Prevention and modulation of aminoglycoside ototoxicity (Review).

More than 60 years after their isolation and characterization, aminoglycoside (AG) antibiotics remain powerful agents in the treatment of severe gram-negative, enterococcal or mycobacterial infections. However, the clinical use of AGs is hampered by nephrotoxicity and ototoxicity, which often develop as a consequence of prolonged courses of therapy, or of administration of increased doses of these drugs. The discovery of non-ototoxic antibacterial agents, showing a wider spectrum of activity, has gradually decreased the use of AGs as first line antibiotics for many systemic infections. However, AGs are now undergoing an unexpected revival, being increasingly indicated for the treatment of severe emerging infections caused by organisms showing resistance to most first-line agents (e.g., multidrug-resistant tuberculosis, complicated nosocomially-acquired acute urinary tract infections). Increasing adoption of aminoglycosides poses again to scientists and physicians the problem of toxicity directed to the kidneys and to the inner ear. In particular, aminoglycoside-induced deafness can be profound and irreversible, especially in genetically predisposed patients. For this reason, an impressive amount of molecular strategies have been developed in the last decade to counteract the ototoxic effect of aminoglycosides. The present article overviews: i) the molecular mechanisms by which aminoglycosides exert their bactericidal activity, ii) the mechanisms whereby AGs exert their ototoxic activity in genetically-predisposed patients, iii) the drugs and compounds that have so far proven to prevent or modulate AG ototoxicity at the preclinical and/or clinical level, and iv) the dosage regimens that have so far been suggested to decrease the incidence of episodes of AG-induced ototoxicity.

Discrepancies between in silico and in vitro data in the functional analysis of a breast cancer-associated polymorphism in the XRCC6/Ku70 gene.

Previous results from our research group have shown that the c.-1310 C↷G single nucleotide polymorphism in the promoter region of the XRCC6/Ku70 gene is significantly associated with breast cancer in a sample human patient population. In an attempt to attribute a functional meaning to this polymorphism, we performed a thorough analysis using a number of established in silico tools that strongly suggested that the c.-1310C↷G transversion would activate a cryptic splicing acceptor located upstream of the canonical promoter of Ku70, but downstream of a putative alternative promoter (PAP) of the same gene. Experimental investigation of alternative transcripts, as well as of the activity of the PAP detected in silico, did not support the initial hypothesis of a functional role of the c.-1310C↷G mutation in alternative splicing. Although a functional role of the SNP has yet to be determined, some evidence points to the linkage disequilibrium of the G variant of the polymorphism, with mutations located at critical sites within the promoter region of Ku70.

Reduction of PSA values by combination pharmacological therapy in patients with chronic prostatitis: implications for prostate cancer detection.

We identified from our clinical database a total of 471 patients affected by cat. II chronic bacterial prostatitis (CBP), cat. III (IIIa and IIIb) chronic pelvic pain syndrome (CP/CPPS), or cat. IV asymptomatic inflammatory prostatitis (AIP), according to NIH criteria. 132 intent-to-treat patients, showing levels of PSA > or =4 ng/mL, were subjected to a 6-week course of combination pharmacological therapy with 500 mg/day ciprofloxacin, 500 mg/day azithromycin (3 days/week), 10 mg/day alfuzosin and 320 mg b.i.d. Serenoa repens extract. At the end of treatment, 111 per-protocol patients belonging to all categories of prostatitis showed a total 32.5% reduction of PSA levels. In the same group, 66 patients (59.4%) showed "normalization" of PSA values under the 4 ng/mL limit. Patients affected by cat. IIIb CP/CPPS showed the highest PSA reduction and normalization rates (40% and 68.4%, respectively). Follow-up data show that, after a marked, significant reduction at completion of therapy, PSA levels, urine peak flow rates and NIH-CPSI symptom scores remained constant or decreased throughout a period of 18 months in patients showing normalization of PSA values. Prostatic biopsy was proposed to 45 patients showing persistently high PSA values (> or = 4 ng/mL) at the end of treatment. Fourteen patients rejected biopsy; of the remaining 31, 10 were diagnosed with prostate cancer. Four months after a first biopsy, a second biopsy was proposed to the 21 patients with a negative first diagnosis and persistently elevated PSA levels. Three patients rejected the procedure; of the remaining 18, four were diagnosed with prostatic carcinoma. In summary, combination pharmacological therapy decreased the number of patients undergoing prostatic biopsy from 111 to 45. Normalization of PSA values in 59.4% of patients--not subjected to biopsy--increased the prostate cancer detection rate from 12.6% (14/111) to 31.1% (14/45). The reduction of PSA after a 6-week course of therapy was calculated in patients affected by cat. II, IIIa, IIIb and IV prostatitis after stratification with respect to the concomitant presence or absence of benign prostatic hyperplasia (BPH). PSA was reduced by 41% in cat. II CBP patients without BPH, compared to a 12.7% reduction in patients affected by BPH. Cat. IIIa CP/CPPS patients without BPH showed a 58.3% reduction of PSA levels, compared to a 20.7% reduction observed in CPPS/BPH patients. These data show that the presence of BPH may prevent the reduction of PSA induced by combination pharmacological therapy, and suggest that care has to be taken in the adoption of PSA as a marker of therapeutic efficacy in the presence of confounding factors like BPH. PSA should in our opinion be used as a significant component of a strategy integrating multiple diagnostic approaches.

Eradication of unusual pathogens by combination pharmacological therapy is paralleled by improvement of signs and symptoms of chronic prostatitis syndrome.

We performed a comparative analysis of microbiological and clinical responses to combination therapy in 104 symptomatic patients showing evidence of infection by traditional uropathogens (TU) or by unusual pathogens (UP) at the prostatic level. Eighty-two pathogens out of a total of 104 isolated microorganisms were eradicated at the end of a 6-week course of combination therapy with ciprofloxacin, azithromycin, alfuzosin and a S. repens extract. The TU and UP groups showed eradication rates of 75.5% and 82.3%, and clinical success rates of 78.8% and 85.7%, respectively. Thus, a similar response to therapy was observed in patients infected by TU or by UP. Intergroup differences were not significantly different, with the exception of higher scores relative to the impact of the disease on quality of life in TU-patients. Long-term improvement of signs and symptoms of prostatitis indicates that combination therapy is beneficial for symptomatic patients showing evidence of infection by unusual pathogens at the prostatic level. Our data support the hypothesis that organisms other than the traditionally recognized uropathogens may play a role in the onset of prostatitis.

Comparative analysis of molecular strategies attenuating positional effects in lentiviral vectors carrying multiple genes.

Efficient, high-level expression of multiple genes is often difficult to achieve in retroviral vectors, due to positional effects affecting transcription of adjacent sequences. Here we describe the comparative analysis of different strategies for co-expressing two model cDNA sequences in the context of a second generation lentiviral vector system. A first option was based on the generation of a polycistronic construct by subcloning an internal ribosome entry site (IRES) sequence between tandem cDNAs. IRES-dependent translation of the cDNA placed downstream (3') of the first transgene was poor, and the protein was barely detectable in transduced cells. A similar result was obtained when both transgenes were placed under the transcriptional control of two independent internal promoters. When these independent transcription units were separated by the 5'HS4 chromatin insulator of the chicken beta-globin locus, a marked increase of the expression of the downstream protein was observed. Similarly, insertion of a polyadenylation sequence between the tandem transcription units fully restored - in transfection experiments - the expression of the downstream sequence, whose protein pattern was identical to the single-gene control, suggesting that in this specific construct transcriptional interference was the likely cause of the observed positional effects. These results indicate that chromatin insulator sequences can be useful molecular tools to overcome positional effects in the context of lentiviral vectors.