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Chronic lymphocytic leukemia (CLL) clones are characterized by loss of a critical region in 13q14.3, (del(13)(q14)) involving the microRNA (miRNA) cluster miR-15a and miR-16-1. We have investigated the effects of replacement of miR-15a and miR-16-1. CLL cells transfected with these miRNA mimics exhibited a decrease in cell viability in vitroand impaired capacity for engraftment and growth in NOD/Shi-scid,γcnull (NSG) mice. No synergistic effects were observed when the two miRNA mimics were combined. The phenomena were not restricted to CLL with the del(13)(q14) lesion. Similar effects induced by miRNA mimics were seen in cells with additional chromosomal abnormalities with the exception of certain CLL clones harboring TP53alterations. Administration of miRNA mimics to NSG mice previously engrafted with CLL clones resulted in substantial tumor regression. CLL cell transfection with miR-15a and miR-16-1-specific inhibitors resulted in increased cell viability in vitroand in an enhanced capacity of the engrafted cells to grow in NSG mice generating larger splenic nodules. These data demonstrate that the strong control by miR-15a and miR-16-1 on CLL clonal expansion is exerted also at the level of full-blown leukemia and provide indications for a miRNA-based therapeutic strategy.

Survival of patients affected by Multiple Myeloma (MM), a B-cell tumor of malignant plasma cells, has dramatically improved, owing to the recent introduction of the proteasome inhibitor (PI) Bortezomib and of the immunomodulatory drugs (IMiDs). This major advance originates from accumulating knowledge on MM biology, leading to the development of drugs targeting not only MM cells, but also their microenvironment. Indeed, the disease develops as a result of genetic abnormalities and of reciprocal interactions between MM cells and the permissive BM microenvironment, which delivers growth- and pro-survival signals and confers resistance to drugs. As for solid tumors, bone marrow (BM) angiogenesis is emerging as a critical component of MM development and progression, and hence as an attractive therapeutic target for the disease. The patho-physiology of MM associated angiogenesis is complex and involves a plethora of soluble factors, cellular players and mechanisms. Moreover, the hypoxic microenvironment inside the BM might significantly contribute to the induction and maintenance of a pro-angiogenic profile, given the well-known role of hypoxia in promoting angiogenesis in all its forms. Here we present an overview of the literature focusing on the mechanisms implicated in the “angiogenic switch”, which corresponds to the transition from the avascular to the vascular phase of the disease. We also review evidence on the anti-angiogenic effects of PI and IMiDs, which substantially contribute to their anti-MM activity. Finally, we summarize possible caveats and perspectives about antiangiogenic strategies that could be addressed to improve the efficacy of treatments for MM patients.

Recently, mutations affecting different domains of dynamin-2 (DNM2) were associated alternatively with autosomal dominant centronuclear myopathy or dominant intermediate (demyelinating and axonal) Charcot–Marie–Tooth disease (CMT) type B.

To identify the molecular basis of a demyelinating Charcot–Marie–Tooth disease type 1 (CMT1) with presumed autosomal recessive inheritance.

The peripheral myelin protein 22 (PMP22) is a tetraspan membrane protein which is localised in the compact myelin of the peripheral nerves. In fibroblasts, where it was originally identified as growth arrest related factor 3 (Gas3), PMP22 has been shown to modulate cell proliferation; in the peripheral nervous system its roles are still debated. The duplication of PMP22 is the most common cause of the demyelinating form of the autosomal dominant Charcot-Marie-Tooth neuropathy (CMT1A); rarer missense mutations of PMP22 also cause CMT1A or severe dehypomyelinating neuropathies of infancy grouped under the heading of Dejerine-Sottas syndrome (DSS). Here, a sporadic patient affected with DSS is described; nerve biopsy disclosed a picture of hypomyelination/amyelination with basal laminae onion bulbs and no florid demyelination and it was consistent with congenital hypomyelination neuropathy (CHN); molecular analysis disclosed a novel point mutation of PMP22 that causes a non-conservative arginine for cysteine substitution at codon 109, in the third transmembrane domain. CHN is the rarest and severest form of DSS and it is thought to reflect dysmyelination rather than demyelination. The reported case suggests that missense point mutations may alter a putative role of PMP22 in modulating Schwann cell growth and differentiation.

The capacity of synthesizing and secreting Ig molecules was studied in 11 patients with B-cell chronic lymphocytic leukemia (B-CLL) whose cells expressed surface IgM, in 3 patients with surface IgG-bearing cells, and in 2 IgM prolymphocyte leukemias (IgM-PLL). Three types of μchains were detected by SDS-polyacrylamide gel electrophoresis analysis of the endogenously labeled molecules isolated by specific immunoprecipitation. Two of them were isolated from the cell lysates and were identified as the membrane μchain and the precursor of the secreted molecules, respectively. The latter also possibly contained precursors of the membrane molecules. The third type of molecule was detected only in the culture medium and was identified as secretory μchain. Not all of the malignant clones possessed the three types of μchains. Only of the IgM-bearing malignant cell clones were capable of secretion, whereas the remaining synthesized the secretory μchains but degraded them intracellularly. Two types of molecules (membrane and secreted) were found in the IgG-bearing CLL cells from three patients. In all of them, secretion was detected. Ultrastructural analysis demonstrated that cells from the secreting clones had the features of more mature lymphocytes than the cells from nonsecreting clones. These features were represented by a developed Golgi apparatus, various types of vesicles (smooth and coated), and strands of the rough endoplasmic reticulum. A certain heterogeneity of the degree of maturation of the cells was observed within these clones. The data are consistent with the hypothesis that CLL clones are heterogeneous and can be distinguished through the different degrees of maturation of their cell components.

Peripheral blood mononuclear cells were fractionated according to the expression of a variety of surface markers, and the fractions obtained were tested for erythroid burst-forming unit (BFU-E) colony formation. BFU-Es were detected in the HLA-DR+non-T cell fraction, but gave rise to optimum colony numbers only in the presence of a nonadherent, relatively radioresistant cell. This accessory cell was found among the HLA-DR−non-T, non-B cells, a fraction that was particularly enriched in large granular lymphocytes (LGLs). Experiments carried out to assess directly the surface markers of the accessory cell revealed an FcR+, OKM1+, Leu 7+, Leu 11+, OKT4−, OKT8−surface phenotype, which is consistent with that of the majority of LGLs. Peripheral blood LGLs, purified by Percoll density gradient, proved very efficient in promoting optimal BFU-E colony formation. All of these results indicate that LGLs have a potent erythroid burst-promoting activity. Such activity is probably mediated through the release of soluble factors, as shown by the observation that LGL culture supernatants were as effective as LGLs in sustaining colony formation. © 1985 by Grune & Stratton, Inc.

Highly purified natural killer (NK) cell suspensions were tested for their capacity to release colony-stimulating activity (CSA) in vitro. NK cell suspensions comprised primarily CD16+ cells and were devoid of CD3+ T cells, CD15+ monocytes, and of B cells. CSA was detected in the NK cell supernatants and sustained the growth of myeloid colonies from both normal peripheral blood and bone marrow. CSA could be in part inhibited by pretreating NK cell culture supernatants with a specific goat anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antiserum. The inhibition, however, was never complete, a finding that suggests that additional factors were responsible for CSA. Incubation of NK cells with K562 cells (an NK-sensitive target) or with normal bone marrow cells resulted in the appearance of a strong colony- inhibiting activity (CIA) in the culture supernatants. Such CIA was demonstrable in an experimental system where bone marrow or peripheral blood progenitors were induced to form myeloid colonies in the presence of conditioned medium by CSA-producing giant cell tumor (GCT) cells. Stimulation of NK cells with NK-insensitive targets failed to induce CIA production. Neutralizing antitumor necrosis factor (TNF) monoclonal antibodies (MoAbs) were found capable of inhibiting CIA present in the supernatants of NK cells stimulated with K562 cells. Following treatment with anti-TNF antibodies, CSA was again detectable in the same supernatants. This finding indicates that induction of TNF production did not concomitantly switch off CSA production by NK cells. Pretreatment of NK cells with recombinant interleukin-2 (rIL-2) or gamma interferon (r gamma IFN) did not change the amount of CSA released. However, treatment with rIL-2 caused the appearance of a factor in the NK cell supernatants capable of sustaining the formation of colonies of a larger size.

Peripheral blood mononuclear cells were fractionated according to the expression of a variety of surface markers, and the fractions obtained were tested for erythroid burst-forming unit (BFU-E) colony formation. BFU-Es were detected in the HLA-DR+ non-T cell fraction, but gave rise to optimum colony numbers only in the presence of a nonadherent, relatively radioresistant cell. This accessory cell was found among the HLA-DR- non-T, non-B cells, a fraction that was particularly enriched in large granular lymphocytes (LGLs). Experiments carried out to assess directly the surface markers of the accessory cell revealed an FcR+, OKM1+, Leu 7+, Leu 11+, OKT4-, OKT8- surface phenotype, which is consistent with that of the majority of LGLs. Peripheral blood LGLs, purified by Percoll density gradient, proved very efficient in promoting optimal BFU-E colony formation. All of these results indicate that LGLs have a potent erythroid burst-promoting activity. Such activity is probably mediated through the release of soluble factors, as shown by the observation that LGL culture supernatants were as effective as LGLs in sustaining colony formation.

The capacity of synthesizing and secreting Ig molecules was studied in 11 patients with B-cell chronic lymphocytic leukemia (B-CLL) whose cells expressed surface IgM, in 3 patients with surface IgG-bearing cells, and in 2 IgM prolymphocytic leukemias (IgM-PLL). Three types of mu chains were detected by SDS-polyacrylamide gel electrophoresis analysis of the endogenously labeled molecules isolated by specific immunoprecipitation. Two of them were isolated from the cell lysates and were identified as the membrane mu chain and the precursor of the secreted molecules, respectively. The latter also possibly contained precursors of the membrane molecules. The third type of molecule was detected only in the culture medium and was identified as secretory mu chain. Not all of the malignant clones possessed the three types of mu chains. Only 7/13 of the IgM-bearing malignant cell clones were capable of secretion, whereas the remaining synthesized the secretory mu chains but degraded them intracellularly. Two types of molecules (membrane and secreted) were found in the IgG-bearing CLL cells from three patients. In all of them, secretion was detected. Ultrastructural analysis demonstrated that cells from the secreting clones had the features of more mature lymphocytes than the cells from nonsecreting clones. These features were represented by a developed Golgi apparatus, various types of vesicles (smooth and coated), and strands of the rough endoplasmic reticulum. A certain heterogeneity of the degree of maturation of the cells was observed within these clones. The data are consistent with the hypothesis that CLL clones are heterogeneous and can be distinguished through the different degrees of maturation of their cell components.

On the long term Parkinson Disease (PD) treatment is often complicated by the occurrence of motor fluctuations. To find out whether early treatment of PD with levodopa, dopaminoagonists or 1-deprenyl is associated with any difference in motor fluctuations occurrence, the Italian Parkinson Study Groups initiated a multicenter, randomized study. Since November 1988, 475 patients cequiring effective treatment for idiopathic PD have been randomized to receive levodopa, dopoamine agonists or deprenyl. After 2 months of therapy, all patients evaluated with the Unified Parkinson Disease Rating Scale showed a significant amelioration. Daily living activities were more impaired in patients treated with deprenyl. Study design is presented and first resuts are discussed.

The expression of CD38 by B cells chronic lymphocytic leukemia (B-CLL) was studied in 20 untreated patients. The cells expressed abundant CD38 (relative fluorescence intensity range, 6 to 15) in 6 cases (group I patients), whereas CD38 expression was low to absent (relative fluorescence intensity range, 0 to 3) in the remaining cases (group II patients). Exposure of the cells from group I patients to goat antihuman mu chain antibodies (Ga mu-ab) resulted in the elevation of intracellular free Ca2+ concentration([Ca2+]i) followed by apoptosis. In contrast, exposure of group II cells to Ga mu-ab was not followed by increased levels of [Ca2+]i, programmed cell death or cell proliferation. No differences in the expression of surface IgM were noted in the two groups of B-CLL cells. Normal peripheral blood B cells, which expressed low to absent CD38, were capable of mobilizing [Ca2+]i and of proliferating after exposure to Ga mu-ab. The collected data suggest that, although group I B-CLL cells were able to transduce the signals delivered by IgM crosslinking, this pathway was severely impaired in group II B-CLL cells. However, unlike that observed in normal circulating B cells, stimulation of group I cells with Ga mu-ab resulted in apoptosis rather than proliferation. CD38 did not appear to be directly involved in [Ca2+]i mobilization induced by Ga mu-ab in group I B-CLL cells because their exposure to anti-CD38 monoclonal antibodies failed to cause [Ca2+]i mobilization or to block the [Ca2+]i response induced by Ga mu-ab. These data indicate that CD38 expression identified a particular subset of B-CLL cells with defined functional properties, including the propensity to undergo apoptosis.

In a previous study, we described a cell line (BRG-P) derived from a woman with Burkitt's lymphoma (BL) and acquired immunodeficiency syndrome that shared the same characteristic cytogenetic abnormalities as the patient's malignant cells. This cell line contained subclones that displayed an isotype switch from IgM to IgA1 and an accumulation of point mutations in the Vh region genes. Because these two features suggested an antigen-driven process, we began a search for the antigen responsible for the stimulation of the malignant B cells. Specifically, we hypothesized that because the patient's tumor had presented as a lymphomatous infiltration of the breast, the malignant B cells were recruited to this site because of the reactivity of their surface lg with breast tissue. A hybridoma (BRG-H) was obtained by fusing BRG-M cells (an IgM producing subclone of the BRG-P cell) with an appropriate cellular partner. The monoclonal antibody (BRG MoAb) produced by this hybridoma reacted strongly with two of five breast cancer cell lines and stained normal and malignant ductal epithelial cells on breast tissue sections. The antigen recognized by the BRG MoAb consisted of a single, minimally glycosylated polypeptide chain of 45 kD (p45). The BRG MoAb failed to react with a panel of human cell lines from different tissues, except for one cell line from a uterine cervical carcinoma. No reactivity was detected for a panel of exogenous antigens from various pathogens, including human immunodeficiency virus and self- antigens frequently recognized by polyspecific antibodies. Experiments were performed to investigate the functional consequences of the interaction of surface IgM with its specific ligand. Coculture of BRG-M cells with p45+, but not with p45-, breast cells caused apoptosis of BRG-M cells. The specificity of the interaction was shown by the observation that apoptosis was prevented by pretreatment of BRG-M cells with a monovalent F(ab′) fragment of rabbit IgG antibody to human mu chains. Moreover, only BRG-M cells, but not other BL cells, underwent apoptosis after exposure to p45+ breast cells. The interaction between the CD40 molecule expressed by BRG-M cells and its specific ligand (CD40L) prevented p45-induced cell apoptosis. Because this interaction mimics that occurring in vivo between T and B cells during immune responses, our data suggest that various events contributed to the emergence of the BL, in this particular patient, including antigenic stimulation possibly assisted by T-cell help.

Normal human B cells were purified from peripheral blood or tonsils and tested for their ability to release colony-stimulating activity (CSA) in short-term cultures. The target cells used in the CSA assays were from peripheral blood or bone marrow. Unstimulated B cells produced CSA in amounts similar to those present in the GCT-conditioned medium used as a positive control. The B cell-derived CSA predominantly promoted the growth of colonies that contained macrophages alone or macrophages and granulocytes. CSA eluted in a single peak from a G-75 Sephadex column with an approximate molecular weight (mw) of 65 to 70 kilodaltons (kd). Fractionation of tonsil B lymphocytes on Percoll density gradients showed that large B cells, probably already activated in vivo, were the main source of CSA. By contrast, small, resting B cells recovered from a different fraction of the Percoll gradient released minimum amounts or no CSA. However, these B cells became CSA producers following stimulation with Staphylococcus aureus Cowan (SAC) in vitro. B cells purified from the peripheral blood of nine out of 12 patients with B-cell chronic lymphocytic leukemia (B-CLL) also released CSA in vitro in the absence of stimuli. These findings suggest that by releasing CSA, B cells may have a role in the regulation of hematopoiesis and in the control of the inflammatory process.

Malignant lymphocytes from 30 B-cell chronic lymphocytic leukemia (B- CLL) patients were studied for the cytochemical localization of two acid hydrolases, alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (AT). The large majority of the cells stained for both ANAE and AP in 7 cases, for AP only in 18 cases, and were negative for both the enzymes in 5 cases. Ultrastructural analysis revealed that the cells that displayed more mature morphological features, such as well developed smooth and rough membrane compartments, were those positive for acid hydrolases. That ANAE and AP are expressed by B cells at late stage of maturation was confirmed by the finding that some lymphocytes and all of the plasmacytoid lymphocytes and plasma cells from Walderstrom's macroglobulinemia, from mixed cryoglobulinemia, and from multiple myeloma patients stained strongly for both ANAE and AP. Using the expression of acid hydrolases and certain ultrastructural features as markers of cell differentiation, it was possible to demonstrate a process of maturation within the single B-CLL clones with accumulation of the cells at stages that differed in the various cases.

This paper describes the experiments conducted in our laboratory on the mode of activation of CD5 + B cells. The data show that these cells are stimulated in a manner that is less dependent of T cell help as compared to CD5- cells and define IL-2 as the major growth and differentiation factor for CD5 + cells. These data are discussed in the light of the supposed function of CD5 + cells that seem represented by the capacity to release polyspecific antibodies reacting with a multiplicity of antigens from various pathogens. These antibodies, also called natural antibodies, probably represent one of the first line of defense against invading microrganisms.

Incidence rates for CNS tumours in children of age 0-14 years in the Region of Lombardy, Italy, during the period 1988-93 were analysed; survival probability updated to December 1995 was also estimated. CNS tumours defined according to the International Classification of Diseases for Oncology codes were actively searched for. CNS tumours were diagnosed in 296 children. The age-standardized rates were 40.0 per million child years for both sexes together, and 45.3 for boys and 34.4 for girls. In all age groups, boys had a higher incidence than girls. The annual incidences were 13.7, 7.0, 5.8 for astrocytoma, medulloblastoma and ependymoma, respectively. The overall survival percentages at 5 and 8 years after diagnosis were 68 and 66, respectively. Prognosis was good for astrocytoma (5-year survival, 81%) and declined in the order: other gliomas (5-year survival, 76%0; ependymoma (5-year survival, 62%), and medulloblastoma (5-year survival, 43%). The histological type of the tumour was the most powerful independent predictor of survival in children with a CNS tumour. Medulloblastoma/primitive neuroectodermal tumours appeared to have the highest risk of a poor prognosis compared with astrocytoma (relative risk, 3.27; 95% confidence interval, 1.81-5.91). Age at diagnosis and sex had no significant effect on survival. The incidence of childhood CNS tumours found in this study is higher than previously reported in Italy, and is one of the highest in the world from population-based data. Survival of children with brain tumours has improved greatly in recent years. These results suggest that children in Lombardy with CNS tumours had a good survival experience compatible with high quality of care.Keywords:childhood CNS tumours; population-based; incidence; survival

Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of CD5-expressing B lymphocytes. Most studies have found that these leukemic CD5+ B cells, like their normal counterparts, use immunoglobulin (Ig) variable (V) region genes that exhibit minimal, if any, somatic diversity. These and other observations have suggested that CD5+ B cells may be incapable of generating Ig V gene diversity, and therefore may not be able to develop higher affinity binding sites that could be selected by antigen. However, most of the studies of CLL and normal CD5+ B cells have focused on IgM-producing cells. Since somatic mutations are most often seen in B cells that have undergone an isotype class switch, we analyzed the Ig heavy (H) and light (L) chain variable region genes of seven IgG+CD5+ CLL B cells to determine if somatic diversification and antigen selection had occurred. The data derived provide evidence for skewed use, somatic diversification, and antigenic selection of the Ig V region genes. Nonrandom use of both H and L chain V region genes was manifested by an overrepresentation of VH4 and VKI family genes and the underrepresentation of the JH4 gene segment. Furthermore, VH4 gene use was restricted to only two family members (4.21 and 4.18). In four of the seven cases, the VH and VL genes displayed > or = 5% difference from the most homologous known germline counterparts. Polymerase chain reaction and Southern blot analyses performed in two of these patients demonstrated that their unique VH CDR2 and adjacent sequences were not present in their germline DNA. In addition, a significant level of diversity was seen in the rearranged DJH segments and at the VL-JL junctions of every patient that occurred both at the time of recombination and subsequently. The localization of replacement changes to complementarity determining regions of some patients suggested that antigen selection had occurred. Furthermore, the mutations identified in the VH and VL genes of each individual patient were strikingly similar, both in number and location. Collectively, the data indicate that a subset of CD5+ CLL B cells can display Ig V region gene mutations. In addition, they are consistent with the notions that in some cases antigen selection of these mutations may have occurred, and that antigen stimulation may be a promoting factor in the evolution of certain CLL clones.

Normal human B cells were purified from peripheral blood or tonsils and tested for their ability to release colony-stimulating activity (CSA) in short-term cultures. The target cells used in the CSA assays were from peripheral blood or bone marrow. Unstimulated B cells produced CSA in amounts similar to those present in the GCT-conditioned medium used as a positive control. The B cell-derived CSA predominantly promoted the growth of colonies that contained macrophages alone or macrophages and granulocytes. CSA eluted in a single peak from a G-75 Sephadex column with an approximate molecular weight (mw) of 65 to 70 kilodaltons (kd). Fractionation of tonsil B lymphocytes on Percoll density gradients showed that large B cells, probably already activated in vivo, were the main source of CSA. By contrast, small, resting B cells recovered from a different fraction of the Percoll gradient released minimum amounts or no CSA. However, these B cells became CSA producers following stimulation with Staphylococcus aureus Cowan (SAC) in vitro. B cells purified from the peripheral blood of nine out of 12 patients with B-cell chronic lymphocytic leukemia (B-CLL) also released CSA in vitro in the absence of stimuli. These findings suggest that by releasing CSA, B cells may have a role in the regulation of hematopoiesis and in the control of the inflammatory process.

The phenotypic and functional features of purified large granular lymphocytes (LGL) from ten patients with LGL population expansions and cytopenias are described. The predominant LGL phenotypes were T3+, T8+, Leu-11+/-; however, in two patients, LGL expressed a T3-, Leu-11+ phenotype. Variable combinations of other LGL markers (OKM1, Leu-7), and HLA-DR were detected in individual cases. In nine of ten cases, freshly isolated LGL did not exert cytolytic activity for K562 target cells, but purified LGL cultured in the presence of recombinant interleukin 2 (rIL2) acquired potent cytotoxic activity in all cases tested. LGL did not proliferate in response to phytohemagglutinin (PHA). However, LGL released variable amounts of IL2 and gamma- interferon (gamma-IFN) after PHA stimulation. In some cases, stimulation of fresh LGL with recombinant IL2 induced production of gamma-IFN. No correlation was found between the functional capabilities and the original phenotype of the expanded LGL populations.

Large granular lymphocytes (LGL) are defined as nonadherent mononuclear cells with cytoplasmic azurophilic granules, avid receptors for the Fc portion of IgG, and cytotoxic functions (NK or ADCC activities). In the present study, the granules of LGL isolated from human peripheral blood have been analyzed by enzyme cytochemistry and electron microscopy. It had been found that: (1) in the single cells, granules at different stages of maturation could be detected: in addition, packaging of the granules took place in the proximity of the Golgi apparatus, which is similar to that seen in secretory cell types. (2) Acid phosphatase (AP) was observed within the granules and the vesicles located in the Golgi area: the Golgi apparatus identified through its thiamine pyrophosphatase-positivity was consistently negative for AP. (3) Alpha naphthyl-acetate esterase (ANAE) activity was localized in the granules as well as on the membrane of LGL and monocytes. (4) The ANAE activity of LGL was of the monocytic and not of the lymphocytic type, as shown by NaF inhibition. (5) The LGL granules, although identifiable as primary lysosomes, were not involved in the process of phagocytosis, since LGL failed consistently to ingest latex particles or opsonized red cells.

Gammadelta T lymphocytes play an important early role in the defense against pathogens. Their function is terminated by acquisition of susceptibility to CD95-triggered apoptosis. Here we show that the regulation of this process depends on the activity of the endothelial NO synthase expressed by gammadelta T lymphocytes, which is modulated in an activation-dependent way. The effects of nitric oxide thus generated, mediated via cGMP generation, are exerted at at least two sites along the CD95 signaling cascade: one at, or upstream, and the other downstream of ceramide generation. At either site, nitric oxide/cGMP action is sufficient for protection from apoptosis. The effect of NO is selective for apoptosis induced by CD95 cross-linking, since it does not affect apoptotic program triggered by other stimuli. The evidence here reported demonstrates a new physiological role for nitric oxide, acting as a survival factor for T lymphocytes.

The phenotypic and functional features of purified large granular lymphocytes (LGL) from ten patients with LGL population expansions and cytopenias are described. The predominant LGL phenotypes were T3+, T8+, Leu-11+/–; however, in two patients, LGL expressed a T3–, Leu-11+ phenotype. Variable combinations of other LGL markers (OKM1, Leu-7), and HLA-DR were detected in individual cases. In nine of ten cases, freshly isolated LGL did not exert cytolytic activity for K562 target cells, but purified LGL cultured in the presence of recombinant interleukin 2 (rIL2) acquired potent cytotoxic activity in all cases tested. LGL did not proliferate in response to phytohemagglutinin (PHA). However, LGL released variable amounts of IL2 and γ-interferon (γ-IFN) after PHA stimulation. In some cases, stimulation of fresh LGL with recombinant IL2 induced production of γ-IFN. No correlation was found between the functional capabilities and the original phenotype of the expanded LGL populations.

A patient is described who presented with a chronic lymphocytic leukemia (CLL) and later developed a lymphoblastic lymphoma. The cells from the CLL were typical mature B lymphocytes as could be assessed by morphologic, cytochemical, and surface marker analyses. The cells from the lymphoblastic lymphoma were immature B cells that expressed CD10, CD20, and HLA-DR markers, but not surface Ig or cytoplasmic mu chains, and were negative for terminal deoxynucleotidyl transferase (TdT). The cells of two continuous cell lines, obtained from the bone marrow and the peripheral blood of the patient, had the same phenotype as the lymphoblastic lymphoma cells, did not contain the Epstein-Barr virus genome, and displayed malignant features in vitro, including the capacity to form colonies in agar. The two cell lines also shared identical chromosomal abnormalities, a finding which suggests that they derived from the same malignant cell already present in vivo. Such chromosomal abnormalities were not seen in the karyotype of the peripheral blood cells at the onset of the disease. Analysis of the Ig heavy chain genes using a DJ-specific probe showed the very same monoclonal rearrangement in the cells from the B-CLL, the lymphoblastic lymphoma and the two cell lines, thus demonstrating their common clonal origin. By contrast, a monoclonal rearrangement of the lambda chain gene locus was found in the B-CLL cells only, a finding consistent with their exclusive capacity to express surface IgM lambda. This patient represents a rare case in whom a chronic lymphoproliferative disorder with mature malignant cells transforms into a lymphoblastic lymphoma characterized by cells frozen at a very early maturational stage. The possible mechanisms leading to such transformation within the same cell clone are discussed.

Malignant lymphocytes from 30 B-cell chronic lymphocytic leukemia (B-CLL) patients were studied for the cytochemical localization of two acid hydrolases, alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (AT). The large majority of the cells stained for both ANAE and AP in 7 cases, for AP only in 18 cases, and were negative for both the enzymes in 5 cases. Ultrastructural analysis revealed that the cells that displayed more mature morphological features, such as well developed smooth and rough membrane compartments, were those positive for acid hydrolases. That ANAE and AP are expressed by B cells at late stage of maturation was confirmed by the finding that some lymphocytes and all of the plasmacytoid lymphocytes and plasma cells from Walderström's macroglobulinemia, from mixed cryoglobulinemia, and from multiple myeloma patients stained strongly for both ANAE and AP. Using the expression of acid hydrolases and certain ultrastructural features as markers of cell differentiation, it was possible to demonstrate a process of maturation within the single B-CLL clones with accumulation of the cells at stages that differed in the various cases.

Human peripheral blood mononuclear cells (PBMC) were stimulated in vitro with anti-lymphocyte globulin (ALG), and the phenotypic and functional properties of the blasts obtained were investigated. When stained with monoclonal antibodies (MoAbs), all of the blasts were identified as T cells that expressed predominantly the CD4 phenotype (70% of the cells). The remaining blasts were CD8+. These findings demonstrate that ALG stimulates both helper-inducer and cytotoxic- suppressor cells at random since the CD4 to CD8 ratio in the stimulated blasts was the same as in resting PBMC. This ratio is different from that observed in short-term cultures of T cells stimulated with phytohemagglutinin (PHA) under the same conditions (CD4 to CD8 ratio less than 1). ALG-stimulated T cells were cloned by limiting dilution in the presence of recombinant Interleukin-2 (rIL-2). The clones obtained were expanded and maintained in long term cultures with rIL-2. Thirty-two clones were tested for their capacity of producing colony stimulating activity (CSA) or burst promoting activity (BPA). Twenty- eight of them produced CSA and 12 produced BPA. No correlation was found between the surface phenotype and the ability of the clones to produce CSA or BPA (ie, both the CD4+ and CD8+ clones released the cytokines). When 16 of the same clones were tested for II-2 and gamma interferon (gamma IFN) production, 12 were found to be gamma INF and IL- 2 producers. All of the gamma IFN producers also released IL-2, whereas in the single clones no correlation was found with the capacity of releasing BPA and CSA. Supernatants from selected T-cell clones were also tested for hematopoietic growth factor activities in the presence of neutralizing antisera to human granulocyte-macrophage colony stimulating factor (GM-CSF) or to Interleukin-3 (IL-3). It was found that most CSA was attributable to GM-CSF, whereas BPA was mainly related to the presence of IL-3.

Peripheral blood mononuclear cells from five patients with IgG+ B-type chronic lymphocytic leukemia (B-CLL) were analyzed for the presence of clone-specific Ig H chain variable region gene mRNA transcripts linked to C mu and/or C alpha. This was assessed by (1) comparing the lengths of portions of the VHDJH of the IgG+ CLL clones with those of the mu and alpha isotype-expressing B cells, (2) performing clone-specific endonuclease digestion studies, and (3) determining the DNA sequences of the mu and alpha isotype-expressing cDNA. Thus, when B-cell mRNA from these five patients were reverse transcribed with C gamma-specific primers and then amplified by polymerase chain reaction, dominant cDNA were found with lengths corresponding to those of the IgG+ CLL B cell. In addition, in four cases, cDNA of lengths identical to those of the CLL B cell were detected when mRNA was reverse transcribed and amplified using c mu- and/or C alpha-specific primers, strongly suggesting clonal relatedness. These CLL-related mu- and alpha- expressing cDNA were present in greater amounts that unrelated (non- CLL) mu- and alpha-expressing cDNA from normal B cells that used genes of the same VH family. When the sequences of these CLL-related C mu- and C alpha-expressing cDNA were compared with those of the IgG+ CLL clones, it was clear that they were derived from the same ancestral gene as the IgG-expressing CLL B cell, thus documenting their common origin. Finally, nucleotide point mutations were observed in the mu- and alpha-expressing cDNA of certain patients, indicating divergence with the CLL. These data suggest that IgM+ B cells, which are precursors of the leukemic B cells, exist in increased numbers in the blood of most patients with IgG+ B-CELL and that these cells may differentiate, accumulate V genes mutations, and undergo isotype switching in vivo. In addition, the data are consistent with a sequential-hit model for the evolution of CLL.

Human peripheral blood mononuclear cells (PBMC) were stimulated in vitro with anti-lymphocyte globulin (ALG), and the phenotypic and functional properties of the blasts obtained were investigated. When stained with mono-clonal antibodies (MoAbs), all of the blasts were identified as T cells that expressed predominantly the CD4 phenotype (70% of the cells). The remaining blasts were CD8 + . These findings demonstrate that ALG stimulates both helper-inducer and cytotoxic-suppressor cells at random since the CD4 to CD8 ratio in the stimulated blasts was the same as in resting PBMC. This ratio is different from that observed in short-term cultures of T cells stimulated with phytohemagglutinin (PHA) under the same conditions (CD4 to CD8 ratio <1). ALG-stimulated T cells were cloned by limiting dilution in the presence of recombinant Interleukin-2 (rlL-2). The clones obtained were expanded and maintained in long term cultures with rlL-2. Thirty-two clones were tested for their capacity of producing colony stimu- lating activity (CSA) or burst promoting activity (BPA). Twenty-eight of them produced CSA and 12 produced BPA. No correlation was found between the surface phenotype and the ability of the clones to produce CSA or BPA (ie, both the CD4 + and CD8 + clones released the cyto-kines). When 16 of the same clones were tested for II-2 and gamma interferon (7IFN) production, 12 were found to be 7INF and IL-2 producers. All of the 7IFN producers also released IL-2, whereas in the single clones no correlation was found with the capacity of releasing BPA and CSA. Supernatants from selected T-cell clones were also tested for hematopoietic growth factor activities in the presence of neutralizing antisera to human granulocyte-macrophage colony stimulating factor (GM-CSF) or to lnterleukin-3 (IL-3). It was found that most CSA was attributable to GM-CSF, whereas BPA was mainly related to the presence of IL-3.1988 by Grune & Stratton, Inc. 0006-4971/88/7203-0011$3.00/0

The specific immune response against the malignant cells was investigated in patients with urinary bladder or larynx cancer. Lymphocytes from lymph nodes that drain the tumor site were tested for their proliferative and cytotoxic capacities against autologous malignant cells isolated from the primary tumor. In no occasion was a proliferative or a cytotoxic response observed. However, when the lymph node cell suspensions were depleted of cells expressing both OKM1 and Leu-7 markers by rosetting with the appropriate mAbs, a proliferative response could be observed. The lymphocytes responded to autologous tumor cells only if IL-2 was added to the cultures. IL-2 alone induced some cell proliferation, which was not, however, comparable to that observed in response to both IL-2 and tumor cells. A panel of allogeneic tumor cells consistently failed to stimulate OKM1-, Leu-7- cells in vitro. Response to autologous tumor cells was not caused by HLA-encoded molecules, as occurs in the autologous mixed lymphocyte reaction, since OKM1-, Leu-7- cells failed to be stimulated by autologous non-T cells. A proliferative response was observed only with cells from lymph nodes that had been classified as invaded by malignant cells according to histopathologic criteria. Cells from noninvaded lymph nodes consistently failed to respond. Cells stimulated with autologous tumor cells could be expanded in short-term lines by continuous addition of IL-2 and malignant cells. One of these lines, which comprised mainly T8+ cells, was stimulated to proliferate only by autologous tumor cells, and its proliferative response was inhibitable by anti-class I and not by anti-class II mAbs. This line showed lytic capacities against autologous malignant targets, while it was inefficient against all of the other allogeneic cells tested. In another set of experiments, the mechanisms whereby exogenous IL-2 had to be added to the cultures to sustain a proliferative response against neoplastic cells were investigated. When cocultured with autologous malignant cells, OKM1-, Leu-7- lymphocytes expressed IL-2 receptors, as could be assessed by anti-Tac fluorescent staining. Under these culture conditions, these cells did not produce IL-2, and no proliferation was observed. Addition of purified IL-1 to the cultures induced IL-2 production and cell proliferation. It is concluded that metastatic lymph nodes contain a T cell population that can be detected in a proliferative assay when both suppressor cells are removed and the appropriate molecular signals are supplied.

Activated c-myc gene was introduced into the cells of three normal Epstein-Barr virus (EBV)-positive lymphoblastoid B cell lines (LCL). The cells were monitored for the appearance of new phenotypic and functional features compared with the control LCL cells transfected with plasmid that did not contain the c-myc gene. The LCL-expressing c-myc constitutively did not arrest growth in low serum concentration. However, the cell number in the cultures failed to increase because of substantial cell death. Death was due to apoptosis as demonstrated by flow cytometric analysis of propidium iodide-stained cells, by typical DNA laddering in gel electrophoresis, and by the inspection of Giemsa-stained cell smears. Apoptosis was also induced by exposing the transfected cells to antibodies directed to the immunoglobulin mu chain (a-mu-ab) irrespective of the serum concentration in the culture. Exposure of the cells to CD40 ligand (CD40L) or CD40 monoclonal antibody prevented cell apoptosis. Upon transfection with c-myc, the LCL cells acquired a vacuolated morphology that was never observed in control cells. Moreover, the expression of CD10 and CD38 was upregulated, while that of CD39 and especially CD23 was downregulated. Unlike that observed in certain Burkitt lymphoma (BL) cell lines that share the same surface phenotype (CD10+CD38+CD23-CD39-), the c-myc-transfected cells expressed lymphocyte function-associated (LFA) 1, LFA-3, and intercellular adhesion molecule 1 and grew in large clumps rather than single-cell layers. Expression of CD10 and CD38 was particularly evident on the cells undergoing apoptosis, thus suggesting a correlation between the presence of these markers and the apoptotic process. Cells placed in conditions favoring in vitro apoptosis displayed downregulation of Bcl-2 protein. Bcl-2 expression was, however, upregulated when the cells were exposed to CD40L. These data indicate that the B cells expressing c-myc constitutively acquire some of the features of normal centroblasts and of BL cells, including the expression of CD10 and CD38, and the propensity to undergo apoptosis, which can be prevented by exposure to CD40L. Therefore, these cells can serve as a model system to study both BL lymphomagenesis as well as the process of B cell selection occurring in the germinal centers.

Both lymphokine activated killer (LAK) cells and specific cytotoxic T lymphocytes appear to play a role in tumour immunity. Tumour infiltrating lymphocytes (TIL) which display a CD56+ phenotype (both CD3+ and CD3-) are also likely to possess anti-tumour activity. We have previously described a 120 kDa surface antigen, termed LAK1, expressed on a subset of human peripheral blood lymphocytes (20-50%) with both NK and LAK activity. The aim of the present study was to determine whether LAK1 antigen is able to distinguish among TIL two populations of effector cells displaying either specific or non MHC-restricted (NK/LAK) activity. We showed that about 25% of freshly derived TIL were weakly stained with anti-LAK1 monoclonal antibody and most of them were also CD3+ CD56-. After culture in recombinant interleukin-2 the majority of TIL were CD3+ CD56- and the percentage of LAK1+ cells increased up to 50%. Among cloned TIL, only those lacking LAK1 antigen displayed a specific cytotoxicity against the autologous tumour, whereas the non-lytic clones were able to produce both tumour necrosis factor and gamma-interferon. Moreover, when TIL from a renal cell carcinoma were fractionated into LAK1- and LAK1+ populations, the specific lytic activity was mainly evident when LAK1- lymphocytes were used as effector cells. Conversely, LAK activity was confined to the LAK1+ subset.

Highly purified natural killer (NK) cell suspensions were tested for their capacity to release colony-stimulating activity (CSA) in vitro. NK cell suspensions comprised primarily CD16+cells and were devoid of CD3+T cells, CD15+monocytes, and of B cells. CSA was detected in the NK cell supernatants and sustained the growth of myeloid colonies from both normal peripheral blood and bone marrow. CSA could be in part inhibited by pretreating NK cell culture supernatants with a specific goat anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antiserum. The inhibition, however, was never complete, a finding that suggests that additional factors were responsible for CSA. Incubation of NK cells with K562 cells (an NK-sensitive target) or with normal bone marrow cells resulted in the appearance of a strong colony-inhibiting activity (CIA) in the culture supernatants. Such CIA was demonstrable in an experimental system where bone marrow or peripheral blood progenitors were induced to form myeloid colonies in the presence of conditioned medium by CSA-producing giant cell tumor (GCT) cells. Stimulation of NK cells with NK-insensitive targets failed to induce CIA production. Neutralizing antitumor necrosis factor (TNF) monoclonal antibodies (MoAbs) were found capable of inhibiting CIA present in the supernatants of NK cells stimulated with K562 cells. Following treatment with anti- TNF antibodies, CSA was again detectable in the same supernatants. This finding indicates that induction of TNF production did not concomitantly switch off CSA production by NK cells. Pretreatment of NK cells with recombinant interleukin-2 (rlL-2) or γ interferon (rγlFN) did not change the amount of CSA released. However, treatment with rlL-2 caused the appearance of a factor in the NK cell supernatants capable of sustaining the formation of colonies of a larger size.

Abnormalities of T-cell subsets in patients with multiple sclerosis are well known; in order to assess whether immunological abnormalities are relevant in the pathogenesis of the disease after its clinical onset, peripheral blood lymphocyte subsets (CD3+, CD4+, CD4+ CD45RA+, CD4+CD45RA-, CD8+, CD8+CD57+, CD57+, CD25+) were analysed serially in 25 patients at the first clinical episode suggestive of inflammatory demyelinating disease and in an equal number of age- and sex-matched controls. During the follow-up period (12–18 months, mean 14) 6 of 25 patients presented new relapses: in this subgroup of patients, significant changes in CD4+ ratio (% CD4+CD45RA-/%CD4+CD45RA-) were detected in comparison both with healthy controls and with clinically stable patients. Patients clinically stable at follow-up did not display immunological abnormalities, regardless of the presence or absence of cerebrospinal fluid and/or magnetic resonance imaging alterations consistent with multiple sclerosis. These findings suggest a possible prognostic role of early T-cell subset imbalance in multiple sclerosis.

Summary Phlogopite and biotite coexist in the ultrapotassic rocks from Cabezo Negro de Zeneta (SE Spain). The compositional range of the early crystallizing phlogopite is comparable to other Spanish lamproitic occurrences, except that it is higher in Al2O3, probably reflecting the higher Al2O3 and/or different oxygen fugacity of the Zeneta magma. Magmatic Al-rich and metamorphic Al-poor biotites also occur in these rocks. The magmatic biotite probably crystallised from intermediate to silicic peraluminous magma(s), whereas the metamorphic type comes from crustal relics of metapelitic rocks entrained and dismembered into the lamproitic melt. It is concluded that the melt of Zeneta was generated through the mixing of a Mg-rich lamproitic component, quantitatively dominant, with a crustal-derived anatectic component, both already partially crystallised before mixing. The “mixed” melt attained chemical homogenization as suggested by the development of late overgrowths of similar composition on the two micas.

Cell lines were established from purified large granular lymphocites (LGL) isolated from the peripheral blood of seven patients with phenotypically homogeneous LGL expansions. LGL were stimulated with phytohemagglutinin (PHA) or recombinant interleukin-2 (rIL-2) and further expandedin vitro in IL-2-containing media. The surface phenotype of LGL, as assessed by monoclonal antibody staining, was T3+ T8+ in five patients, T3- T8- in one, and T3+ T8- in another patient. The cells also expressed Leu 7, Leu 11, and/or OKM 1 markers in various proportions and were identifiable as LGL by their morphological and cytochemical features. The original surface phenotype of the unstimulated LGL was retained in the IL-2-dependent cell lines from each individual patient, i.e., T3+ T8+ cells originated T3+ T8+ cell lines and T3- T8- cells originated T3- T8- cell lines. Other markers, such as Leu 11 and OKM 1, were generally lost in culture. LGL proliferated in response to rIL-2 but did not express detectable IL-2 receptors, even after prolonged periods of culture. All cell lines from each individual patient had the same surface phenotype, and within the single lines, all of the cells expressed the same markers. Cell lines from two patients consistently displayed chromosomal abnormalities. Although different in the two patients, the abnormalities were identical in all of the lines from the same patient and detectable in most of the cells examined, suggesting a clonal origin for the abnormally expanded LGL populations. Freshly isolated LGL did not exert NK activity. However, the IL-2-dependent LGL lines acquired the ability to kill K562 target cells and to produce gamma interferon (?-IFN). No direct correlation was observed between these two properties.

The axonal type 2 Charcot–Marie–Tooth disease (CMT2) is phenotypically poorly characterized. Here the authors report a family with a Pro22Ser mutation in the neurofilament-light gene (NF-L; CMT2E) manifesting electro-physiologically as the demyelinating type 1 CMT (CMT1) and pathologically as an axonopathy with giant axons and accumulation of disorganized NF. NF-L should be investigated in CMT2 as well as in CMT1 not associated with the usual genes PMP22, Cx32, and P0.

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease, as some patients progress rapidly toward the more advanced studies, whereas others survive for a long period without the need for treatment. This heterogeneity of clinical course was somehow unexplained until studies on the CLL cell features disclosed that the CLL clones were heterogeneous and were characterized by different phenotypic and genotypic features in the different patients. On the basis of these observations, it was determined in retrospective studies that clones characterized by unmutated IGHV genes, and/or CD38 and/or ZAP-70 expression conferred a more severe prognosis to the CLL patients. Here, we present data on prospective studies carried out on Binet A-stage patients, in whom the markers were determined at diagnosis and their predictive value was assessed in comparison with chromosomal abnormalities and gene expression or micro RNA profiles. In addition, hypothesis on the potential pathogenetic role of these markers will be presented.

Congenital hypomyelination neuropathy (CHN) has been related with mutations of the MPZ gene that codes for P0, the major structural protein of the peripheral myelin and of EGR2/Krox20gene that codes for a transcription factor essential for the normal development of myelinating Schwann cell. More recently, we reported the association between CHN and a Ser72Leu mutation of the peripheral myelin protein 22 (PMP22), a quantitatively minor component of the compact myelin of peripheral nerves, whose functions are still debated. Here we describe a second patient with CHN associated with a novel mutation of PMP22.

To evaluate the uncertainties for nuclear track detectors used in radon measurements, a full understanding is required of the physical phenomena involved and the behaviour of the instruments utilised in the measuring process. As it concerns the LR115 nuclear track detector, an overall evaluation of uncertainty was given. It was assessed taking into account different contributions and determining their relative weights. Since such detectors are often read by a spark counter device, a model to describe its behaviour was developed and a saturation factor was estimated. Its expression and its associated uncertainty are given. Hence, it has been possible to draw a calibration curve, in which all the uncertainty sources have been considered.