Evidence that liposome incorporation of cyclosporine reduces its toxicity and potentiates its ability to prolong survival of cardiac allografts in mice.

Using liposomes, multilamellar lipid vesicles (MLV), we have found that liposome-incorporated cyclosporine is not only less toxic but also more effective in prolonging survival of cardiac allografts in mice. Following intravenous injection of liposome-incorporated drug, cyclosporine levels in blood were shown to decrease rapidly, while concentrations in spleen were higher (when compared with concentration following administration of commercial preparation). In this context, possible mechanisms of the beneficial effect of liposome-incorporated cyclosporine are discussed.

Local administration of cyclosporin allows reduction of the dose prolonging survival of heart tissue allografts.

In the H-2-incompatible donor-recipient combination (BALB/c----CBA/H) local administration of cyclosporin allows a 4-fold reduction in a single dose prolonging survival of heart tissue allografts. These results suggest that local administration of cyclosporin may be useful for certain grafts.

The effect of chemo- and chemoimmunotherapy on the presence of circulating melanoma cells in peripheral blood. Preliminary results.

Reverse transcription and polymerase chain reaction (RT/PCR) with primers specific for tyrosinase allow for a new method of early detection of individual melanoma cells in peripheral blood. Using this test the effect of chemo- and chemoimmunotherapy on the spread of early micrometastatic cancer cells has been evaluated. No significant correlations have been found between RT/PCR results on the one hand and stage of disease, a kind of the therapy protocol used and usage of the therapy as an adjuvant or palliative on the other hand. Thus, although the RT/PCR test for detection of circulating individual melanoma cells might help in identification of minimal residual disease in some patients, it has no application for routine staging of more advanced disease and in monitoring the response to therapy.

Synergistic antiproliferative activity of tumor necrosis factor alpha (TNF-alpha) and lovastatin.

We assessed the antiproliferative effect of tumor necrosis factor alpha (TNF-alpha) and lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, alone and in combination, on two murine tumor cell lines. Recombinant TNF-alpha inhibited proliferation of murine MmB16 melanoma cells in a concentration-dependent fashion but stimulated growth of murine L1210 leukemia cells at 0.1 ng/ml concentration. Lovastatin inhibited proliferation both of murine MmB16 melanoma cells and of murine L1210 leukemia cells in a concentration-dependent fashion. In combination with tumor necrosis factor alpha lovastatin inhibited synergistically growth of both cell lines as assessed by isobologram analysis. Our data show that lovastatin, a cholesterol synthesis inhibitor, introduced to the clinic to treat hypercholesterolemia, used either as a single or in combination with TNF-alpha inhibits growth of MmB16 melanoma and L1210 leukemia cells.

Allosteric activation of protein phosphatase 2C by D-chiro-inositol-galactosamine, a putative mediator mimetic of insulin action.

Insulin-stimulated glucose disposal in skeletal muscle proceeds predominantly through a nonoxidative pathway with glycogen synthase as a rate-limiting enzyme, yet the mechanisms for insulin activation of glycogen synthase are not understood despite years of investigation. Isolation of putative insulin second messengers from beef liver yielded a pseudo-disaccharide consisting of pinitol (3-O-methyl-d-chiro-inositol) beta-1,4 linked to galactosamine chelated with Mn(2+) (called INS2). Here we show that chemically synthesized INS2 has biological activity that significantly enhances insulin reduction of hyperglycemia in streptozotocin diabetic rats. We used computer modeling to dock INS2 onto the known three-dimensional crystal structure of protein phosphatase 2C (PP2C). Modeling and FlexX/CScore energy minimization predicted a unique favorable site on PP2C for INS2 in a surface cleft adjacent to the catalytic center. Binding of INS2 is predicted to involve formation of multiple H-bonds, including one with residue Asp163. Wild-type PP2C activity assayed with a phosphopeptide substrate was potently stimulated in a dose-dependent manner by INS2. In contrast, the D163A mutant of PP2C was not activated by INS2. The D163A mutant and wild-type PP2C in the absence of INS2 had the same Mn(2+)-dependent phosphatase activity with p-nitrophenyl phosphate as a substrate, showing that this mutation did not disrupt the catalytic site. We propose that INS2 allosterically activates PP2C, fulfilling the role of a putative mediator mimetic of insulin signaling to promote protein dephosphorylation and metabolic responses.

Effect of positioning on discomfort from intramuscular injections in the dorsogluteal site.

An intramuscular injection into a relaxed muscle is believed to result in less discomfort than an injection into a contracted muscle. When the femur is internally rotated, the gluteus maximus muscle is relaxed. The hypothesis that a dorsogluteal injection with the femur internally rotated will cause less discomfort than when the femur is externally rotated was tested in 44 surgical patients who received two injections of preoperative medication. Each patient received an injection of a narcotic medication and one of diazepam. All possible combinations of the factors--position (internal and external rotation), order of injection (first or second injection), and medication (narcotic or diazepam)--were determined, and patients were randomly assigned to one of these conditions. Patients rated their perceived discomfort after each injection on a five-point scale. The hypothesis was supported by discomfort ratings from injections of both types of medications, although diazepam injections caused significantly more discomfort than injections of narcotics. Older patients tended to report less discomfort from diazepam injections than younger patients. Sex, order of injection, and nurse administering the injection did not significantly influence discomfort ratings.

Editing of human alpha-galactosidase RNA resulting in a pyrimidine to purine conversion.

During a study of the gene coding for alpha-galactosidase (EC 3.2.1.22), the lysosomal enzyme deficient in Fabry's disease, RT-PCR amplification of alpha-galactosidase mRNAs obtained from three different tissues isolated from males revealed a substantial number of clones with a U to A conversion at the nucleotide position 1187. Such a modification of the coding sequence would result in an amino acid substitution in the C-terminal region (Phe396Tyr) of the enzyme. Neither PCR analysis of the genomic sequence nor the RT-PCR amplification of RNA obtained by in vitro transcription of the wild-type cDNA showed this change in the sequence. Multiple genes, pseudogenes are allelic variants were excluded. Hence, we propose RNA editing as a mechanism responsible for this base change in the alpha-galactosidase RNA.

[Subcutaneous immunoglobulin infusion in antibody deficiency substitution of a patient sensitized to intravenous immunoglobulins. Case report]. / Podskórne infuzje immunoglobulin w substytucji niedoboru przeciwcial u chorej uczulonej na immunoglobuliny dozylne. Opis przypadku.

Patients with severe form of common variable immunodeficiency require chronic immunoglobulin substitution. However, intravenous Ig administration may not be possible in some of them because of serious anaphylactoid reactions. It has been suggested that such patients may tolerate well Ig administration by subcutaneous infusion. A case is described (originally with IgG level of 53 mg/dl) who reacted with anaphylactic shock to intravenous immunoglobulin and now for more than 7 months at 1-2 week intervals receives immunoglobulin by subcutaneous infusion without any adverse reactions and maintaining IgG level above 400 mg/dl. In contrast to the period proceeding immunoglobulin substitution, the patient remains free of bacterial infection during last 7 months.

[Preliminary evaluation of a method for detection of individual malignant melanoma cells in peripheral blood while monitoring the course of the malignancy and its treatment]. / Wstepna ocena metody wykrywania pojedynczych komórek czerniaka zlossliwego we krwi obwodowej w monitorowaniu przebiegu choroby nowotworowej i jej leczenia.

Using reverse transcription and polymerase chain reaction (PCR) with primers specific for tyrosinase the individual melanoma cells were detected in peripheral blood of patients in different stages of disease, after excision of primary lesion and prior and after chemotherapy. No relation between stage of disease (including situations with overt generalized spread of melanoma) and probability of positive PCR reaction detecting transcript for tyrosinase gene was found. Many patients in III and IV stages were negative for prolonged periods. Therefore, this method cannot be used for monitoring of all patients, because many of them are negative prior as well as after chemotherapy. With regard to the effects of therapy, the patients differed one to another and although some persons positive prior treatment became negative thereafter, a similar number of initially negative patients became positive after treatment.

Donor-specific transfusion via the portal venous route induces prolongation of H-2-compatible but not H-2-incompatible cardiac graft survival.

In the H-2-compatible donor-recipient combination (BALB/c----DBA/2), pretransplant donor-specific blood transfusion (DST) via the portal venous (PV) route significantly prolonged cardiac graft survival. DST via the intravenous (IV) route (systemic circulation) also showed a marked prolongation of heart tissue transplant survival in this model. In the H-2-incompatible combination (BALB/c----CBA/H), DST via the IV - but not via the PV - route resulted in accelerated graft rejection.

Impaired tumor growth in colony-stimulating factor 1 (CSF-1)-deficient, macrophage-deficient op/op mouse: evidence for a role of CSF-1-dependent macrophages in formation of tumor stroma.

Macrophages have been suggested to play a major role in the immune response to cancer. They have also been suggested to stimulate the formation of tumor stroma and to promote tumor vascularization. The availability of the op/op mouse, which has no endogenous colony-stimulating factor 1 (CSF-1) and which possesses a profound macrophage deficiency, provides a new model to verify these notions. Subcutaneous growth of transplantable Lewis lung cancer (LLC) is markedly impaired in the op/op mice compared with normal littermates. Treatment of tumor-bearing op/op mice with human recombinant CSF-1 corrects this impairment. Histological analysis of tumors grown in op/op and normal mice revealed marked differences. Tumors grown in op/op mice display a decreased mitotic index and pronounced necrosis, particularly hemorrhagic. Moreover, particularly in the op/op tumors, peculiar sinusoid-like abortive vessels (not filled with blood) have been observed. These tumors, in contrast to tumors grown in normal mice, are almost deprived of regular arteries and veins. In contrast to tumors grown in normal mice, they exhibit almost no Sirius red-stained collagenous fibers and Gomori silver-stained reticular fibers. Our data suggest that the CSF-1-dependent macrophage subpopulation missing in op/op mice plays a primary role in supporting tumor stroma formation and tumor vascularization in murine LLC tumors.