Quantitative evaluation of liver-specific promoters from retroviral vectors after in vivo transduction of hepatocytes

Hepatic gene therapy could be used to treat a number of inherited blood diseases such as hemophilia or thrombophilia. Although liver-directed retroviral transduction can result in long-term gene expression in vivo, the low level of protein production has limited its clinical application. We reasoned that the insertion of liver-specific promoters into retroviral vectors would increase gene expression in vivo. The 347- bp human alpha 1-antitrypsin (hAAT), the 810-bp murine albumin (mAIb), the 490-bp rat phosphoenolpyruvate carboxykinase (rPECK), and the 596- bp rat liver fatty acid binding protein promoters were inserted into a Moloney murine leukemia retroviral backbone containing the hAAT reporter gene. Vectors that produced appropriately sized RNA and hAAT protein in vitro were tested in vivo by transducing regenerating rat livers. Long-term serum expression of the hAAT reporter gene was normalized to retroviral transduction efficiency as determined by using a polymerase chain reaction-based assay of genomic DNA from transduced rat livers. The hAAT, mAIb, and rPEPCK promoters were, respectively, 35- , 8-, and 0.02-fold as strong as the previously studied constitutive Pol-II promoter. We conclude that the hAAT promoter resulted in the highest expression from a retroviral vector and may result in therapeutically significant expression of other clinically significant blood proteins.     

Liposomal delivery of methylphosphonate antisense oligodeoxynucleotides in chronic myelogenous leukemia [see comments]

Chronic myelogenous leukemia (CML) is a hematologic malignancy characterized by the presence of the Philadelphia (Ph) chromosome. Bcr- abl, the fusion gene associated with the Ph chromosome, expresses a p210bcr-abl protein that promotes a selective expansion of mature myeloid progenitor cells. Methylphosphonate (MP) oligodeoxynucleotides complementary to specific regions of the bcr-abl mRNA were incorporated in liposomes. We studied the effects of liposomal MP (L-MP) on the growth inhibition of CML-like cell lines. L-MP targeted to the breakpoint junctions of the bcr-abl mRNA inhibited the growth of CML cells. Fifty percent inhibition was achieved at approximately 1 mumol/L of L-MP oligonucleotide concentrations. The inhibitory effect was selective because growth inhibition was observed only with CML but not with control cell lines. Moreover, CML cell growth inhibition was dependent on the sequence of the MP oligodeoxynucleotides incorporated in the liposomes. The growth inhibition of CML cells by L-MP resulted from selective inhibition of the expression of the p210bcr-abl protein.     

Mode of action of iron (III) chelators as antimalarials: II. Evidence for differential effects on parasite iron-dependent nucleic acid synthesis

Iron chelation treatment of red blood cells infected with Plasmodium falciparum selectively intervenes with iron-dependent metabolism of malaria parasites and inhibits their development. Highly permeant hydroxamate iron chelator RSFileum2 affects all parasite stages when cultures are continuously exposed to drug, but affects primarily ring stages when assessed for irreversible effects, ie, sustained inhibition remaining after drug removal. On the other hand, the hydrophilic and poorly permeant desferrioxamine (DFO) affects primarily trophozoite/schizont stages when tested either in the continuous mode or irreversible mode. Unlike parasites, mammalian cells subjected to similar drug treatment show complete growth recovery once drugs are removed. Our studies indicate that parasites display a limited capacity to recover from intracellular iron depletion evoked by iron chelators. Based on these findings we provide a working model in which the irreversible effects of RSFs on rings are explained by the absence of pathways for iron acquisition/utilization by early forms of parasites. Trophozoite/schizonts can partially recover from RSFileum2 treatments, but show no DNA synthesis following DFO treatment even after drug removal and iron replenishment by permeant iron carriers. At trophozoite stage, the parasite uses a limited pathway for refurnishing its iron-containing enzymes, thus overcoming iron deprivation caused by permeant RSFileum2, but not by DFO because this latter drug is not easily removable from parasites. Their DNA synthesis is blocked by the hydroxamate iron chelators probably by affecting synthesis of ribonucleotide reductase (RNRase). Presumably in parasites, prolonged repression of the enzyme leads also to irreversible loss of activity. The action profiles of RSFileum2 and DFO presented in this study have implications for improved chemotherapeutic performance by combined drug treatment and future drug design based on specific intervention at parasite DNA synthesis.     

An increased HLA DR2 frequency is seen in aplastic anemia patients

The underlying etiology of aplastic anemia is unknown in the majority of patients, although medications, chemical exposure, or viral infections can be implicated in some. Genetic susceptibility to a variety of diseases has been shown and it has recently been suggested that aplastic anemia is more common in individuals who are HLA DR2+ than in the general population. To examine this question, we retrospectively analyzed the results of HLA-DR typing in 75 aplastic anemia patients who received antithymocyte globulin (ATG) therapy or an HLA-matched sibling bone marrow transplant at UCLA between 1978 and 1989. Thirty-one patients were DR2+, a 1.9-fold higher incidence than the expected number of 16.6 patients (P < .0005). Of the 37 patients who received ATG, 33 were evaluable for a response; 14 patients had either a complete (4 patients) or partial (10 patients) response, for an overall response rate of 42.4%. Of the 14 DR2+ patients who received ATG, 7 responded, for a 50% response rate, which is not significantly higher than the response rate for the DR2- patients (7 of 19 [36.8%]; P = .50). The median survival of patients who are DR2+ was slightly, but not significantly, longer than that of the DR2- patients in the ATG group (P = .19). Although the incidence of HLA DR2 was clearly increased in these patients with aplastic anemia, response rates to ATG were not significantly different in the DR2+ and DR2- patients.     

Anticoagulation therapy and primary care internal medicine

The anticoagulation clinics at the University of Virginia Health Sciences Center and the University of California at Davis Medical Center are nurse-practitioner-operated, are affiliated with the general medicine clinic, and rely on portable prothrombin time (PT) monitors that use whole blood and provide timely as well as accurate results reported in PT seconds or as the international normalized ratio (INR). On-site PT/INR testing at these clinics simplifies anticoagulation, mandates direct patient contact, and facilitates primary as well as comprehensive care for patients requiring multispecialty services in large tertiary care centers. Encounters are relatively brief, averaging 19 minutes; 72% of the encounter time involves anticoagulation care and 28% involves primary care. Anticoagulation results using portable PT/INR monitors are safe and accurate based on comparisons with results from clinics relying on standard instruments. Supported in part by VA grants IIR 87-063 and IIR 90-036.     

Cytogenetic and immunophenotypic analysis of cell lines established from patients with T cell leukemia/lymphoma

Cell lines were established from five patients with T cell malignancies. Two patients had T cell lymphoblastic lymphoma (T-LL), whereas three patients had T cell acute lymphoblastic leukemia (T-ALL). Both T-LL cell lines expressed cell surface antigens characteristic of midthymocytes (Leu 2, 3, 6+). One T-ALL cell line also expressed this immunophenotype, one expressed suppressor/cytotoxic antigens (Leu 2+; Leu 3, 6-), and one expressed antigens of a mature but uncommitted T cell (Leu 4+; Leu 2, 3, 6-). Cytogenetic analysis showed that each cell line had 46 chromosomes with pseudodiploidy. The three T-ALL cell lines had only a few chromosome changes; one cell line had one deletion, another had two deletions, and the third had a translocation and two deletions (including loss of part of 9p). In comparison, both T-LL cell lines had complex chromosome changes, including most notably a rearrangement of band 14q11.2. The immunophenotypes and chromosome breakpoints showed patterns of interlock between the T-LL and T-ALL cell lines because common abnormalities occurred at six distinct chromosome sites. Cell lines with limited and specific chromosomal abnormalities are important because they can provide the basic material for molecular genetic studies that could elucidate the genetic mechanisms involved in neoplasia.     

PEROXYNITRITE: A MEDIATOR OF INCREASED MICROVASCULAR PERMEABILITY?

1. Increased expression of inducible nitric oxide synthase (iNOS) and subsequent elevation of nitric oxide (NO) levels at inflammatory sites have led to the suggestion that peroxynitrite (the reaction product of superoxide and NO) is involved in proinflammatory processes. The present study has investigated the ability of peroxynitrite to induce oedema formation in the rat cutaneous microvasculature. 2. Peroxynitrite was synthesized from hydrogen peroxide and acidified nitrite. Spectrophotometry was used to measure the concentration and breakdown of peroxynitrite. It was also used to determine maximum amounts of hydrogen peroxide and sodium nitrite remaining after synthesis. 3. Oedema formation in response to intradermall. (i.d.) injected peroxynitrite, hydrogen peroxide and sodium nitrite was measured by the extra vascular accumulation of i.v. [¹²⁵I]-albumin in the anaesthetized rat. 4. Peroxynitrite (40,100 and 200 nmol/site) acted in a dose-dependent manner to cause a mean (± SEM) increase in plasma extravasation of 24 ± 2,55 ± 5 and 69 ± 6 μL, respectivel. (n= 4), with resulting inflammatory oedema. Peroxynitrite induced significantly larger plasma extravasation than equivalent vehicle controls at doses of 100 (P > 0.05) and 200 nmol (P > 0.001). This increased extravasation appears to be a direct microvascular response to peroxynitrite administration and not due to either a raised pH, necessary to stabilize the peroxynitrite, or contaminating concentrations of hydrogen peroxide or sodium nitrite from which peroxynitrite is formed. 5. These results suggest that peroxynitrite acts to increase microvascular permeability and oedema formation. Therefore, peroxynitrite may mediate vascular pro-inflammatory effects in addition to its direct cytotoxic activity.     

Status report on ambulatory care and education in the VA Western Region and western medical schools

Since the initial 1988 Ambulatory Care and Education (ACE) conference, reported in the October 1989 supplement to Academic Medicine, the Western Region Veterans Health Administration and its 11 affiliated western medical schools have established several programs and related activities that implement strategic ACE recommendations. This report gives an update on the ACE Advisory Group; the Pilot Ambulatory Care and Education (PACE) Center at the VA Medical Center, Sepulveda, California; other innovative ambulatory care and education projects; the second ACE Development Conference; and future activities in the expanded Western Region of the Department of Veterans Affairs.