Francesco Rivasi

Dirofilariasis due to Dirofilaria repens in Italy, an emergent zoonosis: report of 60 new cases.

Aims: Sixty new cases of human dirofilariasis due to Dirofilaria repens, occurring in Italy between 1990 and 1999, are presented. This is the most extensive case study of this zoonosis reported worldwide by a single study group. The aim is to utilize this large experience to characterize the different histopathological findings in the parasitic lesions in man.

P16INK4a expression and progression risk of low-grade intraepithelial neoplasia of the cervix uteri

The aim of the study was to evaluate the immunohistochemical expression of p16INK4a as a marker of progression risk in low-grade dysplastic lesions of the cervix uteri. p16INK4a immunohistochemistry was performed on 32 CIN1 with proven spontaneous regression of the lesion in the follow-up (group A), 31 (group B) with progression to CIN3 and 33 (group C) that were randomly chosen irrespective of the natural history of the lesion. p16INK4a staining pattern was scored as negative (less than 5% cells in the lower third of dysplastic epithelium stained), as focally positive (≤25%) and as diffuse positive (>25%). A diffuse staining pattern was detected in 43.8% of CIN1 of group A, 74.2% of group B and 56.3% of group C. No p16INK4a staining was detected in 31.3% and 12.9% CIN1 lesions of groups A and B, respectively. Overall, 71.4% and 37.8% of p16INK4a-negative and diffusely positive CIN1 had regressed at follow-up, whereas 28.6% and 62.2% negative and diffusely positive CIN1 were progressed to CIN3, respectively (P<0.05). All CIN3 lesions analyzed during follow-up of group B were diffusely stained for p16INK4a. Although p16INK4a may be expressed in low-grade squamous lesions that undergo spontaneous regression, in this study, CIN1 cases with diffuse p16INK4a staining had a significantly higher tendency to progress to a high-grade lesion than p16INK4a-negative cases. p16INK4a may have the potential to support the interpretation of low-grade dysplastic lesions of the cervix uteri.

Gelatin microspheres crosslinked with D,L-glyceraldehyde as a potential drug delivery system: preparation, characterisation, in vitro and in vivo studies.

To overcome the restriction in using crosslinked gelatin in the pharmaceutical field, D,L-glyceraldehyde (GAL), a non-toxic crosslinking agent, was proposed. Gelatin microspheres crosslinked with different concentrations of GAL (0.5, 1 or 2%, w/v) and for different time periods (1 or 24 h) were prepared. The effect of the preparation variables was evaluated analysing the extent of crosslinking, the morphological aspect, the particle size and the swelling behaviour. To evaluate the pharmaceutical properties, an antihypertensive drug, clonidine hydrochloride, was chosen as drug model and loaded into the microspheres. Either the increase of the crosslinker concentration or of the crosslinking time period decreased both the swelling and the in vitro drug release processes of the microspheres. After the subcutaneous injection, the loaded microspheres crosslinked with the lowest GAL concentration (0.5%, w/v) or for the shortest time period (1 h) showed a reduction of systolic blood pressure (SBP) similar to that recorded with a clonidine hydrochloride solution having the same drug concentration. Instead, the microspheres crosslinked for 24 h with concentrations of GAL higher than 0.5% (w/v) produced a more gradual and sustained SBP reduction and the antihypertensive effect was maintained until 52-72 h. The biocompatibility studies showed that the microspheres crosslinked with GAL are well tolerated in vivo. These results suggest the potential application of gelatin microspheres crosslinked with GAL as a suitable drug delivery system for the subcutaneous administration.

Dirofilarial infections in Europe.

Nematodes of the genus Dirofilaria are currently considered emerging agents of parasitic zoonoses in Europe. Climatic changes and an increase in the movement of reservoirs (mostly infected dogs) have caused an increase in the geographical range of these parasites from the traditionally endemic/hyperendemic southern regions, and the risk for human infection has increased. In the last several years, forecast models have predicted that current summer temperatures are sufficient to facilitate extrinsic incubation of Dirofilaria in many areas of Europe. The global warming projected by the Intergovernmental Panel on Climate Change suggests that warm summers suitable for Dirofilaria transmission in Europe will be the rule in the future decades, and if the actual trend of temperature increase continues, filarial infection should spread into previously infection-free areas. Dirofilaria repens is currently the filarial species that is most commonly reported as spreading from southern to northern areas. This article reviews the zoonotic aspects, effects of climate, and other global drivers on Dirofilaria infections in Europe and the possible implications on the transmission and control of these mosquito-borne nematodes.

Lathosterolosis, a Novel Multiple-Malformation/Mental Retardation Syndrome Due to Deficiency of 3β-Hydroxysteroid-Δ5-Desaturase

We report the clinical, biochemical, and molecular characterization of a patient with a novel defect of cholesterol biosynthesis. This patient presented with a complex phenotype, including multiple congenital anomalies, mental retardation, and liver disease. In the patient’s plasma and cells, we found increased levels of lathosterol. The biosynthesis of cholesterol in the patient’s fibroblasts was defective, showing a block in the conversion of lathosterol into 7-dehydrocholesterol. The activity of 3β-hydroxysteroid-Δ5-desaturase (SC5D), the enzyme involved in this reaction, was deficient in the patient’s fibroblasts. Sequence analysis of the SC5D gene in the patient’s DNA, showing the presence of two missense mutations (R29Q and G211D), confirmed that the patient is affected by a novel defect of cholesterol biosynthesis.

Sialic acid and glycopeptides conjugated PLGA nanoparticles for central nervous system targeting: In vivo pharmacological evidence and biodistribution

Polymeric nanoparticles (Np) have been considered as strategic carriers for brain targeting. Specific ligands on the surface allowed the Np to cross the Blood-Brain Barrier (BBB) carrying model drugs within the brain district after their i.v. administration in experimental animals. It is known that sialic acid receptors are present in several organs, including in the brain parenchyma. Thus, in this paper, we prepared PLGA Np surface modified with a BBB-penetrating peptide (similopioid peptide) for BBB crossing and with a sialic acid residue (SA) for the interaction with brain receptors. This double coverage could allow to obtain novel targeted Np with a prolonged residence within the brain parenchyma, thus letting to reach a long-lasting brain delivery of drugs. The central analgesic activity of Loperamide (opioid drug, unable to cross the BBB) loaded in these novel Np was evaluated in order to point out the capability of the Np to reach and to remain in the brain. The results showed that the pharmacological effect induced by loaded Np administration remained significant over 24h. Using confocal and fluorescent microscopies, the novel Np were localized within the tissue parenchyma (brain, kidney, liver, spleen and lung). Finally, the biodistribution studies showed a localization of the 6% of the injected dose into the CNS over a prolonged time (24h). Notwithstanding an increased accumulation of SA-covered Np in those organs showing SA-receptors (liver, kidney, and lung), the pharmacological and biodistribution results are proofs of the ability of double targeted Np to enter the brain allowing the drug to be released over a prolonged time.

Dirofilarial human cases in the Old World, attributed to Dirofilaria immitis: a critical analysis.

Aims:  To review 28 cases of human dirofilariasis reported in the last 30 years in the Old World and attributed, by their respective authors, to Dirofilaria immitis or a species of Dirofilaria other than D. repens.

P16ink4a and HPV L1 Immunohistochemistry is Helpful for Estimating the Behavior of Low-grade Dysplastic Lesions of the Cervix Uteri

As only a minority of low-grade dysplastic lesions of the cervix uteri will eventually progress to carcinoma, predicting the behavior of these lesions could be of high value in clinical practice. The aim of the study was to evaluate p16ink4a and L1 as immunohistochemical markers of the biologic potentiality of low-grade dysplasia of the uterine cervix. The study included 38 conization specimens with coexisting cervical intraepithelial neoplasia grade 1 (CIN1) and 3 (CIN3) (group A) and 28 punch biopsies from women with CIN1 and proven spontaneous regression in the follow-up (group B). In group A, all CIN3 were p16ink4a positive (p16+) and L1 negative (L1−). The CIN1 of this group were p16+L1− and p16+L1+ in 68.42% and 31.57%, respectively. No other expression pattern was found in this group. In group B, the p16+L1−, p16+L1+, p16−L1+, and p16−L1− patterns were found in 3.57%, 25%, 14.29%, and 57.14%, respectively. Overall, 96.29% p16+L1− CIN1 were found in group A, whereas all the p16−L1+ and p16−L1− CIN1 were found in group B. A significant difference between staining pattern distributions of group A and B was observed (P<0.0001). The results of the study show that p16ink4a and L1 immunohistochemistry can be helpful for estimating the biologic potentiality of low-grade squamous cervical lesions. Particularly in cases in which the grade of the lesion is morphologically difficult to assess, the p16/L1 expression pattern could be useful for planning the clinical management of these women.

Investigation on mechanisms of glycopeptide nanoparticles for drug delivery across the blood–brain barrier

AIM Nanoneuroscience, based on the use polymeric nanoparticles (NPs), represents an emerging field of research for achieving an effective therapy for neurodegenerative diseases. In particular, poly-lactide-co-glycolide (PLGA) glyco-heptapetide-conjugated NPs (g7-NPs) were shown to be able to cross the blood-brain barrier (BBB). However, the in vivo mechanisms of the BBB crossing of this kind of NP has not been investigated until now. This article aimed to develop a deep understanding of the mechanism of BBB crossing of the modified NPs. MATERIALS & METHODS Loperamide and rhodamine-123 (model drugs unable to cross the BBB) were loaded into NPs, composed of a mixture of PLGA, differently modified with g7 or with a random sequence of the same aminoamids (random-g7). To study brain targeting of these model drugs, loaded NPs were administered via the tail vein in rats in order to perform both pharmacological studies and biodistribution analysis along with fluorescent, confocal and electron microscopy analysis, in order to achieve the NP BBB crossing mechanism. Computational analysis on the conformation of the g7- and random-g7-NPs of the NP surface was also developed. RESULTS Only loperamide delivered to the brain with g7-NPs created a high central analgesia, corresponding to the 14% of the injected dose, and data were confirmed by biodistribution studies. Electron photomicrographs showed the ability of g7-NPs in crossing the BBB as evidenced by several endocytotic vesicles and macropinocytotic processes. The computational analysis on g7 and random-g7 showed a different conformation (linear vs globular), thus suggesting a different interaction with the BBB. CONCLUSION Taken together, this evidence suggested that g7-NP BBB crossing is enabled by multiple pathways, mainly membrane-membrane interaction and macropinocytosis-like mechanisms. The results of the computational analysis showed the Biousian structure of the g7 peptide, in contrast to random-g7 peptide (globular conformation), suggesting that this difference is pivotal in explaining the BBB crossing and allowing us to hypothesize regarding the mechanism of BBB crossing by g7-NPs.

Human anisakiasis in Italy: A report of eleven new cases

The authors report on eleven new human cases of anisakiasis occurring in Italy, and emphasize the importance of the infection in clinical medicine, histopathology and public health. For ten of these cases, the diagnosis was based on histological findings: an eosinophilic granuloma associated with a larva of Anisakis sp. For one of them, the larva was removed from the stomach by endoscopy. Nine of the subjects were from Apulia and two from Molise (regions of southern and central Italy, respectively). Ten of them were surgically treated, and in one case the endoscopical extraction of the parasite resolved the situation. In two cases, the gastric wall was affected, in three the intestinal wall, in a further three the omentum, in one the spleen, and in the final two the mesentery and the epiploic appendix. In all the cases, the parasite was discovered unexpectedly during surgical treatment of the patients for supposed illnesses, which had originally been misdiagnosed. In three cases, the patients were also affected by cancer. Human anisakiasis must be taken into account in the differential diagnosis of acute, abdominal synddromes in subjects who have ingested raw fish or squid a few hours to a few days before the onset of symptoms. Histopathologists should consider the possibility of this parasitic infection when confronted with an eosinophilic granuloma of the digestive tract, mesentery or peritoneum. The incidence of anisakiasis in Italy is probably higher than reported, as some cases might not be diagnosed and others might heal spontaneously.