bluuebunny/crossref_metadata_2025 huggingface dataset:
H-2D haplotype-linked expression and involvement of TNF-alpha in Th2 cell-mediated tissue inflammation.
Authors: Müller K M , Lisby S , Arrighi J F , Grau G E , Saurat J H , Hauser C
Published: December 2022

We recently reported that polyclonal anti-CD3 epsilon-pulsed Th2 cells mediate local tissue inflammation (DTH2) when injected into naive syngenic recipient mice, and that this response is entirely dependent on IL-4 in BALB/c (H-2d) mice. We now describe a different cytokine dependence in mice that bear a H-2b MHC haplotype. Injection of either soluble IL-4R (sIL-4R) or anti-TNF Ab partially inhibited swelling that was mediated by Th2 cells from high TNF-producing C57BL/6 mice. Anti-TNF and sIL-4R in combination were required to completely abrogate the swelling reaction and cellular infiltrate. Adoptive transfers across strain barriers showed that the TNF dependence was dictated by the origin of the transferred cells, rather than by the recipient. Experiments with intra-H-2 recombinant C57BL/10 strains indicated that TNF released by Th2 cells was correlated with the involvement of TNF in DTH2: Th2 cells from the H-2Db strains C57BL/10 and B10.A(2R) produced high amounts of bioactive TNF and mediated swelling that was partially inhibited by anti-TNF. In contrast, Th2 cells from B10.D2 and B10.A mice (H-2Dd) produced low levels of TNF, and anti-TNF had no effect on DTH2 in these strains. Our results suggest a linkage between the TNF dependence of DTH2, the capacity of Th2 cells to release TNF upon restimulation, and the donor H-2D haplotype; strain-dependent allelic expression of TNF seems to determine the involvement of this cytokine in DTH2.
Superinduction of cytokine gene transcription by ciprofloxacin.
Authors: Riesbeck K , Sigvardsson M , Leanderson T , Forsgren A
Published: December 2022

In addition to their antibacterial properties, certain antibiotics are also biologic response modifiers. The fluorinated 4-quinolone, ciprofloxacin, for example, up-regulates IL-2 and IFN-gamma production in PBLs stimulated in vitro. In the present study, ciprofloxacin was shown to increase the levels of mRNA for IL-1 alpha, IL-2 and its receptor, IFN-gamma, IL-3, IL-4, granulocyte-macrophage/CSF, TNF-alpha, and lymphotoxin. As investigated with different T cell mitogens and alloantigens, the up-regulation of IL-2 production by ciprofloxacin was found to be independent of the mode of stimulation. Analysis of transcription activity showed that ciprofloxacin enhances IL-2 gene induction. The concentrations of nuclear factor of activated T cells (NF-AT-1) and AP-1 were also found to be increased by ciprofloxacin. Thus, ciprofloxacin interferes with a regulative pathway common to several cytokines.
Analysis of V beta 8-CDR3 sequences derived from central nervous system of Lewis rats with experimental autoimmune encephalomyelitis.
Authors: Buenafe A C , Vainiene M , Celnik B , Vandenbark A A , Offner H
Published: December 2022

We have recently demonstrated that a strong bias for expression of V beta 8.2 is manifested early during the onset of experimental autoimmune encephalomyelitis (EAE) induced by guinea pig basic protein (Gp-BP) immunization of Lewis rats. More importantly, the V beta 8.2 bias was observed in T cells infiltrating the spinal cord (SC) and in cerebrospinal fluid (CSF), but was not present in T cells isolated from the periphery. Here, we report the V beta 8-CDR3 sequences found in unselected SC, CSF, and lymph node (LN) T cell populations at onset of Gp-BP-induced EAE. Striking similarities were observed among sequences derived from SC and CSF. Evidence for oligoclonal expansion of V beta 8.2 sequences associated with previously characterized encephalitogenic clones was observed in both SC and CSF, but not in LN. An AspSer CDR3 motif identified in encephalitogenic clones recognizing the dominant 72-89 epitope of Gp-BP was found in 9/22 SC cDNA clones, 11/24 CSF cDNA clones, and 1/16 LN cDNA clones. Interestingly, J beta 2.7 and J beta 1.3 were also highly represented in SC and CSF, but not in LN. Given that these sequences were derived from T cells present at the site of autoimmune attack and not selected by in vitro manipulation, the data offer compelling evidence that 1) selective recruitment and/or expansion of V beta 8.2+ T cells are occurring in the central nervous system; 2) these events are at least partially dependent on V beta residues which are likely to influence Ag binding; and 3) CSF-derived T cells provide a representative view of CNS events at the onset of EAE.
Soluble CTLA-4 can suppress autoantibody production and elicit long term unresponsiveness in a novel transgenic model.
Authors: Milich D R , Linsley P S , Hughes J L , Jones J E
Published: December 2022

Activation of Ag-specific T cells often requires costimulatory signals in addition to the primary signal mediated through the TCR. The CD28-B7 interaction provides one important costimulatory signal. Previous studies have shown that a soluble CD28 homologue, CTLA4lg, binds B7 with high affinity and can inhibit CD28-B7-mediated costimulation in vitro and in vivo. In this study we examined the ability of soluble human CTLA4lg to inhibit autoantibody production in vivo. For this purpose we used a novel transgenic (Tg) model of autoantibody production. Hepatitis B eAg-expressing Tg mice can be induced to produce autoantibody to the circulating autoantigen (HBeAg) by the injection of a T cell recognition site that fails to elicit T cell tolerance in these mice. Autoimmunity in this model is quantitative because serum autoantibody and autoantigen concentration are inversely correlated and easily measurable by ELISA. In this system a single regimen of CTLA4lg treatment significantly suppressed primary autoantibody production and variably led to long term unresponsiveness. Furthermore, in vivo treatment with CTLA4lg inhibited both T cell activation and T cell-B cell interactions.
CTL responses to the gag polyprotein encoded by the murine AIDS defective retrovirus are strain dependent.
Authors: Schwarz D A , Green W R
Published: December 2022

Murine AIDS (MAIDS) is induced by a mixture of retroviruses, of which a replication defective virus is the proximal agent of disease. This defective virus harbors a single intact gene that encodes an aberrant gag polyprotein. Certain mouse strains are genetically resistant to MAIDS, with several genes, including H-2Dd, contributing to this resistance. Because MHC class I gene products present intracellular Ags to CTL, recombinant viruses were used to determine whether gag-specific CTLs mediate the genetic linkage between H-2Dd and resistance. Interestingly, while genetically resistant BALB/cByJ and C57BL/KsJ mice (H-2d) generated gag-specific CD8+ CTLs, a similar response was not detected in susceptible BALB.B and C57BL/6J mice (H-2b). However, this CTL response does not appear to be responsible for genetic resistance because: 1) a vigorous CTL response could also be generated by susceptible (C57BL/6 x BALB/cBy) F1 mice and 2) the relevant epitope is H-2Kd restricted.
Cross-linking CD40 on B cells rapidly activates nuclear factor-kappa B.
Authors: Berberich I , Shu G L , Clark E A
Published: December 2022

The B cell-associated surface molecule CD40 functions to regulate B cell responses. Cross-linking CD40 on B cells can lead to homotypic cell adhesion, IL-6 production, and, in combination with cytokines, to Ig isotype switching. Tyrosine kinase activity is increased shortly after engagement of this receptor. Little is known about how the very early events induced by CD40 cross-linking link to cellular responses. In this study, we demonstrate that nuclear factor (NF)-kappa B and NF-kappa B-like transcription factors are activated after cross-linking CD40 on resting human tonsillar B cells and on B cell lines. The activation is rapid and is mediated through a tyrosine kinase-dependent pathway. The complexes detected in electrophoretic mobility shift assays contain p50, p65 (RelA), c-Rel, and most likely other components. By using transient transfection assays, we found that cross-linking CD40 supports NF-kappa B-dependent gene expression. Our results define the NF-kappa B system as an intermediate event in CD40 signaling and suggest that the CD40 pathway can influence the expression of B cell-associated genes with NF-kappa B consensus sites.
CD44 triggering enhances human NK cell cytotoxic functions.
Authors: Galandrini R , De Maria R , Piccoli M , Frati L , Santoni A
Published: December 2022

CD44, a major hyaluronate receptor, is involved in a variety of lymphocyte functions including lympho-hemopoiesis, adhesion to high endothelial venules or the extracellular matrix, and T cell activation. Here we investigated the ability of CD44 to affect the cytotoxic functions of human NK cells. Ligation of CD44 by selected mAb (J173 and F10442) resulted in a rapid, dose-response-dependent enhancement of NK cytotoxic activity against a panel of tumor target cells that varied in their sensitivity to NK killing. Neither enhanced killing against NK-resistant target cells nor CD44 mAb-mediated redirected lysis was not observed. CD44 cross-linking also was found to up-regulate CD16-mediated lysis. In an attempt to investigate the early biochemical events that occur after CD44 ligation, we found that optimal cross-linking conditions induce a rapid increase of intracellular free calcium levels, which is abrogated by extracellular Ca2+ chelation. Moreover, enhanced and more sustained Ca2+ rise resulted from CD16 and CD44 coengagement. In contrast, no inositol 1,4,5-trisphosphate generation was found after optimal CD44 cross-linking. These results suggest that although CD44 is not capable of delivering a lytic signal in human NK cells, it coactivates spontaneous or CD16-mediated NK cytotoxicity. The variation in intracellular free calcium may be one of the signals that account for the costimulation of the lytic activity.
Induction by concanavalin A of specific mRNAs and cytolytic function in a CD8-positive T cell hybridoma.
Authors: Gu J J , Harriss J V , Ozato K , Gottlieb P D
Published: December 2022

A previous report from this laboratory described the production of CD8+, class I-specific T cell hybridomas which developed specific cytolytic activity and the ability to secrete IL-2 upon Con A or specific Ag stimulation. Unlike normal lymphocytes or long-term CTL lines for which exposure to Ag triggers both differentiation and proliferation, T cell hybridoma lines can be activated functionally against a background of continuous proliferation. They therefore provide a unique system with which to study the molecular events involved in the induction of cytolytic function. The expression of mRNA from a series of genes was evaluated by Northern hybridization at various times after Con A stimulation of the H-2Ld-specific CD8+ 3D9 hybridoma. Induction of the c-fos proto-oncogene by 45 min poststimulation was followed shortly by c-myc induction. Perforin mRNA was expressed at a low level in the unstimulated hybridomas, but was down-regulated upon Con A stimulation to levels undetectable by PCR. Interestingly, production of granzyme A mRNA was strongly induced by 45 min after Con A stimulation. In the CD8+ RT-1.3G3 hybridoma, which is nonlytic and specific for the HIV-1 envelope glycoprotein, c-fos but not granzyme A mRNA was induced by 45 min poststimulation, and no granzyme A mRNA was detectable at any time. Thus, a significant role for granzyme A in the induction of cytolytic activity is suggested. Cytolysis by the 3D9 hybridoma involved both target cell membrane damage and DNA fragmentation, and both Ca(2+)-dependent and Ca(2+)-independent cytolysis were observed. Although TNF-alpha mRNA was induced by 4 h poststimulation, Ab to TNF-alpha failed to inhibit the Ca(2+)-independent lysis observed, leaving the basis for the observed Ca(2+)-independent lysis unexplained.
Diversity and dominance among TCR recognizing HLA-A2.1+ influenza matrix peptide in human MHC class I transgenic mice.
Authors: Man S , Ridge J P , Engelhard V H
Published: December 2022

The TCR structures of CTL derived from HLA-A2.1 transgenic mice were analyzed to determine features important in the interaction of murine TCR with the HLA-A2.1 + influenza M1(57-68) peptide complex. V beta 8.1 was dominant in 9 of 11 murine CTL lines, although three other V beta segments were also represented. Sequencing of TCR cDNA from a group of six independently derived CTLs that were V beta 8.1-positive demonstrated a restricted set of D-N-J beta sequences and an apparently restricted set of alpha-chains. However, at least five other distinct alpha beta pairs were found among HLA-A2.1 + M1 peptide-specific CTL in the absence of these chains. Consideration of all TCR sequences obtained demonstrated diverse beta-chain CDR3 regions with some restriction in V alpha segment usage and bias in amino acid sequence of alpha-chain CDR3 regions. Nevertheless, the strongest correlation with HLA-A2.1 + M1 specificity was clearly V beta 8.1 usage. Comparison with previously identified human TCR sequences specific for the same Ag-MHC complex revealed that the dominant murine V alpha and V beta segments used were not the homologues of the dominant human V beta and V alpha segments used. These results together with the lack of interspecies conservation in the alpha- and beta-chain CDR3 regions demonstrate that the dominant TCR structures recognizing HLA-A2.1 + M1(57-68) are substantially different between mouse and humans. Different factors may influence Ag-driven selection of the dominant TCRs used in each species.
Nerve growth factor production by lymphocytes.
Authors: Santambrogio L , Benedetti M , Chao M V , Muzaffar R , Kulig K , Gabellini N , Hochwald G
Published: December 2022

Nerve growth factor (NGF) is a neurotrophic protein essential for the maintenance and growth of peripheral sympathetic neurons and basal forebrain cholinergic neurons. Recently, NGF has also been shown to have effects on cells of the immune system. In a search for extra neural sources of NGF, we detected NGF-specific mRNA in mouse T lymphocytes of both the CD4+ and CD8+ phenotypes with the use of an RNase protection assay, PCR, and DNA sequence analysis. In CD4+ cells, NGF was present in both Th1 and Th2 Ag-specific clones, but an increase of NGF-specific message was detected after antigenic stimulation only in Th2 clones. NGF mRNA was also detected in splenic B lymphocytes and in a cell line derived from a murine follicular center cell lymphoma. Translation into protein and secretion of NGF were demonstrated by Western blot analysis. The secreted NGF is in an active form capable of inducing differentiation of PC12 cells into sympathetic-like neurons. Furthermore, conditioned medium from clones or lines positive for NGF mRNA was capable of inducing p140 tyrosine kinase autophosphorylation in 3T3 fibroblasts transfected with cDNA encoding for the tyrosine kinase family NGF receptor. We conclude that lymphocytes synthesize and secrete NGF either as a para-autocrine factor acting on the immune system itself, or as a factor for the maintenance of peripheral neurons.
Essential residues in V(D)J recombination signals.
Authors: Akamatsu Y , Tsurushita N , Nagawa F , Matsuoka M , Okazaki K , Imai M , Sakano H
Published: December 2022

Recombination signal sequences for V(D)J joining consist of a conserved heptamer (CACAGTG) and a nonamer (ACAAAAACC) separated by a spacer of a constant length (12 bp or 23 bp). In the present study, we have analyzed various recombination signal mutations for their effects in V(D)J joining. Using a retroviral vector, we introduced mutant substrates stably into pre-B cells, and assayed recombination using the lacZ gene as a reporter. This method allowed us to study recombination in a single copy within the context of the host cell chromosome. Because this assay did not show any detectable background, it was quite useful in the analysis of low level recombinations. In the heptamer, mutations in the first three residues severely dropped the joining rates. Among them, the first residue adjacent to the recombination site was found to be most essential. Although mutations in the heptamer reduced the joining rate to various extents, they did not lower the site-specificity of recombination. With regard to the nonamer, the presence of three consecutive A residues was necessary for efficient recombination. Furthermore, the nucleotides flanking the A-rich core needed to be other than A residues, probably marking the border of the A-stretch. This may be important when the recombinase measures the distance between the heptamer and the nonamer to satisfy the 12/23-bp spacer rule.
Evidence for a specific post-transcriptional mechanism controlling the expression of HLA-DQ, but not -DR and -DP, molecules.
Authors: De Lerma Barbaro A , Sartoris S , Tosi G , Nicolis M , Accolla R S
Published: December 2022

It is generally believed that the various MHC class II molecules are expressed coordinately in B cells. To investigate this aspect in more detail, interspecies somatic cell hybrids were constructed between Raji or RJ 2.2.5 (a class II-negative derivative of Raji) human B cells and M12.4.1 mouse B cells. In both types of hybrids, HLA-DR and -DP, but not -DQ, molecules were expressed at the cell surface. The specific lack of expression of DQ Ags correlated with undetectability of newly synthesized DQ alpha beta heterodimers, as assessed by biosynthetic labeling and immunoprecipitation with a variety of DQ-specific mAbs. Studies at the mRNA level showed that apparently normal DQ alpha and DQ beta transcripts were present in the hybrids at levels comparable, if not higher, with the levels of DR- and DP-specific transcripts. From these results, we conclude that lack of appreciable amount of DQ molecules in the hybrids is caused by a post-transcriptional block. To date, these findings represent a rather unique example of noncoordinate expression of MHC class II Ags caused by distinct post-transcriptional mechanisms. These data may be relevant to a more correct interpretation of the functional role of the various MHC class II molecules, particularly with regard to the well-known association of HLA-DQ with many autoimmune diseases. Possible mechanisms at the basis of the distinct control of expression within the MHC class II molecular pool are discussed.
Evolution of T cell receptor genes. Extensive diversity of V beta families in the Mexican axolotl.
Authors: Fellah J S , Kerfourn F , Charlemagne J
Published: December 2022

We have cloned 36 different rearranged variable regions (V beta) genes encoding the beta-chain of the T cell receptor in an amphibian species, Ambystoma mexicanum (the Mexican axolotl). Eleven different V beta segments were identified, which can be classified into 9 families on the basis of a minimum of 75% nucleotide identity. All the cloned V beta segments have the canonical features of known mammalian and avian V beta, including conserved residues Cys23, Trp34, Arg69, Tyr90, and Cys92. There seems to be a greater genetic distance between the axolotl V beta families than between the different V beta families of any mammalian species examined to date: most of the axolotl V beta s have fewer than 35% identical nucleotides and the less related families (V beta 4 and V beta 8) have no more than 23.2% identity (13.5% at the amino acid level). Despite their great mutual divergence, several axolotl V beta are sequence-related to some mammalian V beta genes, like the human V beta 13 and V beta 20 segments and their murine V beta 8 and V beta 14 homologues. However, the axolotl V beta 8 and V beta 9 families are not significantly related to any other V beta sequence at the nucleotide level and show limited amino acid similarity to mammalian V alpha, V kappa III, or VH sequences. The detection of nine V beta families among 35 randomly cloned V beta segments suggests that the V beta gene repertoire in the axolotl is probably larger than presently estimated.
Isolation of the Xenopus complement factor B complementary DNA and linkage of the gene to the frog MHC.
Authors: Kato Y , Salter-Cid L , Flajnik M F , Kasahara M , Namikawa C , Sasaki M , Nonaka M
Published: December 2022

C factor B (Bf) is the key component of the C3 convertase of the alternative C pathway, and its gene resides in the class III region of the mammalian MHC. To elucidate the evolution of both the C system and the MHC, we isolated Bf cDNA clones from Xenopus laevis, an ectothermic vertebrate in which the MHC has been well defined at both the biochemical and functional levels. A part of the serine protease domain of the Xenopus Bf mRNA was amplified by reverse transcriptase-PCR, using degenerate primers corresponding to regions encoding the perfectly conserved amino acid sequences found in both the mouse Bf and C2 proteins. A full length Xenopus Bf cDNA clone was isolated from a Xenopus liver cDNA library. The deduced amino acid sequence of 747 residues showed the same domain structure as mammalian Bf and C2: three short consensus repeat domains, a von Willebrand domain and a serine protease domain. Xenopus Bf has 40% and 30% overall amino acid identity to mouse Bf and mouse C2, respectively. Because the amino acid identity between mouse Bf and mouse C2 is 38%, the gene duplication of Bf/C2 probably occurred before the divergence of amphibians and mammals. Southern blotting analysis of the Xenopus Bf gene showed a close linkage to the MHC, indicating that the Bf gene was linked to the class I and class II genes at the time Xenopus shared a common ancestor with mouse and man, 350 x 10(6) yr ago.
Characterization of astrocyte nuclear proteins involved in IFN-gamma- and TNF-alpha-mediated class II MHC gene expression.
Authors: Panek R B , Lee Y J , Itoh-Lindstrom Y , Ting J P , Benveniste E N
Published: December 2022

IFN-gamma is a potent inducer of class II MHC Ags on different cell types, including the astrocyte. TNF-alpha alone has no effect on class II MHC expression, but enhances IFN-gamma-induced class II expression. IFN-gamma acts by inducing transcription of the class II gene, and TNF-alpha enhances the rate of IFN-gamma-induced transcription. We have previously described two factors, IFN-gamma-enhanced factor X (IFNEX) and TNF-alpha induced complex X (TIC-X), whose expression is induced by IFN-gamma and IFN-gamma/TNF-alpha, respectively, which interacted with the X box of the DRA promoter. In this study, we show that IFNEX and TIC-X bind to the X2 core, with contacts extending into the 3' end of X1 and into the spacer region of the DRA promoter. We also show a functional correlation between binding activity and transcriptional activity of the DRA promoter. These results strongly suggest that both IFNEX and TIC-X play important roles in the regulation of class II MHC gene expression in the astrocyte.
E1A repression of IL-6-induced gene activation by blocking the assembly of IL-6 response element binding complexes.
Authors: Takeda T , Nakajima K , Kojima H , Hirano T
Published: December 2022

Some viral products interfere with host antiviral defense mechanisms. Adenovirus E1A represses IFN signal transduction pathways which induces gene activation and an antiviral state. Both IFN and IL-6 activate Jak/Tyk protein tyrosine kinases and the STAT (signal transducer and activator of transcription) family proteins. We showed that 12S E1A repressed IL-6 signals activating the junB promoter and the two IL-6 response elements (REs), JRE-IL6 and type II IL-6 RE (also called acute phase response element), required for IL-6-induced activation of the junB promoter and the type II acute phase reactant genes, respectively, in hepatocytes. Conserved region 1 of the 12S E1A was responsible for the repression. Target molecules of the repression by E1A appeared to be IL-6-inducible DNA-binding proteins acting on the IL-6 REs. In a rat 3Y1 cell line stably expressing E1A, the levels of IL-6-induced IL-6 RE binding proteins were severely reduced compared with those in a parental 3Y1 cell line. Moreover, we found that the levels of the STAT family proteins including Stat1-alpha (p91), Stat1-beta (p84), Stat2 (p113), and Stat3 were decreased by the stable expression of adenovirus E1A. The E1A-induced reduction in the amount of DNA-binding proteins seemed to be partly responsible for the decreased transcriptional activity of the IL-6 RE-driven gene expression in response to IL-6. This repression mechanism may be applicable to the E1A repression of IFN-gamma-induced gene activation.
Influenza and vaccinia viruses expressing malaria CD8+ T and B cell epitopes. Comparison of their immunogenicity and capacity to induce protective immunity.
Authors: Rodrigues M , Li S , Murata K , Rodriguez D , Rodriguez J R , Bacik I , Bennink J R , Yewdell J W , Garcia-Sastre A , Nussenzweig R S
Published: December 2022

We compared the effectiveness of several recombinant influenza and vaccinia viruses to induce a malaria-specific immune response. The CD8+ T cell epitope of the circumsporozoite (CS) protein of Plasmodium yoelii, a rodent malaria parasite, was expressed in two distinct influenza virus proteins, the hemagglutinin and the neuraminidase. These recombinant viruses were found to be equally efficient at inducing CS-specific CD8+ T cells in mice. A third recombinant virus, which expresses a B cell epitope of the CS protein, induced neutralizing anti-sporozoite Abs. Expression in the same recombinant virus of the CD8+ T cell epitope and of the B cell epitope did not impair the capacity of this recombinant virus to induce malaria-specific CD8+ T cells and neutralizing Abs. The immunogenicity of a vaccinia virus, expressing the entire CS protein, was compared with that of a highly attenuated vaccinia strain expressing the same protein and with that of another vaccinia virus expressing only the CD8+ T cell epitope. All three vaccinia virus recombinants elicited CS-specific CD8+ cells and a potent inhibitory response against pre-erythrocytic stages of malaria parasites. Optimal levels of anti-sporozoite Abs, inhibition of liver stage development, and protection against malaria infection resulted from repeatedly immunizing the animals with recombinant influenza viruses followed by boosters with a recombinant vaccinia virus. These findings support the concept that live viral vectors expressing the appropriate proteins and/or epitopes can be used as promising vaccine candidates.
MHC class I gene(s) in the D/L region but not the TNF-alpha gene determines development of toxoplasmic encephalitis in mice.
Authors: Suzuki Y , Joh K , Kwon O C , Yang Q , Conley F K , Remington J S
Published: December 2022

Previous studies revealed that mice with the b or k allele at the H-2D region are susceptible to toxoplasmic encephalitis (TE); those with the d allele are resistant. To determine whether the b or d allele is dominant, F1 hybrids between susceptible C57BL/6 (H-2b) and resistant BALB/c (H-2d) mice were infected with T. gondii. TE was not observed in the F1 hybrids, indicating that the d allele is dominant for protection against development of TE. Mice with a mutation in the D/L region were used to determine whether the D gene or the L gene of MHC class I Ags of the H-2D region is most critical for resistance against development of TE. B10.D2-H2dm1 (dm1) mice that have the mutant D/L hybrid gene formed by fusion of the 5' part of the Dd gene and the 3' part of the Ld gene developed TE in contrast to their background B10.D2 mice. BALB/c-H-2dm2 (dm2) mice, which have a complete deletion of the Ld gene, had significantly more T. gondii cysts in their brains than did dm1 mice and developed large areas of necrosis in their brains that were not observed in dm1 mice. These results indicate that a gene(s) in the D/L region determines whether TE will occur and that the Ld gene plays a critical role in the resistance against development of TE. Polymorphisms in the TNF-alpha gene (located in the H-2D region) have been reported to correlate with resistance against the development of TE. When development of TE was studied in BALB/c and dm2 mice that have the same TNF-alpha gene, only dm2 mice developed TE. This indicates that the TNF-alpha gene is not a determining factor for the development of TE. Transcripts for TNF-alpha were detected in brains of infected dm2 mice but not in BALB/c mice. Injection of neutralizing Abs against TNF-alpha resulted in worsening of the TE in infected dm2 mice but did not induce TE in infected BALB/c mice. Thus, TNF-alpha appears to be produced in the brain after TE has developed and is responsible for preventing the progression of TE.
Epitope mapping of the branched chain alpha-ketoacid dehydrogenase dihydrolipoyl transacylase (BCKD-E2) protein that reacts with sera from patients with idiopathic dilated cardiomyopathy.
Authors: Ansari A A , Neckelmann N , Villinger F , Leung P , Danner D J , Brar S S , Zhao S , Gravanis M B , Mayne A , Gershwin M E
Published: December 2022

Sera from 29 of 48 patients with idiopathic dilated cardiomyopathy (IDCM) and six of six patients with dilated cardiomyopathy (DCM) secondary to suspected viral myocarditis were shown to react with the branched chain alpha-ketoacid dehydrogenase (BCKD) complex mitochondrial proteins. Whereas sera from only 1 of 26 patients with ischemic heart disease showed reactivity against the BCKD complex protein, 0 of 30 sera from normal human volunteers, 0 of 64 sera from patients with lupus, and 0 of 34 sera from patients with rheumatoid arthritis showed detectable reactivity, denoting an element of specificity for the reactivity of sera from IDCM patients. The major reactivity was localized to the dihydrolipoyl transacylase (E2) component of BCKD complex. By using recombinant techniques, the immunodominant BCKD-E2 epitope recognized by sera from IDCM patients was localized to amino acid (aa) sequences 116 to 134. Each of the IDCM sera that reacted with the native BCKD complex was shown to react with the immunodominant peptide, as defined by a peptide inhibition ELISA and by an ELISA using the reactive peptide conjugated to BSA. Sera from IDCM patients that reacted with the native BCKD complex and the reactive peptide also showed inhibition of BCKD enzyme activity. The possible mechanisms for the induction of the Abs and the implications of these findings for the pathogenesis of IDCM are discussed.
Idiotype-cytokine fusion proteins as cancer vaccines. Relative efficacy of IL-2, IL-4, and granulocyte-macrophage colony-stimulating factor.
Authors: Chen T T , Tao M H , Levy R
Published: December 2022

Idiotypic determinants, antigenic sites expressed on the variable region of Ig molecules of malignant B cells, represent tumor-specific Ags but are weak immunogens. We have previously shown that the immunogenicity can be dramatically increased by fusing tumor Id to granulocyte macrophage (GM)-CSF. Here, we demonstrate that fusion proteins with IL-2 or IL-4 can also be highly immunogenic. Co-immunization of these fusion proteins with another Id demonstrated the importance of physical linkage between the cytokine and relevant Ag for this enhancement. All three fusion proteins are capable of eliciting significant levels of specific Abs against the Id without the use of carrier proteins or adjuvants, although the GM-CSF fusion protein appeared to be unique in its ability to induce higher titers of anti-Id Abs in the primary response. Furthermore, the Id-IL-2 fusion protein induced high titers of IgG2a and IgG3 anti-Id Abs, whereas the Id-IL-4 and Id-GM-CSF fusion proteins did not. Despite the differences, tumor protection was comparable in all mice having significant titers of anti-Id Abs, regardless of the fusion protein used. We concluded that Id-cytokine fusion proteins are potent immunogens that can elicit significant antitumor immunity. The general approach of fusing a cytokine to a potential Ag may be applicable to the design of vaccines for immunotherapy of other types of tumors as well as for other pathogens and disease states.
The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes.
Authors: Sette A , Vitiello A , Reherman B , Fowler P , Nayersina R , Kast W M , Melief C J , Oseroff C , Yuan L , Ruppert J , Sidney J , del Guercio M F , Southwood S , Kubo R T , Chesnut R W , Grey H M , Chisari F V
Published: December 2022

The relationship between binding affinity for HLA class I molecules and immunogenicity of discrete peptide epitopes has been analyzed in two different experimental approaches. In the first approach, the immunogenicity of potential epitopes ranging in MHC binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice. In the second approach, the antigenicity of approximately 100 different hepatitis B virus (HBV)-derived potential epitopes, all carrying A*0201 binding motifs, was assessed by using PBL of acute hepatitis patients. In both cases, it was found that an affinity threshold of approximately 500 nM (preferably 50 nM or less) apparently determines the capacity of a peptide epitope to elicit a CTL response. These data correlate well with class I binding affinity measurements of either naturally processed peptides or previously described T cell epitopes. Taken together, these data have important implications for the selection of epitopes for peptide-based vaccines, and also formally demonstrate the crucial role of determinant selection in the shaping of T cell responses. Because in most (but not all) cases, high affinity peptides seem to be immunogenic, our data also suggest that holes in the functional T cell repertoire, if they exist, may be relatively rare.
Neutrophils migrate to delayed-type hypersensitivity reactions in joints, but not in skin. Mechanism is leukocyte function-associated antigen-1-/Mac-1-independent.
Authors: Gao J X , Issekutz A C , Issekutz T B
Published: December 2022

We have previously found that polymorphonuclear leukocyte (PMNL) migration to adjuvant arthritic joints of rats was only partially inhibited by mAbs to the adhesion molecules LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18), suggesting that there is a CD11/CD18-independent mechanism for PMNL migration to inflamed joints. Adjuvant arthritis in rats is believed to be initiated by a T lymphocyte-dependent immune response and maintained by proinflammatory cytokines such as IL-1 and TNF-alpha. Here we studied two types of joint inflammation: that induced by a delayed-type hypersensitivity (DTH) reaction in the joint and that induced by intra-articular (i.a.) injection of cytokines, to explore PMNL migration to inflamed joints and examine the role of CD18. 51Cr-labeled blood PMNL were used to measure PMNL migration in rats to inflammatory reactions in joints and compared with reactions in skin. A large number of PMNL migrated to the carpal and talar joints after i.a. injection of Mycobacterium purified protein derivative in sensitized animals to induce DTH, but there was minimal PMNL migration to this DTH reaction in the skin. This migration to the joints was not inhibited by mAbs to LFA-1 alone or mAbs to LFA-1 plus Mac-1 that almost completely inhibited PMNL accumulation in dermal inflammatory reactions induced by zymosan-activated serum (C5adesArg), endotoxin, IL-1 alpha, or TNF-alpha in the same rats. Intra-articular injection of the cytokines IL-1 alpha and TNF-alpha, but not IFN-gamma, induced marked PMNL accumulation in the joints; this was strongly inhibited by the treatment of rats with anti-LFA-1 and anti-Mac-1. Thus, PMNL migrate to DTH induced in joints but not in skin, and this migration is CD18-independent, but migration to i.a. IL-1 alpha and TNF-alpha is largely CD18 dependent in both joints and skin. This suggests that both the joint microenvironment and the T cell dependence of the inflammatory reaction in the joint governs the mechanism of PMNL recruitment.
Elevated levels of shed type II IL-1 receptor in sepsis. Potential role for type II receptor in regulation of IL-1 responses.
Authors: Giri J G , Wells J , Dower S K , McCall C E , Guzman R N , Slack J , Bird T A , Shanebeck K , Grabstein K H , Sims J E
Published: December 2022

Two types of cellular IL-1Rs have been characterized and cloned from both human and murine sources. The type II IL-1R has a very short cytoplasmic domain and does not seem to participate in IL-1 signaling. We demonstrate that type II IL-1Rs are released from the surface of neutrophils in response to treatment with TNF or endotoxin. In addition, serum from patients with sepsis syndrome contains elevated levels of soluble type II IL-1Rs. Neutrophils isolated from patients with sepsis have greatly enhanced expression of type II IL-1R mRNA and cell surface receptors and are therefore a likely source for the shed receptors in serum. Of the three forms of IL-1, soluble type II IL-1R binds IL-1 beta with highest affinity and also selectively inhibits IL-1 beta activity. We propose that increased cell surface expression and rapid release of preformed type II IL-1R from neutrophils, as a soluble IL-1 beta binding protein, represents a mechanism that has evolved for regulating IL-1 activity in sepsis.
Immunoreactive vasoactive intestinal peptide contributes to the immunosuppressive activity of normal aqueous humor.
Authors: Taylor A W , Streilein J W , Cousins S W
Published: December 2022

Suppression of immune-mediated inflammation within the normal anterior chamber (AC) of the eye is in part the result of active suppression of effector T cell activities by immunosuppressive cytokines found in aqueous humor (AqH), the fluid filling the AC. There are immunosuppressive factors found in the low m.w. fraction (< 5 kDa) of AqH, including the neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH). In seeking other factors, we now report that the neuropeptide vasoactive intestinal peptide (VIP) is also present in normal AqH. VIP immunoreactivity was found in normal rabbit eyes at a concentration of 12 +/- 1 nM. At this intraocular concentration, VIP suppressed Ag-stimulated lymph node cell (LNC) proliferation and IFN-gamma production in vitro. Although suppression of LNC proliferation was not neutralized by absorption of VIP from the low m.w. fraction of AqH, removal of VIP did neutralize suppression of IFN-gamma production by this fraction of AqH. Absorption of both VIP and alpha-MSH from this fraction of AqH permitted production of IFN-gamma by Ag-stimulated LNC that was no different than absorbing VIP alone. The low m.w. fraction of AqH absorbed of either alpha-MSH and VIP lost its ability to suppress local adoptive transfer of delayed-type hypersensitivity. The results suggest that VIP is an important immunosuppressive neuropeptide in AqH. Neuropeptides play an important role in ocular immune privilege and creation of an intraocular immunosuppressive microenvironment.
Inhibition of Plasmodium falciparum in vitro by human gamma delta T cells.
Authors: Elloso M M , van der Heyde H C , vande Waa J A , Manning D D , Weidanz W P
Published: December 2022

As assessed by flow cytometry, human gamma delta T cells were shown here to inhibit replication of blood-stage Plasmodium falciparum in vitro in a dose-dependent fashion; no other leukocyte population tested was suppressive. Replication of intraerythrocytic stages of the parasite (rings, trophozoites, and schizonts) was not affected by coculture with gamma delta T cells nor were erythrocytes damaged by this coculture, indicating that the targets recognized by gamma delta T cells are extracellular merozoites in transit to new host erythrocytes. Moreover, parasite inhibition requires contact between gamma delta T cells and merozoites. These findings suggest that gamma delta T cells may exert a protective effect in immunity to malaria.
Induction of antibodies to the Plasmodium falciparum merozoite surface protein-1 (MSP1) by cross-priming with heterologous MSP1s.
Authors: Hui G S , Hashimoto A C , Nikaido C M , Choi J , Chang S P
Published: December 2022

The merozoite surface protein-1 (MSP1) of Plasmodium falciparum possesses intervening conserved and nonconserved sequences. The relative importance of these sequences in providing T cell help for Ab production was investigated in a series of cross-priming studies using homologous and heterologous parasite MSP1 proteins. Cross-priming with heterologous MSP1s was as efficient as homologous immunizations in inducing anti-MSP1 Abs. Similar to homologous immunization, cross-priming with heterologous MSP1s induced primarily Abs to conserved epitopes. The specificities of the Abs were also similar for the two immunization regimens. Studies were also performed with use of the C-terminal p42 fragment of MSP1 expressed in baculovirus (BVp42). When BVp42 was used either as the priming Ag followed by boosting with homologous (or heterologous) MSP1 or as the booster Ag after priming with homologous (or heterologous) MSP1, much lower anti-BVp42 Ab titers were produced compared with priming/boosting with homologous or heterologous MSP1s or BVp42 alone. Thus, immunization with the complete parasite MSP1 induced a dominant, conserved Th epitope(s) specific for anti-p42 Ab production, and such determinant(s) was either located outside the p42 region or was not provided by the BVp42 because of possible differences in the processing of parasite MSP1 vs BVp42. Our data provided a strong rationale to identify and include conserved Th epitope(s) in MSP1 vaccines. Furthermore, a MSP1 vaccine on the basis of the C-terminal p42 fragment may benefit by the inclusion of additional Th epitopes to achieve effective boosting in the field.
Nitric oxide-independent killing of Francisella tularensis by IFN-gamma-stimulated murine alveolar macrophages.
Authors: Polsinelli T , Meltzer M S , Fortier A H
Published: December 2022

Alveolar macrophages (AMs) were analyzed for ability to support replication of the intracellular bacterium Francisella tularensis live vaccine strain (LVS). AM supported in vitro growth (2 to 3 logs over 5 days) of LVS with a doubling time of 8 +/- 0.8 h. AMs were analyzed for responsiveness to rIFN-gamma for destruction of this lung pathogen. AM treated with 50 U/ml rIFN-gamma allowed early growth of bacteria (six doublings over 48 h) but between 48 and 96 h rIFN-gamma-treated AM eliminated 1.5 logs of LVS. AMs were sensitive to effects of rIFN-gamma; as little as 5 U/ml rIFN-gamma stimulated AM antimicrobial activity, with half-maximal activity 0.3 U/ml. rIFN-gamma-induced antimicrobial effects in AM correlated with amount of nitrites produced, but nitric oxide played only a minimal role in antibacterial effects induced in AM, because NG-MMLA (specific inhibitor of L-arginine-dependent nitric oxide production) failed to block antimicrobial activity of IFN-gamma-stimulated AM. IL-10, TGF-beta 1, and IFN-alpha (cytokines known to regulate effector functions of activated macrophages) also did not block anti-F. tularensis activity of IFN-gamma-stimulated AM. Reactive oxygen metabolites, depletion of tryptophan, and sequestration of iron did not contribute to anti-F. tularensis activity because addition of superoxide dismutase or catalase, excess iron, or tryptophan to IFN-gamma-treated AM did not reverse the anti-F. tularensis activity observed in these cells. Regulation of AM effector activity differed from that of other macrophage populations, in that while rIFN-gamma-stimulated AM produced TNF-alpha (100 U/ml at 72 h), TNF-alpha was not required as a costimulator for induction of antimicrobial activities by rIFN-gamma because anti-TNF-alpha treatment of rIFN-gamma-stimulated AM blocked TNF-alpha but had no effect on either production of nitrites or anti-F. tularensis activity.
Induction of tissue factor-like activity in monocytes by anti-cardiolipin antibodies.
Authors: Kornberg A , Blank M , Kaufman S , Shoenfeld Y
Published: December 2022

Anti-cardiolipin Abs (ACLA) are present in the sera of patients with antiphospholipid syndrome (APLS) and are associated with high incidence of thromboembolic phenomena, fetal loss, thrombocytopenia, and prolongation of the phospholipid-dependent coagulation assays (lupus anticoagulant). Recently, it has been shown that APLS can be induced experimentally by using ACLA. However, the pathophysiology of thrombus formation in this syndrome is unknown. Monocytes generate a potent procoagulant activity (PCA) after stimulation with various substances. Increased PCA has been found in monocytes from patients with diseases that are associated with high incidence of thromboembolic phenomena. In the present study, we report that the monoclonal ACLA that were shown previously by us to induce APLS stimulate mononuclear cells to generate a potent PCA. The PCA resembled tissue factor (TF) in that it accelerated clotting through the extrinsic coagulation pathway, was abolished by phospholipase C, and was inhibited by anti-TF mAbs. The induction of TF-like activity by ACLA in monocytes was dose- and time-dependent. It was induced in monocytes and monocytic cell lines, but not in lymphoid or myeloid cells, and did not require T lymphocytes for expression. The generation of PCA was dependent on protein synthesis inasmuch as it was prevented by adding puromycin to the system and was not affected by cytarabine. The TF-like activity that is induced by ACLA in monocytes may activate coagulation and thereby play a major role in the pathogenesis of thrombus formation in APLS.
Specific stimulation of Graves' disease thyrocytes by the natural anti-Gal antibody from normal and autologous serum.
Authors: Winand R J , Devigne J W , Meurisse M , Galili U
Published: December 2022

Anti-Gal is a natural Ab abundantly produced in humans. It interacts specifically with the carbohydrate epitope Gal alpha 1-3Gal beta 1-4GlcNAc-R (termed the alpha-galactosyl epitope). This epitope is expressed in large amounts on thyrocytes of nonprimate mammals, but not of humans. We have previously found that binding of anti-Gal to alpha-galactosyl epitopes on porcine thyrocytes results in stimulatory effects similar to those exerted by thyroid-stimulating hormone (thyrotropin). In the present study, we tested the hypothesis that anti-Gal may contribute to Graves' disease (GD) pathogenesis by stimulation of the thyrocytes of patients with this autoimmune disorder. Anti-Gal binding and stimulatory effects were assessed in primary thyrocyte cultures. Anti-Gal specifically bound to GD thyrocytes and induced an increase in cAMP synthesis, 125I uptake, and DNA synthesis in these cells. Furthermore, the stimulatory effects of autologous sera on GD thyrocytes were greatly reduced after specific depletion of anti-Gal from these sera. No binding and no stimulatory effects of anti-Gal were observed, however, with normal human thyrocytes and with thyrocytes from thyrotoxic patients who lack thyroid-stimulating Igs or thyrotropin binding inhibiting Igs. These in vitro stimulatory effects of anti-Gal on GD thyrocytes suggest that this natural Ab may contribute to the in vivo continuous stimulation of thyrocytes in GD patients. The possibility that anti-Gal may stimulate GD thyrocytes via interaction with aberrantly expressed alpha-galactosyl epitopes on the thyroid-stimulating hormone receptor is discussed.