood and bearing a T cell receptor (TcR) gamma/delta were analyzed. Cell preparations highly enriched for TcR gamma/delta+ cells were obtained by treatment of E rosette-forming lymphocytes with anti-CD4 and anti-CD8 monoclonal antibodies (mAb) and complement. These preparations consisted of 64-82% TcR gamma/delta+ lymphocytes, as indicated by the sum of cells reacting with the BB3 and A13 mAb which define two distinct, nonoverlapping, TcR gamma/delta+ cell subsets in the peripheral blood. TcR gamma/delta cells were able to form conjugates with the natural killer-sensitive K-562 and with the natural killer-resistant HL-60-R tumor cell lines. The cytochemical localization of lysosomal acid hydrolases showed that 95%-98% of the cells in the TcR gamma/delta+ preparations had the morphologic features of granular lymphocytes. Moreover, electron microscopy analyses showed that TcR gamma/delta+ cells had electron-dense granules dispersed in the cytoplasm and a variety of smooth vesicles, a morphology identical to that of other CD3- or CD3+ granular lymphocyte subsets. Freshly isolated TcR gamma/delta+ cells were unable to lyse K-562 and natural killer-resistant targets, such as HL-60-R and P815. However, low levels of target cell lysis were observed upon triggering of the effectors by anti-CD3 TcR mAb or by lectin. After short-term culture with interleukin 2, TcR gamma/delta+ cells acquired a strong cytolytic activity against K-562 and HL-60-R target cells in the absence of triggering stimuli, and also displayed high levels of cytolytic activity against P815 in the presence of anti-CD3/TcR mAb.

Sonographic evaluation of ovarian volume in postmenopausal women: a screening test for ovarian cancer?

Ovarian cancer is the first cause of death from gynaecological malignancy. The poor over-all five year survival rate requires a specific procedure for early detection of ovarian carcinoma. An evaluation of ultrasonography as a screening test in early ovarian cancer is currently used in our Institute. A group of 500 volunteers without clinical symptoms, older than 45 years, and/or in postmenopausal period, were submitted to the procedure. We used a real-time mechanical sector scanner with 3 mHz transducer. The morphology and size of both ovaries were assessed. Abnormal results were obtained in 11 women. Four (4) postmenopausal patients underwent surgery. At the moment our study proves that ultrasonography is a valid procedure in the investigation of the ovaries in postmenopausal women. We need further evaluations to assess the real effectiveness of ultrasound examination as a screening test for early detection of ovarian cancer.

[The subfornical organ today: morphological aspects and functional role]. / L'organo subfornicale oggi: aspetti morfologici e ruolo funzionale.

The recognition of the role played by the subfornical organ (SFO) in the central regulation of body water balance has recently aroused new interest in this anatomical formation which remained ignored for a long time. The SFO is included in the group of the circumventricular organs. In higher vertebrates it is adherent to the ventral surface of the fornix and protrudes into the third ventricle at the level of the interventricular foramina, partially covered by the choroid plexus. The SFO appears as a small nodule, rounded or ovoidal in shape, consisting of highly vascularized nervous tissue and lined by ependyma at the ventricular surface. Its structural organization is fundamentally constant and presents only minor differences in the various species. The SFO neuronal perikarya show different aspects which have been classified in four types. However, it is not yet clearly defined if such aspects refer to distinct cell types or to different transitional features. Nerve and glial cell processes form a dense plexus through the SFO and the subependymal area, as well as in the connective tissue perivascular spaces. These may be narrow or wide and surround fenestrated and non-fenestrated capillaries, assuming sometimes a labyrinthine aspect. The ependymal lining of the SFO ventricular surface shows large variations and regional differences concerning the cell height, the number and development of microvilli, the cilia distribution. The structural properties of SFO, which is characterized by a rich and highly permeable capillary bed, by a wide surface area of contact and exchange with the cerebrospinal fluid, by direct and indirect neural connections with a number of regulatory structures, have been considered as the basis for the role of neurohumoral integration that SFO plays in regulating physiological and behavioral responses to water-mineral changes. Much experimental evidence substantiates this function. However, the studies on SFO are increasingly enriching the literature with new experimental, especially physiological and cytochemical, data which may suggest for this organ connections even more extensive and functions even more complex than those until now ascertained.