Matriptase autoactivation is tightly regulated by the cellular chemical environments.

The ability of cells to rapidly detect and react to alterations in their chemical environment, such as pH, ionic strength and redox potential, is essential for cell function and survival. We present here evidence that cells can respond to such environmental alterations by rapid induction of matriptase autoactivation. Specifically, we show that matriptase autoactivation can occur spontaneously at physiological pH, and is significantly enhanced by acidic pH, both in a cell-free system and in living cells. The acid-accelerated autoactivation can be attenuated by chloride, a property that may be part of a safety mechanism to prevent unregulated matriptase autoactivation. Additionally, the thio-redox balance of the environment also modulates matriptase autoactivation. Using the cell-free system, we show that matriptase autoactivation is suppressed by cytosolic reductive factors, with this cytosolic suppression being reverted by the addition of oxidizing agents. In living cells, we observed rapid induction of matriptase autoactivation upon exposure to toxic metal ions known to induce oxidative stress, including CoCl2 and CdCl2. The metal-induced matriptase autoactivation is suppressed by N-acetylcysteine, supporting the putative role of altered cellular redox state in metal induced matriptase autoactivation. Furthermore, matriptase knockdown rendered cells more susceptible to CdCl2-induced cell death compared to control cells. This observation implies that the metal-induced matriptase autoactivation confers cells with the ability to survive exposure to toxic metals and/or oxidative stress. Our results suggest that matriptase can act as a cellular sensor of the chemical environment of the cell that allows the cell to respond to and protect itself from changes in the chemical milieu.

Inflammatory monocytes orchestrate innate antifungal immunity in the lung.

Aspergillus fumigatus is an environmental fungus that causes invasive aspergillosis (IA) in immunocompromised patients. Although -CC-chemokine receptor-2 (CCR2) and Ly6C-expressing inflammatory monocytes (CCR2⁺Mo) and their derivatives initiate adaptive pulmonary immune responses, their role in coordinating innate immune responses in the lung remain poorly defined. Using conditional and antibody-mediated cell ablation strategies, we found that CCR2⁺Mo and monocyte-derived dendritic cells (Mo-DCs) are essential for innate defense against inhaled conidia. By harnessing fluorescent Aspergillus reporter (FLARE) conidia that report fungal cell association and viability in vivo, we identify two mechanisms by which CCR2⁺Mo and Mo-DCs exert innate antifungal activity. First, CCR2⁺Mo and Mo-DCs condition the lung inflammatory milieu to augment neutrophil conidiacidal activity. Second, conidial uptake by CCR2⁺Mo temporally coincided with their differentiation into Mo-DCs, a process that resulted in direct conidial killing. Our findings illustrate both indirect and direct functions for CCR2⁺Mo and their derivatives in innate antifungal immunity in the lung.

Proteasome inhibitors act as bifunctional antagonists of human immunodeficiency virus type 1 latency and replication.

BACKGROUND: Existing highly active antiretroviral therapy (HAART) effectively controls viral replication in human immunodeficiency virus type 1 (HIV-1) infected individuals but cannot completely eradicate the infection, at least in part due to the persistence of latently infected cells. One strategy that is being actively pursued to eliminate the latent aspect of HIV-1 infection involves therapies combining latency antagonists with HAART. However, discordant pharmacokinetics between these types of drugs can potentially create sites of active viral replication within certain tissues that might be impervious to HAART. RESULTS: A preliminary reverse genetic screen indicated that the proteasome might be involved in the maintenance of the latent state. This prompted testing to determine the effects of proteasome inhibitors (PIs) on latently infected cells. Experiments demonstrated that PIs effectively activated latent HIV-1 in several model systems, including primary T cell models, thereby defining PIs as a new class of HIV-1 latency antagonists. Expanding upon experiments from previous reports, it was also confirmed that PIs inhibit viral replication. Moreover, it was possible to show that PIs act as bifunctional antagonists of HIV-1. The data indicate that PIs activate latent provirus and subsequently decrease viral titers and promote the production of defective virions from activated cells. CONCLUSIONS: These results represent a proof-of-concept that bifunctional antagonists of HIV-1 can be developed and have the capacity to ensure precise tissue overlap of anti-latency and anti-replication functions, which is of significant importance in the consideration of future drug therapies aimed at viral clearance.

Isolation of multilineage progenitors from mouse brain.

Stem cells are unique cell populations with the ability to undergo self-renewal and differentiation. These cells have been identified in a wide range of tissues and possess varied differentiation potentials. Tissue-specific stem cells have typically been thought to have limited differentiation capabilities. We show here that fibroblast-like cells isolated from mouse brain possess cross-germ layer differentiation abilities. These cells were found to express typical mesenchymal stem cell markers (CD44, CD29, and CD105) and were able to be passaged more than 50 times. When treated under defined conditions, the brain-derived cells were able to generate many different cell types including adipocytes, osteocytes, astrocytes, neurons, and even hepatocyte-like cells. The hepatocyte-like cells not only expressed liver cell-specific markers, but also exhibited the capacity for glycogen storage and low-density lipoprotein uptake. These results demonstrate the existence of cells in the brain with three-germ-layer differentiation potential.

Post-transcriptionally regulated expression system in human xenogeneic transplantation models.

Cells have developed a mechanism to discriminate between premature termination codons (PTCs) and normal stop codons during translation, sparking vigorous research to develop drugs promoting readthrough at PTCs to treat genetic disorders caused by PTCs. It was posed that this concept could also be applied to regulated gene therapy protocols by incorporating a PTC into a therapeutic gene, so active protein would only be made after administration of a readthrough agent. The strengths of the system are highlighted here by results demonstrating: (i) background expression levels were reduced to 0.01% to 0.0005% of wild type in unselected mass populations of cells depending upon the specific stop codon utilized and its position within the gene; (ii) expression levels responded well to multiple "On" and "Off" regulation cycles in vivo in human xenograft systems; (iii) the level of induction approached three logs using aminoglycoside activators including NB54, a newly synthesized aminoglycoside with significantly reduced toxicity; and (iv) expression levels could be appreciably altered when employing different promoters in a variety of cell types. These results strongly support the contention that this system should have important clinical applications when tight control of gene expression is required.

Efficient co-expression of bicistronic proteins in mesenchymal stem cells by development and optimization of a multifunctional plasmid.

INTRODUCTION: Local synthesis of interferon within B16 tumors mediates anti-tumor effects. Based on reports that stem cells are recruited to tumors, and because systemic administration of interferon causes dose-limiting undesirable side effects, we wanted to improve the anti-tumor effects of interferon while simultaneously minimizing its systemic side effects by employing mesenchymal stem cells (MSCs) as tumor-localized ectopic producers of interferon. Many vectors exist to fulfill this purpose, but their transfection efficiency and resulting expression levels vary considerably. METHODS: To follow both the recruitment to tumors and the synthesis of interferon by MSCs, we designed a bicistronic vector system that permits fluorescent visualization of vector-transfected and interferon-producing MSCs. We used Mu-IFNαA cDNA as the first cistron and the cherry fluorescent protein cDNA as the second cistron, whose translation requires the internal ribosome entry sequence (IRES) from the encephalomyocarditis virus 5' untranslated region. Observing inconsistent expression of these cistrons in various vectors and cell lines, especially compared with a control plasmid pmaxGFP, we optimized the expression of this bicistronic message by mutating pcDNA3 to facilitate exchange of the promoter and polyadenylation segments controlling both the gene of interest and the eukaryotic antibiotic resistance gene as well as the eukaryotic antibiotic resistance gene itself, and effectively compare the effects of these exchanges, creating plasmid pc3.5. RESULTS: Murine MSCs stably and ectopically expressing Mu-IFNαA inhibited the establishment of tumors in homogeneic C57/BL6 mice. Mu-IFNαA expressed from the bicistronic message is fully biologically active, but is expressed at only two-thirds of the level observed from a monocistronic message. Cap-dependent translation is threefold more efficient than IRES-driven translation in 293T, B16, and MSC cell lines. Both efficient expression and good transfection efficiency require strong expression of the gene of interest and a chimeric intron. High doses of Mu-IFNαA within tumors inhibited tumor establishment but may not inhibit tumor growth. CONCLUSIONS: Our modified vector and its derived plasmids will find use in stem cell therapeutics, gene expression, mRNA regulation, and transcription regulation. Local release of Mu-IFNαA within tumors may differently affect tumor establishment and tumor growth.

Defective ubiquitin-mediated degradation of antiapoptotic Bfl-1 predisposes to lymphoma.

The antiapoptotic Bcl-2 family member Bfl-1 is up-regulated in many human tumors in which nuclear factor-kappaB (NF-kappaB) is implicated and contributes significantly to tumor cell survival and chemoresistance. We previously found that NF-kappaB induces transcription of bfl-1 and that the Bfl-1 protein is also regulated by ubiquitin-mediated proteasomal degradation. However, the role that dysregulation of Bfl-1 turnover plays in cancer is not known. Here we show that ubiquitination-resistant mutants of Bfl-1 display increased stability and greatly accelerated tumor formation in a mouse model of leukemia/lymphoma. We also show that tyrosine kinase Lck is up-regulated and activated in these tumors and leads to activation of the IkappaB kinase, Akt, and extracellular signal-regulated protein kinase signaling pathways, which are key mediators in cancer. Coexpression of Bfl-1 and constitutively active Lck promoted tumor formation, whereas Lck knockdown in tumor-derived cells suppressed leukemia/lymphomagenesis. These data demonstrate that ubiquitination is a critical tumor suppression mechanism regulating Bfl-1 function and suggest that mutations in bfl-1 or in the signaling pathways that control its ubiquitination may predispose one to cancer. Furthermore, because bfl-1 is up-regulated in many human hematopoietic tumors, this finding suggests that strategies to promote Bfl-1 ubiquitination may improve therapy.

Cross-reactive antibodies induced by xenogeneic IgA can cause selective IgA deficiency.

Selective immunoglobulin A deficiency (sIgAD) is the most common immunodeficiency in humans. Auto-reactive antibodies to human immunoglobulin A (IgA) are found in the serum of 20-40% of individuals with sIgAD. It is unknown whether these antibodies play a role in the pathogenesis of this immunodeficiency and although the prevailing thought is that they are secondary to the onset of sIgAD, there is very little, if any, support for this notion. Here, we propose that anti-IgA antibodies are in fact responsible for the removal of IgA from serum, and that the inducing antigen is most probably a xenogeneic IgA. This hypothesis is based on data obtained from an sIgAD patient in whom changes in dietary consumption of beef and/or bovine dairy products resulted in changes in anti-IgA levels in the serum. To test the hypothesis, the presence of anti-bovine IgA antibodies was tested by a highly specific enzyme-linked immunosorbent assay in serum samples from IgA-deficient and control individuals. All 13 sIgAD individuals with anti-IgA antibodies had a higher titer against bovine IgA than against human IgA. Of 23 control individuals, a surprisingly high proportion (65%) was also found to have IgG anti-bovine IgA antibodies. These results support the hypothesis that the anti-human IgA antibodies found in IgA-deficient individuals are originally produced against bovine IgA. These antibodies are found in many normal individuals, but only in cases where they cross react with endogenous human IgA, sIgAD may develop.

In vivo post-transcriptional regulation of CD154 in mouse CD4+ T cells.

Interactions between CD40 and its ligand CD154 are involved in the progression of both cell mediated and innate immunity. These interactions are brought about by the transient expression of CD154 on activated CD4(+) T cells, which is regulated, in part, at the level of mRNA turnover. Here we have focused on analyzing the pattern of post-transcriptional regulation in mouse CD4(+) T cells in response to activation. Initial experiments identify a region of the murine CD154 mRNA that binds a polypyrimidine tract-binding protein-containing complex (mComplex I), which is activation-dependent and binds to a single CU-rich site within the 3' uTR Subsequent findings demonstrate that in vivo polyclonal activation of T cells leads to a pattern of differential CD154 mRNA stability that is directly dependent on extent of activation. Furthermore, in vitro activation of antigen-primed T cells shows that the CD154 mRNA half-life increases relative to that of unprimed cells. Importantly, this is the first report demonstrating that the regulation of CD154 in vivo is connected to an activation-induced program of mRNA decay and thus provides strong evidence for post-transcriptional mechanisms having a physiological role in regulating CD154 expression during an ongoing immune response.

New strategies for immune-mediated anti-viral drug and vaccine development.

Substantial progress in the development of new anti-viral drugs has taken place in recent years. Most of these new drugs belong to three groups of compounds, nucleoside analogs, thymidine kinase-dependent nucleotide analogs and specific viral enzyme inhibitors. Although these drugs revolutionized the treatment of several viral diseases, the involvement of the immune system is crucial for complete recovery and prevention of re-infection. New advances in the understanding of immune regulation mechanisms, mainly the role of cytokines, led to the development of several new immunologically-based anti-viral drugs and treatments. The most studied group of immunomodulators is the cytokines, some of which were shown to act as potent stimulators of immune responses. Other, non-cytokine immune modulators have also been successfully employed in both humans and experimental animals as anti-viral drugs of which several are currently in clinical trials. Advances in genetic engineering and transgenic mouse technologies facilitated the production of humanized as well as authentic human anti-viral monoclonal antibodies. Some of these antibodies proved to be clinically efficacious and are commercially produced as anti-viral drugs. As is often the case in anti-viral treatments, a combination of conventional and an immune-mediated anti-viral drugs or a combination therapy involving immunomodulators, therapeutic vaccines, immune intervention and even gene therapy might prove most efficacious as a treatment for a particular virus. Most of the advances made in anti-viral treatments have also been applied to the development of new vaccines. Some of the classical attenuated viruses are being replaced by recombinant attenuated viruses. Recombinant viral vaccines containing genes encoding other viral antigens and/or cytokines are being tested as new vaccines. Several chimeric viral vaccines have proven efficacious in experimental animals and are now in different phases of clinical trials. This review will encompass the major new developments in this field, including some that have not yet been subjected to human trials.

Complete protection from relapsing experimental autoimmune encephalomyelitis induced by syngeneic B cells expressing the autoantigen.

Experimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis (MS), is a typical CD4(+) T-cell-mediated autoimmune disease of the central nervous system (CNS) characterized by perivascular inflammation culminating in focal demyelinations. Like MS, EAE induced by proteolipid protein (PLP) usually follows the form of a relapsing-remitting disease. We have previously described an immunotherapy model in which infusion of autologous B cells expressing the PLP encephalitogenic determinant induced PLP-specific unresponsiveness and protected mice from induction of EAE. Here we show that the same treatment when initiated after disease onset, which resembles the clinical situation presented in MS, completely protects all treated animals from further relapses. We also show that protected animals were unresponsive to PLP as measured by delayed-type hypersensitivity (DTH). This represents a novel immunotherapeutic approach that can be exploited to develop treatments for human MS and other T-cell-mediated autoimmune diseases.

Host stem cells can selectively reconstitute missing lymphoid lineages in irradiation bone marrow chimeras.

The regulatory elements governing the process of lymphopoiesis from pluripotential stem cells to mature lymphocytes are not well understood. In this study we found that in bone marrow chimeras made by reconstituting lethally irradiated normal mice with bone marrow taken from genetically B-cell-deficient animals (microMT.B6 --> F1) the B-cell compartment is reconstituted with host-derived B cells. Similarly, in animals reconstituted with bone marrow taken from mice with genetic deficiencies in the development of T cells (TCR-/- --> F1) or both B and T cells (RAG-/- --> F1), the missing lymphocyte lineage(s) was specifically reconstituted from host-derived cells. In all chimeras, all other blood lineages were generated from donor-derived stem cells. Control chimeras (B6 --> F1) had only donor-derived hematopoietic cells as expected. The reconstituted, host-derived lymphoid compartments contained normal functional cell populations as determined by the presence of T cells expressing all 16 common TCR Vbeta families, and the presence of all antibody isotypes in the serum. Reconstituted TCR-/- --> F1 chimeras were also able to mount T-cell proliferative responses to foreign antigens equal to those of control animals. This observation would seem to suggest that during lymphopoietic reconstitution, missing lymphoid lineages can dictate their own reconstitution.

Regulation of gene expression in experimental autoimmune encephalomyelitis indicates early neuronal dysfunction.

Multiple sclerosis is an inflammatory, demyelinating disease of the CNS. Whereas oligodendrocytes have been considered the primary neural cell type most affected, recent evidence indicates that axonal and neuronal degeneration also occurs in both multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), an animal model reproducing many features of multiple sclerosis. The molecular mechanisms underlying neuronal deficits in multiple sclerosis and EAE remain elusive. To address this issue, we have analysed the expression of genes encoding proteins that play critical roles in ion homeostasis, exocytosis, mitochondrial function and impulse conduction in the Lewis rat lumbar spinal cord during the clinical course of acute EAE. Transcript and protein levels of plasma membrane Ca(2+) ATPase 2 (PMCA2), an essential ion pump expressed exclusively in grey matter and involved in Ca(2+) extrusion, synapsin IIa and syntaxin 1B, important regulators of vesicular exocytosis, were dramatically decreased coincident with the onset of clinical symptoms. In contrast, changes in the expression of several other ion pumps, vesicular proteins, mitochondrial enzymes and sodium channels occurred at more advanced disease stages. Moreover, exposure of spinal cord slice cultures to kainic acid significantly reduced PMCA2 mRNA levels. Taken together, our findings suggest that glutamate, which recently has been implicated in EAE pathogenesis, suppresses neuronal PMCA2 expression leading to Ca(2+) dyshomeostasis at initial clinical phases. Consequently, perturbations in Ca(2+) balance and neurotransmitter exocytosis may partially underlie aberrant neuronal function and communication at onset of symptoms. Altered mitochondrial function and impulse conduction may exacerbate neurological deficits at subsequent disease stages.

The 3C protease activity of enterovirus 71 induces human neural cell apoptosis.

The human glioblastoma SF268 cell line was used to investigate the induction of apoptosis by the 3C protease of enterovirus 71 (EV71). Transient expression in these cells of the wild-type 3C protein encoded by EV71 induced morphological alterations typical of apoptosis, including generation of apoptotic bodies. Degradation of cellular DNA in nucleosomes was also observed. When two of the amino acids in the catalytic motif of 3C were changed by mutagenesis, the 3C protein not only lost its proteolytic activity, but also its ability to induce apoptosis in the SF268 cells. Twenty-four hours after 3C transfection, poly(ADP-ribose) polymerase, a DNA repair enzyme, was cleaved, indicating that caspases were activated by the expression of EV71 3C. The 3C-induced apoptosis was blocked by the caspase inhibitors DEVD-fmk and VAD-fmk. Our findings suggest that the proteolytic activity of 3C triggers apoptosis in the SF268 cells through a mechanism involving caspase activation and that this apoptotic pathway may play an important role in the pathogenesis of EV71 infection.