Cre-mediated generation of a VCAM-1 null allele in transgenic mice.

A conditional null allele for VCAM-1 was generated in mice through a one step ES cell selection procedure by flanking the proximal promoter and exons 1 and 2 with loxP sites. The ES cells were used to create chimeric mice, which were then used to produce mice homozygous for the VCAM-1 conditional null, or floxed allele. Although the PGKneo cassette was retained in the promoter, the homozygous mice produced levels of VCAM-1 transcripts similar to that seen in wild-type mice. Homozygous VCAMflox/flox mice were mated to transgenic lines of mice expressing the cre gene under control of the murine platelet endothelial cell adhesion molecule-1 (PECAM-1) promoter. Surprisingly, the VCAMflox allele in all tissues examined from mice that inherited the cre-transgene had underwent complete excision of the floxed VCAM-1 sequences. The 'deleted' VCAM-1 allele (VCAMdel) was stably inherited, even in those mice that did not inherit the cre transgene, indicating the recombination occurs at an early stage of development prior to germ cell development. Thus the cre mice can be used for ubiquitous gene rearrangement in vivo. The data also suggest a novel simplified strategy for using the Cre/loxP system in vivo, in which a single ES cell and line of mice can be used to create mice carrying either a null or conditional null allele.

Identification and characterization of a novel surface antigen gene induced in mast cells activated through the high affinity IgE receptor.

In an effort to isolate novel genes involved in inflammation and/or mast cell activation, we have used a combination of differential screening and subtractive hybridization to isolate genes whose expression are induced upon activation of a transformed rat mast cell line. One of the isolated clones, pMCA-32, contained an open reading frame of 278 amino acids that included a putative hydrophobic transmembrane domain, a cysteine rich Ig-like extracellular domain, and a cytoplasmic domain containing three consensus SH2-domain phosphotyrosine binding sites. The MCA-32 gene is also highly conserved between rat and mouse, with the two coding regions being 73% identical. Although the MCA-32 coding region did not contain an obvious signal peptide, MCA-32 protein was detected on the surface of rat mast cells, and the cloned cDNA produced a cell surface protein when expressed in COS-7 cells. MCA-32 RNA from both mouse and rat undergoes alternative splicing, producing an mRNA containing an in-frame deletion of the TM domain, suggesting that a form of MCA-32 protein may be secreted. MCA-32 mRNA expression was up-regulated upon activation of RBL-2H3 cells and was highly abundant in primary peritoneal mast cells. Expression of MCA-32 RNA was only observed in primary and transformed mast cells from rat, while in the mouse MCA-32, RNA was also produced in significant amounts by a number of transformed monocyte cell lines. Thus, MCA-32 is a novel surface protein whose structure and expression suggest roles in the development and/or activation of mast cells and monocytes.

Murine vascular cell adhesion molecule-1 (VCAM-1) proteins encoded by alternatively spliced mRNAs are differentially targeted in polarized cells.

VCAM-1 is an immunoglobulin (Ig) superfamily member expressed in endothelial cells that mediates adhesion to a variety of leukocytes in a VLA-4 dependent manner. In the mouse, two distinct forms of VCAM are produced. One form, VCAMTM, contains seven Ig domains followed by a single transmembrane region and a short cytoplasmic domain. A second form, VCAMGPI, which is preferentially induced by cytokines and LPS, contains only the first three Ig domains and is attached to the cell surface via a glycosylphosphafidylinositol (GPI) anchor. Both vascular and nonvascular expression of VCAM have been reported in a variety of normal and pathological settings. One possible role for the two VCAM isoforms is to allow for the targeted localization of VCAM to specific cell surface domains of polarized cells. This may be particularly relevant since VCAM is known to be expressed by two different polarized cell types, namely endothelial cells and kidney epithelial cells. In this study, MDCK cells permanently expressing either VCAMTM or VCAMGPI were established and used to examine the targeting of VCAM proteins to different polarized surface domains. VCAMTM was primarily located on the basolateral surface while VCAMGPI was located on the apical surface of polarized MDCK cells. Data is also presented that demonstrates that polarized expression is reversed in endothelial cells where VCAMTM was observed primarily on the apical surface. The differential localization of VCAM isoforms on the cell surface has direct implications for the ability of VCAM to mediate cell adhesion and transmigration.

Characterization of E-selectin-deficient mice: demonstration of overlapping function of the endothelial selectins.

The initial rolling interaction of leukocytes with the blood vessel wall during leukocyte trafficking has been postulated to rely on members of the selectin family of adhesion molecules. Two selectins, E-selectin and P-selectin, have been identified that are expressed on activated endothelial cells. Mice deficient in E-selectin expression have been produced in order to examine the role of this selectin in leukocyte trafficking. Mice homozygous for an E-selectin null mutation were viable and exhibited no obvious developmental alterations. E-selectin-deficient mice displayed no significant change in the trafficking of neutrophils in several models of inflammation. However, blocking both endothelial selectins by treatment of the E-selectin-deficient animals with an anti-murine P-selectin antibody, 5H1, significantly inhibited neutrophil emigration in two distinct models of inflammation. While neutrophil accumulation at early times during thioglycollate-induced peritonitis was dependent on P-selectin, neutrophil accumulation at later time points was blocked by 5H1 only in E-selectin-deficient mice but not in wild-type mice. Similarly, edema as well as leukocyte accumulation in a model of delayed-type hypersensitivity in the skin was almost completely prevented by blockade of P-selectin function with 5H1 in the E-selectin-deficient mice while the same treatment had no effect in wild-type mice. These data demonstrate that the majority of neutrophil migration in both models requires an endothelial selectin but that E-selectin and P-selectin are functionally redundant. These data have important implications in the use of selectin antagonists in the treatment of inflammatory disease.

Cytokine induction of an alternatively spliced murine vascular cell adhesion molecule (VCAM) mRNA encoding a glycosylphosphatidylinositol-anchored VCAM protein.

VCAM-1 is an immunoglobulin superfamily member that mediates adhesion of a variety of leukocytes to endothelial cells. VCAM expression has been associated with a variety of disease states and has been implicated in a number of normal processes. The predominant form of VCAM produced in human endothelial cells is a transmembrane protein containing seven immunoglobulin domains. In this study the murine VCAM gene has been characterized to allow the function(s) of VCAM to be studied in a small genetically accessible animal. While expression of an mRNA encoding a seven-immunoglobulin-domain transmembrane VCAM protein was seen in most tissues, the predominant change in VCAM expression upon interleukin 1 beta treatment was the induction of an alternatively spliced VCAM mRNA containing only the first three immunoglobulin domains. This message encodes a glycosylphosphatidylinositol (GPI)-anchored form of VCAM, VCAMGPI. VCAMGPI was efficiently cleaved from the cell surface by phosphatidylinositol-specific phospholipase C, mediated adhesion to leukocytes in a very late antigen 4-dependent manner, and was produced by mouse endothelial cell lines in culture. These data demonstrate that alternate forms of VCAM are produced under different physiological conditions and suggest that VCAMGPI may have a distinct role in inflammatory processes.

The immune response to short-term nutritional intervention in advanced chronic obstructive pulmonary disease.

Nine patients with advanced chronic obstructive pulmonary disease (COPD) and recent weight loss resulting in a state of mild malnutrition were entered into a refeeding program at a clinical research center. They were divided into two groups, one using a hospital diet and the other a hospital diet with supplementation. Both groups of patients gained significant weight. Refeeding and weight gain were associated with a significant increase in absolute lymphocyte count and with an increase in reactivity to skin test antigens after 21 days of refeeding. Few changes occurred in large numbers of additional serum measurements during the study period. These preliminary observations suggest that dietary and supplementary refeeding may improve the immune responses in patients with COPD.

A conserved cis-acting sequence in the 5' leader of avian sarcoma virus RNA is required for packaging.

The deletion of a conserved sequence of ca. 30 nucleotides in the 5' noncoding leader region of an avian sarcoma virus DNA clone resulted in a loss of infectivity after transfection of chicken embryo fibroblasts. Genetic and biochemical analysis of a representative mutant demonstrated that the env gene was expressed normally. Thus, viral RNA transcription, splicing, and translation were not impaired. The amount of mutant viral RNA encapsidated into virions, however, was severely reduced despite the presence of helper-virus. We conclude that the deleted sequence is an essential cis-acting packaging signal.

Interferon-induced growth modulation: low dose maintenance of the antiproliferative response.

Treatment of the Burkitt's lymphoma-derived Daudi cell line with human beta interferon (HuIFN-beta) results in a dose-dependent antiproliferative response. We have defined three phases including: (1) initiation, (2) maintenance, and (3) termination of the antiproliferative state. Each phase is characterized by specific growth modulatory properties. Initiation of the antiproliferative state with a single interferon dose requires 200 International Reference Units (IRU) per ml, depending on such factors including cell density, serum content in the medium and the length of IFN exposure. Initiation of the IFN response occurred within a 4 h incubation period. Even following this short exposure, the vast majority of cells would be sequestered in the G0/G1 cell cycle compartment. As measured by cytofluorimetry there was a 16-20 h delay in the initiation of the antiproliferative response. The antiproliferative response, as measured by changes in the distribution of cells in the cell cycle, was maximal at 24 h after treatment. This delay was equal to the doubling time of the Daudi cell. The antiproliferative response initiated by 200 IRU/ml of IFN was reversible if the IFN was removed and not replenished. Cells showed renewed growth approximately 40 h after treatment. The antiproliferative state could be maintained by additional interferon, but, even after such treatment, the antiproliferative state decayed in a dose-dependent fashion. This decay was proportional to the decrease in the biological activity of the interferon molecule itself. Finally, we showed that the antiproliferative state could be maintained by using only 40 IRU/ml. These cells were maintained in the antiproliferative state for an extended period of time.

Randomized withdrawal from nifedipine: placebo-controlled study in patients with coronary artery spasm.

A multicenter randomized double-blind withdrawal study was conducted to compare the efficacy of nifedipine to that of placebo in vasospastic angina. Following a 2-week single-blind nifedipine baseline period, during which nifedipine was maintained at prestudy levels, 38 patients, 19 taking placebo and 19 continuing nifedipine therapy, either completed a 4-week randomized phase or were prematurely withdrawn because of therapeutic failure. During the randomized phase, an increase in median anginal frequency (2.8 attacks/wk, p less than 0.003) and nitroglycerin usage (0.5 tablets/wk, p less than 0.03) occurred only in the placebo group. The randomized phase was prematurely terminated because of anginal exacerbation in 7 of 19 placebo patients (37%) (only 1 patient receiving nifedipine [p = 0.02] experienced anginal exacerbation). Double-blind therapy was judged effective in 16 patients (84%) receiving nifedipine and in 3 patients (16%) receiving placebo (p less than 0.001). Nifedipine was well tolerated. This study establishes the efficacy of nifedipine in the treatment of variant and validates previous clinical experience.

Nifedipine therapy in angina pectoris: evaluation of safety and side effects.

Nifedipine, a calcium-channel blocker, differs in structure and mode of action not only from the nitrates and beta blockers but also from the other well-known calcium-channel blockers. These differences have important implications for the treatment of angina pectoris. Our clinical experience over a period of 6 years with all types of angina patients--mostly those with chronic stable angina but also those with Prinzmetal's and unstable angina-corroborates the efficacy reports published in the world literature. Although the safety profile of nifedipine has been generally regarded as favorable, a few reports of clinically significant adverse effects in specific patient groups have appeared in the literature. In order to provide a more comprehensive assessment of safety, the records of over 3000 patients treated with nifedipine in open and controlled multiple-dose studies were tabulated and analyzed. Of special concern were patients with clinically significant adverse experiences, patients with a concomitant diagnosis of congestive heart failure (CHF), patients who were being treated concurrently with beta blockers, and patients who had been taking nifedipine for more than 6 months. Results of this analysis confirm that nifedipine can be safely administered to a broad spectrum of angina patients, including those with a concomitant diagnosis of CHF and those receiving concurrent therapy with beta-blocking agents.

Tumorigenicity of Friend murine erythroleukemia cell lines differing in spontaneous differentiation rates.

The relationship between tumorigenicity and in vitro differentiation was studied in four Friend erythroleukemia cell lines. The cell lines were isolated by repeated subcloning of an unmutagenized population (sib selection). The cell lines differed in their ability to form tumors when 10(3) cells were injected subcutaneously into syngeneic mice (8 to 56% animals with tumors). The ability of cell lines to form tumors was not proportional to their ability to be induced to differentiate by dimethylsulfoxide. The cell lines had indistinguishable chromosome numbers, plating efficiencies, number of cycling cells, population doubling times and spontaneous mutation rates to drug resistance. However, the cell lines had different spontaneous differentiation rates in the absence of dimethylsulfoxide (0.87 to 16.0 X 10(-4) per cell per generation). The ability of cell lines to form tumors was inversely proportional to their spontaneous differentiation rate. Apparently cells which are most efficiently blocked in differentiation have the highest probability of forming tumors.