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Biomedical subjects

G Dighiero

Publications and source records attributed to G Dighiero.

At least 73 records · Page 4Linked to original sources
PubMed1995

Analysis of VH gene expression in CD5+ and CD5- B-cell chronic lymphocytic leukemia.

In contrast to highly mutated follicular lymphomas and multiple myelomas, chronic lymphocytic leukemias (CLLs) frequently express VH genes in germline configuration. It is currently unclear whether this difference is related to the expression of CD5 or to the differentiation stage of the B cell when malignant transformation occurs. We have studied the VH sequence of 11 cases of CD5- B-CLL to address the question whether CD5- B-CLL are derived from naive pregerminal B cells (low mutation pattern) or from germinal center-derived memory B cells (high mutation pattern). Among the 12 detected rearrangements (2 distinct rearrangements in 1 case) VH1 family was found in 2, VH2 in 2, VH3 in 4, and VH4 in 4. Nine different VH genes were detected among the 12 rearrangements, including 2 cases expressing V1-69 (51p1) and 1 case expressing V4-39 (VH4.18), previously reported to be overexpressed in CD5+ B-CLL. A higher mutation pattern, following a random distribution, was observed when compared with classical CD5+ B-CLL. However, as reported in normal B cells, these results appeared to be related to membrane Ig phenotype (less mutations in membrane mu delta-expressing forms in leukemias expressing exclusively membrane mu). Overall, the differences found when comparing the mutational profile with classical CD5+ B-CLL were not clearcut and might be explained more by the membrane isotype (mu v mu delta) than by CD5 expression.

Aged↗
PubMed1995

Flow cytometric analysis of protein-tyrosine phosphorylation in peripheral T cell subsets. Application to healthy and HIV-seropositive subjects.

This paper describes an improved method for detecting tyrosine phosphorylation levels in T cell subsets by flow cytometry early after CD3 crosslinking stimulation. It consists in introducing gentle paraformaldehyde fixation between CD3 crosslinking in cold conditions and the shift to 37 degrees C, which activates downstream signalling machinery. We used the combined properties of monoclonal antibodies for stimulating cells and for detecting surface markers to analyze protein-tyrosine phosphorylation levels in T cells subsets following stimulations which mimic physiological activation. Overall data obtained in healthy subjects, using two- or three-color immunofluorescence, indicated that: (1) most CD3 positive cells phosphorylate tyrosine substrates following CD3 crosslinking stimulation and (2) helper cells phosphorylate tyrosine to a slightly better extent than cytotoxic cells after CD3 crosslinking. Nevertheless, the two subsets follow similar kinetic patterns and tend to retain a homogeneous profile. Processing of samples from HIV-seropositive patients showed heterogeneous phosphorylation levels in both subsets, when compared to normal donors. This assay should, in the future, lead to easy and rapid exploration of the signal transduction pathway in different subsets of T cells under normal and pathological conditions.

CD3 Complex↗
PubMed1995

Evidence for an antigen-driven selection process in human autoantibodies against acetylcholine receptor.

Autoantibodies to the nicotinic acetylcholine receptor (AChR) play a central role in the neurological symptoms associated with myasthenia gravis (MG). A better knowledge of the structural organization and of the mechanisms leading to the production of these antibodies may help in understanding the pathogenesis of the disease. To achieve this, four IgG anti-AChR monoclonal autoantibodies obtained in a previous work were derived from lymphoid cells of MG patients. Two of them (MH1 and MH6) were capable of modulating in vitro the expression of AChR at the surface of TE-671 cells. We report here the complete nucleotide sequence of the heavy and light chains of these four antibodies. Although it is difficult to address the issue of VH gene usage in anti-AChR autoantibodies because of the limited number of clones studied, our results associated with others which have appeared in the literature point to non-stochastic usage by anti-AChR antibody of some defined VH genes belonging to VH2 and VH5 minifamilies overexpressed in the fetal repertoire. The second and major aim of this work was to assess the role of an antigen-driven selection process in the production of anti-AChR autoantibodies. When comparing the expressed sequences to their closest germline counterparts, it appeared that all four studied clones displayed numerous mutations in VH regions. In particular, MH1 and MH6, characterized by their AChR modulating capacity, displayed a higher than expected number of mutations and replacements occurring in CDR regions. These data point to an antigen-driven selection process. On the contrary, the mutational process observed in the MH% clone was borderline and that of MH7 was compatible with a random process. Interestingly, when comparing mutations in heavy and light chains, a significantly lower number of mutations were expressed in light chains for the four clones.

Amino Acid Sequence↗
PubMed1995

Skewed rearrangement of the VH4-21 gene during pre-B acute lymphoblastic leukemia.

Thirty-six pre-B acute lymphoblastic leukemias (ALL) were studied for VH family expression. Among the 35 detected rearrangements, VH1 family genes were expressed in 7, VH2 in 1, VH3 in 18, VH4 in 6 and VH6 in 3. This expression is close to that expected according to the complexity of the system. The complete sequence of the 6 VH4 genes was examined in order to determine whether there is a skewed rearrangement of individual genes in this family. Our results indicate rearrangement of VH4-21 in 3 cases, 71-4 in one, 58P2 in one case and probably of a new germinal VH4 gene for the sixth case. All the genes were displaying an almost complete homology with their germinal VH counterparts. The 6 sequenced genes associated with 6 different D gene segments displaying a close homology with their germinal counterpart. JH4 segment was expressed in 3 cases and JH6 in the remaining 3. These results associated with previous results obtained by others indicate that there is skewed rearrangement of the VH4-21 gene in pre-B ALL. It is presently unknown whether this phenomenon is the consequence of a selective process or whether it reflects what normally occurs in the normal human functional repertoire, which could be more limited than the germline repertoire.

Adult↗
PubMed1994

Characterization of a monoclonal IgMK (IgMGAS) anti-Gd cold agglutinin (CA). Its coexistence with a monoclonal IgG3K (IgGGAS) without CA activity that might be clonally related to IgMGAS.

A monoclonal IgMK (IgMGAS) cold agglutinin (CA) of the infrequent anti-Gd specificity, found in a patient with Waldenström macroglobulinemia, has been characterized. IgMGAS uses a VH gene homologous (93.7%) to the reported VH251 germ-line, one of the two functional genes of the VH5 family, with differences in both framework regions and complementary determining regions (CDR). The VL gene is homologous to the reported 15AVK1 germ-line gene, recently described in an anti-i CA, with differences mostly clustered in CDR. In the patient's serum, IgMGAS coexisted with a monoclonal IgG3K (IgGGAS) that lacked CA activity but expressed the private idiotopes found on IgMGAS. Both Ig lacked reactivity with antibodies detecting VH1 or VHIII or VKIII subgroup regions or the VH4-21 gene product that is expressed by anti-I/i CAs. The K chains from both Igs showed the same isoelectrical mobility. Moreover, the k chains from both serum Igs showed the same N-terminal amino acid sequence. This sequence was identical to that predicted by the nucleotide sequence of VK1GAS gene segment, including one discrepancy at position 15 (Ile for Val) with respect to the consensus VK1 subgroup regions. Although these data do not exclude a possible independent clonal origin, they are consistent with the notion that IgGGAS might be clonally related to IgMGAS.

Aged↗
PubMed1994

Viral superantigen-induced hyporesponsiveness of T cells and polyclonal B cell activation in HIV-1 infection.

The mechanisms of CD4 depletion and hyporesponsiveness during human immunodeficiency virus (HIV) infection are still unknown. Given the ability of superantigens to stimulate a higher number of lymphocytes than conventional antigens, they may play a major role in this process. Recently, a novel superantigen, the rabies virus nucleocapsid (NC), was described in humans. In the present work, we tested the responses of peripheral blood lymphocytes from asymptomatic HIV-infected patients to this superantigen. In contrast to its effect in normal controls, NC failed to expand T cells from HIV-infected individuals expressing the V beta 8 family, and induced a strong decrease in the response to CD3 activation. This absence of response was not the consequence of programmed cell death, and was explained by an anergic state induced by the superantigen. NC superantigen was also able to induce polyclonal activation of B cells, as measured by the secretion of anti-HIV antibodies and autoantibodies. Moreover, V beta 8 depletion experiments showed that induction of autoantibody secretion was V beta 8 dependent, whereas secretion of HIV-1 antibody was not. Interleukin secretion studies showed that NC was able to induce high levels of interleukin-4 and interleukin-10. Taken together, our results suggest a role for exogenous viral superantigens such as NC in the induction of T cell hyporesponsiveness and polyclonal B cell activation during HIV infection. The induction of a Th2 response and the role of these superantigens in the immunopathogenesis of acquired immunodeficiency syndrome are discussed.

Acquired Immunodeficiency Syndrome↗
PubMed1994

The pathogenesis of chronic lymphocytic leukemia: analysis of the antibody repertoire.

CD5+ B cells predominate early in ontogeny and have been associated with autoantibody production. In chronic lymphocytic leukemia (CLL), B lymphocytes express CD5 and frequently produce autoantibodies using developmentally regulated variable (V)-gene segments. Does the self-reactivity observed in CLL reflect transformation of a 'fetal' lineage of cells, or could overexpansion of these B cells occur as a consequence of antigen stimulation? Harry Schroeder and Guillermo Dighiero have reviewed the literature describing antibody sequences in CLL and have compared them with the 'fetal' repertoire. This analysis indicates that CLL cells use a repertoire characteristic of mature cells, and suggests that antigen may play a role in the pathogenesis of this disease.

Animals↗
PubMed1994

Defective protein tyrosine phosphorylation and altered levels of p59fyn and p56lck in CD4 T cells from HIV-1 infected patients.

Early in HIV infection, CD4+ lymphocytes exhibit the properties of an anergic state characterized by unresponsiveness to mitogens or to TCR stimulation and by defective IL-2 production. As tyrosine phosphorylation is the earliest of the biochemical events initiated by stimulation of CD3-TCR, we studied protein tyrosine phosphorylation in purified CD4+ lymphocytes from 25 asymptomatic seropositive patients (CD4 T cells > 350/mm3) previously stimulated in vitro by immobilized anti-CD3 mAb or by co-immobilized anti-CD3 and anti-CD28 mAbs. Purified CD4+ lymphocytes from HIV-infected patients exhibited defective early protein tyrosine phosphorylation in response to CD3 activation when compared with normal subjects. This defect was observed mainly in patients in whom proliferative responses to immobilized anti-CD3 ranged from 2 to 50% of control values obtained in healthy donors, and was frequently associated with increased cellular levels of p59fyn and decreased cellular levels of p56lck. Interestingly, these defects appeared to correlate with the degree of impairment in thymidine incorporation. Since CD28 mAbs have been reported to enhance proliferative responses to the CD3-TCR pathway in cloned murine or human anergic models and to induce tyrosine phosphorylation in human T cells, we studied the role of CD28 mAb as a co-signal. Although anti-CD28 co-stimulation augmented the proliferative responses in both controls and HIV-infected patients, it failed to correct the tyrosine phosphorylation pattern in the latter. Our results suggest a relationship between defective early protein tyrosine phosphorylation and impairment of proliferative responses in CD4 T cells from HIV-infected patients, and evidence is provided that associated altered cellular levels of the fyn and lck tyrosine kinases might play an important role in the anergic response observed early during HIV infection.

CD4-Positive T-Lymphocytes↗
PubMed1994

Potential immunological action of purine nucleoside analogues.

Purine nucleoside analogues are a new class of drugs with activity against nondividing lymphocytes; thus, they should play a major role in the treatment of low grade lymphoid malignancies. As these drugs are active against resting lymphocytes, harmful effects related to this action were expected and have been reported. However, the toxic effects on resting lymphocytes observed during treatment of lymphoid malignancies may potentially be of some benefit in patients with autoimmune diseases. To substantiate this possibility, a considerable amount of work needs to be carried out in order to better define the mechanism of action of these drugs, as well as their potential activity on different immunological effectors. Also, studies in animal models of autoimmune disease should be undertaken.

Animals↗
PubMed1994

Recent progress in chronic lymphocytic leukemia. International Workshop on chronic Lymphocytic Leukemia.

The data we discuss indicate substantial recent progress in understanding and treating CLL. However, despite considerable new information, many of the intriguing issues we posed at previous IWCLL meetings remain unanswered. Prominent among these are the questions of what causes CLL, what is the relation between CLL and normal B-cell development, are T-cell abnormalities a cause of consequence of CLL, why are auto-immune features so prominent and how is CLL best treated? Although these gaps in our knowledge are unfortunate, they give us the opportunity for yet another IWCLL meeting: 1996 in Greece. More to follow.

Antigens, CD↗
PubMed1993

V gene usage by seven hybrids derived from CD5+ B-cell chronic lymphocytic leukemia and displaying autoantibody activity.

We report here the complete heavy and light chain variable region sequences of seven heterohybridomas derived from CD5+ chronic lymphocytic leukemia (CLL) B lymphocytes and displaying natural autoantibody activity. The three hybrids displaying a polyreactive pattern of binding used VH4 family members, ie, the VH4-18 gene in germinal configuration in two cases and a VH4 gene with 90% homology with VH4-21 for the third one. A hybrid expressing anti-Sm activity used a VH3 family member with 95.26% homology with the 30P1 gene. The three hybrids exclusively displaying rheumatoid factor activity expressed VH1 family genes: 51P1 gene for two (in germinal configuration in one, and with 93.2% homology in the other), whereas the third one used the V1-3b gene (98.8% homology). Definitive homology with known germline D segments was found for four of the seven hybrids (DN2 in 3 and DLR4 in 1) and JH use appeared to be random. The three hybrids displaying polyreactive activity expressed V kappa I, V lambda III, and V lambda II genes, all in germinal configuration. Among the three hybrids with rheumatoid factor activity, two used the same V kappa II gene with, respectively, 98% and 96% homology with a gene previously described; the third used a V lambda I gene in germinal configuration. Finally, the clone with anti-Sm activity used a V lambda III gene having 97% homology with a germinal gene. Overall, these results attempt to establish the relationship between frequent self-reactivity observed in CD5+ B-CLL and V gene usage. For VH genes, they confirm overexpression of the 51P1 gene in B-CLL and suggest nonstochastic use of two VH4 genes (4-21 and 4-18). For VL genes, available information is too scarce to lead to firm conclusions.

Amino Acid Sequence↗
PubMed1993

Simultaneous presence, in one serum, of four monoclonal antibodies that might correspond to different steps in a clonal evolution from polyreactive to monoreactive antibodies.

Three monoclonal IgG of different subclasses (IgG1, IgG2, and IgG4) and one IgA1 were isolated from the serum of patient Per suffering from an immunocytic sarcoma. All four monoclonal Ig shared the same N-terminal sequence of their H chains (VH3). Furthermore, their kappa-chains exhibited identical isoelectric charges and N-terminal sequences (VK2) and expressed the same private idiotope. A strong antitubulin activity was found in IgA1Per and in two of the three monoclonal IgGPer. The specificity was restricted to tubulin for IgA1Per and IgG4Per, whereas IgG1Per also displayed significant polyreactive bindings and IgG2Per failed to react with any of the Ag tested. The monoreactive IgG4Per, as well as the polyreactive IgG1Per, bound a large peptide in the central part of both alpha and beta subunits of tubulin (amino acid position 100 to 300). In contrast, the monoreactive IgA1Per bound to a rarely detected epitope close to residue 310 of these subunits. The tubulin epitope recognized by polyreactive IgG1Per was similar to that of germ-line-encoded polyreactive antibodies. It is hypothesized that IgG4Per- and IgA1Per-producing cells derive from the IgG1Per polyreactive clone after somatic events leading to the production of monoreactive antibodies.

Amino Acid Sequence↗
PubMed1993

Biology of the neoplastic lymphocyte in B-CLL.

Immunological markers have identified the proliferating lymphocyte in CLL as a mature B lymphocyte which, unlike lymphocytes in other B cell malignancies, expresses low amounts of surface membrane immunoglobulin (smIg), forms rosettes with mouse erythrocytes and expresses the CD5 marker. It has been postulated that Ly1 B cells (the murine counterpart of human CD5+B cells) constitute a separate B cell lineage. Whether the CD5 marker defines a discrete lineage or is a maturation marker is one of the main issues that might be solved in the near future. Another recent advance has been the discovery that the B lymphocyte in CLL is in an activated state and can be induced to differentiate. Using B cell mitogens and somatic hybridization, it has been demonstrated that the B-CLL lymphocyte is frequently involved in the production of natural autoantibodies and expresses a restricted set of genes. These results may provide a basis for passive immunotherapy using anti-idiotypic antibodies. Hypogammaglobulinaemia is a distinct feature of B-CLL, observed in 60% of patients. It may result from impaired function of residual normal B cells. This could occur as a consequence of progressive dilution of normal non-clonal B cells, or because normal B cells are downregulated by an unknown mechanism. This decrease in or inhibition of normal CD5-B cells could also explain the classical inability of patients with B-CLL to respond to new antigenic challenges, since Ly1 B cells have been claimed to be unable to respond to exogenous antigens. Although regulatory abnormalities in T cells may play a role in the induction of hypogammaglobulinaemia, data concerning helper, suppressive, NK and ADCC cells are contradictory and fail to establish firmly the contribution of these cells in the development of hypogammaglobulinaemia. Associated autoimmune phenomena are a prominent complication in CLL. They are related to the presence of autoantibodies directed mainly against blood components, which in most cases are not the product of the malignant clone. The relationship between autoimmune phenomena and hypogammaglobulinaemia is not definitively substantiated. That hypogammaglobulinaemia may determine the loss of some anti-idiotypic antibodies designed to antagonize autoimmune clones is an attractive hypothesis, which needs to be substantiated. Several recurrent chromosomal abnormalities, such as trisomy 12, structural aberrations of the 13q14 and 14q32 bands, are frequently observed in B-CLL. Less frequently, alterations of chromosomes 11, 6, 18, 3, 17, 7 and 8 have been reported.(ABSTRACT TRUNCATED AT 400 WORDS)

Agammaglobulinemia↗
PubMed1993

In vitro non-productive infection of purified natural killer cells by the BRU isolate of the human immunodeficiency virus type 1.

Highly purified natural killer (NK) cell lines and clones, displaying the typical phenotype, morphology and function and obtained from healthy blood donors, were infected in vitro with the BRU isolate of human immunodeficiency virus type 1 (HIV-1). There was no significant increase in reverse transcriptase activity and levels of p24 antigen in the supernatants, but positive staining was observed using an immunogold technique with polyclonal anti-HIV-1 antibodies. When infected NK cells were co-cultivated with autologous non-infected CD4+ mitogen-activated cells, significant levels of reverse transcriptase activity and p24 antigen in supernatants were detected. Giant syncytial cells and a high number of mature virion particles were also evident. When NK cell lines or clones from HIV-1-infected patients were studied, neither the presence of p24 antigen nor reverse transcriptase activity was detected in the supernatants after stimulation with mitogens, cytokines or co-culture with allogeneic CD4+ mitogen-activated cells. PCR studies did not detect HIV-1 genes in freshly purified NK cells, cell lines or clones from infected patients. Taken together these results suggest that (i) normal NK cells can be infected in vitro by the HIV-1 BRU isolate in a non-productive fashion, (ii) PCR with NK cell DNA of HIV-1-infected patients indicates that in vivo few of these cells, if any, are infected by HIV-1 and (iii) the mechanisms responsible for the impairment of NK cell function during HIV-1 infection remain to be determined and are probably not related to a direct cytopathic effect of the virus.

Cell Line↗
PubMed1993

Differential requirements for HIV-1 replication in naive and memory CD4 T cells from asymptomatic HIV-1 seropositive carriers and AIDS patients.

One of the major routes for modulating HIV-1 expression by infected T cells is through the control of transcription initiation from the HIV-1 long terminal repeat (LTR), which is regulated either by its own viral gene products or by several cellular DNA-binding proteins induced during T cell activation. Previous work reported preferential HIV-1 infection and replication of memory CD4 T cells from infected individuals, which was explained either by a higher viral burden of this subset or by differences between naive and memory cells in the activation of the general transcription machinery involved in HIV-1 replication. In this work, we have studied HIV-1 replication by highly purified naive and memory CD4 T cells from asymptomatic seropositive carriers (ASC) and AIDS patients following different activation signals. Our results demonstrate that viral replication in memory cells from ASC was observed after mitogenic (phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA)) recombinant tumour necrosis factor-alpha (rTNF-alpha) and CD3-mediated activation. In contrast, in naive subsets, early viral replication was almost exclusively observed upon CD3-mediated activation. AIDS patients are characterized by similar levels of viral replication in both subsets after PHA and soluble or immobilized anti-CD3 MoAb activation. However, naive subsets from AIDS patients still displayed differential requirements since they failed to replicate HIV-1 after treatment with PMA and rTNF-alpha. Taken together, these results provide evidence that HIV-1 replication in CD4+ T cells from infected individuals is a function of the differentiation stage of the cells, the disease stage of the patient and the activation signal employed.

Acquired Immunodeficiency Syndrome↗