Correction of autoimmune lymphoproliferative syndrome by bone marrow transplantation.

This report describes a child with a severe phenotype of autoimmune lymphoproliferative syndrome (ALPS) who developed progressive disease requiring stem cell transplantation. This severe form of ALPS was associated with a novel Fas gene splice site mutation that resulted in functional deletion of exons 8 and 9. While this child shared many clinical features with previously described ALPS cases, including massive lymphadenopathy and circulating alphabeta+ CD3+CD4-CD8-T cells, his disease progressed despite immunosuppressive therapy to a clinically aggressive oligoclonal lymphoproliferation which resembled a diffuse large cell non-Hodgkin's lymphoma. After partial remission was achieved with cytotoxic therapy the patient underwent BMT from an unrelated donor. This is the first reported case of ALPS in which BMT was successfully attempted for correction of a Fas deficiency.

Genetic analysis of ozone-induced acute lung injury in sensitive and resistant strains of mice.

Epidemiological studies have found air pollution to be associated with excessive mortality, particularly death from respiratory and cardiovascular causes. Interpretation of these findings is controversial, however, because toxicological mechanisms controlling mortality are uncertain. Susceptibility to many air pollutants entails an oxidative stress response. Accordingly, the best-characterized oxidant air pollutant is ozone, which causes direct oxidative damage of lung biomolecules. An underlying characteristic derived from clinical and epidemiological studies of healthy and asthmatic individuals of all ages is marked variability in the respiratory effects of ozone. This susceptibility difference among humans suggests that genetic determinants may control predisposition to the harmful effects of ozone. Mice also vary considerably in their response to ozone. Moreover, ozone-induced differences in strain responses indicate that susceptibility in mice can be genetically determined. Therefore, we used inbred mice to investigate the genetic determinants of acute lung injury. Recombinant inbred (RI) strains derived from A/J (A) mice (sensitive) and C57BL/6J (B) mice (resistant) showed a continuous phenotypic pattern, suggesting a multigenic trait. Quantitative trait locus and RI analyses suggested three major loci linked to ozone susceptibility. Differences in phenotype ratios among the reciprocal back-crosses were consistent with parental imprinting. These findings implicate various genetic and epigenetic factors in individual susceptibility to air pollution.

Upregulation of endogenous p53 and induction of in vivo apoptosis in B-lineage lymphomas of E(mu)-myc transgenic mice by deregulated c-myc transgene.

E(mu)-myc transgenic mice carry a constitutively overexpressed c-myc oncogene and develop B-lineage lymphomas. Previous studies have shown that c-myc overexpression can lead to in vitro apoptosis. Here, we investigated the in vivo effects of altered c-myc expression on cell proliferation versus death in spontaneously arising E(mu)-myc tumors. E(mu)-myc tumors display extensive in vivo apoptosis confined to small clusters of cells with greatly increased expression of both the c-myc transgene and the endogenous p53 gene as compared with that in normal, pretumor, or surrounding tumor tissue. This restricted overexpression of both the c-myc transgene and the endogenous p53 gene in small clusters of apoptotic tumor cells indicates that overexpression of these genes and apoptosis are not obligatory or uniform during tumor development and suggests that further somatic mutations or microenvironmental influences may be responsible for these properties. Nevertheless, the clear ability of tumor cells to undergo apoptosis in vivo may be exploitable for therapeutic purposes.

Immunodeficient and immunosuppressed mice as models to test anti-Pneumocystis carinii drugs.

Congenitally immunodeficient and immunosuppressed normal mice with naturally acquired Pneumocystis carinii infection were compared as models for testing anti-P. carinii drugs. Among the immunodeficient mice, mice with severe combined immunodeficiency disease (scid), which lack B and T cells, had higher levels of P. carinii pneumonia than did microMT mice, which lack K cells. Normal mice administered dexamethasone in the drinking water had more extensive pneumocystosis than mice administered parenteral methylprednisolone or hybridoma cells making a monoclonal antibody to CD4 cells. The standard anti-P. carinii drugs trimethoprim (TMP)-sulfamethoxazole (SMX), pentamidine, and atovaquone, which work well in rats and humans, worked well in the mice. Clindamycin and primaquine were effective in the scid and microMT mice but not in the immunosuppressed normal mice. High doses of epiroprim, an analog of TMP, appeared to enhance the activities of low doses of SMX and dapsone, while high doses of TMP did not; however, further studies are needed before definitive conclusions about the actions of these drugs can be drawn. Taken together, the data obtained in this study support the growing body of literature suggesting that the mouse is a valid alternative to the rat as a model for testing anti-P. carinii drugs. Additional differences involving the activities of individual drugs in these models will probably emerge as more experience is gained.

Role of c-myc and CD45 in spontaneous and anti-receptor-induced apoptosis in adult murine B cells.

Although adult murine B cells can be stimulated to proliferate by Igm receptor cross-linking, we and others have shown that these cells will undergo apoptosis in vitro in a dose-, time- and temperature-dependent manner with polyclonal but not monoclonal anti-IgM, To test the role of c-myc and cell cycle progression in B cell apoptosis, we examined normal, Sp6 anti-TNP lg and E micro-myc transgenic splenocytes for receptor-mediated apoptosis in vitro. In normal mice, both spontaneous and anti-IgM-induced programmed cell death were specifically blocked by antisense oligodeoxynucleotides for the c-myc proto-oncogene, whereas nonsense myc oligonucleotides and irrelevant oligonucleotides had only a minor effect. Similarly, TNP-dextran-induced apoptosis in Sp6 anti-TNF transgenics was inhibited by antisense c-myc. This effect was not due to the mitogenic effects of unmethylated CpG-containing sequences because ones lacking this motif, as well as methylated oligonucleotides containing this motif, prevented apoptosis, and mitogenic doses of lipopolysaccharide failed to inhibit anti-IgM-driven cell death. Importantly, antisense c-myc also prevented the anti-IgM-induced increase in myc protein species. Moreover, spontaneous apoptosis in vitro was exaggerated in E micro-myc transgenic B cells. To examine the role of CD45 in anti-IgM-induced apoptosis, we treated spleen cells from CD45 knockout mice, which do not proliferate with anti-IgM, and found that B cells from these underwent apoptosis normally despite the lack of entry into S. These data suggest that anti-IgM driven apoptosis does not require CD45. Rather, apoptosis may be due to an overexpression of myc protein in the absence of signals which can drive B cells productively into S, but the failure to proliferate normally is insufficient for apoptosis to occur.

Heterogeneity of B-lymphoid tumors in E mu-myc transgenic mice.

The clinically important issue of tumor heterogeneity was studied in C57BL/6-E mu-myc transgenic mice, which provide a genetically uniform model system in which all animals eventually develop B cell lymphomas after additional genetic changes beyond enforced expression of the transgenic oncogene. Three different approaches were compared for discerning the cellular and genetic homogeneity of these tumors. Analysis of Igh gene rearrangement showed mainly monoclonality and only infrequent oligoclonality in the tumors from a given animal. In contrast, cytogenetic examination indicated a substantial degree of heterogeneity in the tumors from a given animal and showed that a wide variety of secondary genetic changes occur in E mu-myc transgenic mice. Flow cytometry of DNA content also revealed a high degree of heterogeneity within and among the tumor masses from single E mu-myc mice. Estimates of tumor heterogeneity revealed by these three techniques often did not coincide, indicating that these different approaches reflect distinct cellular parameters. Transgenic E mu-myc mice additionally homozygous for the scid mutation displayed enhanced levels of secondary genetic changes that were valuable for the methodological comparisons performed here, and demonstrated that the extent of tumor heterogeneity can be influenced by specific genes other than the primary E mu-myc transgene. In summary, a combination of methodologies appears to be required to reveal the full extent of tumor heterogeneity within a single individual.

Kinetic and organ-specific patterns of cytokine expression in acute graft-versus-host disease.

Although many cytokines have been previously implicated in graft-versus-host disease (GVHD), no study to date has comprehensively evaluated their expression over time or in different tissues affected by GVHD. Using a semi-quantitative reverse transcriptase-PCR technique and a murine model of acute GVHD, we have evaluated the expression levels of mRNA for a wide range of cytokines in spleen, gut and liver tissues at weekly intervals after bone marrow transfer. The earliest cytokine responses seen were increases in IL-2, IL-10, IFN-gamma, MIP-1 alpha and TNF-alpha in the spleen, suggesting a primarily Th1 pathway. Other cytokines (IL-1 alpha, IL-10 and MIP-1 alpha) were persistently elevated in GVHD mice, but were variable depending on the tissue. These data demonstrate that a wide range of cytokines are involved in the GVHD response and that their kinetic pattern of expression is different in various affected tissues.

Apoptosis and macrophage-mediated deletion of precursor B cells in the bone marrow of E mu-myc transgenic mice.

Transgenic mice expressing the c-myc proto-oncogene under the control of the Ig heavy chain enhancer (E mu-myc) all eventually develop clonal pre-B- or B-cell tumors. The preneoplastic period is characterized by increased polyclonal proliferation of pro-B and pre-B cells in the bone marrow (BM) associated with a reduced number of B cells, suggesting a high degree of B-cell loss. To examine the mechanisms of this cell loss, we have identified B220+ B-lineage cells within the BM of pretumorous E mu-myc transgenic mice by in vivo radiolabeling and electron microscope radioautography. Large mitotic B220(+)-labeled cells form prominent clusters in the extravascular compartment of the BM. Some B220+ small lymphocytes, as well as large lymphoid cells, enter BM sinusoids. However, in addition, large numbers of B220+ cells exhibit nuclear chromatin condensation, fragmentation, and other morphologic features characteristic of apoptotic cell death. Propidium iodide staining and flow cytometry of BM cells from pretumorous E mu-myc transgenic mice, as well as agarose gel electrophoresis of DNA, confirm extensive apoptosis. Many B220+ apoptotic cells are closely associated with the extensive processes of prominent macrophages that contain numerous B220+ apoptotic bodies and complex lysosomal systems. These results suggest that the constitutive expression of c-myc oncogene in BM B-lineage cells, which increases the proliferation of precursor B cells, also leads to increased apoptotic cell death and rapid elimination by resident macrophages. Further mutations may be needed to block these protective mechanisms and permit surviving c-myc-dysregulated cells to leave the BM and to initiate tumorigenesis.

Large-scale genomic comparison using two-dimensional DNA gels.

Two-dimensional electrophoresis (2DE) of DNA fragments, in which separation occurs first by size and then by sequence variation, is a method enabling large-scale comparison of complex genomes. Combining 2DE with probing for various classes of repetitive genomic elements allows rapid and efficient comparison of thousands of fragments and millions of base-pairs of DNA distributed across most genomic regions. This approach is demonstrated here by analyzing the extent of genomic relatedness of different inbred strains of mice. Such strains are shown to differ from each other by approximately 0.2-1% of their nucleotides, above which level reproductive speciation occurs. The 2DE method of assessing the overall relationship between two genomes represents an appropriate tool for analyzing members of a single species, but is too sensitive for use in interspecies comparisons.

A specific intercellular pathway of apoptotic cell death is defective in the mature peripheral T cells of autoimmune lpr and gld mice.

Homozygosity for either of the unlinked murine autosomal recessive mutations lpr or gld leads to autoimmunity characterized by peripheral accumulation of CD4-/CD8- "double-negative" T cells, autoantibodies and various forms of tissue pathology. Recently, the gene affected by lpr was identified as fas, whose product acts as a trigger for programmed cell death or apoptosis. Data reported here indicate that the Fas receptor and its ligand, the wild-type form of the gld gene product, are essential for antigen-stimulated peripheral T cell apoptosis. Furthermore, the wild-type gld gene product is a non-cell-autonomous protein that is produced by activated T cells. Apoptotic elimination of antigen-receptor-triggered peripheral T cells appears to be abnormal in lpr and gld mice, and this deficiency causes peripheral T cells to accumulate resulting in lymphadenopathy. These findings support the importance of apoptotic regulation of lymphocyte persistence after antigen encounter in vivo.

Functional distinctions between MRL-lpr and MRL-gld lymphocytes. Normal cells reverse the gld but not lpr immunoregulatory defect.

Homozygosity for either of the autosomal recessive mutations, lpr or gld, confers an autoimmune syndrome characterized by massive lymphoid hyperplasia and extensive autoantibody production. Despite the similarities in disease progression, functional distinctions in these genetic defects have been observed in bone marrow transplantation studies. To elucidate the mechanisms responsible for the aberrant immune phenotype of these strains, we analyzed interactions between normal T cells and T cells from the two autoimmune strains with regard to their in vitro responses to autologous and allogeneic stimuli and in an in vivo bone marrow transplantation model. Despite similar propensities for lpr and gld T cells to spontaneously proliferate in vitro in response to autologous class II Ag, a dramatic difference in their immunoregulatory properties was found when mixtures of normal and autoimmune CD4+ responder cells were challenged with an allogeneic stimulus. T cells from the lpr, but not gld, mice blocked the normal T cell component of the response. In vivo, the ability of lpr stem cells to trigger a wasting syndrome when transplanted into normal irradiated recipients could not be prevented by including normal stem cells in the inoculum; however, the ability of gld stem cells to transfer the gld-lymphoproliferative syndrome to normal recipients could be prevented with the addition of normal stem cells. These results support a model whereby the lpr and gld strains are defective in reciprocal components of a down-regulatory signaling pathway; failure to express either the functional receptor or ligand leads to a dysregulated immune system resulting in systemic autoimmunity. Based on the linkage between the lpr locus and Fas Ag expression, we propose that the failure of lpr mice to express Fas results in overproduction of Fas-ligand, whereas gld mice fail to make either the Fas-ligand or a functionally related protein, presumably belonging to the TNF family.

Ten nucleotide differences, five of which cause amino acid changes, are associated with the Ah receptor locus polymorphism of C57BL/6 and DBA/2 mice.

We have analysed by heteroduplex formation (HF), single stranded conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), and nucleotide sequencing the cDNAs of the Ahrb-1 and Ahrd allelic forms of the aromatic hydrocarbon receptor (AhR) present in inbred strains of mice. The Ahrb-1 allele, found in the C57BL and C57BR strains, encodes a 95 kDa receptor with an affinity for ligand 15-20 times higher than the affinity of the 104 kDa receptor encoded by the Ahrd allele, found in the DBA/2 strain. Five overlapping fragments of the AhR coding sequence were obtained from liver RNA by reverse transcriptase synthesis of a cDNA first strand, followed by polymerase chain reaction amplification of these cDNA sequences (RT-PCR). Analysis by HF and SSCP revealed the presence of sequence differences in three of the five fragments. When the complete nucleotide sequence of the coding regions was determined by PCR sequencing, we found a total of ten nucleotide differences between the two alleles, nine of which localized to the three fragments where differences were detected by HF and SSCP. Five of the differences are silent. Of the other five, one changes the opal termination codon in Ahrb-1 to the codon for Arg in Ahrd, extending translation of the mRNA by 43 amino acids and accounting for the larger size of the AhR peptide in DBA/2 mice. One of the four remaining differences causes the replacement of a leucine residue in Ahrb-1 by a proline residue in Ahrd, and breaks a potential alpha-helix near the AhR Q-rich region; it is likely that structural changes associated with this amino acid change are responsible for the differences in agonist affinity observed between the Ah receptors of these two strains of mice.

Dynamic associations of CD45 and Thy-1 on plasma membranes of C3H-gld/gld and C3H-lpr/lpr. I. Potential effects on proliferation and phosphatase activity.

Spatial associations of individual plasma membrane components have been proposed as being important in the functional coupling of these molecules. CD45 and Thy-1 associate on the membranes of both transformed and normal lymphocytes as measured by several different methods, and both of these molecules have been found to contribute to T cell activation and proliferation. Thy-1, however, lacks both transmembrane and cytoplasmic domains (it is linked to the plasma membrane through a glucosyl-phosphatidylinositol linkage), obliging the activation and proliferation signals that act through Thy-1 to be transduced by neighboring molecules. In the experiments reported here, the association of Thy-1 and CD45 was examined on lymphocytes from two mutant mouse strains, C3H-gld/gld and C3H-lpr/lpr, both of which exhibit lymphocyte activation and proliferation parameters significantly different from the control C3H/HeJ strain. No significant differences in Thy-1/CD45 association (measured by a heterologous epitope blocking assay) to distinguish mutant from wild-type thymocytes were found. However, spatial associations of CD45 and Thy-1 were altered on both mutant lymph node cells and splenic lymphocytes. These abnormal associations suggested possible effects on the membrane protein tyrosine phosphatase (PTPase) activity associated with CD45. Both C3H-gld/gld and C3H-lpr/lpr lymph node cells were found to have significantly elevated levels of membrane PTPase activity, and this elevation correlated with the anergy to mitogens that develops as these animals age. Finally, monoclonal anti-Thy-1 antibody (G7) reduced the PTPase activity of wild-type membrane isolates, suggesting that Thy-1/PTPase interactions may be a mechanism by which G7 provokes a lymphoproliferative response.

Cell populations during tumorigenesis in Eu-myc transgenic mice.

Transgenic mice bearing a c-myc oncogene under control of the immunoglobulin heavy chain (Igh) enhancer (Eu-myc mice) (1, reviewed in 2) undergo a reproducible series of developmental stages and die from malignancies of the B lymphocyte lineage. To investigate the cellular events underlying tumorigenesis in this model, we quantified B lymphoid subpopulations and turnover at various stages of this process. An early stage was characterized by the presence in the blood of many large proliferating B lineage cells marked by surface antigen phenotype IgM+l-, B220low, CD5-, Mac-1low. During a prolonged intermediate 'remission' phase of different duration in each mouse, B lymphocytes in the periphery were non-proliferative, few, and of conventional phenotype (IgM+, B220+, CD5-, Mac-1-), while subsets of precursor B cells were both numerous and highly proliferative in the bone marrow. In the final stage of tumorigenesis, large proliferating cells similar in phenotype to those of the early period reappeared and increased rapidly in numbers. This B cell tumorigenic progression occurred independently of interactions with T lymphocytes. Evidence of massive cell death in the bone marrow during the intermediate phase, plus molecular characterization of the final tumors, suggested that the end of the peripheral 'remission' period and entry into the terminal stage of tumorigenesis may be due to a clone of cells acquiring the ability to circumvent normal processes of cell death and elimination that usually regulate the egress of B cells from the bone marrow to the periphery.

Single and combined humoral and cell-mediated immunotherapy of Pneumocystis carinii pneumonia in immunodeficient scid mice.

Homozygous mutant scid/scid (severe combined immunodeficiency) mice (referred to as scid mice) lack both specific humoral and cell-mediated immune functions and are exemplary in vivo models for analysis of host-parasite relationships. In our colony, scid mice routinely and predictably develop spontaneous Pneumocystis carinii pneumonia (PCP) with high morbidity. Previous studies have identified both T cells (specifically, CD4+ cells) and antibody as independent mechanisms of effective anti-P. carinii resistance; however, CD4+ T cells also cause an often fatal hyperinflammatory reaction. The current study has explored the optimal application of these immune components for conferring protection against P. carinii. Anti-P. carinii hyperimmune serum was highly effective at reducing the number of P. carinii organisms in early, intermediate, and advanced stages of PCP and was capable of increasing the mean life expectancy of P. carinii-infected scid mice by more than threefold if provided on a continuing basis. When a short course of hyperimmune-serum therapy was provided prior to transfer of P. carinii-sensitized normal lymphocytes, scid mice were rendered permanently free of P. carinii without the pathological sequelae of the hyperinflammatory reaction. These findings are discussed in the contexts of mechanism and clinical relevance.

Lethal exacerbation of Pneumocystis carinii pneumonia in severe combined immunodeficiency mice after infection by pneumonia virus of mice.

Mice homozygous for the mutant allele scid (severe combined immunodeficiency) have been described as excellent models for Pneumocystis carinii (Pc) pneumonia (PCP), a major health problem in patients with acquired immune deficiency syndrome (AIDS) and other immunodeficiency states. Other microorganisms have been shown to infect AIDS patients simultaneously with Pc, but whether one opportunist is able to directly influence the pathogenicity of another has not been determined previously. We have deliberately coinfected scid mice (with extent Pc infection) with a variety of primarily pneumotropic viruses and bacteria and have identified pneumonia virus of mice as causing a dramatic increase in the density of Pc organisms and the morbidity due to PCP in immunodeficient scid mice. This finding has clinical significance in the management of PCP, in that the identification and treatment of coinfecting pneumotropic pathogens may be as important as treatment targeted at Pc. A search for other synergistic (or antagonistic) microorganisms and determination of their mechanism(s) of action in altering the progression of PCP is indicated.

Multiple mechanisms of tumorigenesis in E mu-myc transgenic mice.

Transgenic mice bearing a c-myc oncogene under control of the immunoglobulin heavy chain enhancer (E mu-myc mice) reproducibly develop and die from tumors of the B lymphocyte lineage (J.M. Adams, A.W. Harris, C.A. Pinkert, L.M. Corcoran, W.S. Alexander, S. Cory, R.D. Palmiter, and R.L. Brinster, Nature (Lond.), 318: 533-538, 1985; W.Y. Langdon, A. W. Harris, S. Cory, and J.M. Adams, Cell 47: 11-18, 1986; A.W. Harris, C.A. Pinkert, M. Crawford, W.Y. Langdon, R.L. Brinster, and J.M. Adams, J. Exp. Med., 167: 353-371, 1988; reviewed in S. Cory and J.M. Adams, Annu. Rev. Immunol., 6: 25-48, 1988). Analysis of lymphocytes obtained by serial sampling of peripheral blood from individual hemizygous (E mu-myc/0) and homozygous (E mu-myc/E mu-myc) transgenic mice indicates that proliferation in the original host and transplantability into histocompatible recipients are distinct properties that can be acquired independently and in either order. These two types of transgenic mice differ in that homozygous mice have about one-fourth the life span of hemizygous mice and develop polyclonal, non-transplantable tumors in comparison to the oligoclonal, highly transplantable malignancies seen in hemizygous animals. In conclusion, the overall concept of malignancy is best viewed as an aggregate of the separable parameters of cellular proliferation, clonality, tissue invasiveness, metastasis, and (experimental) transplantability. The E mu-myc transgenic mouse represents an attractive model in which to investigate the multistep nature and alternative pathways of tumorigenesis.

Exacerbation of Pneumocystis carinii pneumonia in immunodeficient (scid) mice by concurrent infection with a pneumovirus.

scid mice naturally infected with Pneumocystis carinii and inoculated with a normally apathogenic pneumovirus had significantly higher P. carinii cyst counts and developed significantly more severe P. carinii-related disease than did sham-inoculated, P. carinii-infected scid mice. P. carinii-free, virus-infected scid mice survived for 2 months despite high pulmonary virus titers. These results show that a respiratory virus infection can exacerbate P. carinii disease in an immunocompromised-rodent model.

Differential cytokine expression in acute and chronic murine graft-versus-host-disease.

The relationship between acute and chronic graft-versus-host disease (GVHD) is not well understood. While both syndromes appear to result from recognition of host antigens by donor T cells, their pathological changes differ markedly. In light of the recent concept that helper T cells (Th) may be divided into two types based on their cytokine secretion profile and their ability to mediate cellular (Th1) or humoral (Th2) immunity, and considering the inflammatory nature of acute GVHD and the occurrence of significant B cell activation in chronic GVHD, we hypothesized that acute and chronic GVHD may be associated with differential cytokine production by activated T cells. To evaluate this hypothesis, we assessed expression of a range of cytokines in (C57BL/6 x DBA/2)F1 (B6D2F1) recipients of C57BL/6 (acute GVHD), DBA/2 (chronic GVHD) or B6D2F1 (control) spleen cells. The results reported here indicate that a wide range of cytokines, including interleukin (IL)-4, IL-10, interferon-gamma, tumor necrosis factor beta and macrophage inflammatory protein-1 alpha, are indeed differentially expressed in acute and chronic GVHD and support the concept that the pathology peculiar to acute or chronic GVHD may arise due to differential cytokine expression by activated T cells.