The self antigen heme evades immune recognition by sequestration in some hemoproteins

Heme is a non-protein autoantigen which is ubiquitous in vivo, primarily complexed in various hemoproteins or bound to specialized carrier molecules. Nevertheless, heme is able to stimulate a high frequency of CD4⁺, class II-restricted T cells, freshly explanted from unprimed mice, to proliferate in vitro. In this study, we show that heme incorporated into various species of mammalian cytochrome c (cyt c), including murine cyt c, represents a facultative cryptic determinant, able to be recalled only at high doses of native cyt c. By contrast, avian cyt c is of comparable antigenicity to free heme. Artificially denatured carboxymethylated (CM) mammalian cyt c exhibited greatly increased antigenicity, comparable to that of heme and avian cyt c, indicating that the crypticity of heme in native mammalian cyt c is due to the resistance of the native conformation of this molecule to antigen processing within murine antigen-presenting cells. Thus, tolerance to the heme group of at least some hemoproteins, may be maintained by the crypticity of the heme, rather than by deletion of hemereactive T cells. Given the high frequency of heme-reactive T cells in unprimed mice, these findings suggest that heme may become an important modulator during an inflammatory response.     

Immune alterations in three mouse strains following 2-deoxy- -glucose administration

Using 2-deoxy- -glucose (2-DG)-induced stress, our laboratory has developed studies to define stress effects on immune responses. Here, we report effects of increasing doses of 2-DG on the immune response of BALB/c, C57BL/6 and BDF₁ mice 2 h after three injections of 0 to 2000 mg/kg of 2-DG. Female 4- to 5-week-old mice were euthanized and blood and spleens were collected. A suspension of partially purified mature T splenocytes was obtained by negative selection using J11.d2 antibodies. Glucose and corticosterone levels were measured in the plasma of each mouse. Splenocyte and mature T splenocyte suspensions were tested in in vitro proliferation assays with or without concanavalin A. Splenocytes were analyzed for the following cell-surface markers: CD3, TCR α/β, CD4, CD8 and major histocompatibility complex (MHC) Class II. Significant increases in blood glucose levels were observed in C57BL/6 and BALB/c strains with the highest 2-DG dose (p<0.05). Corticosterone levels were higher in BDF₁ mice and C57BL/6 mice following the administration of 1000 and 2000 mg/kg of 2-DG, respectively (p<0.01). In vitro proliferation of mature T splenocytes in the presence of concanavalin A was decreased in BDF₁ (p<0.05) but not in BALB/c and C57BL/6 mice. In addition, in BDF₁ mice the decrease was highly correlated with an increase of CD3+ and TCR α/β+ cells in the spleen. These results demonstrated high variability in the response of different mouse strains to 2-DG-induced stress.     

Diseases caused by reactions of T lymphocytes to in compatible structures of the major histocompatibility complex. I. Autoimmune hemolytic anemia

The capacities of various lymphoid cells from C57BL/10 donors to induce antibodies against red blood cells (RBC) in (C57BL/10 × DBA/2)F₁ recipients were compared: cortisone-resistant thymocytes were more potent inducers than spleen cells, and bone marrow cells were ineffective. This established a need for parental T lymphocytes in the induction of Coombs-positive hemolytic anemia by the graft-versus-host reaction (GVHR). Since some of the anti-RBC antibodies carried the Ig^c allotype specific for the F₁ host, they were autoantibodies. When antibodies were eluted from the erythrocytes of F₁ mice undergoing the GVHR (GVH F₁ mice) and subsequently tested against normal RBC in the indirect Coombs' test, the Ig^c allotype was demonstrable even on those antibodies found to react with RBC of the donor strain (C57BL/10). In addition, reactions with normal intact RBC of other strains and species were noted. Anti-RBC antibodies belonged to all Ig classes and subclasses that were tested (IgG₁, IgG_2a, IgG_2b, IgA, and IgM). By means of histocompatibility typing it was shown that the overwhelming majority of lymphoid cells from GVH animals were host cells; most of these host cells were Thy-1.2-negative. In several GVH F₁ mice Coombs-positive erythrocytes were demonstrated for periods of more than 5 months; Ig^c-positive anti-RBC antibodies were detected for periods up to 3 months. When an additional injection of parental T cells was administered to GVH F₁ mice, which had become Coombs-negative in the course of the GVHR, new anti-RBC antibodies appeared. In contrast to F₁ B cells, T cells of the F₁ host were not needed for autoimmunization as shown by the induction of anti-RBC auto-antibodies in F₁ recipients that were depleted of autologous T cells prior to the administration of parental T cells. When (C57BL/10 × DBA/2)F₁ mice were back-crossed into strain C57BL/10 and the offsprings injected with C57BL/10 (H-2^bb) lymphoid cells, most of the H-2 heterozygous (H-2^bd) back-crosses developed Coombs'-positive hemolytic anemia, whereas none of the H-2 homozygous (H-2^bb) ones did. The most likely explanation of these results is that anti-RBC autoantibodies were induced by a persistent reaction of parental T helper cells to incompatible H-2 structures on normally present autoreactive F₁ B cells. The hypothesis is proposed that reactions of T lymphocytes against virally or chemically altered structures of the major histocompatibility complex may lead to autoimmunization also in nonchimeric individuals.     

Antigenic Modification, Rosette-Forming Cells, and Salmonella typhimurium Resistance in Outbred and Inbred Mice

To assess the separate contributions of host T cells and the physical state of the antigen in the development of effective. Salmonella resistance, glutaraldehyde-treated and untreated protein- and ribonucleic acid-rich extracts (E-RNA extracts) of virulent Salmonella typhimurium SR-11 or attenuated S. typhimurium RIA were used to immunize Salmonella-resistant Salmonella-susceptible strains of mice for the purpose of determining whether antigen-specific T-cell or B-cell responses were formed and, if so, which responses predominated. The resistance imparted to each mouse strain after vaccination with S. typhimurium RIA was used as the standard for comparison. The inbred mouse strains C57BL/6 and DBA/2 and their F₁ hybrid (strain BDF₁), outbred ICR Swiss mice, and endotoxin-resistant C3H/HeJ mice were examined for the capacity to develop resistance to lethal Salmonella infections, as well as the ability to generate antigen-reactive T cells. Only the BDF₁, C3H/HeJ, and ICR Swiss mice were able to develop resistance to challenge infections mediated by the virulent SR-11 strain of S. typhimurium after vaccination with the living, attenuated RIA strain of S. typhimurium or immunization with E-RNA extracts. We developed an assay to identify the antigen-reactive rosette-forming lymphocytes present in lymph nodes and spleens of immunized mice. Levels of 0.2% or higher of theta antigen-bearing, antigen-reactive rosette-forming cells were found in the lymph nodes or spleens or both of only the BDF₁, C3H/HeJ, and ICR Swiss mice (i.e., in the “Salmonella responder” strains). Mouse strains C57BL/6 and DBA/2, which failed to develop resistance to lethal infections after immunization with the S. typhimurium RIA vaccine or with the E-RNA extracts, lacked effective numbers of antitheta antigen-sensitive rosette-forming cells. Modification of the effective E-RNA extracts by polymerization with glutaraldehyde resulted in a marked diminution in their abilities to induce resistance to salmonellosis in the two responder mouse strains tested (BDF₁ and ICR Swiss), even though detectable levels of antibody were induced.     

Interaction of Y-chromosomal and autosomal gene(s) in the development of intermale aggresiion in mice

It has been suggested that the Y chromosome of DBA/1BBg mice makes an incremental contribution to their aggressive behavior and to that of the C57BL/10 ×DBA/1 F ₁ hybrids. To test this hypothesis, a congenic stock of C57BL/10 with the DBA/1 Y chromosome was developed by the backcross system of breeding; the stock is designated C57BL/10-Y ¹ . There were no significant differences in aggressive behavior between the congenic C57BL/10 and C57BL/10-Y ¹ . However, the hybrid B10D1 F ₁ and D1B10-Y ¹ F ₁ had identical aggression scores, and both of these were more aggressive than the hybrid D1B10 F ₁ . These findings support the hypothesis that there is an interaction between DBA/1 Y chromosomes and autosomes in the development of intermale aggression of these mice.This research was supported by NIMH Grant 28021-02 and NICHHD Grant 11220-01     

The Inhibitory Co-Receptor,PECAM-1 Provides a Protective Effect in Suppression of Collagen-Induced Arthritis

Studies of PECAM-1^–/– mice have identified that PECAM-1 functions as an inhibitory co-receptor to modulate immunological responsiveness. In this study, we describe the in vivo consequences of PECAM-1 deficiency in mouse models of collagen-induced arthritis (CIA) and K/BxN passive transfer model that resembles many of the features of human rheumatoid arthritis. Immunization of PECAM-1^–/– C57BL/6 (H-2^b) mice with chicken collagen type II induced CIA with an incidence of 82% by day 49, while 33%; of wild-type and 100% of DBA/1 mice developed arthritis in a similar time frame. The mean onset of disease for PECAM-1^–/– C57BL/6 mice was day 32 compared to day 51 for wild-type C57BL/6 mice and day 18 for DBA/1 mice (H-2^q susceptible). In terms of disease severity, the mean maximal arthritic index for PECAM-1^–/– C57BL/6 mice was comparable to DBA/1 mice (8.91 ± 0.91 vs 11.67 ± 0.82). This mean maximal index in PECAM-1^–/– C57BL/6 mice was significantly higher than wild-type C57BL/6 mice (5.00 ± 0.73). IgG₁ and IgG_2b antibody responses against CII were elevated in arthritic PECAM-1^–/– C57BL/6 mice compared to wild-type C57BL/6 mice. Histological examination of arthritic paws of PECAM-1^–/– C57BL/6 mice revealed inflammatory infiltrates of lymphocytic/monocytic cells and cartilage/bone destruction similar to CIA-induced DBA/1 arthritic paws. In the K/BxN model, the arthritis was not augmented in PECAM-1^–/– mice compared to wild-type mice. In contrast, in active CIA, PECAM-1^–/– mice developed severe disease comparable to susceptible DBA/1 mice and profoundly more severe than C57BL/6 mice, where only one third developed a mild/moderate disease. Together these observations suggest that PECAM-1 plays a crucial role in the suppression of development of autoimmune arthritis.     

Inheritance of antibody specificity. The IgM anti-lipopolysaccharide response in mice

Mice of different genotypes were immunized with Salmonella anatum. The cross-reactivity patterns of their IgM anti-S. anatum lipopolysaccharide (LPS_AN) antibodies were characterized by their relative avidity toward heterologous LPS. When the LPS from S. cholera suis (LPS_CHS) was used as the heterologous LPS, clear differences between mouse strains were found. DBA/2 and DBA/1 showed cross-reacting IgM, whereas C57BL/10, C57BL/6, BALB/c-Ig^b and B10.D2 had mainly noncross-reacting IgM. In C3H and C57BL/6-Ig^a, individual mice express either the cross-reacting or the noncross-reacting antibodies. The IgM antibodies from individual mice were further characterized for their cross-reactivity toward the LPS from S. strasbourg (LPS_STR) and S. illinois (LPS_ILL). Only individual patterns with no correlation to the cross-reactivity pattern with LPS_CHS were found. This shows that more than one antibody type is characterized by cross-reactivity. (B 10.D2 × DBA/1)F₁ mice showed a biphasic distribution of cross-reactivity. Of the F₁ x DBA backcross mice 21 % had IgM antibodies which showed no cross-reaction with LPS_CHS. This still is in agreement with one locus controlling this phenotype. This locus segregates independently from Ig allotype since no correlation was found between allotype and cross-reactivity pattern in F₁ x DBA backcross mice.     

Host age and H-2 tolerance in chimeric mice: Mixed lymphocyte reactivity,cell-mediated lympholysis and responses to hapten-modified self

In order to further our understanding of the reasons for the increased susceptibility of aged animals to autoimmunity, neoplasms and infectious diseases, experiments were performed to determine the ability of an aged environment to induce and support tolerance to major histocompatibility complex (MHC) determinants as well as to support the development of a specific immune response to modified self-determinants. The degree and mechanisms of tolerance to host and donor histocompatibility antigens were studied in bone marrow chimeras of the type (C57B1/6 × CBA)F₁ → (C57B1/6 × DBA/2)F₁ (BCF₁ and BDF₁, respectively). BCF₁ bone marrow donors were 6 weeks old and BDF₁ hosts were 18 months old at the time of chimerization. Four to ten months later, chimeras were found to be fully tolerant to all three parental haplotypes and competent to respond to fourth-party strains as assessed in both mixed lymphocyte reactions and cell-mediated lympholysis. Tolerance to parental haplotypes could not be attributed to active suppression of reactivity.The aged host environment proved incapable of supporting the development of anti-modified self-reactivity as attested by the fact that neither the senescent BDF₁ mice nor the BCF₁ → BDF₁ chimeras established in aged hosts could respond to trinitrophenol-modified autologous parental cells. In contrast, young BDF₁ mice and BCF₁ → BDF₁ chimeras established in young adult hosts were competent to respond to trinitrophenol-modified autologous and parental cells in an MHC restricted fashion. The significance of these results to the susceptibility of aged animals to intracellular parasitic infections and neoplasia is discussed.     

Antigen presentation is a function of all B cell subpopulations separated on the basis of size

Purified splenic B cells from nonimmune mice were separated by counterflow centrifugal elutriation into 6 subpopulations containing cells of discrete sizes ranging from 119 to 200 μm³. B cells of each subpopulation were competent to process and present a native globular protein antigen, cytochrome c, to a cytochrome c-specific T cell hybrid. In all cases, the B cells' antigen-presenting function was radiation sensitive and did not require T cells or T cell products, since B cells fixed with paraformaldehyde effectively presented a carboxyl-terminal peptide fragment of cytochrome c containing the T cell determinant. Furthermore, the antigen-presenting function of B cells of each subpopulation was augmented by treatment with submitogenic doses of the F(ab')₂ fragment of rabbit anti-mouse Ig antibodies, in that 10-30-fold fewer B cells were required and higher maximal T cell responses were achieved, indicating that B cells of all sizes are capable of being regulated in their antigen presentation function through their surface Ig. In addition, B cells of each subpopulation responded to soluble factors present in the supernatants of activated T cells as evidenced by an increase in volume and by the uptake of [³H]thymidine. These results indicate that B cells, regardless of size, are able to participate in at least two essential phases of T cell-dependent antibody responses, initiating the interaction by processing and presenting antigen to helper T cells and responding to soluble helper factors secreted by activated T cells.     

T Cell Tolerance Induced by Histone Deacetylase Inhibitor is Mediated by P21cip1

MEB [n-butyrate 2-(4-morpholinyl) ethyl butyrate hydrochloride], a histone deacetylase inhibitor and G1 blocker, has been shown to induce unresponsiveness in antigen-activated Th1 cells. MEB was tested for here for its ability to inactivate naive alloantigen-specific T cells from DBA/2 and C57BL/10 mice. Since T cells from these two strains of mice have been shown to differ in their cell cycle regulation, it we hoped that this comparison would provide information concerning the role of cycle regulatory proteins in mediating MEB-induced T cell unresponsiveness. MEB inhibited proliferation in a one-way mixed lymphocyte reaction (MLR) in which spleen cells from DBA/2 mice (H-2^d) or C57BL/10 mice (H-2^b) were stimulated with spleen cells from C57BL/10 or DBA/2 mice, respectively. C57BL/10 responder T cells isolated from the MEB-treated primary MLR remained unresponsive to alloantigen following restimulation in a secondary MLR that did not contain MEB. T cells from DBA/2 mice were less sensitive to MEB-induced unresponsiveness and required a longer exposure or pretreatment with IL-2 to become tolerant. In all cases responsiveness to MEB-induced tolerance in the alloantigen-stimulated T cells corresponded with the levels of the cyclin-dependent kinase inhibitor p21^cip1. Additional experiments showed that T cells from p21^cip1-deficient mice, unlike T cells from p21^cip1 wild-type littermates, were resistant to MEB-induced tolerance. These results underscore the role of p21^cip1 in mediating T cell tolerance induced by the histone deacetylase inhibitor MEB.     

Genetic determinants of lung virus titers and resistance to lethal Sendai virus pneumonia

Summary C 57 BL/6J mice are resistant to lethal Sendai virus pneumonia and have lower lung virus titers than susceptible DBA/2J mice. Linkage between these phenotypes was tested indirectly in segregant hybrids.Sas-1,B2m, andb on chromosomes 1, 2, and 4 were linked to significant (P<.05) differences in virus-induced mortality;d on chromosome 9 was associated with a similar but smaller difference (.1>P>.05). Mean lung virus titers were higher in F₁ × DBA/2 J mice that were homozygous for DBA alleles atB2m, b, andd than in heterozygotes. The difference in lung virus titers was larger between mice that were dihomozygous/diheterozygous for paired combinations;B2m-d (P<.02),B2m-b (P<.06), andb–d (P<.05) and largest between mice that were trihomozygous/triheterozygous forB2m-b–d (P<.001). The distribution of virus titers among 22 recombinant inbred strains derived from C 57 BL/6 J and DBA/2 J progenitors indicated 1) that the loci linked toB2m, b, andd are among at least 4 loci that regulate lung virus titers, 2) thatSas-1 may be linked to a fourth locus, 3) that the C 57 BL/6 J genome contains at least one susceptibility locus, possibly within H-2, and 4) that some of these loci may be expressed through natural killer cell activity.     

Specific cytotoxicity of a mouse thymocyte population sensitized in vitro against H-2 alloantigens

C57BL cortisone-resistant thymocytes were sensitized in vitro against DBA/2 alloantigens and tested for specific cytotoxicity against ⁵¹Cr-labeled DBA/2 mastocytoma cells as targets. T cell sensitization, differentiation, uropod formation and specific cytotoxicity developed in a system of pure T lymphocytes and allogeneic antigenic fibroblasts without any apparent helper mechanism by other cell types or mediators.     

Sensitization for audiogenic seizures in two strains of mice and their F1 hybrids

Acoustic sensitization or priming for audiogenic seizures was studied in 304 “seizure-resistant” mice of C57BL, BALB/c and their F₁ hybirds. The effect of priming was found to be a function of strain, age of the first auditory exposure, and duration of exposure. In BALB/c mice priming at ages 21–28 days induced very high incidence of seizures; in this stran 2-min exposure was more effective than both 60 sec or 30 sec. The same parameters of priming induced very low seizure incidence in C57BL mice. A behavior-genetic analysis indicated that the genotype of each parental strain might contribute equally to the priming induced seizure risk of their F₁ hybrids, but C57BL mice appeared to be dominant to the BALB/c for some components of audiogenic seizures.     

The major histocompatibility complex (MHC) of the secondary transplant tissue donor influences the cross-reactivity of alloreactive memory cells

Memory cells are currently thought to be a major barrier to tolerance induction in transplantation. However, whether alloreactive memory cells resulting from a primary transplant have cross‐reactivity in a second transplant is unclear. Here, we used skin transplantation from BALB/c mice donors to presensitize C₅₇BL/6 (B6) mice. One month later, several strains of mice (including BALB/c, DBA/2, NOD, C3H and B6 mice) were chosen as donors to construct a memory model of heterotopic cardiac transplantation. The higher degree of major histocompatibility complex (MHC) mismatch to sensitizing MHC resulted in longer median survival times (MSTs, BALB/c 3.63 days versus C3H 6.08 days). After 3.5 days of cardiac transplantation, compared with the BALB/c and DBA/2 groups, in the groups of NOD and C3H, the infiltration of inflammatory cells in the grafts, the proportion and proliferation of memory cells in spleens and the function of allogeneic antibodies decreased significantly. The varying degrees of MHC mismatch between the primary and secondary donors influenced the intensity of alloreactive memory cell function, the higher degree of MHC mismatch resulted in better tolerance during secondary transplantation, and these may be related to the changed activation, proliferation and function of the alloreactive memory cells.     

Major histocompatibility complex class I presentation of ovalbumin peptide 257–264 from exogenous sources: protein context influences the degree of TAP-independent presentation

Peritoneal macrophages from C57BL/6 mice process antigens from bacteria or coated on polystyrene beads for presentation by major histocompatibility complex (MHC) class I molecules. To investigate this antigen processing pathway, peritoneal macrophages from homozygous TAP1^−/− mice, which lack the transporter associated with antigen processing (TAP) and are defective in presenting endogenous antigens on MHC class I, were used. TAP1^−/− or C57BL/6 macrophages were co-incubated with either bacteria or polystyrene beads containing the 257–264 epitope from ovalbumin [OVA(257–264)], which binds the mouse class I molecule K^b. The source of the OVA(257–264) epitope was either the Crl-OVA(257–264) (Crl-OVA) fusion protein, the maltose binding protein (MBP)-Crl-OVA fusion protein, native OVA or bacterial recombinant OVA (rOVA); Crl-OVA, MBP-Crl-OVA and rOVA were each expressed in bacteria, and Crl-OVA and MBP-Crl-OVA purified from bacterial lysates and native egg OVA were coated onto polystyrene beads. The data reveal that peritoneal macrophages from C57BL/6 and TAP1^−/− mice can process bacteria expressing Crl-OVA, MBP-Crl-OVA and rOVA as well as beads coated with native OVA, purified Crl-OVA, and purified MBP-Crl-OVA and present OVA(257–264) for recognition by OVA(257–264)/K^b-specific T hybridoma cells, albeit with different relative processing efficiencies. The processing efficiency of TAP1^−/− macrophages co-incubated with bacteria or beads containing Crl-OVA or MBP-Crl-OVA was reduced approximately three to five times compared to C57BL/6 macrophages, but OVA(257–264) was presented 100 times less efficiently when the source of OVA(257–264) was full-length OVA. Chloroquine inhibition studies showed a differential requirement for acidic compartments in C57BL/6 versus TAP1^−/− macrophages, which also depended upon the source of the OVA (257–264) epitope (Crl-OVA versus full-length OVA). These data suggest that TAP1^−/− and C57BL/6 macrophages may process Crl-OVA and full-length OVA in different cellular compartments and that the protein context of the OVA(257–264) epitope influences the extent of TAP-independent processing for MHC class I presentation.     

Pyroptosis of resident macrophages differentially orchestrates inflammatory responses to Staphylococcus aureus in resistant and susceptible mice

The relationship between Staphylococcus aureus and innate immunity is highly complex and requires further investigation to be deciphered. i.p. challenge of C57BL/6 and DBA/2 mice, resistant and susceptible to the infection, respectively, resulted in different patterns of cytokine production and neutrophil recruitment. Staphylococcus aureus infection induced macrophage pyroptosis, an inflammasome‐dependent cell death program, whose rates significantly differed between C57BL/6 and DBA/2 mice. Fast rate pyroptosis of C57BL/6 macrophages released high levels of IL‐1β but limited the synthesis of other cytokines such as TNF‐α, IL‐6, CXCL1, and CXCL2. Conversely, the extended survival of DBA/2 macrophages allowed substantial production of these NF‐κB‐related cytokines. Phenotyping of resting macrophages in different mouse strains revealed differential predisposition toward specific macrophage phenotypes that modulate S. aureus‐mediated inflammasome activation. Treatment of DBA/2 susceptible mice with inflammasome inducers (i.e. nigericin and ATP) artificially increased pyroptosis and lowered the levels of NF‐κB‐related inflammatory cytokines, but restored IL‐1β to levels similar to those in C57BL/6 mice. Collectively, this study promotes the concept that, in association with host genetics, the basal phenotype of resident macrophages influences the early inflammatory response and possibly participates in S. aureus infection outcome via the inflammasome pathway and subsequent pyroptosis.     

Transport of proteins across mitochondrial membranes

The vast majority of proteins comprising the mitochondrion are encoded by nuclear genes, synthesized on ribosomes in the cytosol, and translocated into the various mitochondrial subcompartments. During this process proteins must cross the lipid membranes of the mitochondrion without interfering with the integrity or functions of the organelle. In recent years an approach combining biochemical, molecular, genetic, and morphological methodology has provided insights into various aspects of this complex process of intracellular protein sorting. In particular, a greater understanding of the molecular specificity and mechanism of targeting of mitochondrial preproteins has been reached, as a protein complex of the outer membrane which facilitates recognition and initial membrane insertion has been identified and characterized. Furthermore, pathways and components involved in the translocation of preproteins across the two mitochondrial membranes are being dissected and defined. The energetics of translocation and the processes of unfolding and folding of proteins during transmembrane transfer are closely linked to the function of a host of proteins known as heat-shock proteins or molecular chaperones, present both outside and inside the mitochondrion. In addition, the analysis of the process of folding of polypeptides in the mitochondrial matrix has allowed novel and unexpected insights into general pathways of protein folding assisted by folding factors. Pathways of sorting of proteins to the four different mitochondrial subcompartments — the outer membrane (OM), intermembrane space, inner membrane (IM) and matrix — are only partly understood and reveal an amazing complexity and variation. Many additional protein factors are involved in these latter processes, a few of which have been analyzed, such as cytochrome c heme lyase and cytochrome c ₁ heme lyase, enzymes that catalyze the covalent addition of the heme group to cytochrome c and c ₁ preproteins, and the mitochondrial processing peptidase which cleaves signal sequence after import of preproteins into the matrix. Thus, the study of transport of polypeptides through the mitochondrial membranes does not only contribute to the understanding of how biological membranes facilitate the penetration of macromolecules but also provides novel insights into the structure and function of this organelle. are being dissected and defined. The energetics of translocation and the processes of unfolding and folding of proteins during transmembrane transfer are closely linked to the function of a host of proteins known as heat-shock proteins or molecular chaperones, present both outside and inside the mitochondrion. In addition, the analysis of the process of folding of polypeptides in the mitochondrial matrix has allowed novel and unexpected insights into general pathways of protein folding assisted by folding factors. Pathways of sorting of proteins to the four different mitochondrial subcompartments — the outer membrane (OM), intermembrane space, inner membrane (IM) and matrix — are only partly understood and reveal an amazing complexity and variation. Many additional protein factors are involved in these latter processes, a few of which have been analyzed, such as cytochrome c heme lyase and cytochrome c ₁ heme lyase, enzymes that catalyze the covalent addition of the heme group to cytochrome c and c ₁ preproteins, and the mitochondrial processing peptidase which cleaves signal sequences after import of preproteins into the matrix. Thus, the study of transport of polypeptides through the mitochondrial membranes does not only contribute to the understanding of how biological membranes facilitate the penetration of macromolecules but also provides novel insights into the structure and function of this organelle.Abbreviations OM outer mitochondrial membrane - IM inner mitochondrial membrane - IMS mitochondrial intermembrane space - OMV outer membrane vesicles - AAC ADP/ATP carrier - PiC phosphate carrier - DHFR mouse cytosolic dihydrofolate reductase - F₁ subunit of F₁F₀ ^– ATPase - MPP mitochondrial processing peptidase     

Systemic Treatment with a miR‐146a Mimic Suppresses Endotoxin Sensitivity and Partially Protects Mice from the Progression of Acute Graft‐versus‐Host Disease

Acute GVHD (aGVHD) is driven by interactions between the allogenic T cell response, inflammation, tissue injury and microbial products that enter the circulation when protective barriers such as the intestinal epithelium become compromised. Mice with aGVHD become hypersensitive to LPS, secreting large quantities of inflammatory mediators that exacerbate tissue injury. We hypothesized that microRNA (miR) modulators could be used in vivo to mitigate LPS hypersensitivity, altering the course of aGVHD. Using the C57BL/6 → (C57BL/6 × DBA/2)F₁‐hybrid model of aGVHD, we measured intestinal permeability over time and used a qPCR array to detect concomitant changes in the expression levels of certain microRNAs (miRs) in the intestine. Large increases in permeability were seen on day 15, when endotoxemia becomes detectable and GVHD‐associated histopathological lesions develop. Amongst the miRs with altered expression levels were some that regulate sensitivity to endotoxin. We chose to focus on miR‐146a and treated recipient mice systemically with a miR‐146a mimic early in the GVH reaction. This led to a reduction in the burst of IFNγ that likely plays a priming role in the mechanism underlying heightened sensitivity to endotoxin. LPS‐induced TNFα release and GVHD‐associated weight loss were also diminished and survival was prolonged. In summary, systemic treatment with a miR‐146a mimic dampens the heightened sensitivity to LPS that occurs concomitantly with increased intestinal permeability and provides partial protection from the progression of acute GVHD.     

Immune responses to weakly immunogenic virally induced tumors II. Suppressive effects of the in vivo carried tumor YAC

Unprimed spleen cells from A and C57BL/6 mice could not produce cytotoxic responses to their syngeneic tumors: a Moloney virus-induced in vitro subline YAC-1 and a Rauscher virus-induced in vitro subline RBL5, respectively. Spleen cells from A and C57BL/6 mice immunized with YAC-1 or RBL5 (which cross-react serologically) generated significant syngeneic cytotoxicities after cultivation in vitro. The in vivo carried tumor of A mice, unlike the in vitro sublines, could not stimulate a priming effect. In contrast, YAC stimulated the formation of suppressor cells in both A and C57BL/6 mice. The suppressor cells abrogated the priming effect of the syngeneic tumors, but not the priming effect of the allogeneic tumors. Furthermore, YAC did not suppress normal allogeneic anti-tumor responses. The theoretical and the practical implications of these studies are discussed.