Antibacterial and Antifungal Efficacy of Medium and Low Weight Chitosan-Shelled Nanodroplets for the Treatment of Infected Chronic Wounds.

Purpose: Medium versus low weight (MW vs LW) chitosan-shelled oxygen-loaded nanodroplets (cOLNDs) and oxygen-free nanodroplets (cOFNDs) were comparatively challenged for biocompatibility on human keratinocytes, for antimicrobial activity against four common infectious agents of chronic wounds (CWs) - methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Candida albicans and C. glabrata - and for their physical interaction with cell walls/membranes. Methods: cNDs were characterized for morphology and physico-chemical properties by microscopy and dynamic light scattering. In vitro oxygen release from cOLNDs was measured through an oximeter. ND biocompatibility and ability to promote wound healing in human normoxic/hypoxic skin cells were challenged by LDH and MTT assays using keratinocytes. ND antimicrobial activity was investigated by monitoring upon incubation with/without MW or LW cOLNDs/cOFNDs either bacteria or yeast growth over time. The mechanical interaction between NDs and microorganisms was also assessed by confocal microscopy. Results: LW cNDs appeared less toxic to keratinocytes than MW cNDs. Based on cell counts, either MW or LW cOLNDs and cOFNDs displayed long-term antimicrobial efficacy against S. pyogenes, C. albicans, and C. glabrata (up to 24 h), whereas a short-term cytostatic effects against MRSA (up to 6 h) was revealed. The internalization of all ND formulations by all four microorganisms, already after 3 h of incubation, was showed, with the only exception to MW cOLNDs/cOFNDs that adhered to MRSA walls without being internalized even after 24 h. Conclusion: cNDs exerted bacteriostatic and fungistatic effects, due to the presence of chitosan in the outer shell and independently of oxygen addition in the inner core. The duration of such effects strictly depends on the characteristics of each microbial species, and not on the molecular weight of chitosan in ND shells. However, LW chitosan was better tolerated by human keratinocytes than MW. For these reasons, the use of LW NDs should be recommended in future research to assess cOLND efficacy for the treatment of infected CWs.

Highly specific detection of prostate-specific antigen-positive cells in the blood of patients with prostate cancer or benign prostatic hyperplasia, using a real-time reverse-transcription-polymerase chain reaction method with improved sensitivity.

OBJECTIVE: To assess the presence of circulating prostate-specific antigen (PSA)-expressing cells in patients with prostate cancer or benign prostatic hyperplasia (BPH), and to determine their diagnostic usefulness using a highly sensitive quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) method. PATIENTS, SUBJECTS AND METHODS: Venous blood samples were obtained from 175 patients with prostate cancer (12 metastatic and 163 not metastatic), 49 with BPH, and 50 healthy volunteers. To improve the specificity and sensitivity of the qRT-PCR three innovative features were combined; a primer overlapping two adjacent exons to inhibit nonspecific amplification; a no-end-point first round amplification to increase the sensitivity; and a target-specific primer for the RT phase to increase the specificity. RESULTS: The sensitivity of the method was 1 cell/mL of blood and the interassay coefficient of variation was 10.5%. None of the healthy subjects tested positively, while 9% of those with prostatic cancer and 14% with BPH had PSA-positive cells in the blood. There was a positive association between a positive test and the National Comprehensive Cancer Network classification in the patients with newly diagnosed prostate cancer (P = 0.022). There were no additional statistically significant associations. CONCLUSION: Our results strongly indicate that although there were no false-positive results and the sensitivity of the method was increased to maximal levels, a low frequency of positive results in patients with prostatic cancer and a high frequency of positive results in those with BPH seems to discourage the use of PSA-positive circulating cells in the search for a clinical diagnosis of prostate cancer.