POR polymorphisms are associated with 21 hydroxylase deficiency.

PURPOSE: Genotype-phenotype correlation in congenital 21 hydroxylase deficiency is strong but by no means absolute. Indeed, clinical and hormonal features may vary among patients carrying similar CYP21A2 mutations, suggesting that modifier genes may contribute to the phenotype. Aim of the present study was to evaluate whether polymorphisms in the p450  oxidoreductase (POR) gene may affect clinical features in patients with 21 hydroxylase deficiency METHODS: Sequencing of the POR gene was performed in 96 patients with 21 hydroxylase deficiency (49 classic, 47 non-classic) and 43 control subjects. RESULTS: Prevalence of POR polymorphisms in patients with 21 hydroxylase was comparable to controls and known databases. The rs2228104 polymorphism was more frequently associated with non-classic vs classic 21 hydroxylase deficiency (allelic risk 7.09; 95% C.I. 1.4-29.5, p < 0.05). Classic 21 hydroxylase-deficient carriers of the minor allele in the rs2286822/rs2286823 haplotype presented more frequently the salt-wasting form (allelic risk 1.375; 95% C.I. 1.138-1.137), more severe Prader stage at birth (allelic risk 3.85; 95% C.I. 3.78-3.92), higher ACTH levels, and younger age at diagnosis. CONCLUSIONS: Polymorphisms in the POR gene are associated with clinical features of 21 hydroxylase deficiency both as regards predisposition to classic vs non-classic forms and severity of classic adrenal hyperplasia.

Human collecting lymphatic glycocalyx identification by electron microscopy and immunohistochemistry.

Blood flow is translated into biochemical inflammatory or anti-inflammatory signals based onshear stress type, by means of sensitive endothelial receptors. Recognition of the phenomenon is of paramount importance for enhanced insights into the pathophysiological processes of vascular remodeling. The endothelial glycocalyx is a pericellular matrix, identified in both arteries and veins, acting collectively as a sensor responsive to blood flow changes. Venous and lymphatic physiology is interconnected; however, to our knowledge, a lymphatic glycocalyx structure has never been identified in humans. The objective of this investigation is to identify glycocalyx structures from ex vivo lymphatic human samples. Lower limb vein and lymphatic vessels were harvested. The samples were analyzed by transmission electron microscopy. The specimens were also examined by immunohistochemistry. Transmission electron microscopy identified a glycocalyx structure in human venous and lymphatic samples. Immunohistochemistry for podoplanin, glypican-1, mucin-2, agrin and brevican characterized lymphatic and venous glycocalyx-like structures. To our knowledge, the present work reports the first identification of a glycocalyx-like structure in human lymphatic tissue. The vasculoprotective action of the glycocalyx could become an investigational target in the lymphatic system as well, with clinical implications for the many patients affected by lymphatic disorders.

Panbacterial real-time PCR to evaluate bacterial burden in chronic wounds treated with Cutimed™ Sorbact™.

The impact of polymicrobial bacterial infection on chronic wounds has been studied extensively, but standard bacteriological analysis is not always sensitive enough. Molecular approaches represent a promising alternative to the standard bacteriological analysis. This work aimed to assess the usefulness of a panbacterial quantitative real-time PCR reaction to quantitate the total bacterial load in chronic wounds treated with Cutimed™ Sorbact™, a novel therapeutic approach based on hydrophobic binding of bacteria to a membrane. The results obtained by panbacterial real-time PCR on conserved sequences of the bacterial 16S gene show that the bacterial burden significantly decreased in 10 out of 15 healing chronic wounds, and did not change in 5 out of 5 non-healing chronic wounds. On the contrary, classical culture for S. aureus and P. aeruginosa, and real-time PCR for Bacteroides and Fusobacterium did not show any correlation with the clinical outcome. Our study also shows that quantification of chronic wounds by panbacterial real-time PCR is to be performed on biopsies and not on swabs. These results show that panbacterial real-time PCR is a promising and quick method of determining the total bacterial load in chronic wounds, and suggest that it might be an important biomarker for the prognosis of chronic wounds under treatment.

Vascular endothelial growth factor genetic polymorphisms and haplotypes in female patients with bisphosphonate-related osteonecrosis of the jaws.

OBJECTIVE: To investigate the polymorphisms of the vascular endothelial growth factor (VEGF) gene in relation to female patients who developed bisphosphonate-related osteonecrosis of the jaws (BRONJ). METHODS: Test subjects were 30 Italian female patients with BRONJ (Group A). Control subjects were 30 female patients with a history of intravenous bisphosphonate use without any evidence of osteonecrosis (Group B) and 125 unrelated healthy volunteers (Group C). Three single-nucleotide polymorphisms were investigated: -634 G>C, occurring in 5' untranslated region (UTR); +936 C>T, occurring in 3' UTR; and -2578 C>A of the promoter region. RESULTS: The frequency of the VEGF CAC (+936/-2578/-634) haplotype was increased in patients with BRONJ, compared with female disease-negative controls [odds ratio (OR) = 2.76, 95% CI = 1.09-4.94, P = 0.039; corrected P value: P(c) = 0.117], and was also increased compared with female healthy controls (OR = 2.11, 95% CI = 1.14-3.89, P = 0.024; corrected P value: P(c) = 0.072). The CC homozygotes of -634G>C of VEGF gene and AA homozygotes of -2578C>A have also been significantly correlated in female patients who developed BRONJ compared with healthy controls (OR = 2.04, 95% CI = 1.12-3.70, P = 0.008; corrected P value: P(c) = 0.024). CONCLUSIONS: These results suggest a possible haplotype effect of VEGF polymorphisms expression in BRONJ Italian female patients. Studies with different and larger populations possibly using TagSNP to represent all haplotypes within the VEGF gene are needed to further delineate the genetic contribution of this gene to BRONJ.

Genotype, phenotype and hormonal levels correlation in non-classical congenital adrenal hyperplasia.

Non-classical congenital adrenal hyperplasia (NCAH) is a morbid condition sustained by the reduced function of one of the enzymes involved in the adrenal steroid biosynthesis pathway, mainly the 21-hydroxylase. Different degrees of enzyme activity impairment determine different clinical pictures, with childhood or post-pubertal onset. The aim of this study was to evaluate the relationship between genotype, phenotype, and adrenal hormonal levels in a group of 66 patients affected by NCAH attending outpatient pediatric or endocrinological Clinics. Our findings show that age at pubarche/menarche was significantly younger, height SD score) and Δ bone age-chronological age were significantly higher in patients with a more severe enzyme activity impairment, while cutaneous androgenization and menstrual irregularities in post-pubertal girls were not related to the grading of genotype.

Correlation between genotype and hormonal levels in heterozygous mutation carriers and non-carriers of 21-hydroxylase deficiency.

Congenital adrenal hyperplasia, both in its classic (CCAH) and non-classic form (NCAH), is a morbid condition sustained by the absent or reduced function of one of the enzymes involved in cortisol biosynthesis - mainly 21 hydroxylase - associated with different levels of clinical androgenization. In a wide group of relatives of patients affected by CCAH and NCAH (no.=222) and healthy volunteers (no.=30), a clinical, hormonal and genetic evaluation was performed in order to differentiate between the condition of heterozygous mutation carrier and non-carrier of any among 21-hydroxylase gene (CYP21) mutations. This study shows that clinical presentation and basal 17α-hydroxyprogesterone (17α-OHP) are not able to differentiate between heterozygous carriers and non-carriers, whereas 17α-OHP value after ACTH bolus is significantly different between heterozygous carriers and non-carriers: p<0.001 with a cut-off value of 3 ng/ml (90% sensitivity and 74,3% specificity). Moreover, our data indicate that 17α-OHP response to ACTH may be a useful tool to select subjects for genetic analysis.

Screening for chronic cerebrospinal venous insufficiency (CCSVI) using ultrasound: recommendations for a protocol.

Chronic cerebrospinal venous insufficiency (CCSVI) is a syndrome characterized by stenoses or obstructions of the internal jugular and/or azygos veins with disturbed flow and formation of collateral venous channels. Ultrasound and venographic studies of the internal jugular and azygos venous systems in patients with multiple sclerosis (MS) have demonstrated a high prevalence of CCSVI (mean 71%, range 0-100%; n=1336) associated with activation of collaterals. By contrast, ultrasound and venographic examinations of normal controls and patients without MS have demonstrated a much lower prevalence (mean 7.1%, range 0-22%; n=505). Ultrasound in the form of duplex scanning uses a combination of physiological measurements as well as anatomical imaging and has been used for the detection of CCSVI by different centers with variable results. A high prevalence of obstructive lesions, ranging from 62% to 100%, has been found by some teams in patients with MS compared with a low prevalence (0-25%) in controls. However, others have reported absence of these lesions or a lower prevalence (16-52%). This variability could be the result of differences in technique, training, experience or criteria used. In order to ensure a high reproducibility of duplex scanning with comparable accuracy between centers a detailed protocol with standard methodology and criteria is needed. Also, standardization of the method of reporting of duplex measurements and other findings will facilitate validation of the proposed criteria by different centers. The aim of this document is to produce recommendations for such a protocol and indicate what future research is needed in order to address areas of uncertainty.

Great saphenous varicose vein surgery without saphenofemoral junction disconnection.

BACKGROUND: The aim of this case-control study was to determine whether preoperative duplex imaging could predict the outcome of varicose vein surgery without saphenofemoral junction (SFJ) disconnection. The duplex protocol included a reflux elimination test (RET) and assessment of the competence of the terminal valve of the femoral vein. METHODS: One hundred patients with chronic venous disease who had a positive RET result and an incompetent terminal valve were compared with 100 patients matched for age, sex, clinical class (Clinical Etiologic Anatomic Pathophysiologic (CEAP) class C2-C6) and disease duration, but who had a positive RET result and a competent terminal valve. All patients underwent ligation and proximal avulsion of the incompetent tributaries from the great saphenous vein trunk without SFJ disconnection. Clinical and duplex follow-up lasted for 3 years, and included Hobbs' clinical score. RESULTS: Of legs with a competent terminal valve, 100 per cent were rated as cured (Hobbs' class A or B) and 14.0 per cent developed recurrent varices. Patients with an incompetent terminal valve had significantly worse results: 29.0 per cent had Hobbs' class A or B and 82.0 per cent developed recurrence (P < 0.001). CONCLUSION: Preoperative duplex assessment of the terminal valve could be used to identify patients suitable for varicose vein surgery without the need for SFJ disconnection.

Ethosomes for the delivery of anti-HSV-1 molecules: preparation, characterization and in vitro activity.

This paper describes the production, characterization and in vitro activity of ethosomes containing two molecules with antiviral activity, such as acyclovir (ACY) and N1-beta-D-ribofuranosyl-pyrazole [3,4d]pyridazin-7(6p-chlorine-phenyl)-one nucleoside (N1CP). Ethosomes were prepared and morphologically characterized by Cryo-TEM. The encapsulation efficiency was 92.3 +/- 2.5% for ACY and 94.2 +/- 2.8% for N1CP. The release of the drug from vesicles, determined by a Franz cell method, indicated that both drugs were released in a controlled manner. In order to possibly guarantee the stability during long-term storage ethosome suspensions was freeze-dried. It was found that the freeze-dried ethosomes' cakes were compact, glassy characterized by low density and quick re-hydration. However, the storage time slightly influences the percentage of drug encapsulation within ethosomes showing a drug leakage after re-hydration around 10%. The antiviral activity against HSV-1 of both drugs was tested by plaque reduction assay in monolayer cultures of Vero cells. Data showed that ethosomes allowed a reduction of the ED50 of N1CP evidencing an increase of its antiviral activity. However, ACY remains more active than N1CP. No differences are appreciable between drug-containing ethosomes before and after freeze-drying. Taken together these results, ethosomal formulation could be possibly proposed as mean for topical administration of anti-herpetic molecules.

Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis.

BACKGROUND: The extracranial venous outflow routes in clinically defined multiple sclerosis (CDMS) have not previously been investigated. METHODS: Sixty-five patients affected by CDMS, and 235 controls composed, respectively, of healthy subjects, healthy subjects older than CDMS patients, patients affected by other neurological diseases and older controls not affected by neurological diseases but scheduled for venography (HAV-C) blindly underwent a combined transcranial and extracranial colour-Doppler high-resolution examination (TCCS-ECD) aimed at detecting at least two of five parameters of anomalous venous outflow. According to the TCCS-ECD screening, patients and HAV-C further underwent selective venography of the azygous and jugular venous system with venous pressure measurement. RESULTS: CDMS and TCCS-ECD venous outflow anomalies were dramatically associated (OR 43, 95% CI 29 to 65, p<0.0001). Subsequently, venography demonstrated in CDMS, and not in controls, the presence of multiple severe extracranial stenosis, affecting the principal cerebrospinal venous segments; this provides a picture of chronic cerebrospinal venous insufficiency (CCSVI) with four different patterns of distribution of stenosis and substitute circle. Moreover, relapsing-remitting and secondary progressive courses were associated with CCSVI patterns significantly different from those of primary progressive (p<0.0001). Finally, the pressure gradient measured across the venous stenosies was slightly but significantly higher. CONCLUSION: CDMS is strongly associated with CCSVI, a scenario that has not previously been described, characterised by abnormal venous haemodynamics determined by extracranial multiple venous strictures of unknown origin. The location of venous obstructions plays a key role in determining the clinical course of the disease.

The severity of chronic cerebrospinal venous insufficiency in patients with multiple sclerosis is related to altered cerebrospinal fluid dynamics.

Chronic cerebrospinal venous insufficiency (CCSVI) is a vascular picture that shows a strong association with multiple sclerosis (MS). The aim of this study was to investigate the relationship between a Doppler cerebral venous hemodynamic insufficiency severity score (VHISS) and cerebrospinal fluid (CSF) flow dynamics in 16 patients presenting with CCSVI and relapsing-remitting MS (CCSVI-MS) and in eight healthy controls (HCs). The two groups (patients and controls) were evaluated using validated echo-Doppler and advanced 3T-MRI CSF flow measures. Compared with the HCs, the CCSVI-MS patients showed a significantly lower net CSF flow (p=0.027) which was highly associated with the VHISS (r=0.8280, r2=0.6855; p=0.0001). This study demonstrates that venous outflow disturbances in the form of CCSVI significantly impact on CSF pathophysiology in patients with MS.

HCV infection: pathogenesis, clinical manifestations and therapy.

Chronic hepatitis C virus (HCV) infection is a worldwide public health problem with a global prevalence of 2-3%. It is believed that about 170 million people are currently infected (about 3% of the world's population), and a further 3-4 million are infected each year. HCV is the main reason for liver transplantation in the developed world, and the main cause of liver-related morbidity and mortality in a number of countries, including Italy. It is not only a frequent cause of chronic liver diseases such as hepatitis, cirrhosis and hepatocellular carcinoma, but is also involved in the pathogenesis of various autoimmune and rheumatic disorders (arthritis, vasculitis, sicca syndrome, porphyria cutanea tarda, lichen planus, nephropathies, thyroid diseases, and lung fibrosis), as well as in the development of B-cell lymphoproliferative diseases. Furthermore, patients suffering from C hepatitis tend to produce rheumatoid factor, cryoglobulins and a large series of autoantibodies (ANA, anti-SSA/SSB, SAM, ATG, aCL). The use of glucocorticoids or immuno-suppressant agents in HCV infected individuals, which are needed to treat autoimmune and rheumatic disorders, leads to a risk of worsening the clinical outcome of HCV. Under these conditions, the viral infection often needs to be treated with antiviral agents, mainly pegylated interferon combined with ribavirin. However, cyclosporine A seems to be safe and effective in patients with autoimmune disease (AD) and concomitant chronic HCV infection as is documented by the reduction in viremia and transaminases, particularly in patients with high baseline levels. Finally, HCV is the main trigger of mixed cryoglobulinemia. An attempt at viral eradication is therefore indicated in most patients, and is particularly effective in the case of mild or moderate manifestations. In severe cases, rituximab is an apparently safe and effective alternative to conventional immunosuppression and, specifically, it controls B-cell proliferation.

A novel multicompartimental system based on aminated poly(vinyl alcohol) microspheres/succinoylated pullulan microspheres for oral delivery of anionic drugs.

Poly(vinyl alcohol) (PVA) microspheres were prepared by dispersion reticulation with glutaraldehyde and further aminated. These microspheres were firstly loaded with diclofenac (DF) and then entrapped in cellulose acetate butyrate (CAB) microcapsules by an o/w solvent evaporation technique for intestinal delivery of drug. The encapsulated PVA microspheres due to their low swelling degree in intestinal fluids, do not have enough force to produce the disruption of CAB shell, therefore different amounts of succinoylated pullulan microspheres (SP-Ms) (exchange capacity up to 5.2 meq/g) were co-encapsulated. The SP-Ms do not swell in acidic pH, but swell up to 20-times in intestinal fluids causing the rupture of CAB shell and facilitating the escape of loaded PVA microspheres.

Liposomes and micellar dispersions for delivery of benzoheterocyclic derivatives of distamycin A.

In this article we describe the production and characterization of specialized delivery systems for some distamycin derivatives (DD), namely liposomes and micellar dispersions. All the formulations were designed to increase the solubility of DD in an aqueous environment and to reduce the possible toxicity problems related to the administration of these drugs. For instance, liposomes were prepared by reverse phase evaporation technique followed by extrusion through polycarbonate filters, then characterized in terms of dimensions, morphology, and encapsulation efficacy. The analysis of their in vitro antiproliferative activity on cultured human and mouse leukemic cells demonstrated that liposomes and micellar dispersions containing DD exert quite different effects. These effects were compared with those shown by the free drug depending on type of drug and also cell line used.

Nanosystems for skin hydration: a comparative study.

The present investigation describes a comparative study for the design of innovative topical formulation for skin hydration. In particular, different colloidal forms based on lipidic components have been produced and characterized. Morphology and dimensional distribution have been investigated by means of electron microscopy and photon correlation spectroscopy. Nanoparticulate systems characterized by different morphology and dimensions depending on production procedures have been obtained, namely cubosomes, nanovesicles, solid lipid nanoparticles and liposomes. Hydration power has been studied by means of a corneometer, measuring the skin electrical capacitance before and after the application of opportunely viscosized nanoparticulate systems. It has been demonstrated that nanovesicle gel displayed a pronounced hydration power with respect to the other nanostructured forms, its hydration effect on skin was 3.5-fold higher, with respect to the untreated area, after 5 min from the application and 1.5-fold higher after 2 h.

Investigation of cerebral venous outflow in microgravity.

OBJECTIVE: The gravitational gradient is the major component to face when considering the physiology of venous return, and there is a growing interest in understanding the mechanisms ensuring the heart filling, in the absence of gravity, for astronauts who perform long-term space missions. APPROACH: The purpose of the Drain Brain project was to monitor the cerebral venous outflow of a crew member during an experiment on the International Space Station (ISS), so as to study the compensatory mechanisms that facilitate this essential physiological action in subjects living in a microgravity environment. Such venous function has been characterized by means of a novel application of strain-gauge plethysmography which uses a capacitive sensor. MAIN RESULTS: In this contribution, preliminary results of our investigation have been presented. In particular, comparison of plethysmography data confirmed that long duration spaceflights lead to a redistribution of venous blood volume, and showed interesting differences in the amplitude of cardiac oscillations measured at the level of the neck veins. SIGNIFICANCE: The success of the experiment has also demonstrated that thanks to its easy portability, non-invasiveness, and non-operator dependence, the proposed device can be considered as a novel tool for use aboard the ISS. Further trials are now under way to complete the investigation on the drainage function of the neck veins in microgravity.

Cationic liposomes as potential carriers for ocular administration of peptides with anti-herpetic activity.

In the present study the preparation, characterization and activity of cationic liposomes containing the secretory form of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB1s) or two related polylysine rich peptides, namely DTK1 and DTK2, were described. The immunotherapeutic potential of these HSV antigens containing liposomes was examined with a rabbit ocular model of HSV-1 infection. Our study indicates that the liposomes (i) are able to encapsulate quantitatively gB1s and around 30% the DTK peptides, (ii) are characterized by dimensions compatible with ocular applications and (iii) can release the peptide comparably to the free solution. In addition, neutralization studies demonstrated that an anti-DTK specific polyclonal antiserum can inhibit HSV-1 infection, indicating that such peptides could be a good immunogen/antigen in an anti-HSV vaccine formulation. Although the vaccination protocol did not induce protection against the eye disease, a significative protection against a lethal ocular challenge was detectable together with the absence of reactivation episodes from latency on the survived animals. In this respect, the use of cationic liposomes coupled to gB1s and DTK peptides, as a local ocular vaccine, could represent an interesting approach in order to obtain a possible efficacy in protecting animals against a subsequent HSV-1 ocular challenge.

New insights into immune mechanisms underlying response to Rituximab in patients with membranous nephropathy: A prospective study and a review of the literature.

BACKGROUND: Idiopathic membranous nephropathy (MN) is a common immune-mediated glomerular disease and the main cause of nephrotic syndrome (NS) in Caucasian adults. Rituximab (RTX) has been reported to safely reduce proteinuria in patients with primary MN and severe NS. However, the effects of RTX treatment on T-cells including regulatory T-cells (Treg) in MN have not been fully determined. METHODS: Seventeen patients [mean age 67 (29-86) years, 6 women, 11 men] with biopsy-proven MN, and persistent proteinuria >3.5 g/24h were prospectively enrolled and received RTX, 375 mg/m(2) (iv) on days 1, 8, 15 and 22. Changes in circulating B and T cell homeostasis were examined in the peripheral blood by flow-cytometry studies; serum levels of IL-35 were measured using a high-sensitivity ELISA kits (baseline, at month 3, 6, 9 and 12). RESULTS: Patients had been followed-up for a mean of 36.3 months (24-48). Proteinuria decreased from 5.6 (3.5-8) g/24h to 2.4 (0.06-13) g/24h at 6 months (p<0.05) and to 1.3 (0.06-8) at 12 months (p<0.01), respectively after therapy with RTX. Four patients received a 2nd course of RTX (one at 6 months because of persistent NS, and three at 12, 18, or 30 months for relapse). The three relapsing patients became proteinuria-free (<0.5 g/24h) in the following 6 months. Serum creatinine remained stable during the follow-up: median 1mg/dl (0.7-1.6) at 12 months and 1.1 (0.7-1.7) at 24 months as compared to 1 (0.5-2.4) at baseline. At 6 months after RTX, complete remission (CR) was observed in 7 patients, partial remission (PR) in 4, while 6 were non responders (NR) non responder (NR). At the end of the follow-up, 14 patients were in CR, 1 in PR, while 2 were NR. In the T-cell compartment, upon detection of B cell depletion, there was an increase in Treg up to 10-fold when comparing baseline and at month 12 (mean ± SD 1.2 ± 0.6%, and 5.8 ± 0.7% p=0.02, respectively). When stratifying patients in responders (CR+PR) and NRs at month 12, we observed a significant increase in Treg cells from month 6 which persisted till 12 months only in the responder group (5.5 ± 0.6% and 1.1 ± 0.6%, p=0.04, respectively in responders and NRs). A statistically significant decrease in the levels of active T-lymphocytes (HLA-DR+CD8+ cells) was observed, with a maximum reached at 12 months after treatment with RTX [6 ± 1.1% baseline, 4.7 ± 1.7% at 6 months (p=0.043) and 1.5 ± 1.4% at 12 months (p=0.05)]. A marked increase in IL-35 levels [defined as delta >40% (serum values at 6 months minus baseline values)] was seen in 68% of the patients who achieved clinical response (CR or PR) at 12 month, but in none of the patients who failed to respond (p=0.034). CONCLUSION: Our findings and data from literature support the idea that RTX can be envisaged as a first-line therapy for patients at risk of progression because of persistent NS due to idiopathic MN. Insights into the putative T cell-related mechanisms of action have been discussed.