Antimicrobial effects of murine mesenchymal stromal cells directed against Toxoplasma gondii and Neospora caninum: role of immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs).

Mesenchymal stromal cells (MSCs) have a multilineage differentiation potential and provide immunosuppressive and antimicrobial functions. Murine as well as human MSCs restrict the proliferation of T cells. However, species-specific differences in the underlying molecular mechanisms have been described. Here, we analyzed the antiparasitic effector mechanisms active in murine MSCs. Murine MSCs, in contrast to human MSCs, could not restrict the growth of a highly virulent strain of Toxoplasma gondii (BK) after stimulation with IFN-γ. However, the growth of a type II strain of T. gondii (ME49) was strongly inhibited by IFN-γ-activated murine MSCs. Immunity-related GTPases (IRGs) as well as guanylate-binding proteins (GBPs) contributed to this antiparasitic effect. Further analysis showed that IFN-γ-activated mMSCs also inhibit the growth of Neospora caninum, a parasite belonging to the apicomplexan group as well. Detailed studies with murine IFN-γ-activated MSC indicated an involvement in IRGs like Irga6, Irgb6 and Irgd in the inhibition of N. caninum. Additional data showed that, furthermore, GBPs like mGBP1 and mGBP2 could have played a role in the anti-N. caninum effect of murine MSCs. These data underline that MSCs, in addition to their regenerative and immunosuppressive activity, function as antiparasitic effector cells as well. However, IRGs are not present in the human genome, indicating a species-specific difference in anti-T. gondii and anti-N. caninum effect between human and murine MSCs.

The life-span and recirculation of marrow-derived small lymphocytes from the rat thoracic duct.

These experiments describe the preparation of pure marrow-derived lymphocyte suspensions from the thoracic duct of thymectomized, irradiated rats reconstituted with bone marrow cells. The majority of marrow-derived cells were small lymphocytes morphologically indistinguishable from small lymphocytes in thoracic duct lymph of normal donors. Marrow-derived small lymphocytes (B lymphocytes) were a predominantly long-lived population; the frequency of short-lived B lymphocytes in the thoracic duct was not significantly higher than the frequency of short-lived small lymphocytes in normal lymph. B lymphocytes transferred to normal recipients recirculated from blood to lymph. The first appearance of intravenously injected B lymphocytes in the thoracic duct was delayed relative to lymphocytes from normal donors and there was no clear cut modal recirculation time. Nevertheless their recirculation over a 48 hr period after transfusion was of the same order of magnitude as that of lymphocytes from normal donors.

Specific positive selection of lymphocytes reactive to strong histocompatibility antigens.

Selected populations of thymus-derived (T) rat lymphocytes having specific immunological reactivity to chosen histocompatibility (H) alloantigens are found among the cellular products of the mixed lymphocyte interaction (MLI). Such specific selection seems to depend on (a) the antigen-induced proliferation of specific H antigen reactive cells (HARC), and (b) the disappearance of nonreactive cells from the cultures. When the surviving cells from this lymphocyte-antigen interaction are transferred into thymectomized, X-irradiated, marrow-reconstituted syngeneic recipients (B rats) which lack detectable T-lymphocyte functions, the lymphocyte populations subsequently recovered from the hosts possess the capacity to react in the MLI and in the graft-vs.-host (GVH) reaction, and the reactions have specificity for the original priming alloantigens. In addition, these findings identify the cell that reacts in the MLI with the GVH reactive cell.

The major histocompatibility complex class II-linked cim locus controls the kinetics of intracellular transport of a classical class I molecule.

The dominant trans-acting major histocompatibility complex (MHC)-linked class I modifier (cim) locus, previously recognized through its ability to determine altered alloantigenicity of a rat class I molecule, RT1.A3, is shown here to influence class I intracellular transport. The MHC recombinant laboratory rat strains PVG.R1 and PVG.R8 display unusually long retention of RT1.Aa within the endoplasmic reticulum or cis-Golgi. In appropriate F1 hybrid cells heterozygous for RT1.Aa and another class I MHC allele, RT1.Ac, only the RT1.Aa protein is subject to slow transport. The cim gene product therefore shows class I allele specificity in its action, cim appears to be a polymorphic locus whose product is directly involved in the processes of class I MHC assembly and/or intracellular transport.

Identification of marrow-derived and thymus-derived small lymphocytes in the lymphoid tissue and thoracic duct lymph of normal rats.

These experiments show that small lymphocytes from the thoracic duct of rats are normally a mixture of thymus-derived and marrow-derived cells, and define the traffic areas in lymphoid tissues through which the two populations recirculate. Thoracic duct lymphocytes were labeled in vitro with uridine-(3)H and their histological distribution in the lymphoid tissues of normal recipients was demonstrated by radioautography. Labeled lymphocytes occupied two adjacent areas distinguished by a marked difference in the intensity of labeling; heavily labeled cells were found in thymus-dependent traffic areas of lymphocyte recirculation, while lightly labeled cells localized in the thymus-independent follicular areas around germinal centers. A corresponding heterogeneity of uridine uptake among small lymphocytes from normal donors was demonstrated by sedimentation at 1 g; slowly sedimenting cells incorporated little uridine and localized in follicular areas after transfusion while rapidly sedimenting cells incorporated more uridine and localized in thymus-dependent areas after transfusion. Experimentally prepared marrow-derived small lymphocytes behaved in sedimentation studies and after transfusion like a pure population of the lightly labeled small lymphocytes in normal lymph. Artificially reconstituted mixtures of marrow-derived and thymus-derived lymphocytes were qualitatively indistinguishable from normal lymphocyte populations.

The role of subregions of the rat major histocompatibility complex in the rejection and passive enhancement of renal allografts.

The laboratory recombinant haplotype H-1acl of the Norway rat has been used in studies of the rejection and passive enhancement of kidney allografts. While the full H-1a haplotype provoked acute rejection, neither of the isolated subregions, H-1Aa and H-1Ba, did so. It was also found that alloantisera raised against either the H-1Aa or the H-1Ba antigens would enhance the grafts. It is suggested that both MHC subregions contain a histocompatibility locus (i) for kidney (as they do for skin) and that in the genetic combinations studied only incompatibility for both provokes a response sufficient for rejection. In other combinations, however, single region incompatibilities may be sufficient.

Immune response genes controlling responsiveness to major transplantation antigens. Specific major histocompatibility complex-linked defect for antibody responses to class I alloantigens.

We have identified two major histocompatibility complex (MHC)-linked Ir genes that control the antibody response made by rats against class I major alloantigens. We have named these genes Ir-RT1Aa and Ir-RT1Ac. These Ir genes determine responsiveness of the immunized animal in a typical codominant fashion. There is no evidence so far for trans-complementation between low-responder haplotypes. Detailed studies of Ir-RT1Aa indicate that it controls the antibody response to at least two distinct alloantigenic determinants on RT1Aa molecules. These class I molecules thus behave like hapten-carrier conjugates when the response against the carrier is under Ir gene control. Analysis of the origin of alloantibody-forming cells in tetraparental radiation chimeras indicates that Ir-RT1Aa must control the provision of effective help to B cells. In many respects therefore, the properties of Ir-RT1Aa are broadly similar to those described for Ir genes controlling antibody responses to conventional antigens. The existence of apparently conventional Ir genes controlling the antibody response to major alloantigens strongly suggest that the processing of these transmembrane molecules by host antigen-presenting cells is a prerequisite for immune induction, and that it is the MHC of the responder rather than that of the allograft to which T helper cells are restricted in alloimmune responses in vivo.

A trans-acting major histocompatibility complex-linked gene whose alleles determine gain and loss changes in the antigenic structure of a classical class I molecule.

The RT1.A locus of the rat MHC encodes the H chain of the single classical class I molecule of this species. One of the alleles of this polymorphic locus, RT1.Aa, is present in several laboratory inbred, congenic, and MHC recombinant rat strains. Studies of the RT1.Aa class I molecule from a number of these strains as a target for CTL show that its antigenicity, both as an alloantigen and a restricting element, is subject to gain and loss alterations by the action of a gene mapping in the MHC to the right of RT1.A. This locus is apparently present in two allelic forms (one possibly a null allele) corresponding to the presence or absence of a dominant transacting modifier, and has been named class I modification, or cim. The antigenic change brought about by cim is scarcely detectable serologically but highly immunogenic for CTL. Biochemical investigations show that cim affects the post-translational modification of RT1.Aa.

Cloning and expression of class I major histocompatibility complex genes of the rat.

Little is known about the organization of class I genes in the rat although there is prima facie evidence that it is distinct from that of the mouse. We report the cloning of 61 nonclassical rat class I genes into cosmid clusters with a total mapped length of 1,264 kb. It is certain that the total number of class I genes in the rat must exceed this number. From restriction maps it is possible to identify substantial regions of duplication. By transfection of cosmids into mouse L cells, it has been possible to demonstrate at least seven different nonclassical rat class I genes that are expressible on the cell surface. Crossreaction of a single mouse monoclonal antibody with all of these class I molecules is consistent with sequence homogenization within the rat nonclassical system. Attempts to find rat homologues of the mouse Tla genes by crosshybridization of rat cosmids with a range of different TLa-specific probes were unsuccessful, suggesting that this large group of divergent class I genes is absent or nearly so from the rat. The large number of class I genes in the rat appears to have arisen by expansion of genes more closely related to the classical sequence.

Comparison of the performance of three variations of the Carbapenem Inactivation Method (CIM, modified CIM [mCIM] and in-house method (iCIM)) for the detection of carbapenemase-producing Enterobacterales and non-fermenters.

OBJECTIVE: The objective of this study was to compare the performance of an in-house CIM (iCIM) modification with the CIM and mCIM for the detection of carbapenemase production in 149 well characterised isolates (70 carbapenemase producers and 79 non-carbapenemase producers). METHODS: Isolates were tested using the CIM, mCIM and iCIM procedures. The gold standard was genotypic characterisation by PCR. RESULTS: For Acinetobacter baumannii, the sensitivity was low (10%) for the mCIM, 70% for the CIM but was 100% for the iCIM, with a specificity of 100% for all three. For Enterobacterales, the sensitivity of all three tests was 100% for Ambler class A and B ß-lactamases, while the iCIM also had a sensitivity of 100% for class D ß lactamases. The sensitivity in Enterobacterales was highest for the iCIM at 100% (CIM 98.2%, mCIM 96.2%). The specificity was 100% for the mCIM and 98% for the CIM and iCIM. For Pseudomonas aeruginosa, the sensitivity of the CIM (100%) was higher than the iCIM (85.7%) and the mCIM (71.4%). iCIM exhibited excellent sensitivity (100%) and specificity (98%) for carbapenemase detection in Enterobacterales and was able to detect two OXA-232 producers that the mCIM did not detect and an OXA-181 producer that the CIM did not detect. CONCLUSIONS: In conclusion, iCIM performed better than the CIM and mCIM for carbapenemase detection in A. baumannii and Enterobacterales, however the CIM achieved the highest sensitivity for carbapenemase detection in P. aeruginosa suggesting that different CIM variations should be utilised depending on the organism type.

Inhibition of experimental allergic encephalomyelitis in rats severely depleted of T cells.

Lewis rats depleted of thymus-derived cells (B rats) failed to develop either experimental allergic encephalomyelitis or antibody against myelin basic protein. Lewis B rats reconstituted with 690 x 10(6) thymocytes developed experimental allergic encephalomyelitis and levels of antibody against myelin basic protein comparable to those of controls. The Lewis B rat model should be useful in the analysis of the role of thymus-derived cell populations and antibody in the induction of experimental allergic encephalomyelitis.

Histological demonstration of mosaicism in a series of chimeric rats produced between congenic strains.

Experimental chimeras were produced by aggregating morulae from congenic strains of PVG rats differing in the major histocompatibility complex (RTI). Monoclonal antibodies against variant class I antigens of the two strains were directly conjugated to iodine-125 and applied to tissue sections. Autoradiograms allowed examination of most internal tissues. The proportion of PVG-RTIa cells in the erythrocyte populations of the chimeras varied from 8 to 70 percent, as determined with fluorescence-activated flow cytometry. Digital analysis of autoradiograms demonstrated that the contribution of PVG-RTIa cells to the livers of the chimeras ranged from 34 to 86 percent. Patches of cells of each genotype in the liver were geometrically complex, with large variations in size. The thymus, but not the spleen, showed evidence of oligoclonal development. The adrenal cortex revealed a radially striped pattern, suggestive of clonal expansion of stem cells. With this approach it is possible to measure cell distribution in chimeras through direct histological visualization, which may prove useful in the study of rat organogenesis.

Nucleotide binding by TAP mediates association with peptide and release of assembled MHC class I molecules.

BACKGROUND: Newly synthesised peptide-receptive major histocompatibility complex (MHC) class I molecules form a transient loading complex in the endoplasmic reticulum with the transporter associated with antigen processing (TAP) and a set of accessory proteins. Binding of peptide to the MHC class I molecule is necessary for dissociation of the MHC class I molecule from the complex with TAP, but other components of the complex might also be involved. To investigate the role of TAP in this process, mutations that block nucleotide binding were introduced into the ATP-binding site of TAP. RESULTS: Mutant TAP formed apparently normal loading complexes with MHC class I molecules and accessory components, but had no nucleotide-binding or peptide-transport activity. Nevertheless, whereas wild-type loading complexes in detergent lysates could be dissociated by addition of peptides that bind MHC class I molecules, mutant complexes could not be dissociated in this way. Depletion of nucleotide diphosphates or triphosphates from wild-type lysates blocked peptide-mediated dissociation of MHC class I molecules, which could be reversed by readdition of nucleotide diphosphates or triphosphates. Complexes between mutant TAP and MHC class I molecules remained associated in vivo until they were degraded. Disruption of nucleotide binding also eliminated TAP's peptide-binding activity. CONCLUSIONS: Peptide-mediated dissociation of the MHC class I molecule from the loading complex depends on conformational signals arising from TAP. Integrity of the nucleotide-binding site is required not only for transmission of this conformational signal to the loading complex, but also for binding of peptide to TAP. Thus, the dynamic activity of the loading complex is synchronised with the nucleotide-mediated peptide-binding and transport cycle of TAP.

Distinct functional properties of the TAP subunits coordinate the nucleotide-dependent transport cycle.

BACKGROUND: The transporter associated with antigen processing (TAP) consists of two polypeptides, TAP1 and TAP2. TAP delivers peptides into the ER and forms a "loading complex" with MHC class I molecules and accessory proteins. Our previous experiments indicated that nucleotide binding to TAP plays a critical role in the uptake of peptide and the release of assembled class I molecules. To investigate whether the conserved nucleotide binding domains (NBDs) of TAP1 and TAP2 are functionally equivalent, we created TAP variants in which only one of the two ATP binding sites was mutated. RESULTS: Mutations in the NBDs had no apparent effect on the formation of the loading complex. However, both NBDs had to be functional for peptide uptake and transport. TAP1 binds ATP much more efficiently than does TAP2, while the binding of ADP by the two chains is essentially equivalent. Peptide-mediated release of MHC class I molecules from TAP was blocked only when the NBD of TAP1 was disrupted. A different NBD mutation that does not affect nucleotide binding has strikingly different effects on peptide transport activity depending on whether it is present in TAP1 or TAP2. CONCLUSIONS: Our findings indicate that ATP binding to TAP1 is the initial step in energizing the transport process and support the view that ATP hydrolysis at one TAP chain induces ATP binding at the other chain; this leads to an alternating and interdependent catalysis of both NBDs. Furthermore, our data suggest that the peptide-mediated undocking of MHC class I is linked to the transport cycle of TAP by conformational signals arising predominantly from TAP1.

Co-evolution of rat TAP transporters and MHC class I RT1-A molecules.

The genes for rat major histocompatibility complex (MHC) class I molecules are associated either with those for the A allele of the transporter associated with antigen processing (TAP-A), which can transport peptides with basic carboxy-terminal residues, or with those for TAP-B, which cannot [1-5]. To explore whether these associations have a functional basis, we compared the sequences of 13 rat MHC class la RT1-A cDNAs from nine MHC haplotypes. Of seven TAP-A- linked RT1-A molecules, six possess strongly acidic F pockets, and these bind a high proportion of peptides with basic carboxy-terminal residues. The F pockets of TAP-B-linked molecules, by contrast, were more basic. Furthermore, we identified six positions at the 'righthand end' of the peptide-binding groove, at which a majority of TAP-B-linked molecules diverge from the consensus sequence for class la molecules whereas, at these positions, all the TAP-A-linked molecules reflect the consensus sequence. Our results suggest that the linked rat class la and TAP genes have co-evolved to maximize the supply of appropriate peptides to the presenting molecules.

Supply and transport of peptides presented by class I MHC molecules.

Three observations suggest that the proteasome, transporter associated with antigen processing (TAP) and class I MHC molecules are co-adapted for the generation, transport and loading of specific peptides. Firstly, TAP preferentially transports peptides close in length to the optimum for class I loading; secondly, genetic variation in TAP specificity focusing in the carboxy-terminal of the peptide correlates with preferences among class I molecules for different peptide carboxy-termini; thirdly, TAP associates directly with empty class I molecules and is released by successful peptide loading. This conclusion puts in question the significance for class I loading of proteolytic processing and peptide generation in the endoplasmic reticulum.

Induction of DNA damage by porphyrin photosensitizers.

The photosensitizing effects of hematoporphyrin derivative, meso-tetra(p-sulfonatophenyl)porphine, meso-tetra(p-carboxyphenyl)porphine, and meso-tetra(4-N-methylpyridyl)-porphine on ColE1 supercoiled DNA were studied using agarose gel electrophoresis. Photoinduced single- and double-strand breaks were observed to form under neutral conditions. Singlet oxygen was shown to predominate in the mechanism of the induction of these lesions in the case of hematoporphyrin derivative, meso-tetra(p-sulfonatophenyl)porphine, and meso-tetra(p-carboxyphenyl)porphine and to play a significant role in the case of meso-tetra(4-N-methylpyridyl)porphine. The results suggest the possibility that the risk of photodynamic carcinogenesis may accompany photochemotherapy and fluorescence endoscopic procedures involving porphyrin photosensitizers.

Temporal order and functional analysis of mutations within the Fli-1 and p53 genes during the erythroleukemias induced by F-MuLV.

The erythroleukemias induced by Friend murine leukemia virus (F-MuLV) result from the accumulation of a number of genetic changes, including activation of the Fli-1 proto-oncogene and inactivation of the p53 tumor suppressor gene. We have determined the temporal order of mutation of the genes involved in this multistage malignancy, by serial in vivo transplantation of F-MuLV induced primary erythroleukemias into syngenic Balb/c mice. These primary tumors are capable of growing when transplanted into syngenic mice, but die after several days of in vitro culture. From the transplanted tumors grown in syngenic mice, erythropoietin-dependent cell lines were established in culture that are clonally related to cells in the primary tumors. We show that retroviral insertional activation of the Fli-1 ets family member is the first detectable genetic event in F-MuLV induced primary erythroleukemias. Mutations in the p53 gene were observed in the Epo-dependent cell lines but not in the transplanted erythroleukemias used to establish these cell lines in culture. These data suggest that activation of Fli-1 plays an important role in the early stages of F-MuLV-induced leukemia, perhaps by altering the self-renewal probabilities of erythroid progenitor cells and that p53 mutations immortalize these cells, enabling them to grow in vitro in the presence of Epo.

Activation of the erythropoietin gene in the majority of F-MuLV-induced erythroleukemias results in growth factor independence and enhanced tumorigenicity.

Retroviral insertional activation of Fli-1 is the first detectable genetic alteration associated with F-MuLV-induced primary erythroleukemias, while mutations within p53 are only observed in Epo-dependent (ED) cell lines derived from syngeneic mice serially transplanted with F-MuLV-induced primary erythroleukemias. In this study we have determined the mechanism of growth factor independence in several Epo-independent (EI) cell lines established from adult mice previously injected with ED-erythroleukemia cell lines or serially transplanted primary tumor cells. Here we have shown constitutive expression of the Epo gene in 12 of 15 (80%) EI-erythroleukemia cell lines. Among these 12 cell lines, eight were shown to possess clonal rearrangement of the Epo gene which could be detected in the tumors used to establish the majority of these EI-cell lines. Analysis of the pattern of proviral integration revealed that the activation of the Epo gene in these cell lines is independent of retroviral insertional mutagenesis, but apparently the result of genomic rearrangements. Furthermore, the acquisition of growth factor independence by these leukemic cells confers a selective growth advantage in vivo and is associated with enhanced tumorigenicity. Together these observations suggest that the activation of the Epo gene in the large majority of these F-MuLV-induced erythroleukemic cell lines establishes an autocrine loop resulting in the constitutive activation of the Epo receptor signal transduction pathway, thereby conferring a growth and survival advantage in vito and in vitro.