A dichotomous role for nitric oxide during acute Toxoplasma gondii infection in mice

Production of nitric oxide by macrophages is believed to be an important microbicidal mechanism for a variety of intracellular pathogens, including Toxoplasma gondii. Mice with a targeted disruption of the inducible nitric oxide synthase gene (iNOS) were infected orally with T. gondii tissue cysts. Time to death was prolonged compared with parental controls. Histologic analysis of tissue from infected mice showed scattered small foci of inflammation with parasites in various tissues of iNOS−/− mice, whereas tissue from the parental C57BL/6 mice had more extensive tissue inflammation with few visible parasites. In particular, extensive ulceration and necrosis of distal small intestine and fatty degeneration of the liver was seen in the parental mice at day 7 postinfection, as compared with the iNOS−/− mice where these tissues appeared normal. Serum interferon γ and tumor necrosis factor α levels postinfection were equally elevated in both mouse strains. Treatment of the parental mice with a NO synthase inhibitor, aminoguanidine, prevented early death in these mice as well as the hepatic degeneration and small bowel necrosis seen in acutely infected control parentals. These findings indicate that NO production during acute infection with T. gondii can kill intracellular parasites but can be detrimental, even lethal, to the host.     

T cells cooperate with passive antibody to modify Cryptococcus neoformans infection in mice

Cryptococcus neoformans is an encapsulated fungus that is a major cause of meningitis in patients with AIDS. In immunocompetent mice, administration of IgG1 mAb protects against cryptococcal infection, whereas administration of IgG3 is not protective and can accelerate the infection. In beige mice with impaired natural killer cell function, the effects of IgG1 and IgG3 are similar to those observed in immunocompetent mice, suggesting that natural killer cells are not crucial for antibody-mediated modulation of cryptococcal infection. In mice lacking CD4⁺ T cells, IgG1 is not protective and IgG3 accelerates infection, indicating that CD4⁺ T cells are required for antibody-mediated protection. In mice lacking CD8⁺ T cells, both IgG1 and IgG3 antibodies prolong survival, indicating that acceleration of the disease process by IgG3 involves CD8⁺ T cells. Both IgG1-mediated protection and IgG3-mediated acceleration of infection require interferon γ. These results reveal a functional dependence of passively administered antibody on cellular immunity in cryptococcal infection in mice and have implications for antibody-based therapies in humans in the setting of CD4⁺ lymphopenia.     

A novel role for clathrin in cytokinesis

Using clathrin-minus Dictyostelium cells, we identified a novel requirement for clathrin during cytokinesis. In suspension culture, clathrin-minus cells failed to divide and became large and multinucleate. This cytokinesis deficiency was not attributable to a pleiotropic effect on the actomyosin cytoskeleton, since other cellular events driven by myosin II (e.g., cortical contraction and capping of concanavalin A receptors) remained intact in clathrin-minus cells. Examination of cells expressing myosin II tagged with green fluorescent protein showed that clathrin-minus cells failed to assemble myosin II into a functional contractile ring. This inability to localize myosin II to a particular location was specific for cytokinesis, since clathrin-minus cells moving across a substrate localized myosin II properly to their posterior cortexes. These results demonstrate that clathrin is essential for construction of a functional contractile ring during cell division.     

Genes in the pX region of human T cell leukemia virus I influence Vav phosphorylation in T cells

Human T cell leukemia virus I (HTLV-I) causes acute leukemic disease in a low percentage of infected individuals through obscure mechanisms. Our studies compare two rabbit HTLV-I-infected T cell lines: one, RH/K34, causes lethal experimental leukemia and the other, RH/K30, mediates asymptomatic infection. We show herein that the product of the protooncogene vav is constitutively Tyr-phosphorylated in RH/K34 but not in RH/K30. A role for the retrovirus in phosphorylation of Vav was assigned by transfection experiments with molecular clones of HTLV-I derived from the two lines. The HTLV-I molecular clone from RH/K30, but not that from RH/K34, down-regulates Vav phosphorylation in a Herpesvirus ateles-transformed T cell line. Use of recombinant virus clones revealed that a pX region sequence differing by two nucleotides between the two clones mediates this down-regulation. Because Vav is involved in T cell signaling and Vav phosphorylation occurs upon activation of T cells, control of the activation state of Vav by viral proteins may relate to the leukemogenic potential of certain HTLV-I-infected cells.     

A gene spans the pseudoautosomal boundary in mice

The X and Y chromosomes of the mouse, like those of other mammals, are heteromorphic over most of their length, but at the distal ends of the chromosomes is a region of sequence identity, the pseudoautosomal region (PAR), where the chromosomes pair and recombine during male meiosis. The point at which the PAR diverges into X- and Y-specific sequences is called the pseudoautosomal boundary. We have completed a genomic walk from the X-specific Amelogenin gene to the PAR. Analysis of this region revealed that the pseudoautosomal boundary of mice is located within an intron of a transcribed gene that encodes a novel RING finger protein. The first three of the exons of the gene are located on the X chromosome whereas the 3′ exons of the gene are located on both X and Y chromosomes. This unusual arrangement may indicate that the gene is in a state of transition from pseudoautosomal to X-unique and provides evidence for a process of attrition of the pseudoautosomal region on the Y chromosome.     

Probing the role of packing specificity in protein design

By using a protein-design algorithm that quantitatively considers side-chain packing, the effect of specific steric constraints on protein design was assessed in the core of the streptococcal protein G β1 domain. The strength of packing constraints used in the design was varied, resulting in core sequences that reflected differing amounts of packing specificity. The structural flexibility and stability of several of the designed proteins were experimentally determined and showed a trend from well-ordered to highly mobile structures as the degree of packing specificity in the design decreased. This trend both demonstrates that the inclusion of specific packing interactions is necessary for the design of native-like proteins and defines a useful range of packing specificity for the design algorithm. In addition, an analysis of the modeled protein structures suggested that penalizing for exposed hydrophobic surface area can improve design performance.     

Nitric oxide plays a key role in adaptive control of locomotion in cat

Diverse roles in cellular functions have been ascribed to nitric oxide (NO), and its involvement in induction of long-term depression in cerebellar Purkinje cells has been demonstrated. Manipulations of NO concentration or its synthesis in cerebellar tissues therefore provide a means for investigating roles of NO in cerebellar functions at both cellular and behavioral levels. We tested adaptive control of locomotion to perturbation in cats, and found that this form of motor learning was abolished by application of either an inhibitor of NO synthase or a scavenger of NO to the cerebellar cortical locomotion area. This finding supports the view that NO in the cerebellum plays a key role in motor learning.     

Transgenic mice carrying an imbalance in the native ratio of A to B forms of progesterone receptor exhibit developmental abnormalities in mammary glands

In this report we document the creation of transgenic mice in which the native ratio of A and B forms of progesterone receptor (PR) has been altered by the introduction of additional A form as transgene. We also show that in these mice there is an aberration in mammary development. In ovariectomized prepubertal PR-A transgenic mice, end buds with unusual morphology persist after ovariectomy, and in young adult nonovariectomized mice, mammary glands have extensive lateral branching. The glands of adult mice also exhibit ductal hyperplasia with a disorganized basement membrane and decreased cell–cell adhesion, features commonly associated with neoplasia. Because progesterone is a mitogenic hormone in mammary glands and PR is required for mammary development, these data provide direct evidence that in vivo a regulated expression of the two isoforms of PR is critical for appropriate cellular response to progesterone and that for mammary glands this may have major implications to carcinogenesis.     

T cell receptor restriction of diabetogenic autoimmune NOD T cells

Restricted use of T cell receptor (TCR) gene segments is characteristic of several induced autoimmune disease models. TCR sequences have previously been unavailable for pathogenic T cells which react with a defined autoantigen in a spontaneous autoimmune disease. The majority of T cell clones, derived from islets of NOD mice which spontaneously develop type I diabetes, react with insulin peptide B-(9–23). We have sequenced the α and β chains of TCRs from these B-(9–23)-reactive T cell clones. No TCR β chain restriction was found. In contrast, the clones (10 of 13) used Vα13 coupled with one of two homologous Jα segments (Jα45 or Jα34 in 8 of 13 clones). Furthermore, 9 of 10 of the Vα13 segments are a novel NOD sequence that we have tentatively termed Vα13.3. This dramatic α chain restriction, similar to the β chain restriction of other autoimmune models, provides a target for diagnostics and immunomodulatory therapy.     

A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae

The PKC1–MPK1 pathway in yeast functions in the maintenance of cell wall integrity and in the stress response. We have identified a family of genes that are putative regulators of this pathway. WSC1, WSC2, and WSC3 encode predicted integral membrane proteins with a conserved cysteine motif and a WSC1–green fluorescence protein fusion protein localizes to the plasma membrane. Deletion of WSC results in phenotypes similar to mutants in the PKC1–MPK1 pathway and an increase in the activity of MPK1 upon a mild heat treatment is impaired in a wscΔ mutant. Genetic analysis places the function of WSC upstream of PKC1, suggesting that they play a role in its activation. We also find a genetic interaction between WSC and the RAS–cAMP pathway. The RAS–cAMP pathway is required for cell cycle progression and for the heat shock response. Overexpression of WSC suppresses the heat shock sensitivity of a strain in which RAS is hyperactivated and the heat shock sensitivity of a wscΔ strain is rescued by deletion of RAS2. The functional characteristics and cellular localization of WSC suggest that they may mediate intracellular responses to environmental stress in yeast.     

A critical role of T cell antigen receptor-transduced MHC class I-restricted helper T cells in tumor protection

Adoptive transfer of antigen-specific CD4(+) and CD8(+) T cells is one of the most efficient forms of cancer immunotherapy. However, the isolation of antigen-specific CD4(+) T cells is limited because only few tumor-associated helper epitopes are identified. Here, we used T cell antigen receptor gene transfer to target CD4(+) T cells against an MHC class I-presented epitope of a model tumor antigen. IFN-gamma-producing CD4(+) T cells were unable to expand in vivo and to provide help for tumor rejection. In contrast, CD4(+) T cells producing high levels of IL-2 expanded in vivo, provided help for cytotoxic T lymphocyte-mediated tumor rejection, and developed T cell memory. The data demonstrate in vivo synergy between T cell antigen receptor-transduced CD4(+) and CD8(+) T cells specific for the same epitope resulting in long-term tumor protection.     

A critical role for neutralizing-antibody-producing B cells, CD4+ T cells, and interferons in persistent and acute infections of mice with lymphocytic choriomeningitis virus: Implications for adoptive immunotherapy of virus carriers

This study demonstrates that neutralizing-antibody-producing B cells, CD4⁺ T cells, and interferons (IFNs) are of key importance in virus control both in adoptive immunotherapy of persistent infection and in the late phase of acute infection with the WE strain of lymphocytic choriomeningitis virus (LCMV). We report the following results. (i) Clearance of LCMV-WE from C57BL/6 carrier mice by adoptive transfer of memory spleen cells requires B cells and CD4⁺ T cells but not necessarily CD8⁺ T cells. (ii) At the doses examined, CD8⁺ T cells contribute to the initial reduction of viral titers but are alone not sufficient to clear the virus because they are exhausted. (iii) In the presence of functional IFN-γ, virus clearance correlates well with the generation of neutralizing antibodies in the treated carrier mice. (iv) In the absence of receptors for IFN-γ, virus clearance is not achieved. (v) Adoptive immunotherapy of mice persistently infected with a distinct virus isolate, LCMV-Armstrong, revealed only low levels of neutralizing antibodies; in this case, CD8⁺ T cells were needed for virus clearance in addition to B and CD4⁺ T cells. (vi) After low dose infection of C57BL/6 mice with LCMV-WE, virus is eliminated below detectable levels by CD8⁺ T cells, but long-term (>2 months) virus control is usually not achieved in the absence of B cells or CD4⁺ T cells; reappearance of the virus is paralleled either by exhaustion of virus-specific cytotoxic T lymphocytes or lethal immunopathology. These findings are of importance for adoptive immunotherapy strategies against persistent virus infections in humans.     

Liver repopulation with xenogenic hepatocytes in B and T cell-deficient mice leads to chronic hepadnavirus infection and clonal growth of hepatocellular carcinoma

To investigate host and viral mechanisms determining hepadnaviral persistence and hepatocarcinogenesis, we developed a mouse model by transplanting woodchuck hepatocytes into the liver of mice that contain the urokinase-type plasminogen activator transgene (uPA) and lack mature B and T lymphocytes due to a recombination activation gene 2 (RAG-2) gene knockout. The woodchuck hepatocytes were transplanted via intrasplenic injection and were found to integrate into the recipient mouse liver cord structure. Normal adult woodchuck hepatocytes proliferated and reconstituted up to 90% of the uPA/RAG-2 mouse liver. uPA/RAG-2 mice containing woodchuck hepatocytes were infectable with woodchuck hepatitis virus (WHV) and showed WHV replication for at least 10 months with titers up to 1 × 10¹¹ virions per ml in the peripheral blood. WHV-infected hepatocytes from chronic carrier woodchucks also established a persistent infection in uPA/RAG-2 mice after an 8- to 12-week lag period of viremia. Although WHV envelope, core, and X proteins were produced in the uPA/RAG-2 mice, no inflammatory host immune response was observed in the liver of WHV-replicating mice. A first antiviral test demonstrated a greater than four orders of magnitude drop in WHV titer in response to interferon α treatment. WHV replication was up-regulated by dexamethasone treatment. Comparison of precancerous lesions in donor woodchucks versus recipient uPA/RAG-2 mice revealed an enrichment of dysplastic precancerous hepatocytes in transplanted mice. Clonal amplification of hepatocytes from a woodchuck with hepatocellular carcinomas was demonstrated by the detection of unique WHV DNA integration patterns in hepatocellular carcinomas that arose in uPA/RAG-2 mice. In the absence of B or T cell-mediated immune responses, WHV establishes a persistent noncytotoxic infection of woodchuck hepatocytes in uPA/RAG-2 chimeric mouse livers. Further studies of the kinetics of hepadnavirus infection and replication in quiescent and proliferating hepatocytes should increase our understanding of hepadnavirus spread and aid in the design of therapies to block or cure persistent infection.     

Changes of telomere length cause reciprocal changes in the lifespan of mother cells in Saccharomyces cerevisiae

Budding yeast cells divide asymmetrically, giving rise to a mother and its daughter. Mother cells have a limited division potential, called their lifespan, which ends in proliferation-arrest and lysis. In this report we mutate telomerase in Saccharomyces cerevisiae to shorten telomeres and show that, rather than shortening lifespan, this leads to a significant extension in lifespan. This extension requires the product of the SIR3 gene, an essential component of the silencing machinery which binds to telomeres. In contrast, longer telomeres in a genotypically wild-type strain lead to a decrease in lifespan. These findings suggest that the length of telomeres dictates the lifespan by regulating the amount of the silencing machinery available to nontelomeric locations in the yeast genome.     

CD81 on B cells promotes interleukin 4 secretion and antibody production during T helper type 2 immune responses

Mice lacking CD81 (TAPA-1), a widely expressed tetraspanin molecule, have impaired antibody responses to protein antigens. This defect is specific to antigens that preferentially stimulate a T helper 2 response (ovalbumin or keyhole limpet hemocyanin in alum) and is only seen with T cell-dependent antigens. Absence of CD81 on B cells is sufficient to cause the defect. Also, antigen-specific interleukin (IL) 4 production is greatly reduced in the spleen and lymph nodes of CD81-null mice compared with heterozygous littermates. Thus, expression of CD81 on B cells is critical for inducing optimal IL-4 and antibody production during T helper 2 responses. These findings suggest that CD81 may interact with a ligand on T cells to signal IL-4 production. By using a soluble form of CD81 as a probe, a putative ligand for CD81 was identified on a subset of B and T cells. Two possible models for the interaction of CD81 on B cells with a potential ligand on either B or T cells are proposed.