RESUMO
Aim: To investigate whether and how ten ethical principles are mentioned within documents on critical care resources allocation during public health emergencies. Materials and Methods: We conducted a search of documents con-cerning critical resources allocation during public emergencies publicly available from Google and two specific international websites, up to November 2020. Each document was analyzed by two independent reviewers to assess whether a reference to any of the 10 key ethical principles indicated by the Northern Italy Ethical Committee could be found in the documents. Cohen's K statistic was used. Results: We obtained 34 documents, of which 19 were allocation frameworks, 15 crisis standards of care, 4 clinical triage protocol, 3 clinical guidelines and 2 public health emergency response plans. The principles most frequently mentioned as important for decision-making was "number of lives saved", followed by "transparency", "equity", "respect of person and their autonomy". The most cited tiebreakers were "younger first/life cycle" and "lottery". Conclusions: All documents aim to protect the life and health of the largest number of people and should be objective, ethical, transparent, applied equitably, and be publicly disclosed. It is plausible that short- and long-term prognostic tools can help allocate critical resources, but it remains strong that the decision-making process must be guided by a multi-principle ethical model that is not always easy to apply.
Assuntos
Alocação de Recursos para a Atenção à Saúde , Saúde Pública , Cuidados Críticos , Tomada de Decisões , Emergências , Humanos , Pandemias , TriagemRESUMO
We examined the pathogenic significance of the latent viral reservoir in the resting CD4+ T cell compartment of HIV-1-infected individuals as well as its involvement in the rebound of plasma viremia after discontinuation of highly active anti-retroviral therapy (HAART). Using heteroduplex mobility and tracking assays, we show that the detectable pool of latently infected, resting CD4+ T cells does not account entirely for the early rebounding plasma HIV in infected individuals in whom HAART has been discontinued. In the majority of patients examined, the rebounding plasma virus was genetically distinct from both the cell-associated HIV RNA and the replication-competent virus within the detectable pool of latently infected, resting CD4 + T cells. These results indicate the existence of other persistent HIV reservoirs that could prompt rapid emergence of plasma viremia after cessation of HAART and underscore the necessity to develop therapies directed toward such populations of infected cells.
Assuntos
Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1 , Viremia , Latência Viral , Adulto , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/sangue , Humanos , Pessoa de Meia-Idade , RNA Viral/sangue , Recidiva , Replicação ViralRESUMO
The antigenic polymorphism of HIV-1 is a major obstacle in developing an effective vaccine. Accordingly, we screened random peptide libraries (RPLs) displayed on phage with antibodies from HIV-infected individuals and identified an array of HIV-specific epitopes that behave as antigenic mimics of conformational epitopes of gp120 and gp41 proteins. We report that the selected epitopes are shared by a collection of HIV-1 isolates of clades A-F. The phage-borne epitopes are immunogenic in rhesus macaques, where they elicit envelope-specific antibody responses. Upon intravenous challenge with 60 MID50 of pathogenic SHIV-89.6PD, all monkeys became infected; however, in contrast to the naive and mock-immunized monkeys, four of five mimotope-immunized monkeys experienced lower levels of peak viremia, followed by viral set points of undetectable or transient levels of viremia and a mild decline of CD4+ T cells, and were protected from progression to AIDS-like illness. These results provide a new approach to the design of broadly protective HIV-1 vaccines.
Assuntos
Vacinas contra a AIDS/farmacologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Sequência de Aminoácidos , Animais , Epitopos/administração & dosagem , Epitopos/genética , Anticorpos Anti-HIV/biossíntese , Antígenos HIV/administração & dosagem , Antígenos HIV/genética , Infecções por HIV/imunologia , HIV-1/genética , Humanos , Macaca mulatta , Biblioteca de Peptídeos , Vacinas contra a SAIDS/genética , Vacinas contra a SAIDS/imunologia , Vacinas contra a SAIDS/farmacologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologiaRESUMO
Evolutionary patterns of virus replication and distribution in lymphoid tissue during the early phases of HIV infection have not been delineated. Lymph node (LN) biopsies were excised from patients at different times after the estimated time of primary infection. Within 3 months of the acute viral syndrome, HIV was mostly present in individual virus-expressing cells in LNs; trapping of virions in the follicular dendritic cell (FDC) network was minimal or absent, but was the predominant form of HIV detected in LNs of subjects with chronic infection, either recent (4-20 months after primary infection) or long-term (>2-3 years after primary infection). Plasma viremia was significantly higher in patients during the first 3 months than in those recently infected; however, there were no significant differences in the number of virus-expressing cells per square millimeter of LN tissue in these two groups. Numbers of virus-expressing cells in lymphoid tissue were significantly lower in the subjects with long-term infection than in the other two groups. Therefore, during the transition from primary to chronic HIV infection, the level of HIV replication in lymphoid tissue remains elevated despite the fact that viremia is significantly downregulated. These findings have implications for therapeutic strategies in primary HIV infection and in recent seroconvertors.
Assuntos
Infecções por HIV/virologia , HIV/crescimento & desenvolvimento , Linfonodos/virologia , Doença Aguda , Biópsia , Doença Crônica , Células Dendríticas/virologia , Progressão da Doença , Infecções por HIV/terapia , Humanos , RNA Viral/sangue , Viremia , Replicação ViralRESUMO
The size of the pool of resting CD4+ T cells containing replication-competent HIV in the blood of patients receiving intermittent interleukin (IL)-2 plus highly active anti-retroviral therapy (HAART) was significantly lower than that of patients receiving HAART alone. Virus could not be isolated from the peripheral blood CD4+ T cells in three patients receiving IL-2 plus HAART, despite the fact that large numbers of resting CD4+ T cells were cultured. Lymph node biopsies were done in two of these three patients and virus could not be isolated. These results indicate that the intermittent administration of IL-2 with continuous HAART may lead to a substantial reduction in the pool of resting CD4+ T cells that contain replication-competent HIV.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Interleucina-2/uso terapêutico , Estudos Transversais , Inibidores da Protease de HIV/uso terapêutico , HIV-1/patogenicidade , Humanos , Interleucina-2/farmacologia , Linfonodos/virologia , Contagem de Linfócitos/efeitos dos fármacos , RNA Viral/sangue , Replicação Viral/efeitos dos fármacosRESUMO
In this study we demonstrate that the monocyte/macrophage product, tumor necrosis factor alpha (TNF-alpha), has significant in vitro effects of B cell function. It costimulated with anti-mu in the induction of B cell DNA synthesis, and it prolonged the DNA synthesis initiated in B cell cultures stimulated with the human B cell mitogen, Staphylococcus aureus Cowan strain I (SAC). The addition of either IL-1 or IFN-gamma to TNF-alpha resulted in a substantial further increase in DNA synthesis. The addition of TNF-alpha to IL-2, a known inducer of SAC-activated B cell Ig secretion, resulted in a twofold enhancement in the amount of IL-2 stimulated B cell Ig secretion. Receptor binding studies with 125I-TNF-alpha demonstrate a marked increase in TNF-alpha binding sites after B cell activation (approximately 6,000 sites per cell, with an apparent Kd of 2.0 X 10(-10) M). Thus, TNF-alpha may be an important factor in human B cell function and is likely to interact with other T cell and monocyte derived cytokines in the regulation of human B cell proliferation and Ig production.
Assuntos
Linfócitos B/fisiologia , Glicoproteínas/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , DNA/biossíntese , Glicoproteínas/metabolismo , Humanos , Imunoglobulina M/biossíntese , Interferon gama/farmacologia , Interleucina-1/fisiologia , Interleucina-2/fisiologia , Receptores de Superfície Celular/metabolismo , Receptores do Fator de Necrose Tumoral , Fator de Necrose Tumoral alfaRESUMO
Human immunodeficiency virus (HIV) disease in sub-Saharan Africa generally differs from that observed in the United States and other developed countries in that the risk of seroconversion after exposure is greater and the rate of disease progression to AIDS and death is faster. One theory that could in part explain this difference is the increased state of immune activation associated with a relatively high rate of parasite infestation and other infections among inhabitants of these regions. Using a model based on the cellular microenvironment of lymphoid organs, the role of exposure to HIV during a state of antigen-specific immune activation was investigated. Dendritic cells and CD4+ T cells are the major cellular components of the paracortical region of lymphoid tissue, the primary site of HIV replication. We analyzed cocultures of HIV-pulsed dendritic cells that had matured in the presence of tetanus toxoid and CD4+ T cells before and after inducing an antigen-specific response by in vivo immunization with tetanus toxoid. During antigen-specific immune activation, 100 times less HIV was needed to initiate a productive infection. These findings provide a model system to further delineate the relationship between immune activation and the propagation of HIV infection and suggest a mechanism for the epidemiologic observations of an increased ease of developing HIV infection and faster progression for HIV disease in geographic areas where immune activation is prevalent.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , HIV/crescimento & desenvolvimento , Imunização , Ativação Linfocitária , Apresentação de Antígeno , Linfócitos T CD4-Positivos/citologia , Células Cultivadas , Células Dendríticas/imunologia , Infecções por HIV/etiologia , Humanos , Toxoide Tetânico/imunologiaRESUMO
A model for the detection of single cell antibody production by human tonsillar lymphocytes after stimulation with either sheep red blood cells (SRBC) or polyclonal B-cell activators has been described. The culture system is a modified Mishell-Dutton technique with certain critical factors identified. The assay is a sensitive and resproducible hemolysis-in-gel system employing an ultra-thin layer gel technique measuring plaque-forming cells (PFC) against SRBC targets. Several factors essential for optimal responses are described, but the critical feature of the culture system is the use of selected lots of human AB serum supplements which are extensively absorbed with SRBC. This removes a blocking factor present in most human serum which suppresses the B-cell response to SRBC targets after stimulation with either SRBC or several polyclonal B-cell activators. In addition, absorption of serum with SRBC eliminates the presence of artifactual plaques. Background PFC are extremely low and stimulated cultures show significant and reproducible responses. These studies provide a simple, sensitive, and reproducible model for probing the complex events associated with activation of human B lymphocytes.
Assuntos
Formação de Anticorpos , Linfócitos B/imunologia , Técnica de Placa Hemolítica , Proteínas do Sistema Complemento , Meios de Cultura , Antígenos HLA , Humanos , Ativação Linfocitária , Mitógenos , Tonsila Palatina/imunologiaRESUMO
Activated pneumococcal polysaccharide (PPS)-specific human B lymphocytes have been purified and examined in vitro. They are large cells that are refractory to further activation by antigen, anti-Ig, or Staphylococcus aureus Cowan strain I, but they respond directly by proliferation to B cell growth factors. In addition, they express the isotype pattern of activated cells and a surface marker of activated lymphocytes called 4F2. Finally, they contain a subset of cells that spontaneously secrete PPS-specific antibody.
Assuntos
Antígenos/imunologia , Linfócitos B/imunologia , Epitopos , Ativação Linfocitária , Adulto , Animais , Vacinas Bacterianas/imunologia , Sítios de Ligação de Anticorpos , Separação Celular , Substâncias de Crescimento/fisiologia , Humanos , Interleucina-4 , Camundongos , Vacinas Pneumocócicas , Receptores de Antígenos de Linfócitos B/análise , Formação de RosetaRESUMO
It remains controversial whether human T lymphotropic virus type I (HTLV-I) coinfection leads to more rapid progression of human immunodeficiency virus (HIV) disease in dually infected individuals. To investigate whether HTLV-I infection of certain cells can modulate HIV-1 infection of surrounding cells, primary CD4(+) T cells were treated with cell-free supernatants from HTLV-I-infected MT-2 cell cultures. The primary CD4+ T cells became resistant to macrophage (M)-tropic HIV-1 but highly susceptible to T cell (T)-tropic HIV-1. The CC chemokines RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta in the MT-2 cell supernatants were identified as the major suppressive factors for M-tropic HIV-1 as well as the enhancers of T-tropic HIV-1 infection, whereas soluble Tax protein increased susceptibility to both M- and T-tropic HIV-1. The effect of Tax or CC chemokines on T-tropic HIV-1 was mediated, at least in part, by increasing HIV Env-mediated fusogenicity. Our data suggest that the net effect of HTLV-I coinfection in HIV-infected individuals favors the transition from M- to T-tropic HIV phenotype, which is generally indicative of progressive HIV disease.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Infecções por HTLV-I/imunologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Replicação Viral/imunologia , Linhagem Celular , Quimiocinas/imunologia , Quimiocinas/farmacologia , Produtos do Gene tax/imunologia , Produtos do Gene tax/farmacologia , Humanos , Receptores de Quimiocinas/imunologia , Replicação Viral/efeitos dos fármacosRESUMO
Freshly isolated B lymphocytes from patients infected with human immunodeficiency virus (HIV), in contrast to B cells from normal controls, were shown to induce viral expression in two cell lines: ACH-2, a T cell line, and U1, a promonocytic cell line, which are chronically infected with HIV, as well as in autologous T cells. In 10 out of 10 HIV-infected individuals with hypergammaglobulinemia, spontaneous HIV-inductive capacity was found with highly purified peripheral blood B cells, whereas peripheral blood or tonsillar B cells from six healthy, HIV-negative donors did not induce HIV expression unless the cells were stimulated in vitro. The induction of HIV expression was observed in direct coculture experiments of B lymphocytes and HIV-infected cells, and could also be mediated by supernatants from cultures of B cells. Significantly higher amounts of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) were detected in the B cell culture supernatants from HIV-infected patients with hypergammaglobulinemia (IL-6: mean = 536 pg/ml; TNF-alpha: mean = 493 pg/ml), as compared with normal uninfected controls (IL-6: mean = 18 pg/ml; TNF-alpha: mean = 23 pg/ml). Antibodies against these cytokines abolished the HIV-inductive capacity of B cells. We conclude that in vivo activated B cells in HIV-infected individuals can upregulate the expression of virus in infected cells by secreting cytokines such as TNF-alpha and IL-6, and, therefore, may play a role in the progression of HIV infection.
Assuntos
Linfócitos B/fisiologia , Infecções por HIV/imunologia , HIV-1/crescimento & desenvolvimento , Monócitos/microbiologia , Linfócitos T/microbiologia , Linfócitos B/metabolismo , Linhagem Celular , Células Cultivadas , Infecções por HIV/microbiologia , Humanos , Interleucina-6/metabolismo , Ativação Linfocitária , Fator de Necrose Tumoral alfa/metabolismo , Ativação Viral/imunologiaRESUMO
The present study demonstrates the minimal, optimal, and synergistic signals involved in the activation of normal human peripheral blood and tonsillar B cells to proliferation. Initial activation signals were delivered to B cells by low concentrations of anti-mu antibody which did not induce proliferation by themselves. However, marked synergy was seen when anti-mu antibody was added to cultures in the presence of monoclonal B cell growth factor (BCGF) obtained from a human T-T cell hybrid such that the B cells underwent substantial proliferation. This latter proliferation was seen without maturation into Ig-secreting cells, which indicates that the BCGF is not a differentiation signal but a signal that drives the cell up to but not beyond the proliferative phase. Of note was the fact that B cells reflected differential sensitivity on the basis of size to either the activation signal delivered by anti-mu antibody or the proliferative signal delivered by BCGF. BCGF directly stimulated the larger B cells in the normal tonsillar B cell repertoire to proliferate without the requirement for an in vitro activation signal, which indicates that the cells had already received some form of activation signal in vivo. Indeed, these cells expressed the 4F2 antigen found on activated but not resting lymphocytes. In contrast, the smaller tonsillar B lymphocytes did not express the 4F2 activation antigen and required activation by anti-mu antibody, which did not of itself induce proliferation, but which acted in synergy with BCGF for substantial proliferation of the B cells. These studies thus provide a useful model of human B cell activation, proliferation, and differentiation and allow a more precise delineation of each phase in this cascade.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Linfócitos B/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias mu de Imunoglobulina/imunologia , Ativação Linfocitária , Células Produtoras de Anticorpos/citologia , Células Produtoras de Anticorpos/imunologia , Linfócitos B/classificação , Sítios de Ligação de Anticorpos , Contagem de Células , Células Cultivadas , Células Clonais/imunologia , Substâncias de Crescimento/farmacologia , Humanos , Imunoglobulina M/metabolismo , Interleucina-4 , Tonsila Palatina/citologia , Receptores de Antígenos de Linfócitos BRESUMO
The feasibility of selectively eliminating human antigen-specific B cell responses by treating cells in vitro with antigen covalently linked to a cell toxin was examined. Tetanus toxoid (TT) was conjugated to the toxin ricin via a thioether linkage. Peripheral blood mononuclear cells from recently immunized subjects were preincubated for 2 h with TT-ricin in the presence of lactose. Antigen was then removed, and the cells from recently immunized subjects were preincubated for 2 h with TT-ricin in the presence of lactose. Antigen was then removed, and the cells were stimulated with pokeweed mitogen to induce antibody production. TT-specific antibody production was completely abrogated by preincubation with TT-ricin but not by TT alone or a mixture of TT and ricin. In contrast, polyclonal immunoglobulin production was not diminished by TT-ricin. This selective abrogation was also seen when B cells alone were preincubated with TT-ricin and a source of T cell help was later provided. T cell blastogenic responses to TT remained intact after TT-ricin exposure. Thus, antigen-toxin conjugates are capable of selectively eliminating specific antibody-producing B cell clones, while leaving intact the remainder of the B cell repertoire.
Assuntos
Antígenos , Linfócitos B/imunologia , Ricina , Adolescente , Adulto , Formação de Anticorpos , Separação Celular , Humanos , Monócitos/imunologia , Toxoide Tetânico/imunologiaRESUMO
A highly specific and reproducible antigen-induced, antigen-specific culture and assay system for antibody production by human peripheral blood B lymphocytes has been developed. The system is clearly T cell and monocyte dependent and is independent of exogenous mitogens. The major factors in our ability to trigger specific antibody production with antigen alone have been the use of extremely low concentrations of antigen in vitro (doses several orders of magnitude below those inducing a peak blastogenic response), careful attention to in vitro cell density and culture vessel geometry, and appreciation of the kinetics of the circulating antigen-inducible B cell repertoire. A dichotomy and overlap between antigen-induced, antigen-specific and antigen-induced, polyclonal responses was observed in the study of doubly immunized individuals. Whereas antibody responses highly specific for the antigen in culture were observed under one set of culture conditions (flat-bottomed vessels, 1.5 x 10(6) cells), switching to another culture system (round-bottomed vessels, 5 x 10(5) cells) resulted in polyclonal responses to antigen. Despite these culture condition-related differences in the induction of antibody synthesis, the suppression of specific antibody production that occurred at high concentrations of antigen was specific only for the antigen in culture. The capability to easily and reproducibly look at truly antigen-induced, antigen specific antibody production should be a major tool in furthering the understanding of human B cell activation and immunoregulation.
Assuntos
Formação de Anticorpos , Antígenos , Epitopos , Ativação Linfocitária , Adolescente , Adulto , Linfócitos B/imunologia , Separação Celular , Hemocianinas/imunologia , Humanos , Imunoglobulina M/biossíntese , Cinética , Monócitos , Mitógenos de Phytolacca americana/farmacologiaRESUMO
The success of long-term culture of normal human and murine B cells has been hampered by the limited availability of soluble factors capable of maintaining proliferation of activated B lymphocytes. Previous experiments using various culture-derived supernatants in a human system were unable to separate the activities of B cell growth factor (BCGF) and interleukin 2 (IL-2) by immunochemical means. Thus, purified factors with BCGF activity in the absence of IL-2 activity have not been available for study. In the present study, normal human peripheral blood T cells were fused with the hypoxanthine/aminopterin/thymidine-sensitive human T-leukemic cell line, CEM-6. Supernatants from the resulting hybrid cells were tested for the ability to maintain proliferation of normal human B cells in a recently described assay system for human BCGF. Hybrids demonstrating BCGF activity were cloned by limiting dilution. One hybrid clone, 2B11, continued to support proliferation of B cells in both long-term cultures and 6-d assays at a level significantly above that seen with conventionally produced growth factors. No IL-2 activity was found in the supernatant from hybrid 2B11. The hybridoma supernatant was fractionated by gel filtration, and maximum proliferation of B cells was supported by the 18-20,000 mol wt protein fraction. Thus, a human T-T cell hybridoma that has BCGF activity in the absence of any demonstrable IL-2 activity has been developed. Human T-T cell hybridomas secreting discrete immunoregulatory factors should prove to be powerful tools in dissecting the mechanisms of immunoregulation of human lymphocyte function.
Assuntos
Substâncias de Crescimento/metabolismo , Hibridomas/imunologia , Linfócitos T/imunologia , Linfócitos B/citologia , Divisão Celular , Células Cultivadas , Humanos , Interleucina-4RESUMO
Natural-killer (NK)-cell function was profoundly depressed in donors homozygous for the Chediak-Steinbrinck-Higashi syndrome (C-HS) gene when compared with age- and sex-matched normals. This apparent defect was not simply a result of a delayed response because little cytolysis was evident in kinetics experiments even after 24 h of incubation. NK cells from C-HS donors failed to lyse adherent (MDA, CEM, and Alab) or nonadherent (K562 and Molt-4) tumor cell lines or nontransformed human fetal fibroblasts. Therefore, the apparent C-HS defect was not a result of a shift in target selectivities. In addition, the depressed reactivity did not appear to be a result of suppressor cells or factors because: (a) enriched NK populations (nonadherent lymphocytes bearing receptors for the Fc portion of IgG) from C-HS donors were low in NK-cell function, (b) C-HS mononuclear cells did not inhibit the cytotoxicity of normal cells in coculture experiments, and (c) cells from the C-HS donors remained poorly reactive even after culture for up to 7 d. The nature of the defective NK activity in C-HS patients is not clear but may lie within the lytic mechanism rather than at the level of the recognition structure or population size because the frequency of target-binding cells was normal. In vitro NK activity could be partially restored by interferon treatment. Combined with the results presented in the following paper (4), these observations suggest that the C-HS gene causes a selective immunodeficiency disorder, mainly involving NK cells. This finding, in conjunction with the high incidence of spontaneous possibly malignant, lymphoproliferative disorders in these patients, may have important implications regarding the theory of immune surveillance mediated by NK cells.
Assuntos
Síndrome de Chediak-Higashi/imunologia , Imunidade Inata , Células Matadoras Naturais/imunologia , Adulto , Células Cultivadas , Humanos , Imunidade Celular , Imunidade Inata/efeitos dos fármacos , Interferons/farmacologia , Linfócitos/imunologia , Masculino , Linfócitos T Reguladores/imunologiaRESUMO
The process of B cell growth and differentiation into plasma cells is highly regulated and may be influenced by a large number of inflammatory mediators, including complement components. We have studied the regulatory influence of Bb, a 60-kD peptide created during the cleavage of complement Factor B by Factor D and C3b. Purified Bb alone had no effect on proliferation and differentiation of human splenic or tonsillar B cells. However, when B cells were activated by Staphylococcus aureus Cowan I (SAC), Bb enhanced proliferation in a dose-dependent manner. Bb also enhanced proliferation when cocultured with SAC and suboptimal concentrations of purified 60-kD B cell growth factor (HMW-BCGF), a previously described lymphokine that is known to possess growth-promoting activity. However, Bb had no effect on cells treated with optimal concentrations of HMW-BCGF. Like HMW-BCGF, Bb's major effect was on the larger in vivo activated B cells. Half-maximal enhancement of proliferation was reached at a Bb concentration of 1-10 nM. Of note is the fact that antibody to Factor B recognized HMW-BCGF, and an mAb to HMW-BCGF also recognized Factor B and Bb, but not Ba. Moreover, radiolabeled Bb bound saturably to activated B cells and to an EBV-transformed human B cell line. The binding of Bb was inhibited by HMW-BCGF but not by Ba or IgG. Thus, Bb is antigenically and functionally related to HMW-BCGF, and can act as a B cell growth and differentiation factor at potentially physiologic concentrations. These data suggest that Bb may be important in amplifying the immune response in areas of inflammation. Since complement activation occurs at inflammatory sites long before induction of HMW-BCGF synthesis, Bb may be an early signal for the clonal expansion of antigen-activated B cells.
Assuntos
Linfócitos B/imunologia , Fator B do Complemento/imunologia , Precursores Enzimáticos/imunologia , Substâncias de Crescimento/imunologia , Ativação Linfocitária , Linfócitos B/fisiologia , Diferenciação Celular , Divisão Celular , Ativação do Complemento , Humanos , Monócitos/imunologia , Tonsila Palatina/imunologia , Baço/imunologiaRESUMO
High molecular weight B cell growth factor (HMW-BCGF) produced by a T cell line was purified to homogeneity and demonstrated to bind specifically to activated human B cells. A monoclonal antibody to HMW-BCGF was developed that (a) specifically inhibited the activity of HMW-BCGF in enhancing B cell proliferation, (b) specifically bound to HMW-BCGF in Western blots, (c) specifically absorbed HMW-BCGF activity from culture supernatants, and (d) specifically absorbed an internally labeled protein from T-ALL supernatant which comigrates with HMW-BCGF on sodium dodecyl sulfate-polyacrylamide gels. This antibody should help in cloning the gene for HMW-BCGF and further exploring the physiologic roles of HMW-BCGF.
Assuntos
Anticorpos Monoclonais/imunologia , Linfócitos B/metabolismo , Substâncias de Crescimento/isolamento & purificação , Linfocinas/isolamento & purificação , Animais , Eletroforese em Gel de Poliacrilamida , Substâncias de Crescimento/imunologia , Substâncias de Crescimento/metabolismo , Humanos , Interleucina-4 , Linfocinas/imunologia , Linfocinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Peso MolecularRESUMO
We have cloned a full-length cDNA for the B cell membrane protein CD22, which is referred to as B lymphocyte cell adhesion molecule (BL-CAM). Using subtractive hybridization techniques, several B lymphocyte-specific cDNAs were isolated. Northern blot analysis with one of the clones, clone 66, revealed expression in normal activated B cells and a variety of B cell lines, but not in normal activated T cells, T cell lines, Hela cells, or several tissues, including brain and placenta. One major transcript of approximately 3.3 kb was found in B cells although several smaller transcripts were also present in low amounts (approximately 2.6, 2.3, and 1.6 kb). Sequence analysis of a full-length cDNA clone revealed an open reading frame of 2,541 bases coding for a predicted protein of 847 amino acids with a molecular mass of 95 kD. The BL-CAM cDNA is nearly identical to a recently isolated cDNA clone for CD22, with the exception of an additional 531 bases in the coding region of BL-CAM. BL-CAM has a predicted transmembrane spanning region and a 140-amino acid intracytoplasmic domain. Search of the National Biological Research Foundation protein database revealed that this protein is a member of the immunoglobulin super family and that it had significant homology with three homotypic cell adhesion proteins: carcinoembryonic antigen (29% identity over 460 amino acids), myelin-associated glycoprotein (27% identity over 425 amino acids), and neural cell adhesion molecule (21.5% over 274 amino acids). Northern blot analysis revealed low-level BL-CAM mRNA expression in unactivated tonsillar B cells, which was rapidly increased after B cell activation with Staphylococcus aureus Cowan strain 1 and phorbol myristate acetate, but not by various cytokines, including interleukin 4 (IL-4), IL-6, and gamma interferon. In situ hybridization with an antisense BL-CAM RNA probe revealed expression in B cell-rich areas in tonsil and lymph node, although the most striking hybridization was in the germinal centers. COS cells transfected with a BL-CAM expression vector were immunofluorescently stained positively with two different CD22 antibodies, each of which recognizes a different epitope. Additionally, both normal tonsil B cells and a B cell line were found to adhere to COS transfected with BL-CAM in the sense but not the antisense direction.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos B/genética , Linfócitos B/imunologia , Moléculas de Adesão Celular/genética , Lectinas , Adolescente , Sequência de Aminoácidos , Antígenos CD/biossíntese , Antígenos de Diferenciação de Linfócitos B/biossíntese , Sequência de Bases , Moléculas de Adesão Celular/biossíntese , Criança , Pré-Escolar , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , RNA Mensageiro/análise , Proteínas Recombinantes/biossíntese , Lectina 2 Semelhante a Ig de Ligação ao Ácido SiálicoRESUMO
Although it has been demonstrated that certain cytokines, particularly proinflammatory cytokines, can enhance ongoing viral replication in peripheral blood mononuclear cells (PBMCs) of HIV-1-infected individuals, it is unclear what role these cytokines play in the induction of HIV-1 replication in latently infected, resting CD4(+) T cells. This study demonstrates that the in vitro combination of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha together with the immunoregulatory cytokine IL-2 are potent inducers of viral replication in highly purified, latently infected, resting CD4+ T cells derived from HIV-infected individuals who are antiretroviral therapy-naive as well as those who are receiving highly active antiretroviral therapy (HAART). Viral replication induced by this combination of cytokines was completely suppressed in the presence of HAART in vitro. Given that an array of cytokines, including IL-6, TNF-alpha, and IL-2, are copiously expressed in the microenvironment of the lymphoid tissues, which harbor the latent viral reservoirs, induction of HIV by this combination of cytokines may in part explain the commonly observed reappearance of detectable plasma viremia in HIV-infected individuals in whom HAART was discontinued. Moreover, since it is likely that these infected cells die upon activation of virus and that HAART prevents spread of virus to adjacent cells, the observation that this combination of cytokines can markedly induce viral replication in this reservoir may have important implications for the activation-mediated diminution of the latent reservoir of HIV in patients receiving HAART.