bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential responses to challenge with live and dead Mycobacterium bovis Bacillus Calmette-Guérin.
Authors: Chambers M A , Marshall B G , Wangoo A , Bune A , Cook H T , Shaw R J , Young D B
Published: December 2022

Bacillus Calmette-Guérin (BCG) vaccination has been shown to protect against challenge with virulent Mycobacterium tuberculosis in a range of experimental animal models: in each case, protective efficacy requires vaccination with live bacteria. With the goal of moving to a new generation of safer, nonliving vaccines, efforts have been made to identify the factors that determine the efficacy of live vaccination. We show that injection of live, but not dead, BCG induces localized swelling in the mouse footpad model. Live and dead bacteria induce similar responses during the first week after vaccination as determined by immunohistochemical analysis of the site of injection and of the draining lymph node. The subsequent differential response is characterized by migration of acid-fast bacilli to the draining lymph node in the case of the live vaccine. This is accompanied by an increase in mononuclear cells in the lymph node and by expression of inducible nitric oxide synthase (iNOS) both in the lymph node and at the site of injection. The ability of the bacteria to migrate to the lymph node may be an important element in the efficacy of live BCG vaccination.
A novel CD8 molecule expressed by alveolar and peritoneal macrophages stimulates nitric oxide production.
Authors: Hirji N , Lin T J , Befus A D
Published: December 2022

Macrophages play an essential role in host defense, and we have identified a novel CD8 molecule, on alveolar and peritoneal macrophages, that may be involved in regulating this function. Flow cytometric analysis of bronchoalveolar lavage from normal rats identified a large number of CD8-positive cells that could not be accounted for by T lymphocytes. Within the scatter profile region in which the majority of cells were alveolar macrophages (OX41; 89 +/- 1%), 63 +/- 5% of the cells stained positively for CD8alpha (OX8) and 52 +/- 3% for CD8beta (341). Double-staining of lavage cells confirmed the presence of CD8 on alveolar macrophages. Interestingly, flow cytometry showed differences between CD8 on alveolar macrophages and on T lymphocytes within the ligand binding domain for MHC class I. Reverse transcription-PCR analysis on FACS-enriched alveolar macrophages showed the presence of CD8alpha mRNA, determining that macrophages synthesize CD8. Further studies identified both the alpha (49 +/- 8%)- and beta (37 +/- 4%)-chains of CD8 on peritoneal lavage cells (86 +/- 3% macrophages (OX42, CD11b)). As with alveolar macrophages, there were differences within the ligand-binding domain of CD8 on peritoneal macrophages compared with T lymphocytes. Functional studies determined that anti-CD8alpha (OX8) stimulated a dose-dependent release of nitric oxide, indicating that CD8 can directly regulate macrophage function. Thus, macrophages express an unusual CD8 molecule that differs within its ligand-binding domain, compared with T lymphocytes, and these findings suggest hitherto unknown ligand(s) for CD8. These findings will lead to a greater understanding of macrophage function and regulation.
Human airway smooth muscle cells express and release RANTES in response to T helper 1 cytokines: regulation by T helper 2 cytokines and corticosteroids.
Authors: John M , Hirst S J , Jose P J , Robichaud A , Berkman N , Witt C , Twort C H , Barnes P J , Chung K F
Published: December 2022

RANTES is a basic 8-kDa polypeptide of the C-C chemokine subfamily with strong chemotactic activity for eosinophils, lymphocytes, and monocytes. We determined the regulation of RANTES production by human airway smooth muscle cells in culture. While TNF-alpha, but not IFN-gamma, increased RANTES mRNA expression and protein release, the combination of TNF-alpha and IFN-gamma caused a greater degree of expression and release in a time- and dose-dependent manner. Sequential treatment of airway smooth muscle cells with TNF-alpha and IFN-gamma showed that IFN-gamma sensitized the cells to the stimulatory effect of TNF-alpha. Using a modified Boyden chamber technique, RANTES separated by reverse-phase liquid chromatography from cell culture supernatants of airway smooth muscle cells stimulated by TNF-alpha and IFN-gamma showed a strong chemoattractant effect on human eosinophils, an effect inhibited by an anti-RANTES Ab. RANTES production induced by TNF-alpha and IFN-gamma was inhibited partly by the Th2-derived cytokines, IL-4, IL-10, and IL-13, as well as by dexamethasone. Our studies indicate that, in addition to contractile responses and mitogenesis, airway smooth muscle cells have synthetic and secretory potential with the release of RANTES. They may participate in chronic airway inflammation by interacting with both Th1- and Th2-derived cytokines to modulate chemoattractant activity for eosinophils, activated T lymphocytes, and monocytes/macrophages.
Induction of acute inflammatory lung injury by staphylococcal enterotoxin B.
Authors: Neumann B , Engelhardt B , Wagner H , Holzmann B
Published: December 2022

Superantigens stimulate T lymphocytes at high frequency by interacting with appropriate Vbeta segments of the TCR. Challenge of mice with bacterial superantigens such as staphylococcal enterotoxin B (SEB) induces the systemic release of cytokines resulting in septic shock and death of sensitized animals. Analysis of putative pathogenic mechanisms of T cell-dependent septic shock revealed that administration of SEB results in acute inflammatory lung injury characterized by a profound increase in vascular permeability. Injury was associated with marked leukocyte infiltration of the lung and induction of cell adhesion molecules including VCAM-1, ICAM-1, and P-selectin, but not E-selectin. Lung infiltrating leukocytes consisted of granulocytes, mononuclear phagocytes and NK cells with granulocytes representing the major fraction. Consistent with a role of neutrophils as cellular mediators of inflammatory organ injury, we demonstrate activation of circulating granulocytes in mice treated with SEB. When compared with granulocytes of control mice, peripheral blood granulocytes of SEB-treated mice were found to express increased levels of cell surface Mac-1, to down-regulate expression of L-selectin, and to respond with an increased production of toxic oxygen metabolites upon exposure to FMLP. Interestingly, TNF-alpha further enhanced FMLP-induced oxidant production by granulocytes from SEB-treated but not control mice, suggesting that the systemic response to SEB increases granulocyte sensitivity to TNF-alpha-mediated signals. Together, these results suggest that acute inflammatory lung injury may contribute to the pathogenesis of T cell-dependent lethal shock in mice challenged with bacterial superantigens and indicate common pathogenic mechanisms of lung injury induced by a large number of distinct inflammatory stimuli.
A peptide with unique receptor specificity: stimulation of phosphoinositide hydrolysis and induction of superoxide generation in human neutrophils.
Authors: Seo J K , Choi S Y , Kim Y , Baek S H , Kim K T , Chae C B , Lambeth J D , Suh P G , Ryu S H
Published: December 2022

Previously, we identified peptides that stimulate phosphoinositide hydrolysis in several leukocyte cell lines from mixtures of random hexapeptide sequences. Moreover, the peptides activate phospholipase C via a pertussis toxin-sensitive G protein-coupled receptor. We now investigate the structure-activity relationship of the peptides with the goal of improving the activity of the peptides, as well as the biologic function of the peptides. Substitution of the L-methionine at the C terminus of peptides with D-methionine markedly increased the effectiveness of the peptides. The half-maximal effective concentrations of MKYMPm-NH2 and WKYMVm-NH2 for stimulation of phosphoinositide hydrolysis in U266 cells were 30 and 0.5 nM, respectively. By BIAcore analysis we confirmed the existence of a receptor for WKYMVm-NH2. Furthermore, the intracellular calcium concentration increase induced by WKYMVm-NH2 was not inhibited by several chemoattractants (FMLP, IL-8, platelet-activating factor, C5a, granulocyte-macrophage CSF, and granulocyte CSF) suggests that WKYMVm-NH2 has a unique cell surface receptor on leukocytes. WKYMVm-NH2 stimulated the phosphoinositide hydrolysis in U937, HL60, and U266 cells, as well as in human neutrophils. Moreover, WKYMVm-NH2 is more effective than FMLP in the production of superoxide in human neutrophils. The data suggest that WKYMVm-NH2 may have the ability to activate the microbicidal functions of human neutrophils.
Dominance of a single peptide bound to the class I(B) molecule, Qa-1b.
Authors: DeCloux A , Woods A S , Cotter R J , Soloski M J , Forman J
Published: December 2022

One of the hallmarks of class I(A) molecules is their ability to bind and present a wide array of peptides to CD8 T cells. This diversity is consistent with their ability to restrict a variety of pathogenic peptide epitopes as well as elicit strong transplantation responses. In contrast, class I(B) molecules appear to be involved in presentation of pathogenic epitopes to a relatively lesser extent as well as play a minor role in transplantation responses. Here we have examined the peptides bound and presented by the class I(B) molecule Qa-1b in order to determine if their diversity was similar to that reported for class I(A) Ags. First, we show that bulk-cultured anti-Qa-1b CTL predominantly recognize a single peptide (Qdm) derived from the leader segment of class I(A) alloantigens. These CTL are peptide specific and reflect the activity of previously described CTL clones. Second, we find approximately 4.6 x 10(4) copies of the Qdm peptide/cell. Most of the peptide is Qa-1b associated since the recovery of this peptide from anti-Qa-1b immunoprecipitates is approximately 75% of that seen in whole cell extracts and no detectable activity is observed in Kb or Db extracts from H-2b lymphoblasts. Third, the expression of Qa-1b on lymphoblasts is approximately 1 to 1.25 x 10(4) molecules/cell indicating that the Qdm peptide must be derived from both cell membrane and intracellular compartments. Finally, examination of the diversity of peptides associated with Qa-1b as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry indicates few detectable peptide species associated with this molecule. Taken together, Qa-1b appears to predominantly bind a single peptide species that is recognized by alloreactive CD8 T cells. This feature may account, in part, for the class I(B) properties of this molecule.
Cytokine and chemokine regulation of proMMP-9 and TIMP-1 production by human peripheral blood lymphocytes.
Authors: Johnatty R N , Taub D D , Reeder S P , Turcovski-Corrales S M , Cottam D W , Stephenson T J , Rees R C
Published: December 2022

The production and activation of matrix-degrading proteinases such as the matrix metalloproteinases (MMP) by lymphocytes is likely to be an important factor in facilitating lymphocyte trafficking through the endothelial barrier and the extracellular matrix. Leukocyte infiltration into inflammatory sites occurs as a response to members of the chemokine superfamily and other inflammatory mediators. In the present study, highly purified leukocyte subpopulations were cultured with or without chemokines or cytokines, and their ability to express gelatinolytic MMPs and their inhibitors, the tissue inhibitors of metalloproteinase (TIMPs), was analyzed. In the absence of exogenous stimuli, purified CD4+ T lymphocytes produced similar quantities of proMMP-9 and elevated levels of TIMP-1 compared with PBMC, while purified CD8+ and CD3+ populations exhibited less MMP-9 and TIMP-1 activity. In comparison, CD56+ (NK) cells secreted barely detectable levels of proMMP-9 and TIMP-1. The secretion of proMMP-9 by PBMC and purified CD3+, CD4+, and CD8+ lymphocytes was selectively modulated by beta chemokines and proinflammatory cytokines. ProMMP-9 secretion by CD3+ and CD4+, but not by CD8+ T cells was augmented in response to TNF-alpha and IL-1, and down-regulated by IFN-gamma, while macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated upon activation and normally T cell expressed and secreted) (beta chemokines) up-regulated the secretion of proMMP-9 by all of the lymphocyte subsets tested. These results demonstrate that a number of proinflammatory cytokines and chemokines differentially regulate proMMP-9 secretion from purified CD4+ and CD8+ lymphocytes, while for purified CD3+ T cells (consisting of CD4+ and CD8+ cells in approximately a 2:1 ratio), a predominantly CD4+ lymphocyte response profile was demonstrated.
Patterns of hemopoietic reconstitution in nonobese diabetic mice: dichotomy of allogeneic resistance versus competitive advantage of disease-resistant marrow.
Authors: Kaufman C L , Li H , Ildstad S T
Published: December 2022

Complete replacement of the immune system via allogeneic bone marrow transplantation is sufficient to prevent diabetes in the nonobese diabetic (NOD) mouse model. In the present study we examined whether mixed allogeneic reconstitution would be sufficient to interrupt the autoimmune process with respect to occurrence of overt diabetes, as well as preexisting autoimmune insulitis. NOD mice were lethally irradiated and reconstituted with a mixture of NOD and B10.BR marrow. A relative resistance to allogeneic bone marrow engraftment was noted in NOD recipients of the mixed bone marrow inoculum, compared with disease-resistant controls. Moreover, unlike disease-resistant controls, all animals that initially repopulated as mixed donor/host chimeras became predominantly allogeneic by 4 mo, suggesting a competitive advantage for long term engraftment for disease-resistant marrow. All but one mouse in the group that engrafted with allogeneic marrow remained free of diabetes for the entire follow-up period (n = 22). Moreover, in all animals examined, virtually all islets were free of insulitis. In contrast, 74% of NOD mice that received similar conditioning and failed to engraft with donor marrow developed acute diabetes and intra-islet insulitis was present in all animals examined. These data suggest that NOD mice exhibit a relative resistance to engraftment compared with disease-resistant recipients. Conversely, animals that initially repopulated as a mixture of syngeneic and donor marrow become converted to virtually all donor by 4 mo. These data provide additional support that a defective stem cell is responsible for autoimmune diabetes in this experimental model.
Blocking the CD28-B7 T cell costimulation pathway induces long term cardiac allograft acceptance in the absence of IL-4.
Authors: Lakkis F G , Konieczny B T , Saleem S , Baddoura F K , Linsley P S , Alexander D Z , Lowry R P , Pearson T C , Larsen C P
Published: December 2022

Blocking the CD28-B7 T lymphocyte costimulatory pathway with the recombinant protein CTLA4Ig induces long term allograft survival in rodents. It has been suggested that this results from selective activation of the Th2 immune pathway. To test this hypothesis, we compared vascularized cardiac allograft survival in wild-type (IL-4 +/+) and homozygous IL-4 gene-knockout (IL-4 -/-) mice. We report in this study that long term survival (>100 days) of fully allogeneic grafts can be induced readily in IL-4 -/- recipients treated with a short course of CTLA4Ig. We also demonstrate that IL-4 -/- mice are deficient in Th2-type cytokine expression following in vitro or in vivo allostimulation. These results suggest that IL-4 production and subsequent generation of a Th2-type immune response are not obligatory for CTLA4Ig-induced long term acceptance of vascularized allografts.
Age-associated thymic atrophy in the mouse is due to a deficiency affecting rearrangement of the TCR during intrathymic T cell development.
Authors: Aspinall R
Published: December 2022

Involution of the thymus is a feature of age and precedes inefficient functioning of the immune system. C57BL/10 mice show an 83% reduction in number of thymocytes between 3 and 20 mo of age, with a significant decline in each of the thymic subsets defined by their expression of CD4 and CD8. The similar percentage contribution of each subset to the whole at 3, 12, and 20 mo suggests a lesion in the T cell developmental pathway within an early subset. The CD3- CD4- CD8- subset showed a significant decline in number by 20 mo of age, but despite this reduction, no significant difference was noted in the number of CD44+ CD25- cells, the earliest stage of this subset between 3 and 20 mo of age. A significant decline in the number of their progeny, the CD44+ CD25+, and the progeny of these cells, the CD44- CD25+ cells, was noted by 12 mo of age. Expression of CD25 within this subset is associated with rearrangement of TCR beta-chain genes. F5 transgenic mice, carrying a complete TCR-alphabeta transgene under the control of a CD2 minigene cassette on a C57BL/10 background, showed no age-associated thymic atrophy in any of the defined thymic subsets over the same period as the normal C57BL/10 mice. Similar results were noted with mice carrying the same transgene but which in addition were also RAG-1-. The results indicate that age-associated thymic involution was associated with problems with rearrangement of the TCR beta-chain genes affecting the production of thymocytes.
Adult T cell leukemia (ATL)-derived factor/human thioredoxin prevents apoptosis of lymphoid cells induced by L-cystine and glutathione depletion: possible involvement of thiol-mediated redox regulation in apoptosis caused by pro-oxidant state.
Authors: Iwata S , Hori T , Sato N , Hirota K , Sasada T , Mitsui A , Hirakawa T , Yodoi J
Published: December 2022

Thiol compounds, such as L-cysteine and glutathione (GSH), play crucial roles in the regulation of lymphocyte proliferation. In this study, we analyzed the effect of L-cystine and GSH depletion on lymphocyte survival and investigated the regulatory roles of adult T cell leukemia (ATL)-derived factor (ADF)/human thioredoxin (hTRX) in relation to these low m.w. thiols. MT-1, MT-2, and Jurkat cells underwent apoptosis when cultured in the L-cystine- and GSH-free medium within 18 to 24 h. Dichlorofluorescin oxidation assay indicated that the apoptosis in MT-1 and MT-2 cells was preceded by an increase in the level of intracellular hydrogen peroxide (H2O2). The addition of catalase and recombinant ADF/hTRX (rADF) partially blocked the apoptosis in a dose-dependent manner. rADF has been also shown to enhance the internalization of L-cystine into MT-2 cells in a dose-dependent manner, whereas oxidized rADF or mutated rADF that has no insulin-reducing activity failed to do so. Furthermore, culture in the L-cystine- and GSH-free medium lowered the cellular GSH content of PHA blasts, which was restored dose-dependently by rADF. These data suggest that the inability to neutralize oxidative stress results in the apoptosis of lymphoid cells under L-cystine- and GSH-depleted conditions. The protective effects of rADF may be explained by direct scavenging action on H2O2 (catalase-like activity) or by indirect neutralizing effects on the pro-oxidant status through enhancing the L-cystine internalization and elevating the intracellular GSH content.
TGF-beta stimulates but IFN-gamma inhibits growth-related activation of locomotion of human B cells.
Authors: Komai-Koma M , Wilkinson P C
Published: December 2022

Effects of TGF-beta and IFN-gamma on locomotion of high density B cells from human tonsils were studied using polarization and filter assays. Culture with TGF-beta (10 pg to 1 ng/ml) induced a gradual increase in locomotor morphologies in 40 to 50% of B cells during overnight culture. This was not immediate (<30 min) suggesting that TGF-beta is not a chemotactic factor. The time course suggests that B cells acquired a locomotor phenotype during the first few hours of culture. B cells cultured in TGF-beta increased in size, but to a lesser extent than those cultured in IL-4 used as a control. More cells were polarized in TGF-beta + IL-4 than in either alone. Following culture in TGF-beta, B cells showed vigorous responses to anti-IgD used as a chemoattractant in short-term assays. In contrast, addition of IFN-gamma (20-100 U/ml) to B cells in culture in IL-4, anti-CD40, or TGF-beta inhibited activation of locomotor capacity by the latter agents, and IFN-gamma-cultured B cells showed an even lower response to anti-IgD in a short-term polarization or filter assay than those cultured in medium alone. IFN-gamma also inhibited uridine incorporation by cultured B cells, and cells cultured in IFN-gamma showed no size increase. We suggest that IFN-gamma prevents locomotor activation by inhibiting progress of B cells into the G1 phase of growth.
T cell development in mice expressing splice variants of the protein tyrosine phosphatase CD45.
Authors: Kozieradzki I , Kündig T , Kishihara K , Ong C J , Chiu D , Wallace V A , Kawai K , Timms E , Ionescu J , Ohashi P , Marth J D , Mak T W , Penninger J M
Published: December 2022

The transmembrane protein tyrosine phosphatase CD45 is expressed in multiple isoforms as a result of alternative splicing of variable exons encoding the extracellular domain. CD45 expression is critical for T cell development, and thymocyte maturation is blocked at the immature CD4+ CD8+ double-positive stage in CD45 gene-deficient (CD45 -/-) mice. Moreover, splicing of variable CD45 exons changes during thymocyte selection. To test the role of CD45 extracellular splice variants in T cell selection and development, we introduced CD45RO (a low-m.w. splice variant lacking exons 4, 5, and 6) and CD45ABC (a high-m.w. isoform containing all exons) transgenes under the control of a thymocyte-specific promoter into a CD45 -/- background, generating CD45RO transgene-positive CD45 -/- (CD45RO) and CD45ABC transgene-positive CD45 -/- (CD45ABC) mice. We demonstrate that both CD45 splice isoforms can rescue development of CD4+ and CD8+ TCR-alphabeta+ thymocytes. Neither CD45 isoform rescued positive selection of H-Y TCR transgene thymocytes, and these cells were blocked at a HSA(high) CD69- CD5(low) stage of development. Peripheral T cells from CD45RO and CD45ABC mice proliferated in response to allogeneic stimulator cells and anti-CD3epsilon cross-linking. However, only CD45RO mice, not CD45ABC mice, generated cytotoxic T cell responses and neutralizing, Th cell-dependent IgG Abs after viral infections. In addition, we show that T cells from CD45RO and CD45ABC mice accumulate in lymph nodes but not in the spleen, liver, or skin, indicating that the CD45 phosphatase may control the homing behavior and trafficking of T cells.
CD16 cross-linking induces both secretory and extracellular signal-regulated kinase (ERK)-dependent cytosolic phospholipase A2 (PLA2) activity in human natural killer cells: involvement of ERK, but not PLA2, in CD16-triggered granule exocytosis.
Authors: Milella M , Gismondi A , Roncaioli P , Bisogno L , Palmieri G , Frati L , Cifone M G , Santoni A
Published: December 2022

The phospholipase A2 (PLA2) enzymes play a central role in diverse cellular processes including phospholipid digestion and metabolism, host defense, and cell signaling. We investigated the ability of CD16 clustering to trigger PLA2 and extracellular signal-regulated kinase (ERK) activation in human NK cells, as well as their possible involvement in CD16-stimulated degranulation. Both secretory (sPLA2) and cytosolic (cPLA2) PLA2 were rapidly activated upon CD16 cross-linking; sPLA2 was found in the supernatant and also in a cell-associated form. cPLA2 activation was controlled by the ERK pathway as indicated by the close correlation between their kinetics of activation and by the ability of the specific MEK inhibitor, PD 098059, to abolish cPLA2 activation. CD16 stimulation also resulted in the generation of platelet-activating factor (PAF) and leukotrienes; both phospholipases contributed to their biosynthesis. Using the pharmacologic inhibitors AACOCF3, p-bromophenacyl bromide (pBPB), and PD 098059, which specifically inhibit cPLA2, sPLA2, and MEK, respectively, we demonstrated that the ERK signaling pathway, but not cytosolic or secretory PLA2, is required for CD16-triggered granule release.
An integrin polymorphism that defines reactivity with alloantibodies generates an anchor for MHC class II peptide binding: a model for unidirectional alloimmune responses.
Authors: Wu S , Maslanka K , Gorski J
Published: December 2022

Polymorphic proteins are often the targets of T and B lymphocytes in alloimmune responses. The polymorphic residue 33 of integrin beta3 is responsible for both a B cell response and a T cell response. Leu at position 33 controls the epitopes recognized by alloantibodies generated in individuals homozygous for Pro at 33. The alloantibody response shows a tight class II MHC restriction, and T cells have been identified that respond to the alloantigen. As part of the T cell response, the alloantigen could be recognized as foreign, and the autoantigen would be ignored due to self-tolerance. Alternatively, the polymorphism could change the ability of the peptide to bind. We tested these two possibilities using synthetic peptides corresponding to the polymorphic region and insect cell-derived class II MHC. The Leu33-containing peptide bound to the restricting MHC allele, whereas the Pro33 peptide did not. Thus, the presence of Leu at position 33 generates an anchor specific for the restricting MHC allele and helps define the peptide binding motif of this allele, HLA-DRB3*0101 (DR52a). These data indicate that certain alloresponses can be viewed entirely as foreign because the polymorphism generates a functional anchor residue in a peptide that otherwise would not bind. On the basis of these results, we would predict a unidirectional alloantibody response as the lack of binding of the Pro33 peptide would preclude T cell help. This could explain the observation that alloantibodies to the Pro33 allele are very rare.
Analysis of the genes encoding the mast cell function-associated antigen and its alternatively spliced transcripts.
Authors: Bocek P , Guthmann M D , Pecht I
Published: December 2022

The mast cell function-associated Ag (MAFA) is a C-type lectin that, upon being clustered, inhibits the Fc epsilon RI stimulation-induced mast cell secretory response. We here report that MAFA is encoded by a single-copy gene that spans 13 kb in the rat genome and is composed of five exons. Three separate exons encode the carbohydrate recognition domain of the MAFA, defining its close homology to the genes of CD23, CD69, CD72, NKR-P1, and Ly49. Functional analysis of the 5' flanking region of the gene reveals that a cell type-specific promoter is located within the first 664 bp upstream of the transcription origin. The promoter lacks any obvious TATA box and drives gene transcription originating from multiple start sites. Examination for possible polymorphism of the MAFA transcripts revealed two novel transcripts, generated by alternative splicing. Deletion of the transmembranal exon in one of them does not result in a frameshift and would, upon translation, give rise to a soluble MAFA molecule. Splicing of two exons in a second transcript results in a new reading frame encoding a putative protein containing MAFA's cytoplasmic domain. The transcription of the MAFA gene was detected in normal rat lungs, where both transmembranal and soluble MAFA appear to be expressed. Lung immunohistochemical analysis further suggests that MAFA expression is restricted to mast cells.
Delivery by Trypanosoma cruzi of proteins into the MHC class I antigen processing and presentation pathway.
Authors: Garg N , Nunes M P , Tarleton R L
Published: December 2022

Class I MHC-restricted T cell responses have been shown to be critical for the development of immune resistance to Trypanosoma cruzi in mice. However, to date, no antigenic targets of this anti-parasite response have been characterized. We have analyzed the characteristics of potential T. cruzi CTL target molecules by expression of the model CTL target molecule chicken OVA in different cellular compartments of T. cruzi. OVA (amino acids 139-385) was expressed as a secretory, cytoplasmic, transmembrane, or glycosylphosphatidylinositol-anchored protein in T. cruzi transfectants. Host cells infected with T. cruzi transfectants that secreted or released OVA, but not those producing cytoplasmic or transmembrane forms of OVA, could process and present OVA peptide via the class I MHC pathway, as indicated by the stimulation of OVA-specific CD8+ T cell hybridomas and the cytolysis of host cells infected with OVA-secreting parasites by OVA-specific CTLs. In addition, infection of mice with OVA-secreting parasites elicited the production of OVA-specific CTLs. These studies demonstrate the ability to target proteins to specific cellular compartments in T. cruzi using either trypanosomal or mammalian signal sequences. Furthermore, these results suggest that proteins secreted or released by T. cruzi in infected cells are a major source of peptides for MHC class I presentation and for the generation of parasite-specific CTL.
IFN-gamma is a prerequisite for optimal antigen processing of viral peptides in vivo.
Authors: Geginat G , Ruppert T , Hengel H , Holtappels R , Koszinowski U H
Published: December 2022

The immediate early protein pp89 of mouse CMV is processed into the nonapeptide YPHFMPTNL, which is presented to CD8+ T lymphocytes by the H-2 Ld molecule. The tissue distribution of this peptide was determined during the course of mouse CMV infection. In tissues, there was no general correlation between peptide processing and infectious virus productivity. Immunosuppression by sublethal irradiation resulted in enhanced MCMV replication but did not increase the peptide yield and drastically reduced the peptide to plaque-forming unit rate in infected organs. IFN-gamma administration restored efficient peptide processing in the immunocompromised host, and neutralization of IFN-gamma in the immunocompetent host decreased peptide processing. This suggests that the efficiency of peptide processing after CMV infection in vivo is governed by IFN-gamma rather than by the productivity of virus infection.
Immunodominant and subdominant CTL responses to Listeria monocytogenes infection.
Authors: Vijh S , Pamer E G
Published: December 2022

Protective immunity to infection by intracellular pathogens begins with expansion of Ag-specific, effector T lymphocytes and is followed by persistence of pathogen-specific memory T cells. Infection by Listeria monocytogenes, an intracellular bacterium, induces cytolytic T lymphocytes that mediate systemic sterilization and long term immunity. In cells infected with L. monocytogenes, H2-Kd class I molecules present three nonamer peptides, listeriolysin (LLO) 91-99, p60 217-225, and p60 449-457, to CTL. Herein we show that during the peak CTL response to L. monocytogenes infection, the ratio of T cells specific for LLO 91-99, p60 217-225, and p60 449-457 is approximately 20:10:1, respectively. While the number of Ag-specific T lymphocytes decreases in the weeks after infection, the proportion of T lymphocytes specific for the three epitopes is maintained. Repertoire analysis of a subset of L. monocytogenes-specific T cells, using alanine-substituted variants of p60 217-225, indicates that the range of T cell specificities is maintained by memory cells. These results indicate that the breadth and relative magnitude of T cell specificities initially elicited by an infection are transmitted to the memory compartment. Our results suggest that T lymphocytes with different gross and fine Ag specificities are equally likely to become memory T cells.
Critical residues on HLA-DRB1*0402 HV3 peptide for HLA-DQ8-restricted immunogenicity: implications for rheumatoid arthritis predisposition.
Authors: Zanelli E , Krco C J , David C S
Published: December 2022

Recently, we have proposed that the combination of HLA-DQ and -DR alleles is responsible for the association of the HLA class II region with rheumatoid arthritis (RA). According to this model, some HLA-DQ alleles, namely DQ4, DQ7, DQ8, and DQ9, predispose carriers to severe RA, but a self peptide of sequence KDILEDERAAVDTYC from the third hypervariable (HV3) region of some DRB1 alleles, including DRB1*0402, can protect from the disease if presented by DQ molecules. This model implies that DQ4, DQ7, DQ8, and DQ9 should be able to present a set of common peptides, despite polymorphisms in their Ag binding groove. In the present study, we have further analyzed the immunogenicity of the DRB1*0402 HV3 peptide in DQ8-transgenic mice. We found that the motif DERAA guarantees DQ8-restricted immunogenicity, and that R is the main anchor residue for binding of the DRB1*0402 peptide to DQ. Interestingly, the p1 pocket that probably controls binding of the R residue is identical in all four RA-associated DQ molecules. Our results imply that the association of RA with some DR subtypes can be explained by their linkage with DQ alleles displaying a binding site for similar "arthritogenic" peptides.
A kinase-negative mutation of DNA-PK(CS) in equine SCID results in defective coding and signal joint formation.
Authors: Shin E K , Perryman L E , Meek K
Published: December 2022

The equine SCID defect is more severe than its murine counterpart in that SCID foals are incapable of forming either coding or signal joints, whereas SCID mice manifest normal signal joint formation. To determine the basis of this difference and whether DNA-dependent kinase, catalytic subunit (DNA-PK(CS)), is involved in signal joint formation, equine DNA-PK(CS) transcripts were cloned and sequenced from normal and SCID cell lines. In the mutant allele, a frame-shift mutation truncates the protein N terminal of the domain with homology to the phosphatidylinositol 3-kinase family resulting in complete absence of full length DNA-PK(CS) and accounting for the kinase-negative phenotype of these cells; the mutation in SCID mice allows for some DNA-PK(CS) expression. The difference in DNA-PK(CS) expression in SCID mice and foals explains the more severe phenotype of equine SCID, and definition of DNA-PK(CS) as the defect in equine SCID demonstrates that DNA-PK(CS) is required for both coding and signal joint formation.
Class I processing-defective Burkitt's lymphoma cells are recognized efficiently by CD4+ EBV-specific CTLs.
Authors: Khanna R , Burrows S R , Thomson S A , Moss D J , Cresswell P , Poulsen L M , Cooper L
Published: December 2022

In the present study, we have isolated CD4+ CTLs that recognize an epitope from EBV nuclear Ag 2 in association with two different HLA-DQ Ags, DQA1*0501/DQB1*0201 (DQ2) or DQA1*0501/DQB1*0301 (DQ7). Both the HLA-DQ2 and HLA-DQ7 alleles displayed a similar efficiency in the endogenous and exogenous presentation of this epitope. Since earlier studies have shown that the EBV-associated malignancy, Burkitt's lymphoma (BL), escapes class I-restricted immune recognition by down-regulating the expression of peptide transporter genes, we have explored the possibility that these tumor cells can process class II-restricted CTL epitopes. The data presented in this study clearly demonstrate that BL cells were recognized efficiently by CD4+, MHC class II-restricted EBV-specific CTLs following infection with recombinant vaccinia encoding EBV nuclear Ag 2. Analysis of surface MHC class II expression on BL cells revealed high levels of HLA-DR and HLA-DQ molecules, and most of these molecules were negative for the invariant chain peptide, referred to as CLIP. Moreover, these tumor cells also showed normal levels of HLA-DMB gene expression, which has been shown previously to be an essential component of the class II processing pathway. The present finding of efficient processing function through the class II pathway in BL cells provides a novel mechanism for immune targeting of EBV-positive malignancies.
CD4+ T cell down-regulation in human intestinal mucosa: evidence for intestinal tolerance to luminal bacterial antigens.
Authors: Khoo U Y , Proctor I E , Macpherson A J
Published: December 2022

T cells from the intestinal mucosal proliferate poorly in vitro, and the contribution of Ag-specific recognition to this hyporesponsiveness is unclear, since the Ag repertoire of intestinal mucosal T cells is unknown. In this study, T cell proliferation in response to Ag-prepulsed autologous peripheral blood-derived APC was examined. Whereas T cells from peripheral blood proliferated to inner membrane and cytoplasmic Escherichia coli proteins, T cells from intestinal mucosa responded only to purified component Ags of these proteins and not to their combination. This suggests that the lack of proliferation in response to these Ags presented as a mixture is not due to the absence of E. coli-specific T cells in the mucosa, but, rather, to down-regulation after T cell recognition. Down-regulation was assayed by measuring the inhibition of autologous peripheral blood T cell proliferation in response to Ag-prepulsed APC. Coculture with leukocytes from intestinal mucosa and not from mesenteric lymph nodes, inhibited autologous peripheral blood T cell proliferation in response to E. coli proteins, but not to tetanus toxoid, PHA, or IL-2. Inhibition was independent of cell contact, provided APC were available to the mucosal cell population, and was reversible by neutralization of IL-10 or TGF-beta with mAb or depletion of mucosal CD4+ T cells. Taken together, the data suggest that mucosal T cell unresponsiveness to luminal Ags is mediated by production of inhibitory cytokines after specific Ag recognition by CD4+ T cells.
Adenosine deaminase binding to human CD26 is inhibited by HIV-1 envelope glycoprotein gp120 and viral particles.
Authors: Valenzuela A , Blanco J , Callebaut C , Jacotot E , Lluis C , Hovanessian A G , Franco R
Published: December 2022

CD26, known to be the adenosine deaminase (ADA)-binding protein, has been implicated in HIV infection. Several studies have revealed a correlation between depletion of CD4+/CD26+ T lymphocytes, increased serum levels of ADA, and the evolution of AIDS in infected individuals. We show that in human B and T cell lines, irrespective of CD4 expression, 125I-labeled ADA binding to CD26 is inhibited by recombinant soluble HIV-1 envelope glycoprotein gp120 and by HIV-1 infectious particles. Accordingly, an anti-CD4 mAb, which inhibits the binding of gp120 to CD4 and blocks viral infection, did not affect inhibition of 125I-labeled ADA binding to CD26 by HIV particles. On the other hand, mAbs directed against the V3 loop and the C-terminal region of gp120 abolished completely the inhibitory effect. Overlapping synthetic peptides covering the entire gp120 sequence were tested to map the region in gp120 responsible for ADA binding inhibition. Only peptides in the C3 region significantly inhibited the binding of ADA to CD26. These results provide indirect evidence for the interaction of gp120 with CD26 and indicate that a specific function of gp120 is the inhibition of ADA binding to CD26 in both CD4+ and CD4- cells. Because ADA deficiency leads to severe combined immunodefiency syndrome, it remains possible that HIV particle-mediated blockade of ADA-CD26 interaction may have significant consequences in the pathogenesis of AIDS.
Human cord blood kappa repertoire.
Authors: Feeney A J , Lugo G , Escuro G
Published: December 2022

To determine the Vkappa gene utilization in cord blood, we made libraries of Igkappa sequences from two cord blood cDNA samples. The rearranged sequences were amplified using random amplification of cDNA ends PCR, ensuring unbiased amplification of all Vkappa genes. Although the human kappa locus contains approximately 38 potentially functional V genes, we observed that approximately 75% of the 146 sequences from our two samples used only nine Vkappa genes. Using leader-specific primers, we also amplified VkappaI and VkappaIII rearrangements from genomic DNA from one of these individuals. Nonproductive rearrangements give an approximation of the relative frequency of gene rearrangement. Some of the genes that were overused in the cDNA libraries were also observed to rearrange frequently, but others did not show high rearrangement frequencies, suggesting that cellular selection caused their increase in the periphery. Surprisingly, we observed a high frequency of rearrangements using L9, which has been reported to be a defective Vkappa gene. Sequence analysis of the unrearranged gene revealed two new functional alleles of this gene. We observed that N nucleotides were present in 29% of the productive sequences from cord blood DNA and RNA. To determine the actual rate of N region addition, we analyzed V-J junctions of rearrangements of two nonfunctional V genes. Forty-six percent of those cord blood sequences contained N regions. In comparison, 57% of junctions of the rearranged nonfunctional gene from adult PBMC contained N regions. Finally, we observed that CDR3 length heterogeneity was more pronounced for VkappaIII genes than for all of the other Vkappa families.
Nitric oxide and TNF-alpha production by murine peritoneal macrophages activated with a novel 20-kDa protein isolated from Bacillus thuringiensis var. thuringiensis parasporal bodies.
Authors: Gomez-Flores R , Rodriguez-Padilla C , Mehta R T , Galan-Wong L , Mendoza-Gamboa E , Tamez-Guerra R
Published: December 2022

The effects of a novel 20-kDa protein isolated from Bacillus thuringiensis var. thuringiensis (BTp20) parasporal bodies on macrophage activation were investigated and compared with the activity of LPS. Murine resident or IFN-gamma-primed peritoneal macrophages (50 U/ml of IFN-gamma for 16 h) were treated with 50 microg/ml of BTp20, alone or in combination with LPS (1 to 100 ng/ml). BTp20 was not toxic for macrophages as determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction assay, however, 16% reduction of viability was observed when macrophages were treated with a combination of IFN-gamma, BTp20, and LPS at 100 ng/ml. BTp20 was able to stimulate significant cellular adherence and spreading, and significant production of nitrite and TNF-alpha by resident macrophages after 1.5 h of treatment; nitrite release, however, was induced with only 10 min of macrophage exposure to BTp20. BTp20 activities were significantly potentiated by IFN-gamma pretreatment. It was also observed that nitrite release by IFN-gamma-primed macrophages activated with LPS (1-100 ng/ml) was 20 times lower than that induced by IFN-gamma + BTp20. BTp20 and LPS independently stimulated significant TNF-alpha production by IFN-gamma-primed macrophages, the effect of BTp20 + LPS (1 and 10 ng/ml) combination was additive. In summary, this study demonstrated that BTp20 activates macrophage functions in the absence of IFN-gamma or LPS, and that IFN-gamma enhances BTp20 activities.
Immunoregulatory idiotypes stimulate T helper 1 cytokine responses in experimental Schistosoma mansoni infections.
Authors: Montesano M A , Freeman G L , Secor W E , Colley D G
Published: December 2022

Inbred male CBA/J mice with chronic Schistosoma mansoni infections develop two distinct syndromes. Hypersplenomegaly syndrome (HSS) demonstrates pathologic similarities to the hepatosplenic form of chronic human schistosomiasis, and moderate splenomegaly syndrome (MSS) resembles the asymptomatic intestinal form. Immunoaffinity-purified Abs against S. mansoni soluble egg Ags (SEA) from infected patients' sera differ idiotypically according to the donor's clinical form of the disease. We now show that immunoaffinity-purified anti-SEA Abs (Id) from MSS or HSS mice parallel idiotypic preparations of the analogous human clinical form by their differential ability to stimulate the proliferation of anti-Id T cells. MSS Id preparations stimulate spleen cells from either MSS or HSS animals. In contrast, HSS Id does not stimulate spleen cells from either group. In an anti-SEA ELISA, MSS and HSS Id preparations contained comparable levels of IgM and IgG1. However, the MSS Id preparation had higher levels of SEA-specific IgG2a and IgG2b than did HSS Id. The Ig isotypes of these Id preparations suggested differences in cytokine expression patterns. Studies of the cytokine profiles of the spleen cells responding to anti-SEA Id preparations demonstrated that while Id preparations from acutely infected mice stimulate IL-4 and IL-10 production, Id preparations from chronic MSS mice stimulate IFN-gamma production. HSS Id did not stimulate the production of any of these cytokines. The possibility that distinct immunoregulatory environments may contribute to the development of MSS and HSS correlates with earlier hypotheses that hepatosplenic pathology results at least in part from a lack of development or expression of appropriate regulatory Ids.
Destruction of follicular dendritic cells during chronic visceral leishmaniasis.
Authors: Smelt S C , Engwerda C R , McCrossen M , Kaye P M
Published: December 2022

Follicular dendritic cells (FDCs) play a pivotal role in the germinal center (GC) response and in the development and regulation of B lymphocytes. Pathologic changes in GCs and a loss of FDCs have previously been noted in various viral infections, notably HIV-1. However, such changes have not been formally described in a chronic parasitic infection. In BALB/c mice infected with Leishmania donovani, parasites persist in the spleen for long periods, with associated splenomegaly. To examine the fate of FDC during the course of this chronic infection, we used 1) immunohistology, with FDC-specific mAbs; and 2) passive immunization with immune complexes, followed by light and electron microscopy. This study demonstrates that destruction of FDCs and a concomitant loss of GCs are associated with chronic visceral leishmaniasis. These pathologic effects are notable from 4 wk postinfection. At 8 wk postinfection and beyond, FDC are almost undetectable by both immunohistology and functional immune complex trapping. The loss of FDCs is associated with the infiltration of heavily parasitized macrophages into the GC, and reduction in parasite burden by chemotherapy is able to retard the process of FDC destruction. These data directly demonstrate for the first time the loss of FDCs during a chronic parasite infection and suggest a mechanism underlying the aberrant regulation of B cell function in murine visceral leishmaniasis.
TNF-alpha, unlike other pro- and anti-inflammatory cytokines, induces rapid release of the IL-1 type II decoy receptor in human myelomonocytic cells.
Authors: Orlando S , Matteucci C , Fadlon E J , Buurman W A , Bardella M T , Colotta F , Introna M , Mantovani A
Published: December 2022

The present study was designed to investigate the effect of a series of cytokines on the release of the type II IL-1 decoy receptor, which represents a unique pathway of negative regulation of the IL-1 system. After 20 min, IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IFN-gamma, granulocyte-CSF, macrophage-CSF, and TGF-beta had little or no effect on IL-1 binding by human polymorphonuclear cells. In contrast granulocyte-macrophage-CSF and, to a greater extent, TNF markedly reduced IL-1 binding. The action of TNF was rapid, reaching 50% of its maximum (80%) at 2 min, and plateauing at 20 min with decrease in receptor number and no significant change in affinity. Loss of surface receptor was associated to rapid release of a 45-kDa IL-1-binding molecule identified as the decoy RII. TNF-induced release of the decoy RII was independent of protein synthesis and reactive oxygen intermediates. Monocytes showed a similar response to TNF, except for the size of the released molecule (approximately 60 kDa). TNF induced rapid release of its own receptors. In contrast IL-1beta affected neither its own receptors nor the TNF-R. TNF and, more efficiently, PMA caused release of the decoy RII in fibroblasts transfected with the full-length decoy RII or with a cytoplasmatic deletion mutant. TNF-induced decoy RII release represents an unidirectional pathway of communication in the interplay between the IL-1 and TNF system.