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D Melamed

Publications and source records attributed to D Melamed.

17 recordsLinked to original sources
PubMed1997

Self-antigen does not accelerate immature B cell apoptosis, but stimulates receptor editing as a consequence of developmental arrest.

In pre-B lymphocytes, productive rearrangement of Ig light chain genes allows assembly of the B cell receptor (BCR), which selectively promotes further developmental maturation through poorly defined transmembrane signaling events. Using a novel in vitro system to study immune tolerance during development, we find that BCR reactivity to auto-antigen blocks this positive selection, preventing down-regulation of light chain gene recombination and promoting secondary light chain gene rearrangements that often alter BCR specificity, a process called receptor editing. Under these experimental conditions, self-antigen induces secondary light chain gene rearrangements in at least two-thirds of autoreactive immature B cells, but fails to accelerate cell death at this stage. These data suggest that in these cells the mechanism of immune tolerance is receptor selection rather than clonal selection.

Animals↗
PubMed1997

A functional B cell receptor transgene allows efficient IL-7-independent maturation of B cell precursors.

IL-7 supports the proliferation of B cell precursors, but inhibits their maturation to mature surface IgM+ (sIgM+) B cells. This inhibition is thought to occur by direct or indirect down-regulation of recombinase genes, preventing the B cells from undergoing Ig light chain rearrangements. To directly analyze the IL-7 inhibitory effects, we studied B cell development and maturation in B cells bearing a transgenic (Tg) B cell receptor (BCR). We show here that proliferation of Tg B cell precursors is IL-7 dependent both in vivo and in vitro and is comparable to that of non-Tg B cell precursors. Tg B cell precursors grown on stroma and IL-7 expressed sIgM on >90% of the cells, and a large proportion of these cells coexpressed additional maturation markers such as IgD, CD23, CD21, and L-selectin, indicating that IL-7 does not inhibit maturation of Tg B cell precursors. The presence of the Tg inhibited V(D)J recombination in the cultured cells, as very low levels of recombination activating genes 2 (RAG-2) expression and endogenous V-Jkappa DNA rearrangements were found. Expression levels of RAG mRNAs were not significantly changed after removal of IL-7 from the in vitro Tg B cell cultures. In contrast, we found that IL-7 inhibited maturation of non-Tg B cell precursors and that removal of IL-7 resulted in a significant increase in RAG-2 expression and kappa rearrangements, thus allowing the B cells to express sIgM and to mature. These results suggest that IL-7-mediated inhibition of Ig gene rearrangement blocks maturation of B cell precursors and that the presence of Tg BCR efficiently circumvents this inhibition.

Animals↗
PubMed1996

Peripheral tolerance of Th2 lymphocytes induced by continuous feeding of ovalbumin.

We established conditions for inducing antigen-specific tolerance in Th2 lymphocytes by means of oral tolerance. Mice were continuously exposed to ovalbumin in their drinking water for a minimal period of 20 days and then immunized against antigen in either complete Freund's adjuvant or Al(OH)3. This feeding regimen tolerized both Th2 and Th1 responses as shown by diminished proliferation, cytokine secretion (IL-4, IL-2 and IFN-gamma) and specific cytokine mRNA expression (IL-4, IL-2 and IFN-gamma) in vitro, as well as by absence of specific antibody production (IgG1, IgG2a, IgG2b and IgE) in vivo. Conditions for generating Th2 lymphocyte tolerance were different from those required to generate tolerance in Th1 lymphocytes: these included extended, continuous exposure to high dosages of antigen, rather than a single or intermittent feeding regimen which was sufficient to induce tolerance in Th1 lymphocytes. These findings suggest that continuous oral exposure to a tolerogen may be a biologically relevant strategy to tolerize both Th1- and Th-dependent responses, and extend the potential clinical use of oral tolerance to ailments mediated by Th2 lymphocytes.

Administration, Oral↗
PubMed1996

Alpha interferon suppresses the cyclin D3 and cdc25A genes, leading to a reversible G0-like arrest.

Alpha interferon is a potent growth inhibitor of Daudi Burkitt's lymphoma cells. We show here that alpha-interferon signaling interacted simultaneously with several components of the basic cell cycle machinery, causing cells to enter into a state that had many features characteristic of the G0 state. Within a few hours after alpha-interferon treatment, cyclin D3 mRNA and protein levels dropped to undetectable levels and, in parallel, the activities of cyclin A- and cyclin E-associated kinases were significantly reduced. The latter resulted from the rapid alpha-interferon-mediated elimination of cdc25A, a phosphatase that is required for antagonism of negative tyrosine phosphorylation of cdk2 in cyclin-cdk complexes. This regulation represents a novel mechanism through which an external inhibitory cytokine interacts with the cell cycle machinery. At later time points after alpha-interferon treatment, the levels of the 55-kDa slowly migrating hyperphosphorylated form of cyclin E and of cyclin A were also reduced. The antiproliferative effects were reversible, and cultures from which alpha interferon was removed reentered S phase after a lag that typically corresponded to approximately two doubling times. During this lag period, the expression of cyclin D3 and cyclin A, as well as of the cdc25A phosphatase, continued to be switched off, in spite of the removal of alpha interferon from the cell surface. In contrast, c-myc, which represents another downstream target gene that is subjected to negative regulation by alpha interferon, was relieved from suppression much earlier, concomitant with the decay in early signaling of the cytokine. The delayed pattern of cyclin reexpression provides evidence that alpha-interferon signaling imposes a G0-like state on this system.

Burkitt Lymphoma↗
PubMed1995

The effect of varying dietary concentrations of vitamin A on immune response in the turkey.

1. The effect of dietary vitamin A on antibody production and T-cell proliferative response was determined in poults from 21 to 41 d old. Poults were fed on soyabean meal-sorghum-based diets with concentrations of supplemented vitamin A from 0 to 13.2 micrograms/g retinol equivalents from hatching and were immunised with Newcastle disease virus (NDV) and turkey pox vaccines. T-cell proliferation response to concanavlin A was determined in vitro at 31 d old. Antibodies to NDV and turkey pox in serum were determined at 10 and 20 d after inoculation. 2. Poults receiving the diet with no added dietary vitamin A died by 22 d and had very low concentrations of plasma and liver vitamin A. 3. Increasing dietary concentrations of vitamin A enhanced the proliferative response until the diet contained 6.0 micrograms/g, above which the response began to decrease. The antibody titres to NDV and turkey pox increased as dietary vitamin A increased, with maximal values found 10 d after inoculation with 6.0 micrograms/g. At 20 d after inoculation low antibody titres were found with low vitamin A intake. 4. These data suggest that maximal immune responses in the poult may be achieved at dietary intakes of vitamin A at or higher than those recommended by NRC (1984, 1994).

Analysis of Variance↗
PubMed1994

In vivo tolerization of Th1 lymphocytes following a single feeding with ovalbumin: anergy in the absence of suppression.

Oral tolerance is a biologically relevant pathway for inducing peripheral tolerance to foreign antigens. The mechanisms responsible for the tolerant state following feeding with antigen have been shown to involve both anergy and suppression. The demonstration of anergic T lymphocytes following oral tolerance has so far been limited in in vitro systems, and a primary objective of the present study was to provide evidence, in vivo, for the existence of a state of anergy in mice orally fed with ovalbumin (OVA). In addition, it has been shown that peripheral anergy following the intravenous administration of antigen is selectively induced in Th1 lymphocytes. Thus, a second objective of this study was to investigate whether tolerance induced by a feeding regimen known to cause anergy could be selectively limited to Th1 lymphocytes, and whether tolerance induction could be explained by antigen absorption from the gut into the circulation. Oral tolerance was induced by a single feeding with OVA, and was demonstrated by diminished antibody production in vivo, and by reduced cytokine secretion or proliferation in vitro. Anergy, as a mechanism for tolerance, was demonstrated by the ability to reverse the tolerant state after culturing tolerant cells in recombinant interleukin-2 (rIL-2). Reversal of the tolerant state in vivo was established by antibody production in irradiated mice adoptively transferred with cells cultured in the presence of rIL-2. The possibility that suppression was also an in vivo mechanism for tolerance was studied by adoptive transfer experiments. Our results show: 1) that a single dose of orally administered OVA leads to the selective tolerization of Th1 responses (diminished IgG2a, IL-2 and interferon-gamma production) with intact Th2 responses (IgG1, and IL-4), 2) that tolerance in vivo is explained by anergy in the absence of active suppression, 3) that exposure of tolerant cells to rIL-2 in vitro abrogates the anergic state both in vitro (proliferation and cytokine secretion) and in vivo (IgG2a production), and 4) that the induction of oral tolerance is inhibited by the presence of antibodies specific for the tolerizing antigen. These findings indicate that the induction of anergy via the oral route might depend on the dissemination of antigen absorbed from the gut. It is suggested that tolerance is guaranteed by the fact that this absorbed antigen is presented to Th1 lymphocytes in the absence of inflammatory and co-stimulatory molecules; these foreign antigens are thus not different from self antigens.

Administration, Oral↗
PubMed1994

Retinoic acid receptor-alpha gene expression is modulated by dietary vitamin A and by retinoic acid in chicken T lymphocytes.

The effects of dietary vitamin A and retinoic acid in vitro on the proliferative response and gene expression of retinoic acid receptor-alpha (RAR-alpha) in chicken T lymphocytes were studied. Antigen-specific proliferative responses of T lymphocytes increased with dietary vitamin A intake from 0 to 6.6 mg/kg diet; however, at high dietary vitamin A (13.2 mg/kg diet), the proliferative response declined. RAR-alpha mRNA expression in T lymphocytes peaked in chicks fed low levels of vitamin A (830 and 1500 micrograms/kg diet) and declined at higher intakes. In vitro effects of retinoic acid on the modulation of RAR-alpha mRNA were studied in stimulated T lymphocytes. Retinoic acid (0.01 mumol/L) increased RAR-alpha mRNA levels within 2 or 16 h of incubation with concanavalin A- or beta-casein-stimulated T cells, respectively. This effect was transient. Expression of RAR-alpha mRNA in concanavalin A-stimulated T lymphocytes was up-regulated by retinoic acid in a dose-dependent manner, and maximal expression occurred in response to 1 mumol/L retinoic acid. The proliferative response of these cells was also modulated by retinoic acid in a dose-dependent manner, and highest effects were observed at 0.01 mumol/L retinoic acid. Our results indicate that RAR-alpha mRNA expression and antigen-specific proliferative responses of T lymphocytes are influenced by vitamin A status in vivo, and directly modulated by retinoic acid.

Administration, Oral↗
PubMed1994

The effect of varying levels of dietary vitamin A on immune response in the chick.

The effect of dietary vitamin A on antibody production and T cell proliferative response was determined in broilers from 21 to 39 d old. Chicks were fed soybean meal-sorghum diets with levels of supplemented vitamin A from 0 to 13,200 micrograms/kg retinol equivalents from hatching and were immunized with beta-casein at 21 d of age. T cell proliferation response to beta-casein or an acetone precipitate of antigen to Mycobacterium tuberculosis was determined in vitro at 34 to 37 d old. Antibodies to beta-casein in serum were determined every 5 d from 21 d of age. In chicks with no added dietary vitamin A, antibody production and proliferative response were depressed in comparison with chicks receiving vitamin A. Addition of small amounts of vitamin A enhanced the responses; both antibody production and proliferative responses increased with dietary vitamin A until the diet contained 6,660 micrograms/kg, above which the responses decreased. This suggests that maximal immune responses in the chick may be achieved at dietary intakes considerably higher than NRC recommendations.

Animal Feed↗
PubMed1993

Direct evidence for anergy in T lymphocytes tolerized by oral administration of ovalbumin.

The present study investigated bystander suppression, specific suppression and anergy as mechanisms for oral tolerance. Oral tolerance was induced in mice by a single gastric intubation of 20 mg ovalbumin (OVA) and was evaluated in vitro by the absence of T lymphocyte proliferative responses to OVA after priming by OVA-complete Freund's adjuvant (CFA). T lymphocyte unresponsiveness was antigen specific, systemic and was not affected by the vehicle used for immunization. T lymphocytes derived from tolerant popliteal lymph nodes (PLN) responded to an acetone precipitate (AP) of mycobacteria present in CFA; this response was not suppressed by co-culture with OVA, thereby arguing against a mechanism of bystander suppression in our system. Responses of PLN T lymphocytes derived from OVA-CFA primed, non-tolerant mice, or those of an OVA-specific T lymphocyte line, were not suppressed by PLN or spleen cells derived from OVA tolerant mice. These results excluded the possibility that oral tolerance was induced and maintained by a mechanism of specific suppression. At the cellular level, we found that OVA-tolerant T lymphocytes did not produce interleukin-2 (IL-2) nor express IL-2 receptor in response to OVA stimulation in vitro; both observations are indicative of a state of anergy. Incubation of OVA-tolerant PLN T lymphocytes together with murine recombinant IL-2 for 5 days, released anergic T lymphocytes and a concomitant OVA-specific proliferative response of CD4+ T cells was detected. Taken together, our experimental system excludes the involvement of bystander or specific suppression in the induction of oral tolerance to OVA, and provides direct evidence to show that oral tolerance results from specific T lymphocyte anergy.

Administration, Oral↗
PubMed1993

Modification of the immune response by oral tolerance: antigen requirements and interaction with immunogenic stimuli.

The present study was undertaken to define requirements for antigen in induction and maintenance of oral tolerance and to evaluate the interrelationship between simultaneously administered oral tolerogenic and parenteral immunogenic stimuli. Tolerance was induced by a single gastric intubation of OVA and was assessed by absence of OVA-specific T lymphocyte proliferation in vitro. Tolerance was short termed and held for 21 days in the absence of additional antigen; alternatively, antigen persistence enabled prolonged maintenance of tolerance. Since tolerogenic and immunogenic stimuli reflect two distinct pathways of the immune system, we investigated the consequences of their simultaneous administration. When tolerogenic stimuli preceded immunogenic stimuli up to a minimal period of 24 hr, unresponsiveness dominated. Furthermore, tolerogenic stimuli were able to reduce the level of T lymphocyte proliferation when administered at the time of, and after, immunization. The degree of reduction was inversely related to the interval between parenteral immunization and gastric intubation. Repeated tolerogenic stimuli caused a further substantial reduction in residual T lymphocyte responses. These findings indicate that prevalence of tolerance in the immune response requires rapid dissemination and long term persistence of antigen.

Administration, Oral↗
PubMed1993

Interferons and interleukin-6 suppress the DNA-binding activity of E2F in growth-sensitive hematopoietic cells.

Transcription factor E2F binds to cellular promoters of certain growth- and cell cycle-controlling genes and forms distinct heteromeric complexes with other nuclear proteins. We show here that alpha and beta interferons (alpha, beta) and interleukin-6 abolished the E2F-containing DNA-binding complexes in Daudi Burkitt lymphoma cells and in M1 myeloblastic cells, which responded to the cytokines by suppression of c-myc transcription. Time kinetics studies showed that the abolishment of E2F complexes coincided with reduction of c-myc expression and that both molecular events preceded the cell cycle block in G0/G1 phase. In contrast, the pattern of E2F complexes remained unchanged in an interferon-treated growth-resistant Daudi cell mutant that displayed relaxed regulation of c-myc. All of the DNA-binding E2F complexes, including those containing the retinoblastoma protein (pRB), cyclin A-p33cdk2, and the free forms of E2F, were reduced by interferons or interleukin-6. Their abolishment was unperturbed by pharmacological treatments that alleviated the cyclin A and pRB responses to interferon. Thus, changes in cyclin A expression and pRB phosphorylation are not primary events that influence the pattern of E2F responses to cytokines. Addition of EDTA to cell extracts of interferon-treated Daudi cells restored the DNA-binding activity of E2F, resulting in the appearance of a single E2F complex that exclusively contained pRB. It is suggested that the regulation of E2F by growth-inhibitory cytokines that induce cell cycle exit takes place at the level of the DNA-binding activity, and by that mean it differs basically from the phase-specific regulation of E2F in cycling cells.

Base Sequence↗
PubMed1993

Interleukin 6 induces DNA binding activity of AP1 in M1 myeloblastic cells but not in a growth resistant cell derivative.

The effects that three different growth inhibitory cytokines exert on expression and function of members of the Jun family were studied in this work. M1 myeloblastic cells were chosen for this purpose because of their high growth sensitivity to interleukin 6 (IL-6), transforming growth factor beta 1 and alpha- and beta-interferons. It is reported here that IL-6 elevated the junB and c-jun mRNA levels and induced the formation of a novel DNA-protein complex with high sequence specificity to 12-O-tetradecanoylphorbol-13-acetate response element (TRE) oligonucleotides. This IL-6 induced TRE binding complex was abolished by anti-Jun specific antibodies and was efficiently competed by an oligonucleotide that comprises the mouse homologue of a previously described human c-myc negative DNA element. It persisted in cells for at least 48 h after IL-6 treatment and failed to be induced by alpha- and beta-interferons or by transforming growth factor beta 1, which affected differently the pattern of jun mRNA expression. To further explore regulatory and functional aspects of this induced TRE binding activity, an IL-6 resistant M1 clone was isolated and further analyzed. This clone carried a postreceptor deficiency that abrogated completely the growth inhibitory responses to IL-6 but did not interfere with the induction of two differentiation related cell surface markers. Interestingly, the IL-6 resistant clone had lost two molecular responses to IL-6, induction of TRE binding activity and suppression of the c-myc gene. The data correlate the IL-6 induced AP1 activity with the suppression of c-myc and growth inhibition.

Animals↗
PubMedDate not supplied

A vaccine against avian colibacillosis based on ultrasonic inactivation of Escherichia coli.

Ultrasonic inactivation of Escherichia coli followed by irradiation was found to be the most efficient method for preparation of an effective vaccine against colibacillosis. Challenge experiments revealed that this vaccine provided the best protection compared with other methods of inactivation: heat, formaldehyde, and irradiation. Preparing the ultrasonicated vaccine from O2:K1 strain increased its range and also supported adequate protection against homologous strain O78:K80. The degree of protection conferred by the vaccine was positively correlated with the antibody titer against E. coli as measured on day of challenge. Low antibody titers detected 5 days post-vaccination resulted in only 20% protection. High antibody titers detected at 8 and 15 days post-vaccination correlated with a low number of chicks with lesions. In each challenged group, the live chicks that did not develop lesions had higher antibody titers than chicks with lesions, revealing a correlation between numbers of chicks with lesions and antibody titers as measured by enzyme-linked immunosorbent assay.

Animals↗
PubMedDate not supplied

An enzyme-linked immunosorbent assay for detection of antibodies against Escherichia coli: association between indirect hemagglutination test and survival.

An enzyme-linked immunosorbent assay (ELISA) was modified for detection of antibodies against the two main pathogenic serotypes of Escherichia coli: serotypes O78:K80 and O2:K1. The ELISA was a more sensitive and repeatable test than the indirect hemagglutination test (IHT), which is a common method for detecting antibodies against E. coli. Cross-reactivity between the two strains was measured by reacting antisera of each serotype against homologous and heterologous antigens. The results suggest that aside from similar determinants expressed by the two serotypes, serotype O2:K1 expresses more strain-specific determinants than does O78:K80. Comparison of mean antibody titers of immunized chicks by IHT and ELISA along the primary response revealed that during the first 15 days after immunization with inactivated E. coli, the titers in both tests were parallel. After 15 days post-immunization, antibody titers measured by IHT decreased rapidly, whereas titers measured by ELISA decreased only slightly. In addition, a higher correlation was found between titers detected by ELISA and survival through challenge with E. coli than between titers detected with IHT and survival through challenge. The results suggest that the ELISA is a better test for detection of antibody in flocks suspected of being infected with E. coli.

Animals↗