New self-assembly nanoparticles and stealth liposomes for the delivery of zoledronic acid: a comparative study

Zoledronic acid (ZOL) is a drug whose potent anti-cancer activity is limited by its short plasma half-life and rapid uptake and accumulation within bone. We have recently proposed new delivery systems to avoid ZOL accumulation into the bone, thus improving extra-skeletal bioavailability. In this work, we have compared the technological and anti-cancer features of either ZOL-containing self-assembly PEGylated nanoparticles (NPs) or ZOL-encapsulating PEGylated liposomes (LIPO-ZOL). ZOL-containing NPs showed superior technological characteristics in terms of mean diameter, size distribution, and ZOL encapsulation efficiency, compared to LIPO-ZOL. Moreover, the anti-cancer activity of NPs in nude mice xenografted with prostate cancer PC3 cells was higher than that one induced by LIPO-ZOL. In addition, NPs induced the complete remission of tumour xenografts and an increase of survival time higher than that one observed with LIPO-ZOL. It has also to be considered that PC3 tumour xenografts were almost completely resistant to the anti-cancer effects induced by free ZOL. Both nanotechnological products did not induce toxic effects not affecting the mice weight nor inducing deaths. Moreover, the histological examination of some vital organs such as liver, kidney and spleen did not find any changes in terms of necrotic effects or modifications in the inflammatory infiltrate. On the other hand, NPs but not LIPO-ZOL caused a statistically significant reduction of the tumour associated macrophages (TAM) in tumour xenografts. This effect was paralleled by a significant increase of both necrotic and apoptotic indexes. The effects of the NPs were also higher in terms of neo-angiogenesis inhibition. These results suggest the future preclinical development of ZOL-encapsulating NPs in the treatment of human cancer.     

Five-years cumulative incidence of alcohol consumption in Croatian adult population: the CroHort study

Alcohol consumption is traditionally part of most human cultures, and with the onset of industrial revolution it was recognized as a health and social problem. The aim of this article is to investigate cumulative incidence of alcohol consumption in Croatia. Data were obtained from the Croatian Adult health Survey in 2003 followed by 2008. The cohort consisted of 3229 participants. Questions regarding alcohol consumption were calculated into two factors describing existence or non-existence of risk behavior. Results revealed higher incidence of risk alcohol consumption in man than in women and highest in the 35-65, age group. Due to the some study limitations results might be underestimated. Present problem of alcohol is alarming, even more so, in women's population it might be only the tip of the iceberg. Cultural and regional differences should be taken into account when educational programs are constructed, especially due to the different type of alcohol consumed.     

Could determinants of PCS and MCS serve for public health intervention regarding chronic diseases in Croatia?

The aim of this article was to identify parameters that determinate PCS and MCS values, and analyze 5-year changes in those values according to the age, sex and geographic region. Cohort of 3229 participants was obtained from the CAHS 2003-2008. Results revealed no statistically significant differences between same age group, sex, and different region regarding PCS and MCS. When chronic conditions were in the model difference was present, PCS being more influenced by all conditions but bronchial asthma. The strongest influence comes from musculoskeletal conditions; followed by weak heart. Values for PSC and MSC decreased in 2008 compared with 2003, but only in few cases decrease was statistically significant. Values of PCS and MCS are higher in men in all regions, but they show higher variability than woman. Our results support the findings that data obtained through SF-36 could be the useful for public health interventions regarding chronic diseases.     

Comparison of two PCR methods for detection of Leptospira interrogans in formalin-fixed and paraffin-embedded tissues

In this study we compared two polymerase chain reaction (PCR) methods using either 16S ribosomal RNA (rRNA) or 23S rRNA gene primers for the detection of different Leptospira interrogans serovars. The performance of these two methods was assessed using DNA extracted from bovine tissues previously inoculated with several bacterial suspensions. PCR was performed on the same tissues before and after the formalin-fixed, paraffin-embedding procedure (FFPE tissues). The 23S rDNA PCR detected all fresh and FFPE positive tissues while the 16S rDNA-based protocol detected primarily the positive fresh tissues. Both methods are specific for pathogenic L. interrogans. The 23S-based PCR method successfully detected Leptospira in four dubious cases of human leptospirosis from archival tissue specimens and one leptospirosis-positive canine specimen. A sensitive method for leptospirosis identification in FFPE tissues would be a useful tool to screen histological specimen archives and gain a better assessment of human leptospirosis prevalence, especially in tropical countries, where large outbreaks can occur following the rainy season.     

Genesis of spermatodesms in Tylopsis liliifolia (Orthoptera: Phaneropterinae) and their transit in the male genital tract

The spermatodesms of Tylopsis liliifolia form in the most proximal follicular cysts and are composed of a large number of sperm held together by a cap located in the anterior region of the acrosome. The cap is formed by short thin fibrils, loosely arranged at random, probably derived from secretory activity of cells of the cyst wall. Compared to other Tettigoniidae, a peculiar feature is acrosomal wings that twist gradually around the anterior region of the nucleus; at the end of the twisting process, the region of the sperm acrosome, observed in cross section, shows a typical spiral form. Spermatodesms do not undergo any substantial changes in the spermiduct. The epithelial cells of the wall have secretory activity and many show marked spermiophagic activity, which is conducted by epithelial cell protrusions that envelop the gametes, taking them into the cytoplasm. When removed from seminal vesicles and observed in vivo, spermatodesms show accentuated corkscrew movement, and when observed by SEM, slight torsion. Thus organized, spermatodesms are transferred to the spermatophore during mating, where they are transformed before reaching the seminal receptacle.     

Mitochondrial diabetes in children: seek and you will find it

Maternally Inherited Diabetes and Deafness (MIDD) is a rare form of diabetes due to defects in mitochondrial DNA (mtDNA). 3243 A>G is the mutation most frequently associated with this condition, but other mtDNA variants have been linked with a diabetic phenotype suggestive of MIDD. From 1989 to 2009, we clinically diagnosed mitochondrial diabetes in 11 diabetic children. Diagnosis was based on the presence of one or more of the following criteria: 1) maculopathy; 2) hearing impairment; 3) maternal heritability of diabetes/impaired fasting glucose and/or hearing impairment and/or maculopathy in three consecutive generations (or in two generations if 2 or 3 members of a family were affected). We sequenced the mtDNA in the 11 probands, in their mothers and in 80 controls. We identified 33 diabetes-suspected mutations, 1/33 was 3243A>G. Most patients (91%) and their mothers had mutations in complex I and/or IV of the respiratory chain. We measured the activity of these two enzymes and found that they were less active in mutated patients and their mothers than in the healthy control pool. The prevalence of hearing loss (36% vs 75-98%) and macular dystrophy (54% vs 86%) was lower in our mitochondrial diabetic adolescents than reported in adults. Moreover, we found a hitherto unknown association between mitochondrial diabetes and celiac disease. In conclusion, mitochondrial diabetes should be considered a complex syndrome with several phenotypic variants. Moreover, deafness is not an essential component of the disease in children. The whole mtDNA should be screened because the 3243A>G variant is not as frequent in children as in adults. In fact, 91% of our patients were mutated in the complex I and/or IV genes. The enzymatic assay may be a useful tool with which to confirm the pathogenic significance of detected variants.     

Age‐dependent skewing of X chromosome inactivation appears delayed in centenarians’ offspring. Is there a role for allelic imbalance in Healthy Aging and Longevity?

Recently, it has been proposed that age-related X chromosome inactivation (XCI) skewing can clinically result in late-onset X-linked disorders. This observation leads to hypothesize that age-related skewed XCI might also influence lifespan in women. To investigate this issue, we employed a new experimental model of longevity and healthy aging including 55 female centenarians, 40 of their offspring, 33 age-matched offspring of both non-long-lived parents and 41 young women. Peripheral blood DNA from 169 females was screened for heterozygosity at the HUMARA locus. We confirmed that skewing of XCI is an age-dependent phenomenon. However, skewed XCI was significantly less severe and frequent in centenarians' offspring [degree of skewing (DS) = 0.16 ± 0.02] compared to age-matched offspring of both non-long-lived parents (DS = 0.24 ± 0.02) (P < 0.05). A second goal was to assess whether changes in XCI pattern could be a consequence of loss of methylation on X chromosome. Using a methylation array evaluating 1085 CpG sites across X chromosome and eleven CpG sites located at HUMARA locus, no differences in methylation levels and profiles emerged between all groups analysed, thus suggesting that age-associated epigenetic changes could not influence HUMARA results. In conclusion, the results presented herein highlight for the first time an interesting link between skewing of XCI and healthy aging and longevity. We speculate that the allelic imbalance produced by XCI skewing may compromise the cooperative and compensatory organization occurring between the two cell populations that make up the female mosaic.     

Bovine serum amine oxidase and spm potentiate docetaxel and interferon-alpha effects in inducing apoptosis on human cancer cells through the generation of oxidative stress

It was previously demonstrated that bovine serum amine-oxidase (BSAO) and SPM (SPM) addition to cancer cells induces cell growth inhibition and over-run the multi-drug resistance (MDR) phenotype through the oxidative stress caused by polyamine metabolites. In this study, it is reported that BSAO/SPM enzymatic system antagonizes the survival pathway induced by either docetaxel (DTX) or interferon alpha (IFNalpha) in human epidermoid cancer KB cells. The combination of BSAO/SPM with either DTX or IFNalpha had a synergistic effect on cell growth inhibition through apoptosis in both human epidermoid KB and breast cancer MCF-7 cell lines. The effects of the BSAO/SPM-DTX combination on apoptosis were caspase 3 and 9-dependent and were paralleled by the enhancement of intracellular O(2-), nitric oxide levels and of lipo-oxidation. The scavenger moiety N-acetyl-cysteine antagonized the effects on apoptosis and cell growth inhibition induced by the combination suggesting a role of the oxidative products of SPM. These effects occurred together with a decrease of the physiological scavenger MnSOD and an increase of both p38 kinase activity and DNA damage. The results suggest that DTX and IFNalpha could sensitize tumour cells to the oxidative stress and apoptosis induced by BSAO/SPM through the induction of a survival ras-dependent pathway and the consequent elevation of the intracellular polyamine pool. These data allow the design of new therapeutic strategy based on the use of this combination in human neoplasms.