Selectively expanding subsets of T cells in mice by injection of interleukin-2/antibody complexes: implications for transplantation tolerance.

The biological activity of interleukin (IL)-2 and other cytokines in vivo can be augmented by binding to certain anti-cytokine monoclonal antibodies (mAb). Here, we review evidence on how IL-2/anti-IL-2 mAb complexes can be used to cause selective stimulation and expansion of certain T-cell subsets. With some anti-IL-2 mAbs, injection of IL-2/mAb complexes leads to expansion of CD8 T effector cells but not CD4 T regulatory cells (Tregs); these complexes exert less adverse side effects than soluble IL-2 and display powerful antitumor activity. Other IL-2/mAb complexes have minimal effects on CD8 T cells but cause marked expansion of Tregs. Preconditioning mice with these complexes leads to permanent acceptance of MHC-disparate pancreatic islets in the absence of immunosuppression.

Targeted disruption of the Ceacam1 (MHVR) gene leads to reduced susceptibility of mice to mouse hepatitis virus infection.

The CEACAM1 glycoproteins (formerly called biliary glycoproteins; BGP, C-CAM, CD66a, or MHVR) are members of the carcinoembryonic antigen family of cell adhesion molecules. In the mouse, splice variants of CEACAM1 have either two or four immunoglobulin (Ig) domains linked through a transmembrane domain to either a short or a long cytoplasmic tail. CEACAM1 has cell adhesion activity and acts as a signaling molecule, and long-tail isoforms inhibit the growth of colon and prostate tumor cells in rodents. CEACAM1 isoforms serve as receptors for several viral and bacterial pathogens, including the murine coronavirus mouse hepatitis virus (MHV) and Haemophilus influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis in humans. To elucidate the mechanisms responsible for the many biological activities of CEACAM1, we modified the expression of the mouse Ceacam1 gene in vivo. Manipulation of the Ceacam1 gene in mouse embryonic stem cells that contained the Ceacam1a allele yielded a partial knockout. We obtained one line of mice in which the insert in the Ceacam1a gene had sustained a recombination event. This resulted in the markedly reduced expression of the two CEACAM1a isoforms with four Ig domains, whereas the expression of the two isoforms with two Ig domains was doubled relative to that in wild-type BALB/c (+/+) mice. Homozygous (p/p) Ceacam1a-targeted mice (Ceacam1aDelta4D) had no gross tissue abnormalities and were viable and fertile; however, they were more resistant to MHV A59 infection and death than normal (+/+) mice. Following intranasal inoculation with MHV A59, p/p mice developed markedly fewer and smaller lesions in the liver than +/+ or heterozygous (+/p) mice. The titers of virus produced in the livers were 50- to 100-fold lower in p/p mice than in +/p or +/+ mice. p/p mice survived a dose 100-fold higher than the lethal dose of virus for +/+ mice. +/p mice were intermediate between +/+ and p/p mice in susceptibility to liver damage, virus growth in liver, and susceptibility to killing by MHV. Ceacam1a-targeted mice provide a new model to study the effects of modulation of receptor expression on susceptibility to MHV infection in vivo.

The CEACAM1-L Ser503 residue is crucial for inhibition of colon cancer cell tumorigenicity.

CEACAM1 (also known as biliary glycoprotein, C-CAM or CD66a) is a cell adhesion molecule of the immunoglobulin family behaving as a tumor inhibitory protein in colon, prostate, liver, endometrial and breast cancers. Inhibition of tumor development is dependent upon the presence of the long 71-73 amino acid cytoplasmic domain of the CEACAM1 protein (CEACAM1-L). We have recently defined a number of cis-acting motifs within the long cytoplasmic domain participating in tumor cell growth inhibition. These are Tyr488, corresponding to an Immunoreceptor Tyrosine-based Inhibition Motif, as well as the three terminal lysine residues of the protein. In this study, we provide evidence that treatment with phorbol esters leads to increased phosphorylation of in vivo (32)P-labeled CEACAM1-L in mouse CT51 carcinoma cells, in the mouse 1MEA 7R.1 liver carcinoma cells and in 293 human embryonic kidney cells transfected with the Ceacam1-L cDNA. Basal level Ser phosphorylation was abrogated by treatment with the staurosporine inhibitor, but not by the protein kinase C-specific inhibitor calphostin C or other inhibitors such as H7 or sphingosine. Specific inhibitors of protein kinase A or calmodulin kinase had only minimal effects on the levels of basal or PMA-induced Ser phosphorylation. Furthermore, PMA treatment of the CT51 cells induced cell spreading and cellular relocalization of the CEACAM1-L protein. Since Ser503 has been described as a PMA-induced phosphorylation site in other cell systems, we investigated whether Ser503 was involved in these responses in mouse intestinal cells. No differences were noticed in the basal or the PMA-induced phosphorylation levels, kinase inhibitor sensitivity or the PMA-induced relocalization of the protein between the wild-type and the Ser503Ala mutant CEACAM1-L. However, we provide evidence that Ser503 participates in CEACAM1-L-mediated tumor inhibition as its mutation to an Ala led to in vivo tumor development, contrary to the tumor inhibitory phenotype observed with the wild-type CEACAM1-L protein.

Coexpression of rat glutathione S-transferase A3 and human cytidine deaminase by a bicistronic retroviral vector confers in vitro resistance to nitrogen mustards and cytosine arabinoside in murine fibroblasts.

The transfer of drug resistance genes into hematopoietic cells is an experimental approach to protect patients from drug-induced myelosuppression. Because anti-cancer drugs are often administered in combination to increase their clinical efficacy, vectors that express two drug resistance genes are being developed to broaden the spectrum of chemoprotection. We have constructed a bicistronic vector, MFG/GST-IRES-CD (MFG/GIC) coexpressing rat glutathione S-transferase (GST) A3 isoform (rGST Yc1) and human cytidine deaminase (CD). Murine NIH 3T3 fibroblast cells transduced with this vector were evaluated for their resistance to nitrogen mustards and cytosine nucleoside analogs. GIC-transduced polyclonal cell populations (GIC cells) demonstrated marked increases in selenium-independent glutathione peroxidase (peroxidase) and CD activities, as well as increased resistance to melphalan (2.3-fold), chlorambucil (3.4-fold), and cytosine arabinoside (Ara-C) (8.1-fold). After selection with Ara-C, the peroxidase and CD activities of GIC cells were augmented 2.6- and 2.9-fold, respectively, in comparison with unselected cells, and the resistance to melphalan, chlorambucil, and Ara-C was further increased to 3.7-, 5.9-, and 53-fold, respectively. Melphalan selection of GIC cells likewise augmented their peroxidase (2.3-fold) and CD (1.9-fold) activities. GIC cells proliferated in the simultaneous presence of melphalan and Ara-C at drug concentrations that completely inhibited the growth of untransduced cells. The growth rate of unselected GIC cells exposed to the drug combination averaged 18% that of drug-free cultures. The growth rate of GIC cells exposed to the drug combination increased to 30% of controls after Ara-C selection and to 50% after melphalan selection. Our results suggest that retroviral transfer of MFG/GIC may be useful for chemoprotection against the toxicities of nitrogen mustards and cytosine nucleoside analogs.

Coexpression of cytidine deaminase and mutant dihydrofolate reductase by a bicistronic retroviral vector confers resistance to cytosine arabinoside and methotrexate.

The transfer of a drug resistance gene into hematopoietic cells is an approach being investigated to overcome the problem of myelosuppression produced by anticancer drugs. Chemotherapeutic agents are often given in combination in order to increase their effectiveness. Consequently, there is an advantage in designing vectors for gene transfer that are capable of expressing two drug resistance genes. We have constructed a bicistronic retroviral vector, MFG-DHFR-IRES/CD, which contains the mutated human dihydrofolate reductase (DHFR) cDNA with a phenylalanine-to-serine substitution at codon 31 (F31S) and the human cytidine deaminase (CD) cDNA. Murine fibroblast and hematopoietic cells were transduced with this vector and evaluated for their resistance to methotrexate (MTX) and cytosine arabinoside (ARA-C). The transduced fibroblast cells showed high levels of resistance to MTX and to ARA-C as determined by a clonogenic assay. Using enzymatic assays, we observed a coordinate increase in resistance to MTX and DHFR enzyme activity following an ARA-C selection. In addition, MTX selection produced an increase in CD enzyme activity and ARA-C resistance. Murine hematopoietic cells transduced with the bicistronic vector also showed drug resistance to both MTX and ARA-C. Interestingly, the double-gene construct conferred an equivalent level of drug resistance compared with single-gene vectors bearing only CD or DHFR genes in the hematopoietic cells. These results demonstrate the potential of the MFG-DHFR-IRES/CD vector to confer drug resistance to both MTX and ARA-C and may have future application in chemoprotection of normal hematopoietic cells in patients with cancer.

Retroviral transfer and long-term expression of human cytidine deaminase cDNA in hematopoietic cells following transplantation in mice.

The chemotherapeutic effectiveness of cytosine nucleoside analogues used in cancer therapy is limited by their dose-dependent myelosuppression. A way to overcome this problem would be to insert the drug-resistance gene, cytidine deaminase (CD), into normal hematopoietic cells. CD catalyzes the deamination and pharmacological inactivation of cytosine nucleoside analogues, such as cytosine arabinoside (Ara-C). The objective of this study was to determine if we could obtain long-term persistence and expression of proviral CD in hematopoietic cells following transplantation of CD-transduced bone marrow cells in mice. Murine hematopoietic cells were transduced with an MFG retroviral vector containing CD cDNA and transplanted into lethally irradiated mice. The recipient mice were administered three courses of 10-15 h i.v. infusions of Ara-C (75-110 mg/kg). Blood, marrow and spleen samples were obtained and analyzed for CD proviral DNA by PCR, CD activity by enzyme assay, and drug resistance to Ara-C by clonogenic assay. We detected the presence of the CD proviral DNA in most of the samples examined. Approximately 1 year after transplantation several mice showed increased expression of CD activity in these tissues and some mice displayed signs of Ara-C resistance. These data demonstrate that persistent in vivo expression of proviral CD can be achieved in transduced hematopoietic cells and indicate some potential of this gene for chemoprotection to improve the efficacy of cytosine nucleoside analogues in cancer therapy.

Impact of a decision tree on chronic wound care.

OBJECTIVE: To examine the use of a decision tree and its impact on the accuracy of decision making for chronic wound care. DESIGN: Descriptive comparative design; replication study. SETTINGS AND SUBJECTS: Data were collected from 2 groups of home care nurses in large urban centers. One group was measured after initial contact with the decision tree, and the other group was measured 2 years after implementation of the decision tree. INSTRUMENTS: The chronic wound management decision tree (CWMDT) designed by Knight was used, in combination with pictorial case studies developed by Willey and Swords. METHODS: Edmonton Home Care Program nurses, having used a decision tree for 2 years, completed 3 pictorial case studies without the aid of a written copy of the CWMDT. Current accuracy of wound care staging and treatment was compared with the Melchior-MacDougall and Lander results. After this procedure, Calgary Health Services home care nurses completed 3 pictorial case studies to assess the accuracy of wound care staging and treatment between 2 groups: with the CWMDT and without the CWMDT. A questionnaire was completed on factors identified to influence decision making in wound care. MAIN OUTCOME MEASURE: Accuracy and confidence of decision making in wound care staging and treatment. RESULTS: Accuracy of decision making with use of the CWMDT improved over time, as did accuracy of decision making after initial contact with the decision tree. Age, experience in nursing and home care, number of in-services attended, and number of chronic wounds treated, were not found to correlate with accuracy of decision making. CONCLUSION: A decision tree can assist with decision making by guiding the nurse through assessment and treatment options.

The impact of regionalization on a surgery program in the Canadian health care system.

OBJECTIVE: To examine the impact of the regionalization of health care on the provision of surgical services in the Capital Health Region (Edmonton) of the province of Alberta. DESIGN: A 4-year retrospective descriptive analysis using data from the Canadian Institute for Health Information and from the Capital Health Region data banks. SETTING: To control health care costs, the provincially funded health care system in Alberta reformed its governance structure and service provision model. We studied community hospitals and an academic health sciences center. PATIENTS: All patients undergoing surgical care in the region. INTERVENTIONS: Regionalization of the organizational structure with the elimination of hospital boards, consolidation of services on specific sites within the regional system, and a major reduction in funding. OUTCOME MEASURES: Inpatient and day surgery procedure volumes, average length of hospital stay, relative value units, bed use, and mortality. RESULTS: The Capital Health Region has a population of 723,000 people, with 5 acute care institutions. Eighteen clinical programs now provide care through 2 referral hospitals and 3 community health centers. The reduction in operating dollars for this region was $167.1 million from fiscal years 1992-1993 to 1996-1997. Redistribution of surgical services occurred on July 1, 1995, resulting in an 18% inpatient bed reduction. Regionally, the number of acute care beds has declined from 2.25 to 1.47 per 1000 population (P < .001). Bed use has fallen from 637 to 442 inpatient days per 1000 population (P < .001). The surgery volume (1995-1996) was 44770 procedures (-3.1%). Redistribution of surgical services into high- and low-acuity settings has resulted in most surgeons working on 2 sites. Overall average length of hospital stay has decreased significantly (P < .001); however, it has increased, together with the average relative value units, in the institutions caring for patients with high-acuity surgical illnesses. Mortality remains unchanged. CONCLUSIONS: Regionalization and funding reductions within the surgical program in the Capital Health Region have resulted in a small reduction in surgical volumes. There have been major changes in service provision and the way surgeons practice.

Resistance to cytosine arabinoside by retrovirally mediated gene transfer of human cytidine deaminase into murine fibroblast and hematopoietic cells.

Dose-limiting hematopoietic toxicity produced by the cytosine nucleoside analogue cytosine arabinoside (ARA-C) is one of the major factors that limit its use in the treatment of neoplastic diseases. An interesting approach to overcome this problem would be to insert a gene for drug resistance to ARA-C in normal hematopoietic cells to protect them from drug toxicity. The deamination of ARA-C by cytidine deaminase results in a loss of its antineoplastic activity. The objective of this study was to determine if gene transfer of human cytidine deaminase into murine fibroblast and hematopoietic cells would confer drug resistance to ARA-C. Retrovirally mediated transfer of the human cytidine deaminase gene into 3T3 fibroblasts resulted in efficient expression of the proviral RNA for this gene and in increased cytidine deaminase activity in cytoplasmic extracts. These cells showed marked resistance to ARA-C as determined by the effects of this drug on colony formation, cell growth, and DNA synthesis. The transfer of the human cytidine deaminase gene into murine bone marrow cells by the retroviral vector conferred a high level of drug resistance to ARA-C in clonogenic assays. These studies indicate that the cytidine deaminase gene could be used in cancer gene therapy by protecting normal hematopoietic cells against the cytotoxic effects of ARA-C and related cytosine nucleoside analogues.

Biliary glycoprotein 1 expression during embryogenesis: correlation with events of epithelial differentiation, mesenchymal-epithelial interactions, absorption, and myogenesis.

Biliary glycoprotein (Bgp1), a carcinoembryonic antigen-related family member of the immunoglobulin superfamily, is involved in normal and neoplastic events. Analysis of Bgp1 expression throughout post-implantation mouse embryogenesis using reverse transcription-polymerase chain reactions, immunostaining with anti-Bgp1 monoclonal antibodies, and in situ hybridization with specific Bgp1 cDNA fragments revealed that Bgp1 may be involved in a number of specific embryonic processes. Immunoblot analysis of Bgp1 deletion mutant proteins indicated that distinguishable epitopes of the molecule were preferentially identified by the three Bgp1 antibodies used in this study. This distinction is supported by our immunolocalization studies during mouse embryogenesis in which the three antibodies revealed specific patterns of Bgp1 expression. Bgp1 is not expressed in early post-implantation embryos (7.5 dpc), but is found in the placenta and extra-embryonic tissues (decidual endothelial cells, giant trophoblasts, yolk sac visceral endoderm, and endometrial glands) at this time. The primitive gut epithelium and surface ectoderm were the first embryonic tissues to express Bgp1. Significant Bgp1 expression was also observed later during epithelial-mesenchymal interactions (skin, meninges, lung, kidney, salivary glands, pancreas). A unique epitope of Bgp1, detectable by the monoclonal antibody CC1, was also associated with mesenchymal expression and was prominent during myogenesis (secondary myotube formation) at sites of terminal differentiation. These studies suggest multiple roles for isoforms and glycoforms of the Bgp1 proteins localized in specific sites during prenatal development.

Retrovirus-mediated gene transfer of rat glutathione S-transferase Yc confers in vitro resistance to alkylating agents in human leukemia cells and in clonogenic mouse hematopoietic progenitor cells.

Recently, we have reported that N2Yc, a Moloney-based retrovirus vector expressing the Yc isoform of rat glutathione S-transferase (GST-Yc), conferred resistance to alkylating agents in mouse NIH-3T3 fibroblasts. In this report, we address the feasibility of using rat GST-Yc somatic gene transfer to confer chemoprotection to the hematopoietic system. Human chronic myelogenous leukemia K-562 cells were efficiently transduced with the N2Yc retrovirus vector and showed a significant increase in the 50% inhibitory concentration of chlorambucil (3.2- to 3.3-fold), mechlorethamine (4.7- to 5.3-fold), and melphalan (2.1- to 2.2-fold). In addition, primary murine clonogenic hematopoietic progenitor cells transduced with the N2Yc vector were significantly more resistant to alkylating agents in vitro than cells transduced with the antisense N2revYc vector. The survival of Yc-transduced hematopoietic colonies at 400 nM mechlorethamine and 4 mu M chlorambucil was 39.4% and 42.6%, respectively, compared to 27.2% and 30.4% for N2revYc-transduced cells. Future experiments will determine the level of chemoprotection achievable in vivo, following transplantation of N2Yc-transduced hematopoietic cells in mice.

Retrovirus-mediated gene transfer of rat glutathione S-transferase Yc confers alkylating drug resistance in NIH 3T3 mouse fibroblasts.

A major limitation to successful cancer treatment is the existence of drug resistance. While several mechanisms of drug resistance have now been well characterized, mechanisms of resistance to alkylating drugs have remained less well defined. Several experimental models of alkylator resistance have implicated isoforms of glutathione S-transferase (GST) but transfection experiments using cloned isoforms of GST have yielded conflicting results. While there are several plausible explanations for these apparently contradictory findings, the issue that clonal variability might potentially confound the results of conventional transfection experiments has been raised. To address this issue properly, we have studied rat GST-Yc expression and drug sensitivity to alkylating drugs in populations of mouse NIH 3T3 fibroblasts following either transfection or transduction with an N2-based retrovirus vector. In comparison with cells treated with an antisense vector, Yc-transfected and Yc-transduced populations of NIH 3T3 cells expressed increased levels of GST-Yc mRNA (Northern blot), increased levels of immunodetectable GST-Yc (Western blot), and, respectively, 1.4- and 1.9-fold increases in total GST activity and 6.1- and 8.3-fold increases in glutathione peroxidase activity (associated with the Yc subunit). Yc-transfected and Yc-transduced cell populations were, respectively, 5.8- (P < 0.001) and 2.4-fold (P < 0.05) resistant to chlorambucil and 10.8- (P < 0.01) and 5.4-fold (P < 0.001) resistant to mechlorethamine. The range of resistance of clonal isolates from either population was 1.8-6.0-fold for chlorambucil and 4.6-6.1-fold for mechlorethamine (P < 0.05). In contrast, these cells showed unaltered sensitivity to the antimetabolite methotrexate, a nonalkylating drug. These results clearly demonstrate that the rat GTS-Yc is able to confer alkylating drug resistance in mouse fibroblasts. The ability to confer alkylating drug resistance following retrovirus-mediated gene transfer also raises the possibility of using GST-Yc somatic gene transfer to confer protection to the hematopoietic system in a gene therapy strategy applicable to cancer.

Isolation and characterization of an Escherichia coli strain with a high frequency of C-to-T mutations at 5-methylcytosines.

We used a genetic selection system to isolate a strain of Escherichia coli with a high frequency of C-to-T transition mutations at the second C of the sequence CCAGG. Cytosines in other sequences do not mutate to thymine at a high frequency in this strain, and the frequencies of other base substitution mutations are not increased to the same extent. The gene responsible for the mutator phenotype has been mapped to 43 min on the E. coli chromosome. Several lines of evidence indicate that this gene is distinct from the very short patch repair gene vsr.