Stable, stoichiometric delivery of diverse protein functions.

As contemporary "genomics" steadily reveals an increasing number of novel gene sequences, the need for efficient methodologies to functionally characterize these genes in vivo increases significantly. Reliable coupling of target gene expression to a variety of surrogate reporter functions is critical to properly assay novel gene function in complex cell populations. Ideally, independent target and reporter proteins would be derived from a single open reading frame creating a stoichiometric relationship without obscuring subcellular localization. We report here effective strategies for assaying gene function through the stable production of chimeric polyproteins, processed intracellularly by inclusion of an intervening 19-amino-acid sequence from the 2A region of the Foot and Mouth Disease virus. Using drug-resistance and flow cytometry-based assay systems, we demonstrate that diverse protein functions are effectively delivered to various cell types by retroviral constructs as single 2A-cleaved polyproteins. For example, cells infected with a retrovirus encoding a nuclear cell cycle regulator, linked via the 2A-motif to a marker membrane protein, showed a direct correlation between cell cycle arrest and surface marker level. This demonstrates the utility of this methodology for stable and stoichiometric delivery of distinctly localized protein functionalities. In particular, the ability to exploit multiple cellular functions will serve to accelerate the functional characterization of gene products and facilitate novel gene therapy approaches.

Characterization of F21.A, a monoclonal antibody that recognize a leucocyte surface antigen for killer whale homologue to beta-2 integrin.

The specificity of F21.A, a monoclonal antibody raised against bottlenose dolphin leucocytes, was characterized in killer whale on the basis of immunoprecipitation of a protein of 94 kDa, as well as flow cytometric analysis. While minimally expressed on resting cells, F21.A labeled a homologue to beta-2 integrin in 89-97% of PMA-activated neutrophils, 53-66% of activated monocytes, and activated B cells but not T cells. Activation of neutrophils reached its maximum 10 min after PMA stimulation. F21.A did not label intracellular stores as did both cross-reacting anti-canine CD11b and CD18, suggesting that an activation-induced conformational change would expose a neoepitope recognized by F21.A. F21.A labeling was largely inhibited by pre-incubation with plasma, suggesting a binding site closely related to that for fibrinogen. In vitro phagocytosis and respiratory burst were almost fully inhibited upon pre-incubation with F21.A, demonstrating its functional importance. This antibody is foreseen as a possible valuable diagnostic and research tool in cetacean immunology.

Retroviral delivery of peptide modulators of cellular functions.

Stable transduction of genetic material, in combination with sensitive methodologies for in vivo study of cell physiology, provides an opportunity to efficiently evaluate the functions of regulatory proteins. To dissect the minimal therapeutic function of such proteins, we have stably expressed protein microdomains as fusions, composed of short peptides, and detected specific subfunctions distinct from holoprotein function, using flow cytometry and other techniques. We demonstrate that retroviral delivery of the 24-amino-acid proliferating cell nuclear antigen-binding motif (p21C), derived from the C-terminus of the cell cycle inhibitor protein, p21, is sufficient to induce cell cycle arrest. Cells expressing this peptide motif reversibly execute both G1- and G2-checkpoint controls that are normally activated subsequent to interference with DNA synthesis. The p21C effect is distinct from results obtained with an intact p21 protein that also binds cyclin-CDK complexes and arrested cells exclusively at the G1/S transition. Thus, microdomains can exert unique biological effects compared to the parental molecules from which they were derived. To further evaluate the peptide delivery strategy, we analyzed the role of various kinases in IgE-mediated stimulation of mast cell exocytosis. Primary bone marrow-derived mast cells were transduced with retroviral constructs encoding short-kinase inhibitor motifs and analyzed by flow cytometry for effects on exocytosis. We found that a specific protein kinase A (PKA) inhibitor peptide suppressed IgE-mediated stimulation of mast cell exocytosis. This anti-exocytotic effect was mimicked by a small molecule inhibitor of PKA (KT5720). Thus, the ability to express protein microdomains can be a powerful means to subtly perturb cellular physiology in manners that reveal new paths for therapeutic intervention. We believe that such approaches might allow for new forms of gene therapy to become available.

Role of T cells and cytokines in fatal and resolving experimental babesiosis: protection in TNFRp55-/- mice infected with the human Babesia WA1 parasite.

We characterized the cytokine response and T-cell requirements of mice infected with the intraerythrocytic parasites Babesia microti and WA1. WA1 infections were fatal, whereas B. microti infections were resolved. We measured production of tumor necrosis factor (TNF)-alpha, interferon-gamma, interleukin (IL)-10, and IL-4 by splenic CD4+, CD8+, and gammadelta+ T cells using flow cytometry. WA1 inoculation stimulated TNF-alpha production, whereas resolving B. microti infections were characterized by increased IL-10 and IL-4. The role of TNF-alpha in WA1 infections was further investigated by inoculating TNFRp55-/- mice with a lethal dose of WA1. A survival rate of 90% in the TNFRp55-/- mice indicated that a disruption in the TNF-alpha pathway abrogated the pathologic mechanism of WA1. Inoculation of WA1 into CD4-/- and CD8-/- mice resulted in survival rates of 60% and 78%, respectively, whereas WA1 infection in gammadelta-/- and control mice was fatal. These results suggest that CD8+ T cells may contribute to the WA1-associated disease. Babesia-infected CD4-/- mice experienced a longer duration of parasitemia, indicating that CD4+ T cells participate in parasite elimination. These studies demonstrate differences in immune responses during fatal or resolving Babesia infections, and they identify TNF-alpha as an important mediator of the WA1-associated pathogenesis.

Up-regulation of tumor necrosis factor-alpha and interferon-gamma expression in the spleen and lungs of mice infected with the human Babesia isolate WA1.

We analyzed cytokine expression in mice infected with the intraerythrocytic parasites Babesia microti and WA1. In C3H/HeN mice, WA1 infections were fatal, whereas B. microti infections were resolved. We propose that the proinflammatory cytokines tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) contribute to the WA1-associated disease. WA1 infection was characterized by up-regulation of TNFalpha and IFNgamma mRNA in the spleen. Previous studies in WA1-infected mice showed that pathologic lesions occurred primarily in the lungs, including pulmonary edema and intravascular margination of leukocytes. Analysis of cytokine expression in the lungs is important for an understanding of the disease process in WA1-infected mice. Expression of both TNFalpha and IFNgamma mRNA was increased in the lungs of WA1-infected mice. Immunohistochemical staining confirmed the upregulation of these proinflammatory cytokines in the lungs. Expression of TNFalpha and IFNgamma was not up-regulated in the lungs of B. microti-infected mice. The results implicate TNFalpha and IFNgamma in the pathogenesis of WA1-associated disease.

Murine T cell determination of pregnancy outcome.

At the fetomaternal interface, maternal effector cells come in intimate contact with fetal trophoblast cells which express paternal antigens. Failure of fetal trophoblast cells to activate maternal Th1 immune responses has been attributed in part to the absence of classical Class I and Class II major histocompatibilty complex (MHC) antigen expression and elaboration of factors which reduce TcR expression and shift any immune responses which may occur to Th2. Classical TcR alphabeta(+) T cells have not been found to be able to respond to trophoblasts. Recently, TcR gammadelta(+) T cells have been characterized in the low-abortion-rate pregnant C57Bl/10 mouse decidua, and the Vgamma1(+) subset may be able to respond to trophoblasts in a non-MHC-dependent manner. Trophoblast-recognizing T cells with Vgamma1 receptors are also present in the decidua of CBA/J mice pregnant by DBA/2, an abortion-prone mating combination. To test the role of the Vgamma1 subset of decidual gammadelta T cells in abortion-prone pregnancies, we altered this subset by injecting monoclonal anti-Vgamma1.1 antibody on gestation day 5.5, 1 day after implantation. This reduced detectability of a Vgammadelta subset producing TNF-alpha and reduced the abortion rate. Anti-Vgamma2, which reacts with a similar proportion of decidual gammadelta T cells as anti-Vgamma1.1, failed to prevent abortions. Vdelta6.3(+) cells are prominent at the fetomaternal interface, and anti-Vdelta6 antibody injected on day 5.5 prevented abortions. TGF-beta2(+) gammadelta cells first appear on day 8.5 of pregnancy; anti-Vgamma1.1 antibody injection on day 8.5 depleted these cells and boosted abortions; anti-Vdelta6.3 given on day 8.5 boosted abortions to the same level. These results suggest that two populations of Vgamma1.1(+)delta6.3(+) T cells may arise in the decidua: an early population that is Th1, abortogenic, and present during the time of implantation, and a Th2/3 cell subset that is present in the decidua later during pregnancy and which is pregnancy-protective.

Cutting edge: protective response to pulmonary injury requires gamma delta T lymphocytes.

Gamma delta intraepithelial lymphocytes are thought to coordinate responses to pathogens that penetrate the epithelial barrier. To directly test this, mice were inoculated with Nocardia asteroides. At doses that were nonlethal for control mice, gamma delta-deficient mice became severely ill and died within 14 days. Histologic examination of these lungs demonstrated the presence of severe tissue damage and unimpeded bacterial growth in the gamma delta-deficient mice compared with neutrophilic lesions and clearance of the organism in control mice. Interestingly, ozone exposure that targets a comparable lung region also resulted in diffuse epithelial necrosis associated with a similar lack of neutrophil recruitment in gamma delta-deficient mice. These data demonstrate that gamma delta intraepithelial lymphocytes can protect the host from pathogenic and nonpathogenic insults by targeting the inflammatory response to epithelial necrosis.

Antigenic and nucleotide characterization of a herpesvirus isolated from Pacific harbor seals (Phoca vitulina richardsii).

This report describes the antigenic and nucleotide characterization of a herpes-like virus that has been isolated from the adrenal tissues of neonatal Pacific harbor seals. Infection with this virus has been previously implicated as a major cause of death of animals undergoing rehabilitation. Comparison and phylogenetic analysis of sequenced fragments of the DNA polymerase, glycoprotein B and glycoprotein D genes, and immunofluorescence assay using herpesvirus-specific monoclonal antibodies, demonstrated close similarity of the Pacific harbor seal herpesvirus to European isolates of phocid herpesvirus-1 (PHV-1) and other alpha-herpesviruses affecting terrestrial carnivores.

Characterization of a monoclonal antibody that recognizes a lymphocyte surface antigen for the cetacean homologue to CD45R.

As part of our current efforts to develop assays and reagents to study the immune system of marine mammals, and in view of the effort currently made to develop monoclonal antibodies to cell surface proteins of lymphocyte subsets in different species, the present paper reports on the characterization of a monoclonal antibody against the homologue of CD45R on cetacean lymphocytes. The specificity of this antibody has been characterized on the basis of immunoprecipitation of the antigen it recognized, immunoperoxidase staining on cetacean lymph node and thymus sections, as well as one and two-colour flow cytometric analysis of cetacean peripheral blood mononuclear cells and single-cell suspensions of thymus, lymph node and spleen. Anticetacean CD45R (F21.H) immunoprecipitated proteins of 180, 200 and 220 x 10(3) MW, with the 180 x 10(3) MW from being predominantly expressed on T cells and the 220 x 10(3) MW form expressed predominantly on B cells and thymocytes F21.H labelled all B cells and a proportion of T cells on single-cell suspensions of spleen cells. CD45R- killer whale peripheral blood lymphocytes expressed a higher density of CD2 than CD45R+, a characteristic of memory T cells. Killer whale T lymphocytes also lost the expression of CD45R upon activation with concanavalin A (Con A) and phytohaemagglutinin (PHA). This is the first report of a monoclonal antibody to CD45R in cetaceans, and this antibody is foreseen as a possible valuable diagnostic and research tool to assess immune functions of captive and wild cetaceans as part of the evaluation of their health status.

The induction of a protective response in Leishmania major-infected BALB/c mice with anti-CD40 mAb.

A protective immune response to the intracellular parasite Leishmania major requires the development of a Th1 CD4+ T cell phenotype. We demonstrate herein that BALB/c mice, which normally develop a susceptible Th2 response to L. major infection, are protected when co-injected with an agonistic anti-murine CD40 mAb. Anti-CD40 mAb-mediated protection in this system was found to be T cell dependent, since it was not observed in C57BL/6 x 129 mice that were rendered T cell deficient (TCR beta-/- x TCR delta-/-) and L. major susceptible. Anti-CD40 mAb stimulation of L. major-infected BALB/c mice was accompanied by increased IL-12 and IFN-gamma production in draining lymph nodes, analyzed either by direct expression, or in an antigen-specific in vitro recall assay. The protective role of these cytokines was indicated by the finding that anti-CD40 mAb-mediated protection of L. major-infected BALB/c mice could be reversed by co-treating the animals with neutralizing anti-IL-12 and/or anti-IFN-gamma mAb. Collectively, these data suggest that BALB/c mice develop a protective Th1 CD4+ T cell response to L. major infection when co-injected with anti-CD40 mAb. While the CD40-CD40L interaction has been previously shown to be vital in the control of murine Leishmaniasis, the current study establishes in vivo that anti-CD40 mAb treatment alone is sufficient to protect BALB/c mice from L. major infection and raises the possibility of utilizing this approach for vaccination strategies.

Murine T cell determination of pregnancy outcome: I. Effects of strain, alphabeta T cell receptor, gammadelta T cell receptor, and gammadelta T cell subsets.

PROBLEM: T cells bearing alphabeta T cell receptor (TcR) and gammadelta TcR are present at the fetomaternal interface, and the latter, which express surface activation markers, can react with fetal trophoblast cell antigens. What is the role of these cells? METHOD: Using stress-abortion-prone DBA/2-mated CBA/J and abortion-resistant C57/B16 mice, alphabeta, gammadelta, and CD8+/- T cell subsets were measured in spleen and uterine decidua. The effect of immunization against abortion and administration of anti-TcR antibody in vivo was examined. Cytokine synthesis was measured by intracellular staining of Brefeldin A-treated cells. RESULTS: Abortion-prone matings showed an unexpected accumulation of gammadelta T cells beginning in the peri-implantation period and this was suppressed by immunization against abortion. The immunization deleted gammadelta T cells producing the abortogenic cytokines, TNF-alpha and gamma-interferon, and increased production of the anti-abortive cytokines, IL-10 and transforming growth factor-beta2 (TGF-beta2). Immunization also boosted the number of alphabeta T cells which were present in the decidua as early as 2 days after implantation. In vivo injection of GL4 (anti-delta) depleted gammadelta T cells producing Th1 cytokines in the peri-implantation period, and prevented abortions, whereas H57 (anti-beta) decreased the number of alphabeta T cells and led to 100% abortions. CD8+ T cells present in peri-implant decidua before onset of abortions were mostly alphabeta TcR+, although some were gammadelta+. Changes in gammadelta and alphabeta T cells in pregnancy were most dramatic in uterine tissue. CONCLUSION: Although decidual gammadelta T cells after formation of a distinct placenta and fetus produce anti-abortive TGF-beta2-like molecules and IL-10, prior events can lead to abortion. High local production of TNF-alpha and gamma-interferon develop during the peri-implantation phase because of an excessive increase in the Th1 cytokine+ subset of gammadelta cells; these cytokines may be contributed by other tissues in decidua, and the contribution of bioactive factors by gammadelta T cells may augment the cytokine pool. In contrast, alphabeta T cells (which may be inactivated by stress that causes abortions) may mediate the anti-abortive effect of alloimmunization. Alloimmunization involves a shift from a Th1 to a Th2 pattern in the gammadelta T cells in decidua.

Bovine cytokine expression during different phases of bovine leukemia virus infection.

The potential role of aberrant cytokine production in the pathogenesis of bovine leukemia virus (BLV) was studied by analyzing cytokine mRNA expression in pokeweed-stimulated PBMLs of cows in different phases of disease progression. To analyze the mRNA, a semi-quantitative RT-PCR assay was developed. The RT-PCR assay was developed for detection of IL-2, -4, -6, -10, -12, IFN-gamma and actin using cDNA derived from phorbol-stimulated peripheral blood mononuclear leukocytes. Using a PCR specific for BLV tax, agar gel immunodiffusion and white blood cell counts, BLV-negative, BLV-positive aleukemic (AL), and BLV-positive persistently lymphocytotic (PL) cattle were identified. Peripheral blood lymphocytes cultured in vitro for 24 h in pokeweed mitogen were analyzed for cytokine production using the RT-PCR assay. Consistently elevated levels of IL-2 and IL-12 in AL and PL cattle in pokeweed mitogen-stimulated cells was detected, while IFN-gamma was elevated in the AL but not the PL cattle.

Characterization of horse (Equus caballus) immunoglobulin mu chain-encoding genes.

Horse (Equus caballus) immunoglobulin mu chain-encoding (IgM) variable, joining, and constant gene segments were cloned and characterized. Nucleotide sequence analyses of 15 cDNA clones from a mesenteric lymph node library identified 7 unique variable gene segments, 5 separate joining segments, and a single constant region. Based on comparison with human sequences, horse variable segments could be grouped into either family 1 of immunoglobulin (Ig) clan I or family 4 of Ig clan II subclan IV. All horse sequences had a relatively conserved 16 base pair (bp) segment in framework 3 which was recognized with high specificity in polymerase chain reaction by a degenerate oligonucleotide primer. Horse complementarity determining regions (CDR) had considerable variability in predicted amino acid content and length but also included the presence of relatively conserved residues and several canonical sequences that may be necessary in formation of the beta chain main structure and conformation of antigen-binding sites through interaction with light chain CDR. Sequence analysis of joining regions revealed the presence of nearly invariant 3' regions similar to those found in human and mouse genes. A single horse IgM constant region comprising 1472 bp and encoding 451 residues was also identified. Direct comparison of the horse constant region predicted amino acid sequence with those from eleven other species revealed the presence of 53 invariant residues with particularly conserved sequences within the third and fourth exons. Phylogenetic analysis using a neighbor-joining algorithm showed closest similarity of the horse mu chain-encoding constant region gene to human and dog sequences. Together, these findings provide insights into the comparative biology of IgM as well as data for additional detailed studies of the horse immune system and investigation of immune-related diseases.

Regulation of abortion by gamma delta T cells.

PROBLEM: T cells are present at the feto-maternal interface, but their function during pregnancy has not been fully elucidated. T cells bearing gamma delta T-cell receptor (TCR) may be particularly important, as some subsets can react to trophoblast cells by producing cytokines, such as interleukin-2 (IL-2). METHOD: We depleted T cells bearing the gamma delta receptor by injecting monoclonal antibodies (mAb) into females of the abortion-prone animal model CBA x DBA/2. We investigated the percentage and number of gamma delta T-cell receptor positive (TCR)+ cells in decidua and spleen during pregnancy in control and gamma delta-depleted female mice. Pregnant females were also exposed to ultrasonic sound stress to boost the abortion rate. RESULTS: Stress failed to increase the abortion rate in the gamma delta TCR-depleted mice. FACScan analysis show that the ratio of cells bearing the gamma delta TCR dramatically decreased after injection of mAB to the gamma delta TCR in spleen and decidua, these cells recovered six days after depletion, showing a change in cytokine pattern. Levels of TNF-alpha in decidual gamma delta T cells decreased; similar effects of decreasing Th1 cytokines could be observed in splenic gamma delta T cells. We further identified increased levels of intracellular TNF-alpha in the V delta 4 subset in the decidua, compared to spleen. CONCLUSIONS: Trophoblast recognition by the V delta 4 T-cell subset in the decidua may cause the release of abortogenic cytokines such as TNF-alpha. Depletion of such gamma delta TCR T cells during early pregnancy may promote successful pregnancy outcome in normal pregnancy and prevent stress-induced abortions.

Cytokine transcription in lymph nodes of cattle in different stages of bovine leukemia virus infection.

Bovine leukemia virus (BLV) is a transforming oncovirus that contains no oncogenes or preferred site of proviral integration. The role of cytokines in the disease process of BLV is potentially important due to the similarity of BLV with other retroviruses in which cytokines play a role, such as HTLV-I and -II. Mesenteric and supra-mammary lymph nodes were obtained from a panel of nine cattle. Three were non-infected controls, three were BLV-positive aleukemic (AL), and three were BLV-positive persistent lymphocytotic (PL). Mononuclear cells were perfused from the organs and total RNA extracted from either 1 x 10(8) unseparated cells or 1 x 10(7) purified CD4/CD8 T-cells. cDNA was generated and subjected to RT-PCR to analyze cytokine transcription during disease progression. cDNA levels were normalized using beta-actin PCR at sub-plateau cycle number, enabling a semi-quantitative assessment of cytokine gene transcripts. Using this approach, IL-2, IL-10 and IFN-gamma message was detected in the T-cell fractions of all of the BLV-infected animals, but not in the non-infected controls.

CD40 signaling induces interleukin-4-independent IgE switching in vivo.

T cell-deficient T cell receptor (TCR) beta-/- x TCR delta-/- knockout mice lack circulating IgE and fail to produce antigen-specific IgE in response to stimulation with T cell-dependent antigens. We show here that these animals are able to produce significant levels of circulating polyclonal IgE when injected with an agonistic anti-mouse CD40 monoclonal antibody. CD40-mediated induction of circulating polyclonal IgE in T cell-deficient mice was only partially reduced when the animals were co-treated with neutralizing anti-interleukin-4 (IL-4) antibody. The IL-4 independence of this response was further supported by experiments showing that anti-CD40 antibodies induced circulating IgE when injected into IL-4 knockout mice, and sterile RNA epsilon transcript production when cultured with purified B cells from the same mice. These data strongly suggest that CD40 signaling causes IL-4-independent IgE switching in mice.

Identification of a novel selD homolog from eukaryotes, bacteria, and archaea: is there an autoregulatory mechanism in selenocysteine metabolism?

Escherichia coli selenophosphate synthetase (SPS, the selD gene product) catalyzes the production of monoselenophosphate, the selenium donor compound required for synthesis of selenocysteine (Sec) and seleno-tRNAs. We report the molecular cloning of human and mouse homologs of the selD gene, designated Sps2, which contains an in-frame TGA codon at a site corresponding to the enzyme's putative active site. These sequences allow the identification of selD gene homologs in the genomes of the bacterium Haemophilus influenzae and the archaeon Methanococcus jannaschii, which had been previously misinterpreted due to their in-frame TGA codon. Sps2 mRNA levels are elevated in organs previously implicated in the synthesis of selenoproteins and in active sites of blood cell development. In addition, we show that Sps2 mRNA is up-regulated upon activation of T lymphocytes and have mapped the Sps2 gene to mouse chromosome 7. Using the mouse gene isolated from the hematopoietic cell line FDCPmixA4, we devised a construct for protein expression that results in the insertion of a FLAG tag sequence at the N terminus of the SPS2 protein. This strategy allowed us to document the readthrough of the in-frame TGA codon and the incorporation of 75Se into SPS2. These results suggest the existence of an autoregulatory mechanism involving the incorporation of Sec into SPS2 that might be relevant to blood cell biology. This mechanism is likely to have been present in ancient life forms and conserved in a variety of living organisms from all domains of life.