Mechanisms of Establishment and Maintenance of RNA Virus Persistence in Primary Lymphocytes: a Dissertation

RNA virus persistence in lymphocytes has been studied extensively in vitro, but the influence of lymphocyte homeostatic mechanisms and antiviral immunity on persistence has not been well studied in an in vivo system. It is demonstrated here that vesicular stomatitis virus (VSV), a negative-strand RNA virus, is maintained in B lymphocytes in vivo despite the existence of homeostatic mechanisms that drive the cells to proliferate under conditions of B cell deficiency and a strong antibody response to the virus. It is also shown that antiviral antibodies inhibit VSV reactivation from persistently infected primary B cells in vitro. A model is proposed for virus persistence in vivo in which B cell homeostatic signals drive virus expression in some infected cells, resulting in an antibody response, which maintains virus persistence in B cells. In the course of conducting experiments to define the homeostatic signals that might act on persistently infected B cells in vivo, it was found that a fraction of small, resting splenic B cells proliferates after adoptive transfer into B cell deficient hosts (sublethally irradiated, xid, or SCID). This process, termed homeostatic proliferation, is driven by B cell deficiency since proliferation is limited in B cell sufficient hosts. This reveals the existence of a mechanism by which B cells sense their own numbers. The proliferation is unique in that the replicating cells do not upregulate cell surface markers, such as CD25 and B7-2, associated with antigen or mitogen induced proliferation. They do, however, show transient increases in other activation markers (CD69, CD71), demonstrating the action of an inductive signal. Homeostatic proliferation is a property of both mature and immature B cells, but in competition experiments, only mature B cells inhibit proliferation. xid B cells express a defective form of Bruton\u27s tyrosine kinase (Btk); as a result, these cells proliferate poorly in response to stimulation through a number of cell surface receptors including the BCR, IL-5R, IL-10R, the toll-like receptor RP-105, and CD38. Homeostatic proliferation is severely reduced in xid B cells; thus, this process is regulated by a Btk-dependent inductive signal, which is counterbalanced by an inhibitory signal provided by mature B cells. B cell homeostatic proliferation does not rely on transcription factors (c-rel and p50) critical for conventional proliferation induced by antigen or mitogen (c-rel), or for peripheral B cell survival (p50), suggesting that multiple signals drive this process and that survival and proliferation signals are not identical. VSV persists in small, resting primary B cells for several weeks in vitro, and virus replication is restricted at multiple levels depending on the activation state of the cells. After adoptive transfer of infected B cells into B cell deficient (xid) recipients, viral RNA, but not infectious particles, can be detected by RT-PCR in recipient spleens for at least 72 days. RT-PCR analysis of FACS sorted donor cells stained with CFSE reveals that viral RNA is maintained in transferred B cells but can also found in recipient cells. Infected B cells can undergo homeostatic proliferation and an antibody response is generated to the virus, suggesting that homeostatic signals induce virus expression in some transferred cells. Virus persistence is maintained despite an active immune response to the virus. In fact, persistence may be maintained by antiviral antibody since in vitro treatment of infected primary B cells with anti-VSV antibody inhibits virus reactivation at multiple levels (transcription, protein synthesis, assembly/release of infectious particles). This inhibition is reversible upon antibody removal, demonstrating that functional virus is maintained in antibody treated cells. Antibody specific for a single viral protein (VSV G) is sufficient since inhibition is mediated by monoclonal antibodies specific for a VSV G; neutralizing activity is not required because inhibition occurs with non-neutralizing monoclonal antibodies to VSV G. It is proposed that antibody binding to VSV G on infected B cells generates inhibitory signal(s) that suppress signaling pathways required for virus replication in B cells. Finally, a model of RNA virus persistence in B cells is proposed in which lymphocyte homeostatic signals promote virus expression, leading to the production of antiviral antibodies, which suppress virus replication inside infected B cells and help to maintain persistence

Biosorption potential of dead and living Arthrobacter viscosus biomass in the removal of Cr(VI): Batch and column studies

Batch experiments were conducted with dead and living Arthrobacter viscosus biomass for Cr(VI) removal from aqueous solution. Both dead and living cells successfully reduced Cr(VI) to Cr(III) from aqueous solution in highly acidic pH (pH 1 and 2) with an efficiency of 100% for aqueous solutions having the initial concentrations of Cr(VI) lower than 100 mg/L. Langmuir isotherm and kinetic models based on reduction could simulate chromium removal at 5 and 8 g/L biosorbent dosages and in highly acidic pH conditions (pH = 1-2). Further, the potential use of the Arthrobacter viscosus biomass was examined in an open system, where Cr(VI) removal from aqueous solution was performed by a bacterial biofilm supported on a new type of polyethylene supports. The experiment showed a favorable uptake of chromium ions bound to the biomass, of 20.37 mg/g, with high potential for scaling up. This study showed that the reduction of toxic Cr(VI) to the less toxic Cr(III) by Arthrobacter viscosus, in batch and continuous modes is an efficient and promising technique for wastewaters polluted with chromium.This paper was elaborated with the support of BRAIN project Doctoral scholarships as an investment in intelligence - ID 6681, financed by the European Social Found and Romanian Government and with the support of a grant of the Romanian National Authority for Scientific Research, CNCS – UEFISCDI, project number PN-II-ID-PCE-2011-3-0559, Contract 265/2011. H. Figueiredo is thankful to “FCT – Fundação para a Ciência e Tecnologia” for the financial support through the concession of PhD grant SFRH/BD/28201/2006.info:eu-repo/semantics/publishedVersio

S1P1 receptor directs the release of immature B cells from bone marrow into blood

S1P1 receptor expression is required for the egress of newly formed T cells from the thymus and exit of mature T and B cells from secondary lymphoid organs. In this study, we deleted the expression of the S1P1 receptor gene (S1pr1) in developing B cells in the bone marrow. Although B cell maturation within the bone marrow was largely normal in the B cell–specific S1pr1 knockout (B-S1pr1KO) mice, their newly generated immature B cells appeared in the blood at abnormally low numbers as compared with control mice. In the bone marrow of B-S1pr1KO mice, immature B cells in contact with the vascular compartment displayed increased apoptosis as compared with control mice. Forced expression of CD69, a negative regulator of S1P1 receptor expression, in developing bone marrow B cells also reduced the number of immature B cells in the blood. Attenuation of CXCR4 signaling, which is required for the proper retention of developing B cells in bone marrow, did not release immature B cells into the blood of B-S1pr1KO mice as effectively as in control mice. Our results indicate that the S1P1 receptor provides a signal necessary for the efficient transfer of newly generated immature B cells from the bone marrow to the blood

Expansion of immunoglobulin-secreting cells and defects in B cell tolerance in Rag-dependent immunodeficiency

The contribution of B cells to the pathology of Omenn syndrome and leaky severe combined immunodeficiency (SCID) has not been previously investigated. We have studied a mut/mut mouse model of leaky SCID with a homozygous Rag1 S723C mutation that impairs, but does not abrogate, V(D)J recombination activity. In spite of a severe block at the pro–B cell stage and profound B cell lymphopenia, significant serum levels of immunoglobulin (Ig) G, IgM, IgA, and IgE and a high proportion of Ig-secreting cells were detected in mut/mut mice. Antibody responses to trinitrophenyl (TNP)-Ficoll and production of high-affinity antibodies to TNP–keyhole limpet hemocyanin were severely impaired, even after adoptive transfer of wild-type CD4+ T cells. Mut/mut mice produced high amounts of low-affinity self-reactive antibodies and showed significant lymphocytic infiltrates in peripheral tissues. Autoantibody production was associated with impaired receptor editing and increased serum B cell–activating factor (BAFF) concentrations. Autoantibodies and elevated BAFF levels were also identified in patients with Omenn syndrome and leaky SCID as a result of hypomorphic RAG mutations. These data indicate that the stochastic generation of an autoreactive B cell repertoire, which is associated with defects in central and peripheral checkpoints of B cell tolerance, is an important, previously unrecognized, aspect of immunodeficiencies associated with hypomorphic RAG mutations

Investigations of the Mechanism of the 12-Molybdophosphoric Acid Reactionand Application to a Rapid Quantitative Reaction-Rate Procedure for Phosphate

167 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1969.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Transesterification of soybean oil with menthanol and acetic acid at lower reaction severity under subcritical conditions

Soybean oil (56–80 g) was reacted with methanol (40–106 mL) to produce fatty acid methyl ester in the presence of 1–6% acetic acid under subcritical condition at 250 _C. Stirring and loading of the reaction system affected the yield and severity of the process. The presence of acetic acid improved the yield of FAME from 32.1% to 89.5% at a methanol to oil molar ratio of 20 mL/g. Acetic acid was found to act strongly as an acid catalyst and to some extent improved the solubility between oil and methanol. Reaction pressure higher than the supercritical pressure of methanol (7.85 MPa) was not required to achieve high FAME yield (89.5–94.8%) in short time (30–60 min)

Expression of a human coxsackie/adenovirus receptor transgene permits adenovirus infection of primary lymphocytes

Replication-defective adenoviruses are effective vehicles for gene transfer, both for the repair of defective genes and for studies of gene function in primary cells. Many cell types, including lymphocytes, are refractory to adenovirus infection because they lack the Coxsackie/adenovirus receptor (CAR) needed for virus attachment. To extend the advantages of adenovirus-mediated gene transfer to primary lymphoid populations and other cell types lacking endogenous CAR, we produced a mouse that expresses human (h) CAR as a transgene under control of a murine MHC class I promoter. hCAR protein is expressed on T and B lymphocytes from a variety of organs (spleen, lymph node, bone marrow, thymus, and peritoneum). These lymphocytes are susceptible to adenovirus infection, as demonstrated by reporter green fluorescent protein gene expression, with the fraction of expressing cells as high as 70%. Some lymphocyte subpopulations required stimulation subsequent to adenovirus infection for reporter expression. This activation requirement is a restriction imposed by the promoter used in the adenovirus construct. In subpopulations requiring activation, the elongation factor 1 promoter was far superior to a hCMV promoter for directing green fluorescent protein production. We also find that hCAR mRNA is produced in nonlymphoid tissues from all founder lines, including tissues that do not express endogenous murine CAR, suggesting the opportunity for effecting gene delivery to and testing gene function in a wide variety of primary cell types previously resistant to gene transfer