Strength of conjugate binding to plasmid DNA affects degradation rate and expression level in vivo.

In vitro assays have demonstrated the capability of poly-L-lysine to protect plasmid DNA from serum nucleases and cellular lysates. Our purpose was to evaluate the stability and potency of poly-L-lysine-DNA polyplexes after intravenous injection into mice. Polyplexes consisted of 32P-radiolabeled plasmid DNA complexed with poly-L-lysine at specified charge ratios. Variations in conjugate hydrophobicity and levels of modification with polyethylene glycol were investigated. Our results show that, in contrast to in vitro studies, the systemically administered polyplexes exhibited marked DNA degradation in the vascular compartment within 5 min. Substitution of poly-L-lysine epsilon-amino sites with polyethylene glycol or hydrocarbon chains resulted in faster degradation even when complexed at higher charge (+/-) ratios. Use of excess cationic charge in the polyplexes (+/- 2.5) diminished degradation rates only slightly. An analysis was made of the strength of the poly-L-lysine:DNA interaction by competition with poly-aspartic acid. Polyplexes with the strongest binding between conjugate and DNA in the competition assay were also the most stable in blood. However, tighter binding was not enough to fully protect the polyplex in vivo and polyplex DNA was substantially degraded within 10 min. Increased polyplex stability did not correlate with improved in vivo transfection efficiency.

Comparison of flavoxate hydrochloride in daily dosages of 600 versus 1200 mg for the treatment of urgency and urge incontinence.

Flavoxate hydrochloride at a daily dosage of 600 mg was compared to a daily dosage of 1200 mg for the treatment of unstable bladder. Twenty-seven patients were treated for 4 weeks in a double-blind, randomized, parallel-group trial. Clinically, both schedules were equally successful. In urodynamic terms, however, particularly with respect to uninhibited detrusor contractions, 1200 mg/day was significantly superior to 600 mg/day. Tolerability was excellent for both regimens. The side-effect free treatment of urgency and urge incontinence is of paramount importance for a patient's quality of life.

Flavoxate hydrochloride for urinary urgency after pelvic radiotherapy: comparison of 600 mg versus 1200 mg daily dosages.

This preliminary communication reports on a non-randomized pilot type trial of 34 females with urgency after pelvic radiotherapy who were treated with flavoxate hydrochloride for 4 weeks. A dosage of 600 mg/day was given to 21 patients and 1200 mg/day to 13 patients. Clinically, both regimens achieved comparable results. Urodynamically (first desire volume, bladder capacity and pressure at capacity) treatment with 1200 mg/day was significantly superior to 600 mg/day. Both schedules were equally well tolerated by patients and no treatment interruption occurred. A randomized double-blind trial comparing 600 and 1200 mg/day flavoxate hydrochloride is currently underway the results of which will be reported in due course.

[An echocardiographic study of left ventricular diastolic function in patients with diabetes mellitus type 2]. / Studio ecocardiografico della funzione diastolica del ventricolo sinistro in pazienti con diabete mellito di tipo 2.

AIM OF THE STUDY: To evaluate left ventricular diastolic function by Doppler echocardiography in patients with type 2 (non-insulin-dependent) diabetes mellitus, without coronary artery disease. BACKGROUND: Previous studies suggest that the velocity curve obtained by Doppler echocardiography of the mitral inflow may reflect the filling pattern of the left ventricle. METHODS AND RESULTS: To evaluate the presence of diastolic impairment of the left ventricle in diabetic patients without evidence of coronary artery disease, 30 patients with non-insulin-dependent diabetes mellitus and 20 normal control subjects underwent M-mode, two-dimensional (2-D) and Doppler echocardiography. In the group of diabetic patients (Diabetics), the peak E wave velocity was 0.70 +/- 0.11 m/sec, while in the control group (Controls) it was 1.1 +/- 0.23 m/sec (mean values, +SD, p < 0.001). The peak A wave velocity was 0.89 +/- 0.17 in Diabetics, versus 0.60 +/- 0.34 in Controls. Consequently, E/A ratio was 0.81 +/- 0.18 in Diabetics, versus 1.73 +/- 0.29 in Controls (p < 0.001). Isovolumic relaxation time was 0.08 +/- 0.021 sec in Diabetics, while in Controls it was 0.04 +/- 0.02 sec (p < 0.001). Left atrium diameter was 41 +/- 11 mm in Diabetics, and 37 +/- 4 mm in Controls (p = NS). Left ventricular volumes, ejection fraction, interventricular septal and posterior wall thickness were similar in both groups. No correlation was found between diabetes duration and diastolic function indexes. In Diabetics no correlation was found between age and E/A ratio (correlation coefficient +/- 0.11) while in Controls E/A ratio was lower in the older subjects (r = +/- 0.75). This ratio was 1.89 +/- 0.20 in Controls aged < 65 years, and 1.6 +/- 0.33 in Controls aged > or = 65 years. (p = 0.06). These data suggest that 1) E/A ratio and isovolumic relaxation time are significantly altered in non-insulin-dependent diabetic patients without coronary artery disease; 2) Doppler echocardiography is a useful technique to detect left ventricular diastolic impairment; 3) diastolic impairment seems not to correlate with disease duration; 4) systolic function is normal in our group of type 2 diabetic patients.

Identification of differentially expressed mRNA species during HIV infection by RNA arbitrarily primed PCR.

BACKGROUND: A number of strategies, such as subtractive cDNA libraries and high through-put sequencing, have been devised to assess differential gene expression. Most of these approaches, however, are cumbersome and/or require tremendous technological power. In this paper, we describe a method, RNA fingerprinting using arbitrarily primed polymerase chain reaction (RAP-PCR), that is rapid, less cumbersome and can differentiate low levels of mRNA expression. OBJECTIVES: To identify genes that are differentially expressed following human immunodeficiency virus type 1 (HIV-1) infection in different cell types by RAP-PCR. STUDY DESIGN: RNA was extracted from both HIV-1-infected and uninfected HUT78 cells and peripheral blood mononuclear cells (PBMCs), reverse transcribed, and RAP-PCR amplified using numerous primer sets. RESULTS: Three genes, gamma-actin, the HIV-1 nef and an unknown sequence, were identified as being differentially expressed in HUT78 cells. The level of gamma-actin mRNA expression is increased after HIV infection and, as expected, the nef gene was solely expressed in HIV-infected cells. In contrast, the unknown mRNA is down-regulated by HIV. Northern blot analysis and/or specific PCR confirmed the differential expression of these three genes. RNA fingerprinting using phytohemagglutinin (PHA)-activated PBMCs infected by HIV in vitro, revealed that gamma-actin is still up-regulated by HIV, whereas the unknown product no longer shows down-regulation. CONCLUSIONS: These results illustrate the usefulness of the RAP-PCR method for isolating and identifying differentially expressed genes during HIV-1 infection of primary lymphocytes.

T cell receptor V beta repertoire in HIV-infection individuals: lack of evidence for selective V beta deletion.

The gradual decline of CD4+ T lymphocytes in HIV-infected individuals culminates in the lethal immunosuppression of AIDS. The mechanism of CD4+ T cell loss is currently unknown, but has recently been suggested to occur as a result of an HIV-encoded superantigen which facilitates a selective deletion of T cells expressing specific V beta genes. To verify and extend such observations, peripheral blood leucocytes (PBL) from 15 HIV+ individuals, 10 of which had very low CD4 T cell counts (< 200/mm3), were analysed for T cell receptor (TCR) V beta gene expression. In contrast to a recent study, the results presented here fail to provide evidence that selective loss of V beta-bearing T cells occurs in HIV+ individuals. Furthermore, when PBL from HIV+ individuals were stimulated with Staphylococcal enterotoxin B (SEB), T cells expressing V beta subfamilies known to engage this superantigen were expanded, indicating that such cells were not deleted and were responsive to stimulation by a bacterial superantigen. Collectively, these data suggest that CD4 loss in HIV patients does not occur in a V beta-selective, superantigen-mediated fashion.

Novel membrane-bound GM-CSF vaccines for the treatment of cancer: generation and evaluation of mbGM-CSF mouse B16F10 melanoma cell vaccine.

Cancer vaccines composed of tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) are currently being clinically evaluated. To enhance the immunogenicity of GM-CSF-secreting tumor cell vaccines, a novel approach expressing GM-CSF as a membrane-bound form (mbGM-CSF) on the tumor cell surface was investigated. The intent was to enhance antigen presentation by increasing interactions between the tumor cell lines in the vaccine and GM-CSF receptor positive antigen presenting cells (APC), notably the patient's Langerhans cells residing within the intradermal injection site. B16.F10 cells engineered to express either membrane-bound or secreted GM-CSF were compared in the B16.F10 mouse melanoma model. We observed that mbGM-CSF on the tumor cell surface retarded growth and induced protective immunity to subsequent wild-type tumor challenge more effectively than tumor cells secreting GM-CSF. Vaccination with irradiated mbGM-CSF B16.F10 also provided strong protection from wild-type tumor challenge, improved therapeutic effects against established tumors, and retarded lung metastases. These results demonstrate that mbGM-CSF B16.F10 cells can induce strong systemic immunity that protects against and therapeutically treats B16.F10 melanoma more effectively than analogous vaccines containing only secreted GM-CSF. These data warrant further development and clinical testing of mbGM-CSF tumor cell vaccines.