Relationship between adenovirus-mediated aquaporin 1 expression and fluid movement across epithelial cells

AdhAQP1, a recombinant adenovirus encoding the human water channel aquaporin 1 (AQP1), has been shown to be useful for gene therapy of salivary glands rendered hypofunctional following irradiation. Here we utilized AdhAQP1 to examine the relationship between AQP1 expression and fluid movement across a polarized salivary epithelial cell monolayer. In response to a 440 to 340 mosm gradient, net fluid movement across cells infected with AdhAQP1 was approximately 10-fold that seen in uninfected cells or cells infected with a control virus. At a multiplicity of infection (MOI) of 5, fluid movement was linear for 15-30 min. Increasing the osmotic gradient resulted in a proportional increase in fluid movement. At low virus MOIs (0.1-1.0), fluid movement was markedly enhanced relative to that seen at higher MOIs (approximately 5.10), where the level of AQP1 expression and number of cells transduced were considerably greater. We conclude that significant, osmotically-obliged fluid movement in a salivary cell monolayer with low basal water permeability does not require high levels of AQP1 expression.     

Time course of chronic diazepam effects on the auditory evoked potential of the rat

We have analyzed 30 human tumor specimens (two breast, six lung, and 21 ovarian carcinomas and one malignant melanoma) for the expression of the transporter associated with antigen processing, TAP-1. Cell suspensions judged to contain negligible contamination with non-tumor cells were tested for reactivity with the antibody directed to TAP-1 in Western blot. According to these results all lysates prepared from the tumor samples contained the protein, but with considerable quantitative variations. Tumors exposed in vitro for a short time to IFN-gamma and TNF-alpha had elevated TAP-1 levels in 12 out of 30 experiments. In accordance with previous results, the tumor cell populations were heterogeneous with regard to MHC class I expression, as analyzed by indirect immunofluorescence on viable cell suspensions. Short term in vitro treatment with IFN-gamma and TNF-alpha elevated MHC class I expression in several tumors. In several mixed cultures, cytokine treated tumor cells induced cytotoxic activity in autologous or allogeneic lymphocyte populations. In vitro treatment with IFN-gamma and TNF-alpha upregulated thus the expression of MHC class I and TAP-1 in a fraction of the tumor samples. However the potentiation of the capacity to interact with T cells induced by the cytokines was not always parallel with the changes in these two parameters. It is therefore likely that the cytokine treatment induced additional changes in the tumors which contributed to their capacity to elicit the T cell response.     

Renal dysfunction after myocardial revascularization: risk factors, adverse outcomes, and hospital resource utilization. The Multicenter Study of Perioperative Ischemia Research Group

Preclinical studies with murine tumor models have demonstrated that tumor cell vaccines engineered to secrete certain cytokines in a paracrine fashion elicit systemic immune responses capable of eliminating small amounts of established tumor. In particular, tumors that express the cytokine GM-CSF produce potent systemic antitumor immune responses against poorly immunogenic murine tumors. These results have encouraged the development of paracrine-cytokine secreting tumor vaccines for gene therapy of human cancer. GM-CSF recruits professional antigen-presenting cells, which in turn activate effector T cells. These findings suggest that allogeneic as well as autologous tumor cells can be used as the tumor source for developing cancer vaccines. A major obstacle to creating genetically modified human allogeneic tumor vaccines is the absence of stable cell lines required for efficient gene transfer, because most human tumors isolated from primary surgical specimens fail to proliferate in long-term culture. We have developed a method for the routine generation of in vitro cell lines from primary tumors of the pancreas. This method overcomes the common problem of stromal and fibroblast overgrowth that can inhibit the in vitro expansion of many histologic types of tumors. In addition, we have analyzed 12 of these cell lines for cytokeritin and mutated K-ras expression to demonstrate that they derive from the original epithelial tumor tissue. The lines can be genetically modified to stably express the cytokine GM-CSF. These methods should be helpful to investigators attempting to establish cell lines from other histologic tumor types for the development of allogeneic genetically modified tumor vaccines.     

Ex vivo culture of CD34+/Lin-/DR- cells in stroma-derived soluble factors, interleukin-3, and macrophage inflammatory protein-1alpha maintains not only myeloid but also lymphoid progenitors in a novel switch culture assay

We have demonstrated that long-term culture initiating cells (LTC-IC) are maintained in a stroma noncontact (SNC) culture where progenitors are separated from stroma by a microporous membrane and LTC-IC can proliferate if the culture is supplemented with interleukin-3 (IL-3) and macrophage inflammatory protein-1alpha (MIP-1alpha). We hypothesize that the same conditions, which result in LTC-IC proliferation, may also maintain lymphoid progenitors. Natural killer (NK) cells are of lymphoid lineage and a stromal-based culture can induce CD34+/Lin-/DR- cells to differentiate along the NK cell lineage. We developed a three-step switch culture assay that was required to demonstrate the persistence of NK progenitors in CD34+/Lin-/DR- cells assayed in SNC cultures supplemented with IL-3 and MIP-1alpha. When CD34+/Lin-/DR- progeny from the SNC culture were plated sequentially into "NK cell progenitor switch" conditions (contact with stromal ligands, hydrocortisone-containing long-term culture medium, IL-2, IL-7, and stem cell factor [SCF]) followed by "NK cell differentiation" conditions (contact with stromal ligands, human serum, no hydrocortisone, and IL-2), significant numbers of CD56+/CD3- NK resulted, which exhibited cytotoxic activity against K562 targets. All steps are required because a switch from SNC cultures with IL-3 and MIP-1alpha directly to "NK cell differentiation" conditions failed to yield NK cells suggesting that critical step(s) in lymphoid commitment were missing. Additional experiments showed that CD34+/CD33- cells present after SNC cultures with IL-3 and MIP-1alpha, which contained up to 30% LTC-IC, are capable of NK outgrowth using the three-step switch culture. Limiting dilution analysis from these experiments showed a cloning frequency within the cultured CD34+/CD33- population similar to fresh sorted CD34+/Lin-/DR- cells. However, after addition of FLT-3 ligand, the frequency of primitive progenitors able to develop along the NK lineage increased 10-fold. In conclusion, culture of primitive adult marrow progenitors ex vivo in stroma-derived soluble factors, MIP-1alpha, and IL-3 maintains both very primitive myeloid (LTC-IC) and lymphoid (NK) progenitors and suggests that these conditions may support expansion of human hematopoietic stem cells. Addition of FLT-3 ligand to IL-2, IL-7 SCF, and stromal factors are important in early stages of NK development.     

Effects of species raw material source, ash content, and processing temperature on amino acid digestibility of animal by-product meals by cecectomized roosters and ileally cannulated dogs

We conducted experiments to determine amino acid (AA) digestibility of nine animal by-product meals using precision-fed cecectomized roosters and ileally cannulated dogs. The products initially evaluated in roosters were meat and bone meals (MBM) containing 24 or 34% ash, poultry by-product meals (PBP) containing 7 or 16% ash, lamb meals (LM) containing 15 or 24% ash, a LM analog containing a mixture of LM and turkey meal, and two MBM processed at either a low or high temperature. The MBM and PBP differing in ash, low-ash LM, and low-temperature MBM then were incorporated into extruded dry dog foods and evaluated in cecectomized roosters and ileally cannulated dogs. True digestibility of total AA in roosters averaged 76% for the nine meals fed alone, with the low-temperature MBM being highest at 84% and the low-ash LM being lowest at 66% (P < .05). No consistent differences in rooster AA digestibility were observed between pairs of meals differing in ash content. Digestibilities of AA were higher in the low-temperature MBM than in the high-temperature MBM. Differences in rooster AA digestibility values among the six extruded dog foods containing selected animal meals were similar to those observed when the animal meals were fed alone. The ileally cannulated dog assay yielded results for AA digestibilities that were highly correlated (r = .87 to .92) with those of the rooster assay, whereby the high-ash MBM and low-temperature MBM foods had the highest mean AA digestibility at 82% and the low-ash LM food had the lowest mean AA digestibility at 62% (P < .05). Again, no consistent differences in AA digestibilities for dogs were observed between pairs of dog foods containing MBM or PBP differing in ash content. Results of this study indicated that processing temperature influenced AA digestibility of MBM, but species raw material source and ash content had no consistent effect on AA digestibility. Results also indicated that the precision-fed cecectomized rooster assay could be used to predict differences in AA digestibility among animal by-product meals for dogs.     

In vitro and in vivo activity of two Pt(IV) salts against leishmania donovani

The activities of 8 platinum drug complex salts were determined against Leishmania donovani promastigotes. The three most active salts were selected: [PtIVBr6]H2 (pentamidine); [PtIVBr6]H2 (stilbamidine), and [PtIVCl6]H2 (2-piperazinyl(1) ethyl amine), which induced growth-inhibition rates of more than 50% at 24 h of treatment and at the maximum dosage tested. The cytotoxicity assays on the macrophage cell line J-774 showed high cytotoxicity for the salt [PtIVBr6]H2 (stilbamidine) with a percentage of specific 51Cr release of 58.2% at 24 h of incubation and 100 microg/ml. Meanwhile, assays of the other compounds showed practically no cytotoxicity. The salt [PtIVBr6]H2 (pentamidine) notably inhibited the incorporation of 3H-thymidine in the treated parasites. The ultrastructural alterations observed in the flagellates treated with the salts [PtIVCl6]H2 (2-piperazinyl(1)ethyl amine) and [PtIVBr6]H2 (pentamidine) suggest that both act preferentially at the nuclear level and at the kinetoplast-mitochondrion complex. Both compounds showed a high in vivo activity in parasitized Wistar rats.     

Synthesis and potent antifolate activity and cytotoxicity of B-ring deaza analogues of the nonpolyglutamatable dihydrofolate reductase inhibitor Nalpha-(4-amino-4-deoxypteroyl)-Ndelta-hemiphthaloyl- L-ornithine (PT523)

Six new B-ring analogues of the nonpolyglutamatable antifolate Nalpha-(4-amino-4-deoxypteroyl)-Ndelta-hemiphthaloy l-L-ornithine (PT523, 3) were synthesized with a view to determining the effect of modifications at the 5- and/or 8-position on dihydrofolate reductase (DHFR) binding and tumor cell growth inhibition. The 5- and 8-deaza analogues were prepared from methyl 2-L-amino-5-phthalimidopentanoate and 4-amino-4-deoxy-N10-formyl-5-deaza- and -8-deazapteroic acid, respectively. The 5,8-dideaza analogues were prepared from methyl 2-L-[(4-aminobenzoyl)amino]-5-phthalimidopentanoate and 2, 4-diaminoquinazoline-6-carbonitriles. The Ki for inhibition of human DHFR by the 5-deaza and 5-methyl-5-deaza analogues was about the same as that of 3 (0.35 pM), 11-fold lower than that of aminopterin (AMT, 1), and 15-fold lower than that of methotrexate (MTX, 2). However the Ki of the 8-deaza analogue was 27-fold lower than that of 1, and that of the 5,8-dideaza, 5-methyl-5,8-dideaza, and 5-chloro-5,8-dideaza analogues was approximately 50-fold lower. This trend was consistent with the published literature on the corresponding DHFR inhibitors with a glutamate side chain. In colony formation assays against the human head and neck squamous carcinoma cell line SCC25 after 72 h of treatment, the 5- and 8-deaza analogues were approximately as potent as 3, whereas the 5,8-dideaza analogue was 3 times more potent. 5-Methyl and 5-chloro substitution was also favorable, with the 5-methyl-5-deaza analogue being 2. 5-fold more potent than the 5-deaza analogue. However the effect of 5-methyl substitution was less pronounced in the 5,8-dideaza analogues than in the 5-deaza analogues. The 5-chloro-5,8-dideaza analogue of 3 was the most active member of the series, with an IC50 = 0.33 nM versus 1.8 nM for 3 and 15 nM for MTX. The 5-methyl-5-deaza analogue of 3 was also tested at the National Cancer Institute against a panel of 50 human tumor cell lines in culture and was consistently more potent than 3, with IC50 values in the low-nanomolar to subnanomolar range against most of the tumors. Leukemia and colorectal carcinoma cell lines were generally most sensitive, though good activity was also observed against CNS tumors and carcinomas of the breast and prostate. The results of this study demonstrate that B-ring analogues of 3 inhibit DHFR activity and tumor cell colony formation as well as, or better than, the parent compound. In view of the fact that 3 and its B-ring analogues cannot form polyglutamates, their high cytotoxicity relative to the corresponding B-ring analogues of AMT is noteworthy.