In vitro and in vivo characterization of a murine cytomegalovirus with a mutation at open reading frame m166

We have recently generated a pool of murine cytomegalovirus (MCMV) mutants by using a Tn3-based transposon mutagenesis approach. In this study, one of the mutants, Rvm166, which contained the transposon sequence at open reading frame m166, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. The viral mutant replicated as well as the wild-type Smith strain in vitro in NIH 3T3 cells, whereas the transposon insertion precluded the expression of >65% of the m166 open reading frame. Compared to the wild-type strain and a rescued virus that restored the m166 region, the viral mutant was significantly attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. At 21 days postinfection, the titers of the viral mutant in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice were lower than the titers of the Smith strain and the rescued virus by about 30000-, 10000-, 1000-, 300-, and 800-fold, respectively. Moreover, the virulence of the mutant virus appears to be severely attenuated because no death was found in SCID mice infected with the viral mutant up to 90 days postinfection, whereas all of the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results suggest that m166 probably encodes a virulence factor and is required for MCMV virulence in killing SCID mice and for optimal viral growth in vivo.     

In vitro and in vivo characterization of a murine cytomegalovirus with a transposon insertional mutation at open reading frame M43

We have recently generated a pool of murine cytomegalovirus (MCMV) mutants by using a Tn3-based transposon mutagenesis approach. In this study, one of the MCMV mutants, RvM43, which contained the transposon inserted in open reading frame M43, was characterized. Our results provide the first direct evidence to suggest that M43 is not essential for viral replication in vitro in NIH 3T3 cells. Moreover, RvM43 exhibited a titer similar to that of the wild-type virus in the lungs, livers, spleens, and kidneys of both BALB/c and SCID mice and was as virulent as the wild-type virus in killing SCID mice that had been intraperitoneally infected with the viruses. In contrast, titers of the mutant virus in the salivary glands of the infected animals at 21 days postinfection were significantly (100 to 1,000-fold) lower than those of the wild-type virus and a rescued virus that restored the M43 region and its expression. Thus, M43 appears to be not essential for viral growth in vivo in the lungs, livers, spleens, and kidneys of infected animals and is also dispensable for virulence in killing SCID mice. Moreover, our results suggest that M43 is an MCMV determinant for growth in the salivary glands. Studies of viral genes required for replication in the salivary glands are important in understanding the mechanism of viral tropism for the salivary glands and shedding in saliva, which is believed to be one of the major routes of CMV transmission among healthy human populations.     

In vitro and in vivo gene delivery by recombinant baculoviruses

Although recombinant baculovirus vectors can be an efficient tool for gene transfer into mammalian cells in vitro, gene transduction in vivo has been hampered by the inactivation of baculoviruses by serum complement. Recombinant baculoviruses possessing excess envelope protein gp64 or other viral envelope proteins on the virion surface deliver foreign genes into a variety of mammalian cell lines more efficiently than the unmodified baculovirus. In this study, we examined the efficiency of gene transfer both in vitro and in vivo by recombinant baculoviruses possessing envelope proteins derived from either vesicular stomatitis virus (VSVG) or rabies virus. These recombinant viruses efficiently transferred reporter genes into neural cell lines, primary rat neural cells, and primary mouse osteal cells in vitro. The VSVG-modified baculovirus exhibited greater resistance to inactivation by animal sera than the unmodified baculovirus. A synthetic inhibitor of the complement activation pathway circumvented the serum inactivation of the unmodified baculovirus. Furthermore, the VSVG-modified baculovirus could transduce a reporter gene into the cerebral cortex and testis of mice by direct inoculation in vivo. These results suggest the possible use of the recombinant baculovirus vectors in combination with the administration of complement inhibitors for in vivo gene therapy.     

Cloning of the complete human cytomegalovirus genome in cosmids

Purified virion DNA (155 X 10(6) Mr) of human cytomegalovirus (CMV) strain Ad169 was partially cleaved with restriction endonucleases HindIII and EcoRI and cloned in the respective cleavage sites of cosmid pHC79. A complete gene library was established in a set of clones containing the viral DNA in long overlapping segments. Restriction maps for HindIII (29 fragments) and EcoRI (36 fragments) were constructed from the linkage of cosmid-cloned fragments, from double digestions of cloned DNA, and from blot hybridization of labeled cloned viral DNA with restriction fragments of virion DNA and singly or doubly cleaved cosmid clones.     

In vivo and in vitro processing of recombinant pro-von Willebrand factor

Von Willebrand factor (vWF) is synthesized in endothelial cells as pre-pro-vWF and processed intracellularly to propeptide (vWFpp) and mature vWF. Recombinant pro-vWF when infused into animals can also be processed extracellularly in vivo. Within 1 h of infusion in a dog and mice the multimer pattern changed to that typically seen in mature vWF indicating that propeptide cleavage from unprocessed vWF occurs extracellularly in the circulation. Incubation of a recombinant pro-vWF preparation with canine and human vWF-deficient plasma induced a time-dependent decrease in pro-vWF antigen and an increase in vWFpp antigen without changing total vWF antigen or collagen-binding activity. Multimer analysis showed the gradual transformation of the pro-vWF multimers to mature vWF multimers and cleaved vWFpp was visualized on autoradiograms of SDS-polyacrylamide electrophoresis gels using (125)I-labeled pro-vWF. When recombinant pro-vWF was incubated with increasing amounts of purified thrombin, the extent of pro-vWF processing was dose dependent. The specific cleavage of vWFpp was confirmed by immunoblots using an anti-vWFpp antibody and by amino-terminal amino acid analysis. Hirudin preconditioning of vWF-deficient mice attenuated processing of infused recombinant pro-vWF suggesting that thrombin plays a part in the processing events in vivo.     

In vitro and in vivo characterization of neural stem cells

Neural stem cells are defined as clonogenic cells with self-renewal capacity and the ability to generate all neural lineages (multipotentiality). Cells with these characteristics have been isolated from the embryonic and adult central nervous system. Under specific conditions, these cells can differentiate into neurons, glia, and non-neural cell types, or proliferate in long-term cultures as cell clusters termed "neurospheres". These cultures represent a useful model for neurodevelopmental studies and a potential cell source for cell replacement therapy. Because no specific markers are available to unequivocally identify neural stem cells, their functional characteristics (self-renewal and multipotentiality) provide the main features for their identification. Here, we review the experimental and ultrastructural studies aimed at identifying the morphological characteristics and the antigenic markers of neural stem cells for their in vitro and in vivo identification.     

In vitro and in vivo modifications of recombinant and human IgG antibodies

Tremendous knowledge has been gained in the understanding of various modifications of IgG antibodies, driven mainly by the fact that antibodies are one of the most important groups of therapeutic molecules and because of the development of advanced analytical techniques. Recombinant monoclonal antibody (mAb) therapeutics expressed in mammalian cell lines and endogenous IgG molecules secreted by B cells in the human body share some modifications, but each have some unique modifications. Modifications that are common to recombinant mAb and endogenous IgG molecules are considered to pose a lower risk of immunogenicity. On the other hand, modifications that are unique to recombinant mAbs could potentially pose higher risk. The focus of this review is the comparison of frequently observed modifications of recombinant monoclonal antibodies to those of endogenous IgG molecules.     

In vitro and in vivo modifications of recombinant and human IgG antibodies

Tremendous knowledge has been gained in the understanding of various modifications of IgG antibodies, driven mainly by the fact that antibodies are one of the most important groups of therapeutic molecules and because of the development of advanced analytical techniques. Recombinant monoclonal antibody (mAb) therapeutics expressed in mammalian cell lines and endogenous IgG molecules secreted by B cells in the human body share some modifications, but each have some unique modifications. Modifications that are common to recombinant mAb and endogenous IgG molecules are considered to pose a lower risk of immunogenicity. On the other hand, modifications that are unique to recombinant mAbs could potentially pose higher risk. The focus of this review is the comparison of frequently observed modifications of recombinant monoclonal antibodies to those of endogenous IgG molecules.     

In vivo and in vitro characterization of TEV protease mutants

Tobacco etch virus protease (TEVp) is frequently applied in the cleavage of fusion protein. However, production of TEV protease in Escherichia coli is hampered by low yield and poor solubility, and auto-cleavage of wild type TEVp gives rise to the loss-of-function. Previously it was reported that TEVp S219V displayed more stability, and TEVp variant containing T17S/N68D/I77V and double mutant L56V/S135G resulted in the enhanced production and solubility, respectively. Here, we introduced T17S/N68D/I77V in TEVp S219V to generate TEVpM1 and combined five amino acid mutations (T17S/L56V/N68D/I77V/S135G) in TEVp S219V to create TEVpM2. Among TEVp S219V, and two constructed variants, TEVpM2 displayed highest solubility and catalytic activity in vivo, using EmGFP as the solubility reporter, and the designed fusion protein as in vivo substrate containing an N-terminal hexahistidine tagged GST, a peptide sequence for thrombin and TEV cut and E. coli diaminopropionate ammonia-lyase. The purified TEVp mutants fused with double hexahistidine-tag at N and C terminus showed highest yield, solubility and cleavage efficiency. Mutations of five amino acid residues in TEVpM2 slightly altered protein secondary structure conformed by circular dichroism assay.     

In vivo and in vitro activation of alveolar macrophages by recombinant interferon-gamma

In vivo administration of recombinant interferon-gamma (rIFN-gamma) was previously shown to result in activation of the microbicidal activities of peritoneal macrophages (PM phi). Because macrophages at different anatomical sites vary in their functional capacities, we considered it of interest to determine whether administration of murine rIFN-gamma, either in vitro or in vivo, can enhance the microbicidal activity of resident alveolar macrophages (AM phi) and to compare the effects of rIFN-gamma on AM phi and PM phi. After incubation in vitro with rIFN-gamma, the antimicrobial activities of both murine AM phi and PM phi were enhanced, as assessed by their ability to inhibit replication of the intracellular parasite, Toxoplasma gondii. This effect was dose dependent for AM phi over a range of 0.1 to 1 U/ml and for PM phi over a range of 0.5 to 1000 U/ml. In this assay, the minimum dosage required for in vitro activation of AM phi was one-half that required for activation of PM phi, suggesting a greater sensitivity of AM phi to the in vitro activity of rIFN-gamma. Macrophages from both anatomical sites were also activated when rIFN-gamma was administered in vivo. This effect was dose dependent over a range of 10(3) to 10(5) U/mouse. Freshly harvested AM phi and PM phi from mice injected 24 hr earlier with 10(4) U rIFN-gamma by either the i.v. or i.p. routes markedly inhibited intracellular multiplication of Toxoplasma. In contrast, AM phi and PM phi from control mice permitted fourfold to ninefold increases in numbers of intracellular Toxoplasma. The anti-toxoplasma activity of AM phi and PM phi gradually diminished over a period of 3 days when assayed at successive 24 hr periods after a single i.v. injection of rIFN-gamma. At 3 days after injection, a substantial loss of anti-toxoplasma activity was observed with PM phi as compared with controls; residual anti-toxoplasma activity was still demonstrable in AM phi at 3 days. These results demonstrate that in vitro as well as in vivo treatment with rIFN-gamma confers on AM phi an enhanced antimicrobial activity. These findings provide a rationale for evaluating rIFN-gamma in the treatment of pulmonary infections, especially those due to opportunistic pathogens against which AM phi play a major role in host defense.     

In vitro and in vivo characterization of retinoid synthesis from beta-carotene

Retinoids are indispensable for the health of mammals, which cannot synthesize retinoids de novo. Retinoids are derived from dietary provitamin A carotenoids, like β-carotene, through the actions of β-carotene-15,15′-monooxygenase (BCMO1). As the substrates for retinoid-metabolizing enzymes are water insoluble, they must be transported intracellularly bound to cellular retinol-binding proteins. Our studies suggest that cellular retinol-binding protein, type I (RBP1) acts as an intracellular sensor of retinoid status that, when present as apo-RBP1, stimulates BCMO1 activity and the conversion of carotenoids to retinoids. Cellular retinol-binding protein, type II (RBP2), which is 56% identical to RBP1 does not influence BCMO1 activity. Studies of mice lacking BCMO1 demonstrate that BCMO1 is responsible for metabolically limiting the amount of intact β-carotene that can be absorbed by mice from their diet. Our studies provide new insights into the regulation of BCMO1 activity and the physiological role of BCMO1 in living organisms.     

Conditional cytomegalovirus replication in vitro and in vivo

We have established a conditional gene expression system for cytomegalovirus which allows regulation of genes independently from the viral replication program. Due to the combination of all elements required for regulated expression in the same viral genome, conditional viruses can be studied in different cell lines in vitro and in the natural host in vivo. The combination of a self-sufficient tetracycline-regulated expression cassette and Flp recombinase-mediated insertion into the viral genome allowed fast construction of recombinant murine cytomegaloviruses carrying different conditional genes. The regulation of two reporter genes, the essential viral M50 gene and a dominant-negative mutant gene (m48.2) encoding the small capsid protein, was analyzed in more detail. In vitro, viral growth was regulated by the conditional expression of M50 by 3 orders of magnitude and up to a millionfold when the dominant-negative small capsid protein mutant was used. In vivo, viral growth of the dominant-negative mutant was reduced to detection limits in response to the presence of doxycycline in the organs of mice. We believe that this conditional expression system is applicable to genetic studies of large DNA viruses in general.     

In vivo and in vitro analyses of recombinant baculoviruses lacking a functional cg30 gene.

The cg30 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes two sequence motifs, a zinc finger-like motif and a leucine zipper, found in other polypeptides known to be involved in gene regulation. To gain insight into the function of the cg30 product, CG30, we constructed and characterized recombinant viruses lacking a functional cg30 gene. We found that cg30 mutants had no striking phenotype in cell lines derived from Spodoptera frugiperda or Trichoplusia ni or in T. ni larvae. Although cg30 is known to be transcribed as an early monocistronic RNA and as the second cistron of an abundant late bicistronic RNA, production of a CG30-beta-galactosidase fusion protein was observed mainly at early times postinfection. Viruses containing cg30 had a subtle growth advantage over those lacking cg30 after several viral passages in cell culture. We employed transient expression assays to determine whether cg30 and pe-38, an AcMNPV gene that encodes a polypeptide with zinc finger-like and leucine zipper motifs similar to those of cg30, have redundant functions. Although pe-38 may have a role in AcMNPV gene expression, there was no indication that cg30 and pe-38 are functionally redundant.     

In vitro and in vivo characterization of a novel CCR3 antagonist, YM-344031

Eosinophils play a prominent proinflammatory role in a broad range of diseases, including atopic dermatitis and asthma. Eotaxin-1 and its receptor CCR3 are implicated in the recruitment of eosinophils from blood into inflammatory tissues, therefore inhibition of Eotaxin-1/CCR3 interaction may have therapeutic potential for allergic inflammation with eosinophil infiltration. YM-344031, a novel and selective small molecule CCR3 antagonist, potently inhibited ligand binding (IC(50)=3.0nM), ligand-induced Ca(2+) flux (IC(50)=5.4nM), and the chemotaxis of human CCR3-expressing cells (IC(50)=19.9nM). YM-344031 (1-10mg/kg) orally administered to cynomolgus monkeys significantly inhibited Eotaxin-1-induced eosinophil shape change in whole blood. Additionally, orally administered YM-344031 (100mg/kg) prevented both immediate- and late-phase allergic skin reactions in a mouse allergy model. YM-344031 therefore has potential as a novel and orally available compound for the treatment of allergic inflammation, such as atopic dermatitis and asthma.     

In vitro and in vivo regulation of tumor antigen expression by human recombinant interferons

In vitro treatment with either type I or type II Interferon (IFN) can selectively enhance the expession of several tumor antigens, such as the carcinoembryonic antigen (CEA) and the tumor-associated glycoprotein-72 (TAG-72) in different human carcinoma cell lines and result in enhanced level of monoclonal antibody (MAb) binding to the cell surface. In vivo animal studies demonstrated that treatment of athymic mice with a type I interferon [i.e. interferon-α(A)]significantly increased the expression of a 90 kDa tumor antigen which improved the targeting of a MAb to the carcinoma xenograft. More recent studies reported that in vitro IFN treatment of human adenocarcinoma cells isolated from human malignant serous effusions selectively increased the expression of TAG-72 and CEA. One can envision that the ability of these cytokines to upregulate the level of expression of human tumor antigens presents an important experimental model in which to study the regulation of markers often correlated with epithelial cell differentiation. In addition, the increase of selective MAb-defined antigens may also be exploited in an adjuvant setting to localize higher amounts of MAbs to the tumor cell surface and, thereby, improve the effectiveness of a MAb for tumor diagnosis and, possibly, therapy.     

Expression and characterization of recombinant murine cytomegalovirus protease.

The protease domain of the murine cytomegalovirus (MCMV) M80 open reading frame was expressed in and purified from Escherichia coli. The recombinant enzyme was recovered as a mixture of active one- and two-chain forms. The two-chain enzyme was formed by internal cleavage of the one-chain enzyme at the I site. Activity measurements showed that MCMV protease cleaves R- and M-site peptide mimics with kinetics similar to those of recombinant human cytomegalovirus (HCMV) protease. Both the MCMV and HCMV proteases cleave I-site peptide substrates very poorly, but the crystal structure of the HCMV protease indicates that the cytomegalovirus I site likely resides on a solvent-exposed loop close to the active site.     

In vivo and in vitro studies of Mgs1 suggest a link between genome instability and Okazaki fragment processing

The non-essential MGS1 gene of Saccharomyces cerevisiae is highly conserved in eukaryotes and encodes an enzyme containing both DNA-dependent ATPase and DNA annealing activities. MGS1 appears to function in post-replicational repair processes that contribute to genome stability. In this study, we identified MGS1 as a multicopy suppressor of the temperature-sensitive dna2Δ405N mutation, a DNA2 allele lacking the N-terminal 405 amino acid residues. Mgs1 stimulates the structure-specific nuclease activity of Rad27 (yeast Fen1 or yFen1) in an ATP-dependent manner. ATP binding but not hydrolysis was sufficient for the stimulatory effect of Mgs1, since non-hydrolyzable ATP analogs are as effective as ATP. Suppression of the temperature-sensitive growth defect of dna2Δ405N required the presence of a functional copy of RAD27, indicating that Mgs1 suppressed the dna2Δ405N mutation by increasing the activity of yFen1 (Rad27) in vivo. Our results provide in vivo and in vitro evidence that Mgs1 is involved in Okazaki fragment processing by modulating Fen1 activity. The data presented raise the possibility that the absence of MGS1 may impair the processing of Okazaki fragments, leading to genomic instability.