Regulation of human glioma cell migration, tumor growth, and stemness gene expression using a Lck targeted inhibitor.

Migration of human glioma cells (hGCs) within the brain parenchyma makes glioblastoma one of the most aggressive and lethal tumors. Studies of the cellular and molecular mechanisms underlying hGC migration are hindered by the limitations of existing glioma models. Here we developed a dorsal root ganglion axon-oligodendrocyte-hGC co-culture to study in real time the migration and interaction of hGCs with their microenvironment. hGCs interact with myelinated and non-myelinated axons through the formation of pseudopodia. Isolation of pseudopodia-localized polysome-bound RNA reveals transcripts of Lck, Paxillin, Crk-II, and Rac1 that undergo local translation. Inhibition of Lck phosphorylation using a small-molecule inhibitor (Lck-I), blocks the phosphorylation of Paxillin and Crk-II, the formation of pseudopodia and the migration of hGCs. In vivo intraventricular administration of the Lck-I using an orthotopic xenograft glioma model, results in statistically significant inhibition of tumor size and significant down-regulation of Nanog-targeted genes, which are associated with glioblastoma patient survival. Moreover, treatment of human glioma stem cells (hGSCs) with Lck-I, results in significant inhibition of self-renewal and tumor-sphere formation. The involvement of Lck in different levels of glioma malignant progression, such as migration, tumor growth, and regulation of cancer stemness, makes Lck a potentially important therapeutic target for human glioblastomas.

Defining the risk of developing grade 2 proctitis following 125I prostate brachytherapy using a rectal dose-volume histogram analysis.

OBJECTIVE: To determine the rectal tolerance for developing Grade 2 radiation proctitis after 125I prostate implantation based on the rectal dose-volume histogram. METHODS AND MATERIALS: Two hundred twelve patients with T1-T2 prostate cancer underwent 125I implantation without external beam irradiation. One month after the procedure, all patients underwent CT-based postimplant dosimetry (3-mm abutting slices). The rectal volumes, defined by an inner and outer wall, were determined from 9 mm above the seminal vesicles to 9 mm below the prostate apex. All doses were calculated by TG43 formalism. The prostate prescription dose was 160 Gy. A dose response analysis was undertaken for volumes of rectal tissue receiving a given dose. Dose levels examined were 80 Gy, 100 Gy, 120 Gy, 140 Gy, 160 Gy, 180 Gy, 200 Gy, 220 Gy, and 240 Gy. Grade 2 proctitis was defined as rectal bleeding occurring at least once a week for a minimum period of one month. The risk of proctitis was calculated using actuarial methods. For each dose level, a critical volume cutpoint was chosen to define a low and high volume group of patients. The cutpoint was determined based on two goals: minimizing thep value and finding a < or =5% risk of proctitis in the low volume group. Patients were followed from 12 to 61 months (median: 28 months) after implantation. RESULTS: Twenty-two patients developed Grade 2 proctitis: 14% within the first year, 72% between the first and second year, and 14% during the third year after the implant date. After the third year postimplantation, no cases of proctitis were reported. Proctitis was found to be significantly volume dependent for a given dose. The prescription dose (160 Gy) delivered to < or =1.3 cc of rectal tissue resulted in a 5% rate of proctitis at 5 years vs. 18% for volumes >1.3 cc (p = 0.001). Similar results were found for all doses examined. As the rectal volume receiving the prescription dose (160 Gy) increased, so did the proctitis rate: 0% for < or =0.8 cc, 7% for >0.8-1.3 cc, 8% for >1.3-1.8 cc, 24% for >1.8-2.3 cc, and 25.5% for >2.3cc (p = 0.002). CONCLUSIONS: Rectal dose-volume histogram analysis is a practical and predictive method of assessing the risk of developing Grade 2 proctitis after 125I prostate implantation. Delivered dose should be kept below defined rectal volume thresholds to minimize this risk. This information can allow one to decrease rectal morbidity by modifying prostate implant technique.

Interferon-gamma suppresses T-cell proliferation to mitogen via the nitric oxide pathway during experimental acute graft-versus-host disease.

The development of graft-versus-host disease (GVHD) is associated with long-lasting and profound deficits in immune function that lead to increased morbidity and mortality after bone marrow transplantation (BMT). We investigated a mechanism of T-cell immunodeficiency in response to mitogen or alloantigen in an experimental model of acute GVHD by analyzing the roles of two immunosuppressive moieties: interferon gamma (IFN-gamma) and nitric oxide (NO). Splenocytes from mice with GVHD did not proliferate either to the T-cell mitogen, concanavalin A (Con A), or to host alloantigens, but only mitogen-activated cultures produced increased levels of NO. The abrogation of NO synthesis with LG-mono-methyl-arginine (NMMA) restored mitogen-induced proliferation but not the response to host antigens. The mechanism of impared proliferation to mitogen was dependent on IFN-gamma because blockade of this cytokine in culture inhibited NO production and restored proliferation to Con A to levels similar to those in transplanted control mice without GVHD. NMMA did not substantially reduce IFN-gamma levels, demonstrating that NO acted distally to IFN-gamma in the pathway of immunosuppression in response to mitogen. Furthermore, the prevention of IFN-gamma production in vivo after allogeneic BMT, by transplantation of polarized type 2 donor T cells (secreting interleukin-4 but not IFN-gamma), also prevented NO production and restored splenocyte responses to mitogen. Our data demonstrate the existence of NO-dependent and NO-independent pathways involved in suppression of T-cell proliferation during acute GVHD. Excess NO synthesis appears to be one mechanism by which IFN-gamma induces immunodeficiency after allogeneic BMT.

Suppression of B-cell proliferation to lipopolysaccharide is mediated through induction of the nitric oxide pathway by tumor necrosis factor-alpha in mice with acute graft-versus-host disease.

Graft-versus-host disease (GVHD) is associated with impaired B-cell responses. We investigated the mechanism of impaired proliferation of B cells in response to the mitogen lipopolysaccharide (LPS) by analyzing the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO), both of which have independently been described as important effector mechanisms in the pathogenesis of acute GVHD. A threefold decrease of mature surface Ig-positive (slg+) B cells was observed in GVHD spleens isolated 2 weeks after transplant. However, proliferation of these cells in response to LPS was suppressed by more than 35-fold. Activated GVHD splenocytes secreted large amounts of TNF-alpha and NO in culture. Neutralization of TNF-alpha with anti-TNF-alpha antibody (Ab) both abrogated NO production and restored LPS-induced proliferation of B cells to levels found in non-GVHD control mice. The specific inhibition of NO synthesis with LG-monomethyl-arginine (NMMA) restored splenocyte responses but did not significantly reduce TNF-alpha levels, showing that TNF-alpha per se did not cause immunosuppression. These data show that, during GVHD, induction of the NO pathway is an important mechanism that mediates B-cell hyporesponsiveness to LPS and that this pathway is induced by TNF-alpha.

Polarized type 2 alloreactive CD4+ and CD8+ donor T cells fail to induce experimental acute graft-versus-host disease.

Acute graft-vs-host disease (GVHD) is thought to be mediated by alloreactive T cells with a type 1 cytokine phenotype. To prevent the development of acute GVHD, we have successfully polarized mature donor T cells toward a type 2 cytokine phenotype ex-vivo by incubating them with murine rIL-4 in a primary MLC. Polarized type 2 T cells were then transplanted with T cell-depleted bone marrow cells into irradiated recipients across either MHC class II (bm12-->C57BL/6) or class I (bm1-->C57BL/6) barriers, and the intensity of GVHD was measured by assessment of several in vitro and in vivo parameters. The injection of polarized type 2 T cells abrogated the mitogen-induced production of IFN-gamma by splenocytes from transplanted hosts on day 13 after bone marrow transplantation (BMT). Injection of polarized type 2 T cells failed to induce secretion of the effector phase cytokine TNF-alpha by splenocytes stimulated with LPS both in vitro and in vivo, and survival of transplanted mice after i.v. injection with LPS was significantly improved. Furthermore, cell-mixing experiments revealed that polarized type 2 T cells were able to inhibit type 1 cytokine responses induced by naive T cells after BMT. These data demonstrate that both polarized CD4+ and CD8+ type 2 alloreactive donor T cells can be generated in vitro from mature T cell populations. These cells function in vivo to inhibit type 1 T cell responses, and such inhibition attenuates the systemic morbidity of GVHD after BMT across both MHC class II or class I barriers in mice.

Difloxacin metabolism and pharmacokinetics in humans after single oral doses.

By using high-performance liquid chromatography, the metabolism and pharmacokinetics of difloxacin were characterized in humans after single oral doses of 200, 400, and 600 mg. Group mean peak levels in plasma were obtained 4 h after administration. The means of the individual peak levels for the 200-, 400-, and 600-mg groups were 2.17, 4.09, and 6.12 micrograms/ml, respectively. The mean respective terminal-phase half-lives were 20.6, 27.1, and 28.8 h; the mean half-life for all subjects was 25.7 h. Within the dose range studied, the behavior of difloxacin could be well described by a set of linear pharmacokinetic parameters with a one-compartment open model. Levels of unconjugated metabolites in plasma were negligible. The major urinary components were difloxacin and its glucuronide, each accounting for roughly 10% of the dose. Also present were the N-desmethyl and N-oxide metabolites, accounting for 2 to 4%. Trace levels of other metabolites were observed. Group mean renal clearances ranged from 4.1 to 5.6 ml/min, indicating extensive reabsorption from the glomerular filtrate. As a result, the terminal phase half-life and the dose-normalized area under the curve were substantially greater than those of other members of the class.

P1 plasmid replication: multiple functions of RepA protein at the origin.

Replication functions of a bacteriophage P1 miniplasmid are carried on a 1.2-kilobase pair (kb) segment that can be subdivided into a 245-base pair (bp) replication origin and a 959-bp region that encodes a protein required for replication (RepA). The origin region contains five 19-bp direct repeats. By using primer extension and gene-fusion assays, we mapped the promoter of the repA gene within the repeated sequences and showed that the promoter is repressed by RepA. Regulation of RepA synthesis is apparently achieved by the binding of RepA to the repeat sequences. This regulation might be a key step in the replication-control circuit, as we found that overproduction of RepA (from a foreign promoter) inhibits replication. Thus, in addition to being an autoregulated activator of replication, the protein also can have a negative regulatory role.

P1 plasmid replication: replicon structure.

Bacteriophage P1 lysogenizes Escherichia coli as a unit-copy plasmid. We have undertaken to define the plasmid-encoded elements implicated in P1 plasmid maintenance. We show that a 2081 base-pair fragment of the 90,000 base P1 plasmid confers the capacity for controlled plasmid replication. DNA sequence analysis reveals several open reading frames in this fragment. The largest is shown to encode a 32,000 Mr protein required for plasmid replication. The corresponding gene, repA, has been identified genetically. A set of five 19 base-pair repeats is located upstream from repA; a set of nine similar repeats is located immediately downstream from repA. Each set of repeats, when cloned into pBR322, exerts incompatibility towards a P1 replicon. The upstream set, designated incC, consists of direct repeats that are spaced about two turns of the DNA helix apart; the downstream set, designated incA, consists of nine repeats arranged three in one orientation and six in the other. Spacing between incA repeats were three or four turns of the helix apart. The organization of the plasmid maintenance regions of P1 and the unit-copy sex factor plasmid, F, is strikingly similar. Although the DNA sequences of this region in the two plasmids exhibit little homology, a 9 base-pair sequence that appears four times in the origin region of members of the Enterobacteriaceae also occurs twice as direct repeats in similar positions in P1 and F. This sequence, where it occurs in E. coli, has been postulated to be the binding site for the essential replication protein determined by dnaA. The dnaA protein appears not to be essential for the replication of either plasmid; therefore, the function of the sequence in P1 and F may be regulatory.

Legionnaires' disease: report of sixty-five nosocomially acquired cases of review of the literature.

Sixty-five cases of nosocomially acquired Legionnaires' disease are reported and the world literature is reviewed. The etiologic agent, Legionnella pneumophila, has been isolated from several environmental sources at outbreak sites. Legionnaires' disease appears to be acquired by inhalation and is primarily manifested by severe, potentially fatal, pneumonia. Characteristic clinical disease consists of high fever with relative bradycardia, dry cough, chills, diarrhea, and pleuritic pain. Although no single feature is pathognomonic, the clinical presentation is usually sufficiently characteristic to suggest the diagnosis. The diagnosis of Legionnaires' disease during acute illness may be established by culture of Legionella pneumophila, or by demonstration of the bacterium using special stains. However, in most instances, the physician must make a presumptive diagnosis based on the clinical presentation in order to institute appropriate antimicrobial therapy. Retrospective confirmation of the diagnosis may be made by serologic studies in most instances. Erythromycin is, at this time, the drug of choice for the treatment of Legionnaires' disease. A prompt salutory response following institution of erythromycin therapy is typical.

Legionnaires' disease: clinical features of 24 cases.

Twenty-four cases of Legionnaires' disease were diagnosed at the Wadsworth Veterans Administration Hospital during a 5-month period. All cases occurred in persons exposed to the hospital environment during the usual incubation period of Legionnaires' disease. The clinical illness was quite characteristic. All patients complained of weakness, malaise, anorexia, and cough. Rigors, diarrhea, and pleuritic pain were frequent symptoms. All patients had a maximum temperature of greater than or equal to 39.4 degrees C. Thirteen of 22 patients had relative bradycardia. Chest roentgenograms documented pneumonia in all patients. Leukocytosis, hyponatremia, hypophosphatemia, and abnormal liver-function test results were typical. Diagnosis was made by serologic criteria in 20 patients, postmortem examination of tissue in two, and both serology and tissue examination in two. Four patients in whom the disease was not suspected died of Legionnaires' disease. One patient died of unrelated causes. Fifteen of 19 survivors received erythromycin therapy. The presentation of Legionnaires' disease was characteristic enough to allow early, specific therapy.

Legionnaires' disease in renal-transplant recipients.

Legionnaires' disease is reported in five renal-transplant recipients. All had febrile respiratory illnesses with pulmonary infiltrates and one died. The diagnosis was made on clinical features and by indirect fluorescent antibody titres. Symptoms started after maximum immunosuppressive therapy.