Construction and immune response characterization of a recombinant pseudorabies virus co-expressing capsid precursor protein (P1) and a multiepitope peptide of foot-and-mouth disease virus in swine

Foot-and-mouth disease (FMD) is the most contagious and devastating disease of livestock. Our previous studies demonstrated that TK⁻/gG⁻/P1⁺, a recombinant expressing the FMDV capsid precursor protein (P1) based on attenuated pseudorabies virus (PRV) TK⁻/gG⁻, could be used as a recombinant vaccine to protect pigs against both pseudorabies and FMD. However, because the P1 expression cassette is inserted into the gG locus of the genome of PRV TK⁻/gG⁻, this bivalent vaccine cannot be used in conjunction with the PRV gE-ELISA, an extensively used discriminatory serological test in eradication programs for pseudorabies, which limits the clinical use of this bivalent vaccine. To circumvent this shortcoming, in this study, an expression cassette containing synthetic multiepitope gene “FHG” consisting of six potential B cell epitopes and two potential T cell epitopes of FMDV, under the control of CMV promoter, was further inserted into the gE/gI locus of genome of TK⁻/gG⁻/P1⁺, resulting in a new recombinant FHG/P1/PRV. The immunogenicity of FHG/P1/PRV was evaluated and compared with TK⁻/gG⁻/P1⁺ in piglets. Our results clearly showed that FHG/P1/PRV performed better than or comparable with TK^-/gG^-/P1⁺, as demonstrated by comparable PRV-specific neutralizing antibodies, enhanced FMDV-specific neutralizing antibodies, and cellular immune responses. More importantly, no gE- and gG-specific antibodies could be detected in pigs immunized with FHG/P1/PRV. These data indicate that FHG/P1/PRV is a promising bivalent vaccine candidate with more extensive potential application than TK⁻/gG⁻/P1⁺ against both pseudorabies and FMD.     

Analysis of spontaneous mutations in a chromosomally located hsv-1 thymidine kinase (TK) gene in a human cell line

We have developed a system for studying spontaneous mutations at a chromosomally located single-copy HSV-1 thymidine kinase (TK) gene in the human 143TK ⁻ cell line. The neo gene, which confers resistance to the antibiotic G418, was placed next to theTK gene for the purpose of screening out gross chromosomal alterations.TK ⁻ mutations were selected using the anti-TK nucleotide analogs trifluorothymidine, acyclovir, and DHPG 9-(1,3 dihydroxy-2-propoxymethyl)-guanine either separately, or in combination to eliminate leaky mutations. Analysis of theTK ⁻ mutations by Southern blotting revealed that the majority had undetectable alterations of less than 50 base pairs. The results using the methylationsensitive enzymes HpaII,AvaI, andSmaI suggest that the inactivation of the TK gene was not due to extensive methylation, although specific methylation of a limited number of MspI sites cannot be ruled out. Reversion studies, however, showed that of 16 mutants analyzed, about half had a very high reversion frequency (approximately 10⁻² This suggests that inactivation of theTK gene may have occurred by a variety of mutational events.     

Transformation of the gene for hypoxanthine phosphoribosyltransferase

Purified DNA from wild-type Chinese ovary (CHO) cells has been used to transform three hypoxanthine phosphoribosyltransferase (HPRT) deficient murine cell mutants to the enzyme positive state. Transformants appeared at an overall frequency of 5×10^–8 colonies/treated cell and expressed CHO HPRT activity as determined by electrophoresis. One gene recipient, B21, was a newly isolated mutant of LMTK^– deficient in both HPRT and thymidine kinase (TK) activities. Transformation of B21 to HPRT⁺ occurred at 1/5 the frequency of transformation to TK⁺; the latter was, in turn, an order of magnitude lower than that found in the parental LMTK^– cells, 3×10^–6. Thus both clonal and marker-specific factors play a role in determining transformability. The specific activity of HPRT in transformant extracts ranged from 0.5 to 5 times the CHO level. The rate of loss of the transformant HPRT⁺ phenotype, as measured by fluctuation analysis, was 10^–4/cell/generation. While this value indicates stability compared to many gene transferents, it is much greater than the spontaneous mutation rate at the indigenous locus. The ability to transfer the gene for HPRT into cultured mammalian cells may prove useful for mutational and genetic mapping studies in this well-studied system.     

Stimulation of thymidine kinase activity in baculovirus infected cells is not due to a virus-coded enzyme

Summary A polyhedrin-positive recombinant autographa californica nuclear polyhedrosis virus (AcNPV) carrying a herpes simplex virus thymidine kinase gene under the control of theSyn XIV promoter, a fusion of synthetic and linker-modified polyhedrin promoters, has been constructed. When this recombinant baculovirus was used to infect a variant ofSpodoptera frugiperda cells deficient in thymidine kinase (TK^–), a high level of TK activity was detected. These results, in conjunction with the demonstration that AcNPV could replicate in TK^– S. frugiperda cells and no TK activity was found throughout infection, imply that the wild type virus-stimulated TK activity observed inS. frugiperda (TK⁺) cells is not contributed by a virus-coded enzyme.     

Influence of double infections on the induction of thymidine kinase by UV-irradiated herpes simplex virus types 1 and 2 and pseudorabies virus

Summary Simultaneous infection of primary rabbit kidney cells with HSV type 1 TK⁺ and a TK^– strain results in a mutual influence of both viruses on the induction of thymidine kinase (TK). TK⁺ virus has an enhancing and TK^– virus a depressing effect on TK induction by a superinfecting TK⁺ virus. The enzyme induction depends on the ratio of multiplicities of both viruses. The mutual influence on TK induction depends further on the time of addition of the superinfecting virus: the effect of the second virus can still be observed when given 6 hours after primary infection. Identical phenomena can be observed using combinations with HSV type 2 or Pseudorabies viruses.The ability of HSV to induce TK is progressively inactivated with increasing the time of UV-irradiation. The depressing effect of a TK^– strain and the stimulating effect of a TK⁺ strain on superinfecting TK⁺ strains is UV-sensitive: after 6 minutes of UV-irradiation neither inhibition nor stimulation of TK induction by a superinfecting TK⁺ strain can be observed. Infection by long-term (20 minutes) UV-irradiated TK⁺ strains results in a depression of TK induction by a superinfecting TK⁺ virus. Long-term irradiation of the TK^– virus does not show this effect.Cytosine-arabinoside has no effect on the mutual influence of TK induction by TK⁺ and TK^– strains; the phenomenon of mutual depression therefore has to be considered an early process.With 5 Figures     

The mutagenicity of sodium fluoride to L5178Y [wild-type and TK+/- (3.7.2c)] mouse lymphoma cells

L5178Y wild-type and TK+/– (3.7.2c) cells were treatedwith sodium fluoride over a range of concentrations (10—500µg ml^–1) and treatment times (4, 16 and 48 h) covering< 10 —100% survival. The mutant frequency at five geneticloci (resistance to ouabain, 6–thioguanine, excess thymidine,methotrexate and 1–3–D-arabinofuranosyl cytosine)was assayed in wild-type cells and trifluorothymidine in TK⁺/–cells.No significant induced mutation at any locus was observed after4 h of treatment. Sixteen hours of treatment with high concentrationsof sodium fluoride did not induce resistance to ouabain, butresulted in some significant induction of 6-thioguanine, lS-D-arabinofuranosylcytosine and methotrexate resistance, although the results werevariable between experiments and no dose-response was observed.At the thymidine kinase locus, a dose-related increase in mutantfrequency to excess thymidine and trifluorothymidine resistancewas observed. The maximum induction was approximately eighttimes the control frequency after TK⁺/–cells were treatedwith the highly toxic concentration of 500 % ml of sodium fluoridefor 16 h. These observations, and an analysis of the colonysize of trifhiorothymidine-resistant mutants in TK⁺/–cells, suggest that sodium fluoride is clastogenk to dividingcultured mammalian cells at high, toxic concentrations. Furtherwork is desirable to investigate the mechanism by which chromosomesare damaged at high concentrations of fluoride, since withoutsuch a mechanistic understanding, extrapolation of our datato the human situation must be insecure. Nevertheless, the knowledgeavailable at present gives no reason to expect any genotoxiceffects in human tissues at levels of fluoride ions to whichthey are currently exposed in the general population.     

DNA-mediated restoration of phenylalanine hydroxylase gene expression in enzyme-deficient derivatives of enzyme-constitutive mouse cell hybrids

Phenylalanine hydroxylase (PH) gene expression is not extinguished in hybrids between PH^– mouse A9 cells, or its neomycin-resistant derivative A9Neo-3, and PH⁺ mouse erythroleukemia (MEL) cells, PHC-3A, in contrast to its extinction in hybrids between A9Neo-3 and PH⁺ rat hepatoma cells, FT-2. Two different types of 6-thioguanine (TG)-resistant derivatives of these A9 x PHC-3A hybrids (LP), are generated in regard toPH gene expression. In regular growth medium supplemented with 10^–4 M TG (Tyr⁺/TG), TG^r derivatives, all of which continue to express PH, occur with high frequency (10^–3). In contrast, in tyrosine-deficient selective medium, supplemented with 10^–4 M TG (Tyr^–/TG), no actively growing colonies are observed. Nevertheless, small colonies containing quiescent cells can be rescued by supplementing the medium with tyrosine. The rescued TG^r clones do not express any detectable level of PH. Biochemical, hybridization, and cybridization analyses of one such rescued clone, LPTG-3, showed that these cells lack the regulatory factor capable of activatingPH gene in PH^– MEL cells. The PH^– phenotype of LPTG-3 cells can be converted to the PH⁺ phenotype by transfection with restriction enzyme-digested or -undigested PHC-3A or mouse liver DNA. Therefore, these cells could be used to clone a fragment of DNA involved inPH gene regulation through DNA-mediated gene transfer methods.     

Ultraviolet-induced reactivation,amplification, and hypomethylation of a herpes simplex virus thymidine kinase gene

A line of mouse cells containing a methylated and inactive herpes simplex virus thymidine kinase(TK) gene was irradiated with ultraviolet (UV) light in an attempt to induce expression of the inactiveTK gene. UV irradiation was shown to be capable of inducing expression of the viralTK gene in a dose-dependent manner. Analysis of the methylation pattern of the viralTK gene indicated that the activeTK gene in three UV-induced TK⁺ cell lines was methylated to a lesser extent than was the inactive viralTK gene in the parental cells. Analysis of the copy number of the viralTK gene in parental and UV-induced cell lines also revealed that the viralTK gene was amplified 3- to 20-fold in three of four UV-induced TK⁺ cell lines tested.     

Transfer of the Herpes simplex thymidine kinase gene from human cells to mouse cells by means of metaphase chromosomes

Thymidine kinase (TK)-deficient human cells were infected with ultraviolet light-inactivated Herpes simplex virus type 1, and transformed cells that expressed Herpes TK activity were isolated. Purified metaphase chromosomes were isolated from the transformed human line and incubated with TK-deficient mouse cells. TK ⁺ cells were selected, and it was shown that these cells were gene transferents which expressed Herpes TK activity, identical to that found in the transformed human cells. The gene transferents contained no intact human chromosomes. When removed from selective pressure, the gene transferents rapidly lost the TK ⁺ phenotype. However, upon continued growth in nonselective medium, a subpopulation in which the TK ⁺ phenotype had become more stabilized appeared. These results suggest that the Herpes gene for thymidine kinase has integrated into the genome of the HSV-transformed human cells and that it can be transferred to other cells by means of purified metaphase chromosomes.     

Herpes simplex virus thymidine kinase expression in infection of the trigeminal ganglion.

Infection of the trigeminal ganglion was investigated using standard thymidine kinase-positive (TK⁺) herpes simplex virus (HSV) and two TK^? mutants. After corneal inoculation of mice with TK^? HSV, the incidence of acute and latent trigeminal ganglion infection was markedly decreased compared to TK⁺ virus. When cell-free virus was titered from mice 1 hr to 5 days post-corneal inoculation, ocular replication of TK^? HSV was found to be similar to TK⁺ HSV, but whereas TK⁺ HSV replicated well and was found in substantial amounts in trigeminal ganglia (2 × 10³ PFU/mg ganglion tissue), TK^? HSV did not replicate in the ganglia. Mean TK^? trigeminal ganglion virus titers were 10,000-fold less than TK⁺ titers. When a TK⁺ revertant of TK^? mutant virus was tested, however, trigeminal ganglion virus replication was similar to that with the parental TK⁺ virus. The results obtained were interpreted as being consistent with impaired axonal transport of TK^? HSV from cornea to trigeminal ganglion neurons or more likely, with impaired replication of TK^? HSV in ganglionic neurons.     

Herpesvirus-associated nuclear antigen(s) in cells biochemically transformed by fragments of herpesvirus DNA and in somatic cell hybrids

Human and mouse cells biochemically transformed by ultraviolet light (UV)-irradiated HSV-1 express HSV-1 thymidine kinase (TK) activity and also express type-specific herpesvirus-associated nuclear antigen(s) (HANA). To identify the HSV-1 DNA sequences coding for HANA and their location on the viral genome, studies were carried out on: (i) somatic cell hybrid clones obtained by fusing mouse [LM(TK^?)] cells with UV-irradiated HSV-1-transformed human [HeLa(BU25)/KOS 8-1] cells; and (ii) LM(TK^?) cells biochemically transformed with restriction endonuclease fragments of DNA which code for HSV-1 TK. Molecular hybridization experiments were also carried out and demonstrated that HSV-1 DNA sequences coding for TK were integrated in the biochemically transformed cells. The human-mouse somatic cell hybrid clones (LH81) which were HSV-1 TK+ were also HANA⁺, while clones counterselected in bromodeoxyuridine which had lost HSV-1 TK activity and DNA sequences likewise lost HANA. Previous studies had shown that the HSV-1 TK gene of LH81 hybrid clones was associated with a marker chromosome, designated M7, which consists of a human chromosome 17 translocated to the short arm of chromosome 3, or a modified M7 chromosome containing a translocation from a mouse chromosome. The present results indicate that at least one HANA gene was integrated in the same chromosome as the HSV-1 TK gene. LM(TK^?) cells biochemically transformed by HSV-1 DNA restriction nuclease fragments of diminishing size, which map in the HpaI-I region (26.2 to 31.7) of the HSV-1 genome, were HANA⁺ as well as TK⁺. The HANA⁺ cells included LM(TK^?)/TF pAGO PP and LM(TK^?)/TF pAGO PS clones. The latter are clones of LM(TK^?) cells biochemically transformed, respectively, by a PvuII fragment (1.35 × 10⁶ daltons) and a PvuII-SmaI fragment (0.9 × 10⁶ daltons; 30.2 to 31.1 map units) of HSV-1 DNA derived from Escherichia coli plasmid, pAGO. Since the PvuII-SmaI DNA fragment has only enough genetic information to code for a polypeptide of about 53,000 daltons and the HSV-1 TK polypeptide is about 40,000 daltons, the findings indicate that the genes for HSV-1 TK and one herpesvirus-associated nuclear antigen are either contiguous or overlapping, or HSV-1 TK and one HANA gene are identical.     

Herpes simplex virus thymidine kinase expression in trigeminal ganglion infection: correlation of enzyme activity with ganglion virus titer and evidence of in vivo complementation

Experimental trigeminal ganglion and corneal infection in mice was studied with three thymidine kinase-positive (TK⁺) strains of herpes simplex virus type 1 (HSV-1) and eight TK^? HSV-1 mutants. Viruses were extensively tested in cell culture to determine whether any were temperature sensitive (ts) for virus replication or for TK activity. TK^? mutants were no more ts than were TK⁺ viruses. By arabinosylthymine testing and measurement of thymidine phosphorylation, it was apparent that some TK^? mutants were, in fact, intermediate for TK activity (TK^±). After corneal inoculation of individual viruses it was observed with one exception that TK^?, TK^±, and TK⁺ viruses replicated in ocular tissues (3 days postinoculation, 2.2 × 10³–1.1 × 10⁵ PFU/eye). However, TK^? mutants were rarely isolated from trigeminal ganglia (<2.5–<5.6 × 10^?1 PFU/mg), whereas TK⁺ and to a lesser degree TK^± viruses were frequently (1.2 × 10³–1.2 × 10⁴ PFU/mg and 3.2 × 10⁰–2.1 × 10³ PFU/mg). In vivo complementation studies were performed by corneal inoculation of TK^??TK^? and TK^??TK^? virus mixtures. TK⁺ HSV complemented TK^? virus since significant amounts of TK^? HSV were isolated from trigeminal ganglia (complementation indices were >60–>1600). In addition, after inoculation of certain TK^?–TK^? pairs, complementation in ganglia was observed (complementation indices were >2.4–>120). These studies support the hypothesis that HSV-1 TK expression is necessary for sensory ganglion (neuron) infection in three ways: HSV-1 TK mutants that replicated in ocular tissues and were not ts mutants did not replicate in vivo in trigeminal ganglia; (ii) there was a correlation between level of viral TK activity and trigeminal ganglion virus titer; and (iii) when complemented by TK⁺ or TK^? HSV, TK^? virus replicated in trigeminal ganglia.     

Identification,mapping and cloning of the thymidine kinase gene of fish lymphocystis disease virus

The thymidine kinase (TK) gene of fish lymphocystis disease virus (FLDV) was identified by biochemical transformation of 3T3 TK negative (TK⁻) to 3T3 TK positive (TK⁺) cells using specific viral DNA sequences. DNA fragments of the viral genome used in this study were obtained from a defined gene library of FLDV genome containing the complete viral DNA sequences. The selection of the converted cells was carried out under the condition of the HAT selection procedure. The results of these experiments revealed that the EcoRI FLDV DNA fragment C (11.2 kbp; 0.611 to 0.718 map units) is able to transform 3T3 TK⁻ to 3T3 TK⁺ cells. Additional experiments using the subclones of EcoRI DNA fragment C revealed that DNA sequences of 4.1 kbp size between the coordinates 0.669 to 0.718 of the FLDV genome possessed the ability for biochemical transformation, indicating that the TK gene locus is located in this particular region.     

Herpes simplex virus thymidine kinase gene is stably maintained and expressed in cells transformed by protoplast fusion

We examined a series of transformed cell lines resulting from transfer of the herpes simplex virus type 1 thymidine kinase gene to Ltk^– cells by protoplast fusion gene transfer. We show that multiple copies of the transforming plasmid DNA, ranging from a minimum of two to greater than 20, were present in one or at most a few integration sites in each cell line. The TK⁺ phenotype was stable in five independent transformed cell lines after growth in nonselective medium for over a year. Transforming plasmid DNA was stable in one cell line containing from two to five copies after a year of growth in nonselective medium. In another cell line initially containing about 20 copies, the transforming DNA became rearranged soon after growth to mass culture, resulting in a decrease to two to five copies which then remained stably maintained. This suggests that TK⁺ transformants resulting from protoplast fusion are stable when the input DNA has integrated in a relatively low copy number.     

Major histocompatibility complex class I- and II-deficient knock-out mice are resistant to primary but susceptible to secondary Eimeria papillata infections

Two distinct mechanisms seem to function in reducing oocyst output during Eimeria papillata infections in mice. For naive mice, immunity was afforded␣by␣a T-cell-independent gamma-interferon (IFN-γ) response mediated by natural killer (NK) cells. On reinfection, resistance was associated with T-cells and, to a lesser extent, perforin. To determine if antigen presentation with major histocompatibility complex␣(MHC) molecules was required to control oocyst production by NK cells during primary infection or by T-cells during secondary infection, mutant mice that lacked H2-IAβb (Aβb^−/−) or β₂-microglobulin (β2m^−/−) were used. Since MHC molecules are required for the maturation of αβ T-cells, Aβb^−/− and β2m^−/− mutant mice are also deficient in functional αβ⁺CD4⁺ or αβ⁺CD8⁺ T-cells, respectively. As compared with wild-type control mice, oocyst output by mutant mice was not significantly affected during primary infection, suggesting that the ability of NK cells to control parasite replication is not dependent on the expression of MHC molecules. On reinfection, differences were observed for mutant mice as compared with controls. Aβb^−/− mice were found to be more susceptible than β2m^−/− mice, suggesting that the αβ⁺CD4⁺ T-cell subset plays a greater role in resistance to reinfection than does the αβ⁺CD8⁺ T-cell subset. The mechanism of resistance depends on the immune status of the host and requires the coordinated interaction of both αβ⁺ T-cell subsets for optimal parasite control during subsequent infections. Received: 6 August 1997 / Accepted: 24 October 1997     

Association of Graves’ Disease and Prevalence of Circulating IFN-γ-producing CD28<Superscript>−</Superscript> T Cells

Background  Peripheral blood CD4⁺ and CD8⁺ T-cell subsets lacking surface CD28 have been suggested to predispose patients to immune-mediated disorders. Materials and Methods  To determine the role of CD28⁻ T-cell subset in Graves’ disease (GD), we characterized peripheral blood CD4⁺CD28⁻ and CD8⁺CD28⁻ T cell from early onset GD patients. Results and Discussion  GD patients had significantly higher percentages of CD4⁺CD28⁻ and CD8⁺CD28⁻ T cells than did healthy donors. Both CD28⁻ T cells expressed mostly CD45RO, suggesting that they are activated and/or are memory T cells. GD patient-derived CD4⁺CD28⁻ and CD8⁺CD28⁻ T cells produced more intracellular IFN-γ than their counterparts from healthy donors. Furthermore, CD4⁺CD28⁻ and CD8⁺CD28⁻ T cells from GD patients with Graves’ ophthalmopathy (GO) secreted higher level of intracellular IFN-γ than those CD28⁻ T cells from GD patients without GO. Retrospective analysis showed that the increased levels of CD4⁺CD28⁻ T cells and their IFN-γ-producing subgroups were positively correlated to the serum anti-thyrotropin receptor (TSHR) autoantibodies (TRAb). Our observations suggest that increased IFN-γ-producing CD28⁻ T cells in GD patients may play an important role in the pathogenesis of GD. Zhiping Sun and Weixue Zhong contributed equally to this paper.     

Sodium and chloride absorptive defects in the small intestine in Slc26a6 null mice

PAT1 (Slc26a6) is located on the apical membrane of the small intestinal villi, but its role for salt absorption has not been studied. To ascertain the role of Slc26a6 in jejunal sodium and chloride absorption, and its interplay with NHE3, muscle-stripped jejuna from Slc26a6+/+ and −/− and NHE3 +/+ and −/− mice were mounted in Ussing chambers and electrical parameters, and ³⁶Cl⁻ and ²²Na⁺ fluxes were measured. In parallel studies, expression of the apical Na⁺/H⁺ exchanger (NHE3) was examined by immunofluorescence labeling and immunoblot analysis in brush border membrane (BBM). In the basal state, net Cl⁻ and Na⁺ fluxes were absorptive in Slc26a6−/− and +/+ jejuni, but significantly decreased in −/− animals. Upon forskolin addition, net Na⁺ absorption decreased, Isc strongly increased, and net Cl⁻ flux became secretory in Slc26a6−/− and +/+ jejuni. When luminal glucose was added to activate Na⁺/glucose cotransport, concomitant Cl⁻ absorption was significantly reduced in Slc26a6 −/− jejuni, while Na⁺ absorption increased to the same degree in Slc26a6 −/− and +/+ jejuni. Identical experiments in NHE3-deficient jejuni also showed reduced Na⁺ and Cl⁻ absorption. Results further demonstrated that the lack of NHE3 rendered Na⁺ and Cl⁻ absorption unresponsive to inhibition by cAMP, but did not affect glucose-driven Na⁺ and Cl⁻ absorption. Immunoblotting revealed comparable NHE3 abundance and distribution in apical membranes in Slc26a6−/− and +/+ mice. The data strongly suggests that Slc26a6 acts in concert with NHE3 in electroneutral salt absorption in the small intestine. Slc26a6 also serves to absorb Cl⁻ during glucose-driven salt absorption.     

Role of Thymic Stromal Lymphopoietin (TSLP) in Palifermin-Mediated Immune Modulation and Protection from Acute Murine Graft-Versus-Host Disease

Using the C57BL/6→(C57BL/6 x DBA/2)F₁-hybrid model of acute graft-versus-host disease (GVHD), we previously showed that treating the donor mice with palifermin provides protection from morbidity and a shift from Th1 to Th2 cytokine production. To determine whether thymic stromal lymphopoietin (TSLP) is involved in palifermin-mediated immune modulation, we used donors from the following groups: (1) untreated wild-type donors, (2) palifermin-treated wild-type donors, (3) untreated TSLPR^−/− donors, and (4) palifermin-treated TSLPR^−/− donors. Survival in the recipients was 0%, 100%, 31%, and 0%, for groups 1–4, respectively, indicating that TSLP responsiveness is required for palifermin-mediated protection from GVHD. We also found that the increases in Th2 cytokine levels that are induced by palifermin treatment are obviated in TSLPR^−/− donors, and that protection from GVHD (group 2) is associated with a higher percentage of CD4⁺CD25⁺Foxp3⁺ cells in the graft. Collectively, our findings show that when palifermin and TSLP act in concert, the predominant effect is protection in this model.     

Biochemical transformation of LM(TK-) cells by hybrid plasmids containing the coding region of the herpes simplex virus type 1 thymidine kinase gene

Recombinant plasmid pAGO codes for herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) and consists of a 2-kbp HSV-1 DNA fragment inserted at the unique PvuII cleavage site of plasmid pBR322. A hybrid plasmid, designated pMH110, has been derived from plasmid pAGO by deleting the 1689-bp pBR322 nucleotide sequence of pAGO, which extends from the BamHI to the PvuII cleavage site, and the 250-bp HSV-1 nucleotide sequence of pAGO, which extends from the PvuII to the BglII cleavage site. Plasmid pMH110 biochemically transformed LM(TK^?)cells to the TK⁺ phenotype. The biochemically transformed cell lines had the following properties: (i) they were resistant to the growth-inhibiting effects of 1 mM thymidine; and (ii) they expressed an HSV-1-specific TK activity. This HSV-1 TK activity was purified after labeling biochemically transformed cell lines [LM(TK^?)/TF pMH110 E2 and LM(TK^?)/TF pMH110 Hc2] with [³⁵S]methionine. Immunoprecipitation experiments revealed that the TK polypeptides made in the biochemically transformed cells had molecular weights of about 39,000 to 40,000, which are about the same as the molecular weights of the TK polypeptides previously purified from HSV-1-infected LM(TK^?) cells and other biochemically transformed cell lines. The experiments support the hypothesis that the functional coding region of the HSV-1 TK gene is 3′ to the BglII cleavage site, and they also suggest that the HSV-1 TK messenger RNA may have been initiated in cells transformed by HincII- and EcoRI-cleaved pMH110 DNA at a site in cellular (or plasmid) DNA upstream from the HSV-1 DNA BglII cleavage site.