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R Carsetti

Publications and source records attributed to R Carsetti.

17 recordsLinked to original sources
PubMed2004

Insight into B cell development and differentiation.

The main topic of this article is B cell development and differentiation, with a special focus on the mechanisms and molecules that regulate the expression of humoral immunity. Molecular epidemiological analysis was performed on the genes responsible for the X-linked agammaglobulinemia (XLA) phenotype of the majority of Italian patients and their distinct mutations were characterized. Mutations in Bruton's tyrosine kinase (BTK), a member of Tec Family of protein tyrosine kinases, have been found to be mainly responsible for XLA disease. The exact function of BTK in signal transduction is not yet known; thus, the specific role of BTK in receptor-dependent calcium signaling and the pro-antiapoptotic regulatory activity was addressed by transfecting RAMOS-1, a BTK-deficient human Burkitt's/B cell leukemia line with wild-type and mutant constructs. This work may provide clues about critical sites in the molecule and give support for gene therapy as a potential successful approach to XLA. Another aspect of this research is the identification and dissection of the molecular events that are likely to be directly related to the ability to express various isotypes of immunoglobulin with differing function and certain B cell immunodeficiency, mainly common variable disease and non-X-linked hyperIgM. B cell development and maturation steps in different compartments of the immune system are tracked by the analysis of cell-surface molecules and components of the signal transduction pathways, i.e. CD40, CD30, CD27, CD38, CD22 and CD24. A few components involved in B cell development, maturation and differentiation and their specific functional role are at least partially known, but these are far from fitting into an understandable pathway at present.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2001

Abnormal bone marrow stroma in mice deficient for nemo-like kinase, Nlk.

The stromal compartment of the bone marrow is composed of various cell types that provide trophic and instructive signals for hematopoiesis. The mesenchymal stem cell is believed to give rise to all major cellular components of the bone marrow microenvironment. Nemo-like kinase, Nlk, is a serine-threonine kinase that connects MAP kinase and Wnt signaling pathways; its in vivo function in mouse is unknown. We have generated mice with a targeted disruption of Nlk and find that the complex phenotype significantly varies with the genetic background. Whereas C57BL/6 mice lacking Nlk die during the third trimester of pregnancy, the 129/Sv background supports survival into adolescence; such mice are growth retarded and suffer from various neurological abnormalities. We show here that the Nlk deficiency syndrome includes aberrant differentiation of bone marrow stromal cells. Varying degrees of morphological abnormality, such as increased numbers of adipocytes, large blood sinuses and absence of bone-lining cells are observed in the bone marrow of mutant mice. Nlk deficient mice thus provide a novel model to study the genetic requirements for bone marrow stromal differentiation.

Animals↗
PubMed1999

B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals.

Only mature B lymphocytes can enter the lymphoid follicles of spleen and lymph nodes and thus efficiently participate in the immune response. Mature, long-lived B lymphocytes derive from short-lived precursors generated in the bone marrow. We show that selection into the mature pool is an active process and takes place in the spleen. Two populations of splenic B cells were identified as precursors for mature B cells. Transitional B cells of type 1 (T1) are recent immigrants from the bone marrow. They develop into the transitional B cells of type 2 (T2), which are cycling and found exclusively in the primary follicles of the spleen. Mature B cells can be generated from T1 or T2 B cells. Mice with genetic deletions of elements participating in the B cell receptor signaling cascade display developmental arrest at the T1 or T2 stage. The analysis of these defects showed that the development of T2 and mature B cells from T1 precursors requires defined qualitative and quantitative signals derived from the B cell receptor and that the induction of longevity and maturation requires different signals.

Animals↗
PubMed1998

The molecular mechanism of B cell activation by toll-like receptor protein RP-105.

The B cell-specific transmembrane protein RP-105 belongs to the family of Drosophila toll-like proteins which are likely to trigger innate immune responses in mice and man. Here we demonstrate that the Src-family protein tyrosine kinase Lyn, protein kinase C beta I/II (PKCbetaI/II), and Erk2-specific mitogen-activated protein (MAP) kinase kinase (MEK) are essential and probably functionally connected elements of the RP-105-mediated signaling cascade in B cells. We also find that negative regulation of RP-105-mediated activation of MAP kinases by membrane immunoglobulin may account for the phenomenon of antigen receptor-mediated arrest of RP-105-mediated B cell proliferation.

Animals↗
PubMed1997

CD22 is a negative regulator of B-cell receptor signalling.

BACKGROUND: . Antibody responses are triggered by binding of antigen to the B-cell antigen receptor (BCR). The strength of the resulting signal determines the outcome of the response, which may vary from the induction of tolerance to the antigen, to the production of specific high-affinity antibodies. Additional cell-surface proteins assist the BCR in its function, and can facilitate or inhibit an antibody response. CD22 is a BCR-associated transmembrane protein, the cytoplasmic tail of which contains three immunoreceptor tyrosine-based inhibitory motifs. These motifs are phosphorylated upon BCR-crosslinking, and can bind the tyrosine phosphatase SHP-1, a putative negative regulator of signalling from the BCR. In order to assess the role of CD22 in vivo, we have generated CD22(-/-) mice by targeted gene inactivation. RESULTS: . In CD22(-/-) mice, B-cell development is normal. There are normal numbers of peripheral B cells, but these have a more mature phenotype. In addition, recirculating B cells are absent from the bone marrow. However, the distribution of the two B-cell subtypes, B-1 and B-2, is normal. After BCR-crosslinking in vitro, splenic CD22(-/-) B cells show an increased Ca2+ influx and a lower survival due to an increased induction of apoptosis. In contrast, there is an increased proliferative response to the B-cell mitogen lipopolysaccharide (LPS). A shorter average lifespan in the B-cell compartment is also found in vivo. Furthermore, T-cell independent immune responses are impaired, whereas T-cell dependent responses are normal. CONCLUSIONS: . The absence of CD22 expression lowers the signalling threshold for BCR-crosslinking and can thus influence the fate of the B cell. We propose that the low threshold leads to hyperresponsiveness of the B cells and a chronic basal activation. In this model, engagement of the receptor without T-cell help leads to an increased induction of apoptosis, thus explaining the shorter lifespan of CD22(-/-) B cells and the low response to T-cell independent antigens. The alteration in B-cell phenotype and the higher levels of LPS-reactivity are attributable to the chronic basal stimulation.

Animals↗
PubMed1996

Immunoglobulin-mediated signal transduction in B cells from CD45-deficient mice.

CD45 expression is essential for immunoglobulin (Ig)-mediated B cell activation. Treatments with either anti-Ig or anti-CD45 suggest that CD45 may facilitate early signaling events such as calcium mobilization, and phosphoinositide hydrolyis as well as later events leading to transcription of genes such as c-myc. To examine the role of CD45 more extensively, CD45-deficient mice were generated by disruption of exon 6. Although normal numbers of B cells were found in peripheral lymphoid tissues, CD45-deficient cells failed to proliferate upon IgM crosslinking. In the present study, we demonstrate that the fraction of high buoyant density B cells is reduced while low buoyant density cells are increased. Moreover, there is a significant decline in the number of splenic B cells of the mature IgDhi, IgMlo phenotype. Although both the basal and anti-Ig-induced levels of phosphorylation of Ig-alpha and phospholipase C gamma 2 are indistinguishable from that observed in CD45+ control B cells, a major distinction was found in Ca2+ mobilization. While anti-Ig-induced mobilization of intracellular Ca2+ stores was normal, influx from extracellular sources was abrogated. This finding reveals a novel pathway of regulating B cell responses mediated by CD45.

Animals↗
PubMed1996

Induction of CD14 expression in Lpsn, Lpsd and tumor necrosis factor receptor-deficient mice.

The involvement of CD14 in lipopolysaccharide (LPS) recognition and signaling has been demonstrated in several studies. For this reason, we investigated whether the resistance of Lpsd mice to LPS might be related to an impaired CD14 expression. We compared the in vivo and in vitro expression of CD14 in Lpsn (LPS sensitive) and Lpsd mice, and its modulation by LPS, killed gram-negative and gram-positive bacteria and double-stranded (ds)RNA. Untreated Lpsn and Lpsd cultured macrophages (M phi), expressed similar amounts of CD14 mRNA and membrane-bound (m)CD14. LPS enhanced CD14 expression only in Lpsn M phi, while all bacteria, or dsRNA, enhanced CD14 in Lpsn and Lpsd M phi. Similarly, in vivo administration of LPS induced or enhanced CD14 mRNA in different organs of Lpsn mice only, while bacteria or dsRNA in both types of mouse. Furthermore, exogenous recombinant tumor necrosis factor (TNF) induced in vivo and in vitro enhanced CD14 expression in Lpsn, Lpsd and also in TNF receptor 2-deficient (TNFR2-/-) mice, but failed to do so in TNFR1-/- mice, showing that TNFR1 mediates the effect of TNF on CD14. However, LPS, bacteria and dsRNA induced CD14 in both TNFR2-/- and TNFR1-/- mice to a similar extent, revealing that this induction does not require TNF signaling.

Animals↗
PubMed1996

A novel mouse thymocyte antigen (F3Ag): down-regulation during the CD4+CD8+ double-positive stage indicates positive selection.

We describe a novel mAb (F3) which reacts with a 65 kDa thymocyte surface protein, expressed on approximately 80% of thymocytes, referred to as F3Ag. In ontogeny, F3Ag expression begins in the CD4(-)CD8(-) double-negative (DN) CD25(+) population and is maintained through approximately 85% of the CD4(+)CD8(+) double-positive (DP) stage. DP cells with high TCR expression and CD4(+) single-positive (SP) cells are predominantly negative for F3Ag, whereas many CD8(+) SP thymocytes express F3Ag. F3Ag-DP thymocytes show a reduced expression of RAG-1 and RAG-2 compared with F3Ag+ DP cells. The shutdown of F3Ag expression during the DP stage is related to positive selection: mice deficient for MHC class I and class II molecules maintain F3Ag expression in almost all DP cells. Transgenic (tg) mice carrying TCR restricted for MHC class II show a more pronounced down-regulation of F3Ag in the DP compartment than normal mice, depending on the presence of a positively selecting MHC. The size of the F3Ag- DP subset is positively correlated with the efficacy of positive selection into the CD4(+) SP compartment. Because some CD8(+) SP cells express F3Ag, the relationship between F3Ag down-regulation and positive selection is less obvious in DP cells of mice carrying MHC class I-restricted tg TCR. However, in reaggregate thymic organ cultures, sorted F3Ag- DP cells differentiate into CD8(+) SP cells more rapidly than do F3Ag+ DP cells. Thus, after down-regulation in the DP stage, a proportion of CD8(+) SP cells appears to re-express F3Ag. In addition, the proportion of F3Ag-CD8(+) SP cells depends on the efficacy of positive selection into the CD8 lineage. Taken together, the regulation of the expression of the F3Ag appears to be associated with signals that control thymic repertoire selection.

Animals↗
PubMed1996

Role of c-myc and CD45 in spontaneous and anti-receptor-induced apoptosis in adult murine B cells.

Although adult murine B cells can be stimulated to proliferate by Igm receptor cross-linking, we and others have shown that these cells will undergo apoptosis in vitro in a dose-, time- and temperature-dependent manner with polyclonal but not monoclonal anti-IgM, To test the role of c-myc and cell cycle progression in B cell apoptosis, we examined normal, Sp6 anti-TNP lg and E micro-myc transgenic splenocytes for receptor-mediated apoptosis in vitro. In normal mice, both spontaneous and anti-IgM-induced programmed cell death were specifically blocked by antisense oligodeoxynucleotides for the c-myc proto-oncogene, whereas nonsense myc oligonucleotides and irrelevant oligonucleotides had only a minor effect. Similarly, TNP-dextran-induced apoptosis in Sp6 anti-TNF transgenics was inhibited by antisense c-myc. This effect was not due to the mitogenic effects of unmethylated CpG-containing sequences because ones lacking this motif, as well as methylated oligonucleotides containing this motif, prevented apoptosis, and mitogenic doses of lipopolysaccharide failed to inhibit anti-IgM-driven cell death. Importantly, antisense c-myc also prevented the anti-IgM-induced increase in myc protein species. Moreover, spontaneous apoptosis in vitro was exaggerated in E micro-myc transgenic B cells. To examine the role of CD45 in anti-IgM-induced apoptosis, we treated spleen cells from CD45 knockout mice, which do not proliferate with anti-IgM, and found that B cells from these underwent apoptosis normally despite the lack of entry into S. These data suggest that anti-IgM driven apoptosis does not require CD45. Rather, apoptosis may be due to an overexpression of myc protein in the absence of signals which can drive B cells productively into S, but the failure to proliferate normally is insufficient for apoptosis to occur.

Animals↗
PubMed1995

Transitional B cells are the target of negative selection in the B cell compartment.

B lymphocytes recognize antigen through membrane-bound antigen-receptors, membrane IgM and IgD (mIgM and mIgD). Binding to foreign antigens initiates a cascade of biochemical events that lead to activation and differentiation. In contrast, binding to self-antigens leads to death or to inactivation. It is commonly believed that the B cells acquire the ability to discriminate between self and nonself in the early phases of development. We report here that immature B cells, which have just emerged from the mIgMneg, B220pos pool, are not deleted upon binding of self-antigen. In vivo, developing B cells become sensitive to tolerance induction in a relatively late window of differentiation, when they are in transition from the immature (HSAbright, B220dull) to the mature (HSAdull, B220bright) stage. In the transitional B cells, early markers of differentiation such as Pgp1 (CD44) and ThB reach the highest level of expression, while the expression of CD23 and mIgD, late markers of differentiation, and expression of class II MHC, progressively increases. Most of the transitional B cells, but only few of the mature and of the immature B cells, express the fas antigen, while mature B cells, but not immature and transitional B cells, express bcl-2 protein. mIgM is present in low amounts in immature B cells, reaches the highest level of expression in transitional B cells and is down-regulated in mature resting B cells, where it is coexpressed with mIgD. The high expression of mIgM, the presence of the fas antigen and the absence of bcl-2 protein is compatible with the high sensitivity of transitional B cells to negative selection. In vitro, immature B cells die rapidly by apoptosis after cross-linking of mIgM. This result, combined with the resistance of immature B cells to elimination in vivo, suggests that early in development the stroma cell microenvironment modulates signals transduced through mIgM. The functional and phenotypic division of IgMpos bone marrow B cells in three compartments not only allows to define the target population of physiological processes like negative selection, but will also be a helpful tool for an accurate description of possible developmental blocks in mutant mice.

Animals↗
PubMed1993

Regulation of thymocyte development through CD3. II. Expression of T cell receptor beta CD3 epsilon and maturation to the CD4+8+ stage are highly correlated in individual thymocytes.

Recent studies have shown that maturation of CD4-8- double negative (DN) thymocytes to the CD4+8+ double positive (DP) stage is dependent on expression of the T cell receptor (TCR)-beta polypeptide. The exact mechanism by which the TCR-beta chain regulates this maturation step remains unknown. Previous experiments had suggested that in the presence of some TCR+ thymocytes, additional DN thymocytes not expressing a TCR-beta chain may be recruited to mature to the DP stage. The recent demonstration of an immature TCR-beta-CD3 complex on early thymocytes lead to the alternative hypothesis that signal transduction through an immature TCR-CD3 complex may induce maturation to the DP stage. In the latter case, maturation to the DP stage would depend on the expression of TCR-beta-CD3 in the same cell. We examined these two hypotheses by studying the expression of the intra- and extracellular CD3 epsilon, CD3 zeta, and TCR-beta polypeptides in intrathymic subpopulations during embryogenesis. CD3 epsilon and CD3 zeta were expressed intracellularly 2 and 1 d, respectively, before intracellular expression of the TCR-beta chain, potentially allowing immediate surface expression of an immature TCR-beta-CD3 complex as soon as functional rearrangement of a TCR-beta gene locus has been accomplished. Calcium mobilization could be induced by stimulation with anti-CD3 epsilon mAb as soon as intracellular TCR-beta was detectable, suggesting that a functional TCR-beta-CD3 complex is indeed expressed on the surface of early thymocytes. From day 17 on, most cells were in the DP stage, and over 95% of the DP cells expressed on the TCR-beta chain intracellularly. At day 19 of gestation, extremely low concentrations of TCR-beta chain and CD3 epsilon were detectable on the cell surface of nearly all thymocytes previously thought to be TCR-CD3 negative. These findings strongly support the hypothesis that maturation to the DP stage depends on surface expression of and subsequent signal transduction through an immature TCR-beta-CD3 complex and suggest that maturation to the DP stage by recruitment, if it occurs at all, is of minor relevance.

Animals↗
PubMed1993

Molecular mimicry of the antigen receptor signalling motif by transmembrane proteins of the Epstein-Barr virus and the bovine leukaemia virus.

BACKGROUND: Many transmembrane proteins of eukaryotic cells have only a short cytoplasmic tail of 10 - 100 amino acids, which has no obvious catalytic function. These tails are thought to be involved either in signal transduction or in the association of transmembrane proteins with the cytoskeleton. We have previously identified, in the cytoplasmic tails of components of B and T lymphocyte antigen receptors, an amino-acid motif that is required for signalling. The same motif is also found in the cytoplasmic tails of two viral proteins: the latent membrane protein, LMP2A, of Epstein Barr virus and the envelope protein, gp30, of bovine leukaemia virus. Interestingly, both viruses can activate infected B lymphocytes to proliferate, as does signalling by the B-cell receptor. RESULTS: In this study, we show that the cytoplasmic tails of the two viral proteins, and the cytoplasmic tail of the B-cell receptor immunoglobulin-alpha chain, when linked to CD8 in chimeric transmembrane proteins, can transduce signals in B cells. Cross-linking of these chimeric receptors activates B-cell protein tyrosine kinases and results in calcium mobilization. Furthermore, these cytoplasmic sequences are also protein tyrosine kinase substrates and may interact with cytosolic proteins carrying SH2 protein-protein interaction domains. CONCLUSION: Our findings suggest that viral transmembrane proteins can mimic the antigen-induced stimulation of the B-cell antigen receptor and thus can influence the activation and/or survival of infected B lymphocytes.

Journal Article↗
PubMed1993

A role for immunoglobulin D: interference with tolerance induction.

We have studied the induction and maintenance of tolerance in the B lymphocyte compartment. Neonatal and adult transgenic mice which expressed either surface IgM (sIgM) or sIgM and sIgD anti-2,4,6-trinitrophenyl (TNP) were treated with soluble mono- and multivalent forms of TNP-modified carriers. We compared the B cell compartment of mice treated with antigen and of littermates injected with phosphate-buffered saline. Antigen-mediated cross-linking of membrane-bound IgM (sIgM) caused deletion of B cells both in neonatal and adult mice with mu and kappa transgenes. Deletion was the result of apoptosis. In mice that carried an additional delta transgene sIgD interfered with tolerance induction. The stage in which the cells were sensitive to deletion was characterized as a transitional stage between immature (sIgMdull, heat-stable antigenbright, B220dull, sIgD-) and more mature (IgMbright, heat-stable antigendull, B220bright, sIgD-) B cells. Surviving cells were functional as measured by receptor-mediated changes in the intracellular free Ca2+ concentration. We propose that when the immature B cells have reached the final stages of maturation IgM always transmits negative signals in the absence of T cell help. When B cells need to be screened against self reactivity IgM is the only antigen receptor expressed. The presence of sIgD protects resting B cells from deletion and allows them to initiate an effective immune response.

Animals↗
PubMed1989

Immune status of a mu, kappa transgenic mouse line. Deficient response to bacterially related antigens.

We have examined the immune repertoire and immune response of a mouse that carries transgenes for a mu heavy chain and kappa light chain. The expression of these genes is under the regulation of their own controlling elements. The transgenes are expressed early in ontogeny and are easily detectable from day 13 of gestation onwards. The pre-B cells seem to function normally as they generate IgM-secreting colonies at normal frequencies. Colonies show predominantly the transgenic specificity. Expression of the transgenes is not limited to B cells since around 10%-20% of peripheral T cells and 50% of thymocytes express the mu transgene as an intracellular protein. Ectopic expression of kappa was not seen. The spleen size of the transgenic mouse is decreased by around 20%; this reduction is largely caused by a reduction of the B cell pool. Almost all B cells express the transgenes, only 30% co-express endogenous heavy chain genes and all co-express endogenous light chain genes. Serum Ig levels for IgM and IgA were normal, 20% of the IgM consist of the transgenic product. Serum IgG levels were decreased. T cell functions (helper and cytotoxic) were normal. Immune responses to conventional antigens were impaired, especially in the early phases of the immune response, but after boosting they were virtually normal, except for IgG3 which remained low. Primary antibody responses to T cell-independent antigens of the class II type (bacterially related antigens) were absent, although precursor frequencies for these antigens were within the expected range. The significance of this finding, as it relates to allelic exclusion of Ig genes, is discussed.

Animals↗
PubMed1988

Viral oncolysates in patients with advanced ovarian cancer.

Viral oncolysates (VO) derived from two cultured ovarian carcinoma cell lines infected with influenza A/PR8/34 were administered intraperitoneally (IP) to 40 patients with advanced ovarian carcinoma, including 31 with late-onset ascites and 5 with pleural effusions. PR8 virus-specific antigens and ovarian tumor-associated antigens have been demonstrated on two oncolysates designated OVO1 and OVO2. Thirty-five patients received 9 mg of a 1:1 mixture of OVO1 and OVO2, 5 patients received one or the other. During the first month three IP schedules were evaluated, i.e., single, biweekly, and weekly, which were followed by monthly injections. Intrapleural (IP1) injections of a 3.0-mg 1:1 mixture of OV1 and OV2 were administered to 3 patients concurrently with initial IP injections and to 2 patients following later development of pleural effusions. In 7 patients ascites disappeared; in 5 of these the number of cytologically detected malignant cells was markedly reduced, in 1 pleural effusion disappeared, and in 3 tumor masses were reduced. Tumor masses shrank also in 2 patients without ascites. Tumor reduction conformed to standard response criteria in 2 of the 5 patients. Response duration in the 9 responding patients lasted from 3 to 19 months and survival durations 4 to 42 months. Disease symptoms in 7 patients improved noticeably. Two of the 9 responders later developed unilateral pleural effusions that responded for 7 and 15+ months to a single IP1 injection. Seventeen patients experienced one or more treatment side effects including fever, nausea or anorexia, malaise, abdominal pain, and arthralgia, but in only 2 patients, both on the weekly schedule, was toxicity severe enough to require treatment withdrawal. Humoral responses to viral and tumor cell-surface antigens were frequently observed in patients demonstrating clinical activity.

Adult↗
PubMedDate not supplied

Immunoglobulin M memory B cell decrease in inflammatory bowel disease.

BACKGROUND & OBJECTIVES: Memory B cells represent 30-60% of the B cell pool and can be subdivided in IgM memory and switched memory. IgM memory B cells differ from switched because they express IgM and their frequency may vary from 20-50% of the total memory pool. Switched memory express IgG, IgA or IgE and lack surface expression of IgM and IgD. Switched memory B cells derive from the germinal centres, whereas IgM memory B cells, which require the spleen for their survival and/or generation, are involved in the immune response to encapsulated bacteria. Since infections are one of the most frequent comorbid conditions in inflammatory bowel disease, we aimed to verify whether IgM memory B cell pool was decreased in Crohn's disease and ulcerative colitis patients. PATIENTS & METHODS: Peripheral blood samples were obtained from 22 Crohn's disease patients, 20 ulcerative colitis patients, 22 healthy controls and 18 splenectomized patients. To analyse peripheral blood lymphocytes, flow cytometry was performed using anti-CD19, anti-CD22, anti-CD27, anti-IgM, anti-IgD and anti-CD38 monoclonal antibodies. RESULTS: Circulating IgM memory B cells were significantly lower in Crohn's disease (median 7.1%, range 1.8-20.7) and ulcerative colitis patients (median 8.1%, range 2.1-18.8) in comparison to control subjects (median 14.0%, range 6.8-31.1). As expected, there was a highly significant difference in the proportion of IgM memory B cells between splenectomized patients (median 2.4%, range 0.9-6.9) and healthy controls. Crohn's disease patients with abscesses showed the lowest frequency of IgM memory B cells. DISCUSSION: Our findings show that peripheral IgM memory B cells are reduced in inflammatory bowel disease patients. Further studies are necessary to answer the question of whether high risk of infection (abscess development) is promoted by the reduction/depletion of IgM memory B-cell pool in inflammatory bowel disease.

Adult↗