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Biomedical subjects

A Salerno

Publications and source records attributed to A Salerno.

At least 91 records · Page 5Linked to original sources
PubMed1994

Dominant V beta 8 gene usage in response to TNP: failure to use other V beta chains following removal of V beta 8+ T cells by monoclonal antibody in vivo.

This paper investigates the V beta usage of lymph node cells from mice immunized with TNP and of cell lines made from them. In cell lines stimulated weekly with TNP in vitro for 1 month, about 87% of the cells were V beta 8+ and further analysis showed that these cells were actually V beta 8.2+. This was also true for the cells that proliferated in lymph nodes in response to TNP 4 days after primary immunization, i.e. proliferation occurred mainly in the V beta 8+, and in particular in the V beta 8.2+, population while much less proliferation occurred when the V beta 8- or V beta 8.2- T-cell populations are used. This was not due to non-specific damage during separation, as the response to concanavalin A and alloantigen was intact. In a separate series of experiments, mice were acutely depleted of V beta 8+ T cells by treatment with F23.1 or a control monoclonal antibody (mAb) in vivo given before immunization. Treatment with the relevant mAb virtually abolished the response to TNP. In contrast, SJL mice, which lack the gene segment coding for the V beta 8 family and several other V beta chains, made a normal proliferative and delayed-type hypersensitivity (DTH) response to TNP. This poses the problem, which may be important in the study of the T-cell repertoire, of why acute removal of V beta 8+ T cells, which are dominantly used in the response to TNP, does not allow T cells using other chains to substitute in the response, while the absence of this population over a long period of time, because of a deletion in the genome, allows the use of T cells bearing other V beta chains.

Animals↗
PubMed1993

Covalent modification with concomitant inactivation of the cAMP-dependent protein kinase by affinity labels containing only L-amino acids.

Affinity labels for proteins that process other proteins (e.g. proteinases and protein kinases) are an amalgam of two components, an active site-directed peptide carrier and a non-peptidic electrophilic appendage. We have synthesized several affinity labels for the cAMP-dependent protein kinase that are composed solely of L-amino acids and therefore contain only functionality present in naturally occurring proteins. We have found that 2 adjacent cysteine residues, when covalently linked via a peptide bond and an intramolecular disulfide loop (abbreviated as Cys<-->Cys), serves as a potent electrophile. The peptides Leu-Arg-Arg-Cys<-->Cys-Leu-Gly (1), Leu-Arg-Arg-Ala-Cys<-->Cys-Gly (2), and Leu-Arg-Arg-Ala-Ala-Cys<-->Cys-Gly (3) inactivate the cAMP-dependent protein kinase in a time-dependent fashion. Since dialysis does not restore activity, but dithiothreitol does, this strongly suggests that covalent modification of the target enzyme has occurred at a cysteine residue. Although there are 2 cysteine moieties contained within the protein kinase, the 14C-acetylated affinity labels modify the enzyme only once. In addition, since ATP blocks inactivation of the protein kinase, this implies that it is the active site cysteine residue (Cys-199) that has undergone covalent modification. Based on the KI(inact) values obtained from inactivation kinetics, we conclude that the optimal site on the affinity label for the electrophilic Cys<-->Cys is 1 amino acid removed from the 2 arginine residues (i.e. 2). In addition, the efficacy of these inhibitors is also dependent upon the size of the disulfide ring. The eight-membered disulfide ring-containing peptides 1-3 are relatively poor affinity labels compared to the 12-membered ring-containing inhibitor, [formula: see text]

Affinity Labels↗
PubMed1993

ATPase-promoting dead end inhibitors of the cAMP-dependent protein kinase.

The cAMP-dependent protein kinase is a bifunctional enzyme, catalyzing the phosphorylation of the serine and threonine residues in peptides and proteins (kinase activity) as well as the phosphorylation of water (ATPase activity). We have found that several peptides, which serve as inhibitors of the kinase reaction, will either maintain or enhance the ATPase reaction catalyzed by the enzyme. Positively charged dipeptides (e.g. Arg-Arg), as well as small guanidino-containing compounds (e.g. guanethidine) block protein kinase activity yet enhance ATPase activity up to 3.5-fold over that exhibited by the enzyme in the absence of these compounds. In contrast, several nonphosphorylatable peptides, whose primary sequences are based on that of a known substrate (i.e. Leu-Arg-Arg-Ala-Ser-Leu-Gly), such as Leu-Arg-Arg-Ala-Ala-Leu-Gly, Leu-Arg-Arg-Ala-Phe-Leu-Gly, and Leu-Arg-Arg-Ala-Tyr-Leu-Gly, have little or no effect on the rate of the kinase-catalyzed hydrolysis of ATP. An exception to the latter observation is Leu-Arg-Arg-Ala-Cys-Leu-Gly, a cysteine-containing peptide that promotes the protein kinase-catalyzed ATPase reaction by 2.2-fold. We have also found that peptides that possess relatively large amino acid side chain moieties immediately following the arginine dyad (i.e. such as Phe, Tyr, Cys, or Asn at Xaa in Leu-Arg-Arg-Xaa-Ala-Leu-Gly) sharply reduce the rate of enzyme-catalyzed ATP hydrolysis. This suggests that in the presence of peptides containing an -Arg-Arg-Ala- sequence, the enzyme-bound gamma-phosphate of ATP is relatively accessible to water. In contrast, when the latter alanine moiety is replaced by a larger residue, access by water to ATP appears to be hindered. These results indicate that certain structural features associated with the substrate or substrate analog have a profound influence on the manner by which these species interact with the protein kinase. Furthermore, the work described herein demonstrates that it is possible to block the physiologically important kinase reaction and simultaneously promote the energetically wasteful ATPase reaction.

Adenosine Triphosphatases↗
PubMed1993

The active site substrate specificity of the cAMP-dependent protein kinase.

cAMP-dependent protein kinase substrates have been synthesized employing an unusually efficient method that allows the alcohol-bearing residue to be incorporated into the peptide after solid phase peptide synthesis. These peptide substrates have been utilized to map the active site substrate specificity of the protein kinase. Only alpha- or beta-substituted alcohol-bearing residues containing the proper absolute configuration are phosphorylated by the enzyme. However, the cAMP-dependent protein kinase will phosphorylate achiral residues. The implications of the observed protein kinase substrate specificity with respect to inhibitor design are discussed.

Alcohols↗
PubMed1993

Major histocompatibility complex regulation of interleukin-5 production in the mouse.

Lymph node cells of CBA (H-2k), but not of BALB/c (H-2d) mice immunized epicutaneously with picryl chloride secrete interleukin (IL)-5 when stimulated with the specific antigen in vitro. The low IL-5 production in BALB/c mice persists when either picryl chloride or the unrelated antigen oxazolone are used, when the amount of antigen in vitro is varied and when a secondary response is studied. The difference in IL-5 production maps to the major histocompatibility complex (MHC) in the congenic BALB/b, BALB/c and BALB/k mice. Furthermore, lymph node cells from (k x d) F1 mice produce IL-5 when stimulated by antigen presented on H-2k but not on H-2d antigen-presenting cells. Finally, the low IL-5 production in vitro in BALB/c mice is correlated with low picryl-specific IgA levels in vivo, which otherwise are ten times greater in CBA and BALB/k mice. The influence of MHC on IL-5 production and IgA secretion in the mouse might be a possible basis for the association of MHC with IgA deficiency in humans.

Animals↗
PubMed1993

Natural killer and lymphokine-activated killer activity in HLA-B8,DR3-positive subjects.

The haplotype HLA-B8,DR3 is over-represented in several autoimmune diseases, implying that genes predisposing people to these disorders are linked to this haplotype. In these diseases, various dysfunctions reflecting an impairment of the immune system have been found. Several reports indicate also that in HLA-B8,DR3-positive healthy subjects similar disorders may be demonstrated. In the present work, we have evaluated NK and LAK activity in these subjects. The study has been performed on monocyte-depleted peripheral blood MNCs by using, the K-562 cell line as a target for NK activity and the HL-60 cell line for as a target LAK activity. LAK cells were obtained by incubating MNCs for 3 days with 100 U/ml of rIL-2. The results of our experiments demonstrate that NK cell activity is significantly decreased in healthy subjects bearing the HLA-B8,DR3 haplotype. Since the number of circulating CD16+ cells is not significantly different between HLA-B8,DR3-positive subjects and negative ones, it is unlikely that this defect is due to a decreased number of NK cells in effector cell preparations. The observation that the treatment with rIL-2 can restore the killer activity of MNCs from these subjects suggests instead that the reduced NK activity may be due at least in part to the imbalance of cytokine network that has been demonstrated in HLA-B8,DR3-positive subjects. Finally, since a decreased NK activity has been reported in the autoimmune diseases linked to this haplotype, our results support the suggestion that immunologic changes observed in autoimmune diseases reflect systemic regulatory disorders that have a genetic basis.

Adult↗
PubMed1993

Augmented passive transfer of contact sensitivity in severe combined immunodeficiency mice and its dependence of V beta 8+ cells in the picryl system.

The passive transfer of contact sensitivity using picryl chloride immune cells from H-2 syngenic BALB/c donors was analyzed in severe combined immunodeficiency (SCID) mice which lack functional T and B lymphocytes. H-2-restricted and antigen-specific contact sensitivity was transferred to SCID mice, and comparison between the level of contact sensitivity and the number of transferred cells showed a significantly more efficient transfer to SCID than to BALB/c mice. The cells passively transferring contact sensitivity were shown to carry the V beta 8 phenotype. Moreover, chromium-labeled cells from BALB/c PC1-primed donors localize normally in peripheral lymphoid organs and an increased percentage of cell arrival in the ears is clearly observed in SCID after challenge with picryl chloride.

Animals↗
PubMed1992

A nonspecific inhibitor of contact sensitivity elaborated by macrophages: genetic restriction in its production but not in its action.

It is known that macrophages armed with hapten-specific T suppressor factor (TsF) and then exposed to antigen (haptenized spleen cells) liberate a nonspecific inhibitor of the transfer of contact sensitivity (CS). This is called macrophage suppressor factor (MSF). This paper shows that MSF is only released when the source of the TsF and the haptenized spleen cells share the same I-J subregion. This is based on the comparison of B10.A(3R) and B10.A(5R) mice. In contrast, the action of MSF is antigen nonspecific and genetically unrestricted. In these respects it resembles the antigen-nonspecific inhibitor (nsTsF-1) made by the T acceptor cell when armed with TsF. However, it differs from nsTsF-1 in acting directly on the I-A- population which transfers contact sensitivity and not indirectly via I-A+ T cells. In vitro, MSF fails to inhibit the proliferative response of lymph node cells to specific antigen and their production of IL-3 activity, IFN-tau, and IL-2. This indicates that MSF is not a global inhibitor of T cell activity. The finding that MSF inhibits systemic passive transfer of contact sensitivity, but has no effect on local passive transfer strongly supports the view that MSF affects the arrival of certain cells critical for the development of the reaction to the skin challenge site.

Animals↗
PubMed1992

The effect of age on mitogen responsive T cell precursors in human beings is completely restored by interleukin-2.

It is well known that the function of T lymphocytes is significantly impaired by advancing age. In the present study, attempts have been made to further characterize the T cell impairment of elderly subjects. Thus, we have performed limiting dilution microculture analysis to evaluate the precursor frequency of T lymphocytes responding to a mitogenic stimulus in old and young subjects. Furthermore we have evaluated the activity of recombinant interleukin-2 (rIL-2) on these cells. The results demonstrate that in older subjects the frequency of these precursors is significantly decreased. The in vitro treatment with rIL-2 increased the frequency of mitogen responsive T lymphocyte precursors in both groups so that the difference between the two groups was not significant. Thus present results extend the findings demonstrating that older subjects display an impairment of T cell functions and that IL-2 treatment may correct these alterations. In particular, they confirm the hypothesis that age-associated functional changes are more likely due to diminished numbers of reactive cells, than to a decline in the activity of all cells.

Adult↗
PubMed1992

Induction of contact sensitivity by cell-associated immunocomplexes requires activation of the early complement components.

Lymph node cells collected from CBA/J mice 4 days after painting the skin with picryl chloride behave like antigen presenting cells and induce contact sensitivity when injected into naive recipient mice. The immunizing capacity of these '4-day' cells is due to T cells which carry on their membrane hapten-IgM immunocomplexes. Incubation of the cells with complement from mouse strains that express high C4 serum levels (C4H), abolishes their immunizing capacity. This effect is related to the activation of the early components of the classical complement pathway, as supported by experiments using C3 and C4-depleted or C5 and C6-genetically deficient mouse sera. The detection of different amounts of C3b and C4b on the surface of 4-day T cells after incubation with C4L and C4H sera supports the possibility that membrane bound activated complement components could modify the immunizing capacity of these cells. Results herein reported suggest that membrane-bound C3b and C4b are not per se inhibitory but interfere with the residual complement activating capacity of 4-day T cells. The role of complement activation by 4-day T cells is pivotal as complement depletion of recipient mice by cobra venom factor (CVF) inhibits the immunizing capacity of untreated 4-day T cells, while 4-day T cells treated with complement in vitro and injected together with C4a anaphylatoxin are able to immunize recipient mice.

Animals↗
PubMed1992

Impairment of contrasuppressor activity in mice infected with the paramyxovirus of Newcastle disease.

Spleen cells from mice infected with the virus of Newcastle disease (NVD) fail to mediate the passive transfer of contact sensitivity to simple chemical haptens such as picryl chloride (Pcl) and oxazolone (Ox). The inhibitory effect of NDV can be bypassed by treating recipient mice with low doses of cyclophosphamide (Cy), suggesting that the T-effector cell which mediates the passive transfer of contact sensitivity is not affected by NDV infection. Vicia Villosa-adherent cells from immune mice display contrasuppressor activity and restore the ability of cells from NDV-infected mice to transfer contact sensitivity to naive recipients. In contrast, Vicia Villosa-adherent cells from NDV-infected mice fail to exert any contrasuppressor activity. Furthermore, contrasuppressor activity can also be detected in the culture supernatants of Vicia Villosa-adherent cells from uninfected, sensitized mice, but not in culture supernatants of Vicia Villosa-adherent cells from NDV-infected mice. The present results suggest that a Vicia Villosa-adherent contrasuppressor cell population is impaired by NDV infection.

Animals↗
PubMed1991

Soluble interleukin-2 receptor release defect in vitro in elderly subjects.

The evidence from several studies indicates that as individuals age, they may display immune dysfunctions, mostly T cell dysfunctions. Recently, a soluble form of the receptor for interleukin-2 (IL-2) (sIL-2R) has been demonstrated in human sera and in vitro stimulated culture supernatants from human T lymphocytes. In the present paper, we report in vitro sIL-2R production from peripheral blood mononuclear cells in elderly subjects. The results show that no difference exists for unstimulated cultures, whereas after mitogen stimulation the elderly subjects showed the lowest values compared with young ones. These findings suggest that sIL-2R may provide a new tool for the study of T lymphocyte dysfunctions in old age.

Aged↗
PubMed1991

Reduction in the number of UCHL-1+ cells and IL-2 production in the peripheral blood of patients with visceral leishmaniasis.

PBMC from patients with visceral leishmaniasis (VL), before and after successful antimony therapy, were analyzed for their phenotypes and for their ability to produce IL-2 and IFN-gamma and to proliferate against PHA and leishmanial Ag. In agreement with results of earlier studies, PBMC from active VL patients showed a markedly reduced proliferative response and IL-2 and IFN-gamma production, compared with those of healthy controls. The levels of CD4+ and CD8+ T cells were within the normal range, but there was a significant decrease in UCHL-1+ cells (helper-inducer), compared with healthy individuals. The inhibited cellular responses, and lymphokine secretion and decreased level of UCHL-1+ cells in the PBMC of the VL patients returned to the normal range after successful chemotherapy. PBMC from active VL patients were fractionated into adherent cells and nonadherent cells, and the non-adherent were further fractionated into UCHL-1+ and UCHL-1- subpopulations. Results from cell depletion and reconstitution experiments suggest that the IL-2 production by nonadherent cells stimulated with PHA was inhibited by adherent cells, but the IL-2 production by nonadherent cells in response to specific Ag was not. In contrast, UCHL-1- cells seem to mediate the inhibition of Ag-driven IL-2 production by nonadherent cells but not mitogen-stimulated IL-2 secretion by nonadherent cells. Ag-specific IL-2 production principally involves UCHL-1+ cells.

Adult↗
PubMed1990

Noncovalent active site interactions enhance the affinity and control the binding order of reversible inhibitors of the cAMP-dependent protein kinase.

The peptides, Leu-Arg-Arg-Ala-Ala-Leu-Gly-NH2, Leu-Arg-Arg-Gln-Ala-Leu-Gly-NH2, and Leu-Arg-Arg-Asn-Ala-Leu-Gly-NH2, serve as active site-directed inhibitors of the cAMP-dependent protein kinase from bovine cardiac muscle. The Asn-containing peptide is a 10-fold more potent inhibitor than its Ala- and Gln-containing counterparts. All three peptides are linear competitive inhibitors versus a peptide-based substrate and uncompetitive inhibitors versus MgATP. The enhanced inhibitory potency of the Asn-peptide, in conjunction with the observed loss of ATP-ase activity of the enzyme in the presence of the inhibitor, suggests that asparagine may serve as a through-space isostere of serine. The uncompetitive inhibition pattern displayed by amide-capped peptides versus MgATP indicates that these species bind in an ordered fashion to the cAMP-dependent protein kinase, with MgATP binding first.

Adenosine Triphosphatases↗
PubMed1989

Role of the fourth complement component (C4) in the regulation of contact sensitivity. III. In vivo effect of purified C4.

Lymph node cells obtained from CBA/J mice 4 days after painting with contact sensitizing agents such as picryl chloride or oxazolone ("4-day" cells), induce contact sensitivity into naive recipient mice by membrane-associated immunocomplexes. This immunizing capacity is abolished after incubation of the cells in serum from mice with high C4 levels (C4H), but not in serum from mice with low C4 levels (C4L), and the inhibitory activity of C4H serum is due to the activation of the early components of the classical complement pathway. The presence of 4-day cells depends on C4 levels: in fact, C4H mice lack these cells because they activate their own complement in vivo, whereas C4L mice fail to activate complement in vivo and possess 4-day cells. CBA/J (C4L) mice injected with purified C4 preparations from the C4H mice BALB/c, lose 4-day cells and show a short-term contact-sensitivity reaction, exactly as BALB/c mice, thus indicating that C4 levels play a role in the control of contact-sensitivity reaction to simple chemical haptens.

Animals↗