High rate of detection of ultrasound signs of prostatitis in patients with HPV-DNA persistence on semen: role of ultrasound in HPV-related male accessory gland infection.

BACKGROUND: Papillomavirus (HPV) often occurs in the semen of patients with male accessory gland infection (MAGI). Ultrasound (US) evaluation has been suggested as a promising diagnostic tool for patients with HPV-related MAGI. No data on the spontaneous clearance of HPV-DNA have been reported so far in HPV-related MAGI. PURPOSE: The primary aim of the study was to assess the percentage of early HPV-DNA spontaneous clearance in patients with prostatitis. The secondary aim was to evaluate the frequency of spontaneous clearance of HPV-DNA among patients with prostatitis associated with the presence or absence of US abnormalities. METHODS: Patients with inflammatory MAGI and at least one suspicious criterion for HPV infection underwent semen HPV-DNA detection and prostate US. The presence of HPV-DNA was further investigated after a 6-month-long follow-up. MAIN RESULTS: Eighty patients satisfied the inclusion criteria and were recruited in the study. 69% of patients (55/80) showed HPV-DNA persistence in the semen. Among them, 82% (45/55) was positive for US signs of prostatitis, while they occurred only in 12% (3/25) of those patients with no sign of HPV-DNA persistence (p < 0.001). All patients with persistent high-risk HPV genotype (n = 30) showed at least two US signs of prostatitis. In 73% of patients (22/30), E6 and E7 mRNAs were detected. CONCLUSION: US signs of prostatitis more frequently occurred in patients with evidence of HPV-DNA persistence on semen, especially in those with high-risk genotypes. This highlights the importance of US in the framework of HPV-related MAGI.

Evaluation of isavuconazole MIC strips for susceptibility testing of Aspergillus and Scedosporium species.

Isavuconazole is a new triazole with an expanded-spectrum and potent activity against moulds and yeasts. It has been authorized for use in adults for the treatment of invasive aspergillosis and for mucormycosis. The only commercially available isavuconazole susceptibility test is the minimum inhibitory concentration (MIC) strip isavuconazole test. The objective of this study was to assess the in vitro activity of isavuconazole using gradient concentration MIC strips, compared with the EUCAST broth microdilution reference method. A total of 147 clinically relevant fungal isolates comprising 120 Aspergillus sp. and 27 Scedosporium apiospermum complex were tested for susceptibility to isavuconazole using the EUCAST broth microdilution method and by the MIC strip isavuconazole test. The percent essential agreement between the two methods was calculated within a 1-fold dilution. The geometric means for the MICs using the EUCAST reference methods and the strip test were respectively: 0.60 mg/l and 0.65 mg/l for A. fumigatus, 0.70 mg/l and 0.77 mg/l for A. flavus, 1.50 mg/l and 1.25 mg/l for A. niger, 0.41 mg/l and 0.38 mg/l for A. terreus, 1.22 mg/l and 1.08 mg/l for S. apiospermum complex. The isavuconazole MIC strips showed good agreement with the EUCAST reference method. Isavuconazole MIC strips could be useful for susceptibility testing of Aspergillus sp. and S. apiospermum complex.

Papillary squamous cell carcinoma of the palatine tonsil: a rare cancer of the head and neck.

Papillary squamous neoplasms of the upper respiratory tract are rare variants of squamous cell carcinomas. They are characterised by an exophytic, papillary growth and generally have favourable prognosis. The tumour has been described in the upper aerodigestive tract. In this context, most common sites of involvement are the larynx and hypopharynx, and rarely the oral cavity and oropharynx. The limited studies and small number of published cases of papillary squamous cell carcinoma of the palatine tonsil led us to make a complete analysis of this tumour by analysing the clinical, histological, radiological, virological and therapeutic aspects that are not always present in the literature. A case of papillary squamous cell carcinoma of the palatine tonsil is reported. The lesion (T2N0M0) was located into the left palatine tonsil that hung towards the oral cavity. Both HPV 16 DNA and E6/E7 mRNA were detected in the lesion. The clinicopathological profile of the neoplasm is presented and a comprehensive review of recent literature was made by analysing all aspects of interest of this neoplasm.

CD55 is a HIF-2α marker with anti-adhesive and pro-invading properties in neuroblastoma.

CD55 has been revealed to have an important role in tumor genesis, and presence of small populations of cells with strong CD55 expression would be sufficient to predict poor prognosis of several tumors. In our study we revealed that CD55 is a novel target of hypoxia-inducible factor HIF-2α in neuroblastoma (NB) cells. We show that HIF-2α expression is sufficient to sustain stem-like features of NB cells, whereas CD55 protein upon HIF-2α expression contributes to growth of colonies and to invasion of cells, but not to stemness features. Interestingly, in NB tissues, CD55 expression is limited to quite a small population of cells that are HIF-2α positive, and the gene expression of CD55 in the NB data set reveals that the presence of CD55(high) affects prognosis of NB patients. The functional characterization of CD55-positive populations within heterogeneous NB monoclonal cell lines shows that CD55 has pro-invading and anti-adhesive properties that might provide the basis for the ability of solid tumors to survive as microscopic residual disease. The easy accessibility to CD55 membrane antigen will offer the possibility of a novel antibody approach in the treatment of recurrent tumors and will provide a ready target for antibody-based visualization in NB diagnosis and prognosis.

Outcomes of transcatheter aortic valve implantation in high surgical risk and inoperable patients with aortic stenosis: a single Australian Centre experience.

BACKGROUND: Degenerative aortic stenosis is the most common valvular heart disease in the elderly, and many patients are not suitable for aortic valve replacement surgery. Transcatheter aortic valve implantation (TAVI) is a new therapeutic option for selected patients at high risk for surgery. AIM: To evaluate the safety and efficacy of TAVI in Australian patients. METHODS: This is a prospective study of patients undergoing TAVI for severe symptomatic aortic stenosis at The Prince Charles Hospital, Brisbane, Australia between August 2008 and July 2013. Patients were at high risk of surgical aortic valve replacement, or inoperable, as deemed by a multidisciplinary 'heart team'. Outcomes include procedural success and complications, 30-day and 1-year mortality and stroke, combined end-points as outlined by the Valve Academic Research Consortium 2 consensus document. RESULTS: Two hundred and nine patients underwent TAVI during the study period. The mean age was 83.7 ± 6.7 years, and 101 (48%) were men. The valve systems utilised were as follows: Edwards-SAPIEN valve in 104 (49.5%), Medtronic CoreValve in 86 (41.2%) and Boston Scientific Lotus valve in 19 (9.3%) patients. Thirty-day and 1-year mortality rates were 5.7% and 11.5% respectively. Thirty-day and 1-year stroke rates were 4.3% and 6.2% respectively. The composite end-points of device success, early safety and clinical efficacy occurred in 80.4%, 27.3% and 68.4%. CONCLUSIONS: TAVI with various valve systems, delivered through several approaches, is feasible in high surgical risk and inoperable patients with severe aortic stenosis, with acceptable outcomes at short-term and intermediate-term follow up.

Prevalence of human papilloma virus infection in patients with male accessory gland infection.

The frequency of human papillomavirus (HPV) infection in the semen of patients with male accessory gland infection (MAGI) was evaluated. One hundred infertile patients with MAGI were classified into group A: patients with an inflammatory MAGI (n = 48) and group B: patients with a microbial form (n = 52). Healthy age-matched fertile men (34.0 ± 4.0 years) made up the control group (n = 20). Amplification of HPV DNA was carried out by HPV-HS Bio nested polymerase chain reaction for the detection of HPV DNA sequences within the L1 ORF. Ten patients in group A (20.8%) and 15 patients in group B (28.8%) had a HPV infection; two controls (10.0%) had HPV infection. Patients with MAGI had a significantly higher frequency of HPV infection compared with controls; patients with a microbial MAGI had significantly higher frequency of HPV infection compared with patients with an inflammatory form (both P < 0.05). Patients with MAGI and HPV had a slight, but significantly lower sperm progressive motility and normal morphology compared with patients with MAGI HPV-negative (P < 0.05). Elevated frequency of HPV infection occurred in patients with MAGI, suggesting that HPV should be investigated in the diagnostic work-up of these patients.

Development of new photon-counting detectors for single-molecule fluorescence microscopy.

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level.

Percutaneous ASD closure in a large Australian series: short- and long-term outcomes.

AIM: To assess the clinical and echocardiographic outcomes in patients referred for device closure of atrial septal defects in a tertiary referral hospital in Australia. METHODS: A prospective follow-up study was performed on all patients who had device closure of a secundum atrial septal defect (ASD) from June 1999 to December 2007. Clinical and echocardiographic data at the time of implantation and follow-up is presented. RESULTS: 176 patients were referred for shunt closure of ASD. All patients had a significant shunt defined as a shunt with right heart dilatation and/or a shunt ratio of at least 1.5:1. The majority were female (67%) and the average age was 36.5 ± 22.7 years; age range 3-84. The average hospital admission time was 2.5 ± 1.7 days. The average follow-up occurred at 3.7 ± 3.6 months for the first follow-up and 26.3 ± 18.2 months (range 3 months-7.8 years) for the long-term follow-up. Baseline echocardiogram findings showed the majority had a normal left ventricular ejection fraction (99%); average LVEF=63.2 ± 7.2% while the right ventricle was dilated in 61% of patients. Procedure information: The average procedure time was 94.8 ± 36.4 min. Procedural imaging was performed using Transoesophageal echocardiography (TOE) in 107 cases (61%); Intracardiac Echocardiography (ICE) in 69 (39%). Device use was as follows: Amplatzer=156 cases, Helex=18, and Starflex=2. Postprocedure shunt assessment by transthoracic echocardiography showed successful closure (no shunt or trivial shunt) in 99% cases. Two patients were referred for inpatient surgery due to a significant residual shunt in one case and an unstable device in another. One patient who had an unstable device had their device repositioned successfully. Atrial arrhythmia was the most common complication occurring in the peri-implantation period in 12 cases (6%) with four further cases at final up. The high prevalence of right ventricular dilatation in 65% patients at baseline had improved significantly at the first and long term follow-up to 2% (p=0.0001). CONCLUSION: Device closure of secundum atrial septal defects in this large Australian cohort demonstrates a high procedural success rate with a low incidence of complications in the short and long term.

Polychromatic flow cytometry analysis of CD34+ hematopoietic stem cells in cryopreserved early preterm human cord blood samples.

During the last decades, extended characterizations were performed of human full-term cord blood (hTCB) cells, but little information is available on human early preterm cord blood (hEPCB) hematopoietic stem cells (HSCs). In our study, we analyzed by flow cytometry 19 hEPCB and 17 hTCB samples. First, we observed that the percentage of CD34(Pos)CD45(Dim) cells was higher in hEPCB compared with hTCB and that it decreased during 16th-20th week of pregnancy. Within the CD34(Pos)CD45(Dim) population, we examined the expression of CD29, CD31, CD38, CD90, CD117, CD133, CD135, CD200, CD243, and CD338. We found that CD135 intensity and CD243(Pos) cells percentage were lower in hEPCB compared with hTCB. As to CD38, we observed that hEPCB samples were richer in undifferentiated CD34(Pos)CD45(Dim)CD38(Neg) HSCs compared with hTCB counterparts. We also compared the expression of the above-mentioned molecules in undifferentiated and committed HSCs residing in hEPCB and hTCB. In particular, although CD34(Pos)CD45(Dim)CD38(Pos) HSCs from both hEPCB and hTCB expressed relatively higher amounts of CD29, CD71, and CD135 compared with CD34(Pos)CD45(Dim)CD38(Neg) cells, a higher expression of CD31 was restricted to CD34(Pos)CD45(Dim)CD38(Pos) cells from hEPCB samples, and a higher expression of CD117 was demonstrated in CD34(Pos)CD45(Dim)CD38(Pos) cells from hTCB samples. Moreover, our data showed that CD34(Pos)CD45(Dim) cell population from hEPCB displayed higher percent of undifferentiated CD38(Neg)CD133(Pos) cells compared with hTCB samples. Finally, analyzing monocytes and lymphocytes within the two samples, we observed that T-cell percentages were higher in hTCB, whereas B-cell percentages were higher in hEPCB. We, therefore, studied the B-cell lineage maturation and found a higher percent of pro-B and pre-B cells in hEPCB compared with hTCB samples. Taken together, these results evidence the hematopoietic peculiarity of hEPCB, potentially useful for highlighting early steps of human hematolymphopoiesis as well as for developing novel strategies of stem cell-based therapy.

New photon-counting detectors for single-molecule fluorescence spectroscopy and imaging.

Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. Two typical geometries can be used for these experiments: point-like and widefield excitation and detection. In point-like geometries, the basic concept is to excite and collect light from a very small volume (typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy. Therefore the advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence measurements. In widefield geometries, the same issues of background reduction and single-molecule concentration apply, but the duration of the experiment is fixed by the time scale of the process studied and the survival time of the fluorescent probe. Temporal resolution on the other hand, is limited by signal-to-noise and/or detector resolution, which calls for new detector concepts. We will briefly present our recent results in this domain.

Elastic scattering and reaction mechanisms of the halo nucleus 11Be around the Coulomb barrier.

Collisions induced by (9,10,11)Be on a 64Zn target at the same c.m. energy were studied. For the first time, strong effects of the 11Be halo structure on elastic-scattering and reaction mechanisms at energies near the Coulomb barrier are evidenced experimentally. The elastic-scattering cross section of the 11Be halo nucleus shows unusual behavior in the Coulomb-nuclear interference peak angular region. The extracted total-reaction cross section for the 11Be collision is more than double the ones measured in the collisions induced by (9,10)Be. It is shown that such a strong enhancement of the total-reaction cross section with 11Be is due to transfer and breakup processes.

A comparison of transcutaneous Doppler corrected flow time, b-type natriuretic peptide and central venous pressure as predictors of fluid responsiveness in septic shock: a preliminary evaluation.

Aortic corrected flow time (FTc) is easily measured by Doppler techniques. Recent data using transoesophageal Doppler suggest that it may predict fluid responsiveness in critical care. This use of FTc has not previously been evaluated in septic shock, nor have any studies incorporated transcutaneously measured FTc. Furthermore, no comparison has been made between FTc, plasma B-type natriuretic peptide concentration (BNP) or central venous pressure. The aim of this preliminary study was to compare FTc, BNP and central venous pressure as predictors of fluid responsiveness in septic shock patients without cardiac dysrhythmia. This was a prospective study of 10 consecutive adult septic shock patients (in sinus rhythm; 60% mechanically ventilated) treated with intravenous fluid challenge (4% albumin 250 ml over 15 minutes) in a mixed medical/ surgical tertiary intensive care unit. Mean + SD Acute Physiological and Chronic Health Evaluation II score was 21.8 +/- 12.7. Haemodynamic assessment incorporating transcutaneous aortic Doppler (USCOM) occurred before and five minutes after fluid challenge. Concurrent with initial assessment, blood samples were collected for BNP assay (ADIVA Centaur). Four patients demonstrated an increase in stroke volume > or = 15% (responders). Percent change in stroke volume strongly correlated with baseline FTc (r = -0.81, P = 0.004) but not BNP (r = -0.3, P = 0.4) or central venous pressure (r = -0.4, P = 0.2). Baseline FTc < 350 ms discriminated responders from non-responders (P = 0.047). Our data support FTc as a better predictor of fluid responsiveness than either BNP or central venous pressure in septic shock. Transcutaneous aortic Doppler FTc offers promise as a simple, completely non-invasive predictor of fluid responsiveness and should be evaluated further

High-throughput single-molecule fluorescence spectroscopy using parallel detection.

Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. The basic concept of this technique is to excite and collect light from a very small volume (typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy. Therefore the advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence measurements obtained with different novel multipixel single-photon counting detectors.

Surveillance of human papilloma virus infection and cervical cancer in kidney transplant recipients: preliminary data.

INTRODUCTION: Development of cancer after transplantation has rapidly became one of the leading causes of death in kidney transplant recipients with functioning grafts. Anogenital malignant neoplasms may occur with a 14-fold increased incidence, and human papilloma virus (HPV) infection has been recently identified as the leading cause of cervical carcinoma. We report the preliminary findings of a prospective study that evaluated the incidence of HPV infection and cervical carcinoma in a population of kidney transplant recipients. PATIENTS AND METHODS: The study included 35 female recipients of a deceased donor kidney with at least 6 months of follow-up. All patients underwent a cervicovaginal brushing, an HPV DNA test, and a Papanicolaou test. RESULTS: Twenty-two patients (62.8%) were positive for HPV DNA. Thirteen of 22 HPV DNA-positive recipients (59%) demonstrated a high-risk HPV genotype. No cytologic anomalies were detected in Papanicolaou smears. CONCLUSIONS: These preliminary data demonstrated a high incidence of HPV infection in renal transplant recipients. Most of our recipients exhibited a high-risk HPV genotype, which suggests higher aggressiveness of such infection in immunosuppressed patients. The HPV test is useful to monitor patients at higher risk of anogenital malignant neoplasms by identifying the cytologic anomalies at an earlier stage. This ongoing study will investigate the rate of progression of HPV infection and the clinical patterns of HPV-positive cytologic anomalies in renal transplant recipients.

Evaluation of the platelet counting by Abbott CELL-DYN SAPPHIRE haematology analyser compared with flow cytometry.

The Abbot Cell-Dyn Sapphire is a new generation haematology analyser. The system uses optical/fluorescence flow cytometry in combination with electronic impedance to produce a full blood count. Optical and impedance are the default methods for platelet counting while automated CD61-immunoplatelet analysis can be run as selectable test. The aim of this study was to determine the platelet count performance of the three counting methods available on the instrument and to compare the results with those provided by Becton Dickinson FACSCalibur flow cytometer used as reference method. A lipid interference experiment was also performed. Linearity, carryover and precision were good, and satisfactory agreement with reference method was found for the impedance, optical and CD61-immunoplatelet analysis, although this latter provided the closest results in comparison with flow cytometry. In the lipid interference experiment, a moderate inaccuracy of optical and immunoplatelet counts was observed starting from a very high lipid value.

An optimized method to separate reticulocytes from peripheral blood for molecular analysis.

A method based on immunomagnetic sorting of reticulocytes from peripheral blood was set up and combined to a commercial extraction kit for the isolation of total RNA from whole blood. This procedure resulted in high-quality RNA samples suitable for molecular analysis. We used this procedure to analyse erythroid-specific transcripts, starting from peripheral blood samples, to search for differently expressed mRNAs in patients with hereditary persistence of foetal haemoglobin. After erythrocyte lysis, CD15(+)and CD45(+) peripheral cells were negatively sorted to remove leucocyte populations that could have affected the subsequent screening procedure. The cell sorting and RNA extraction procedure was completed within 1-2 h of erythrocyte lysis, which represents a consistent saving of time compared with other procedures. Moreover, it produced 1 microg of total RNA per ml of blood samples, which is sufficient for molecular analysis. Therefore, our method is a reliable and efficient tool to isolate RNA from specific cell subpopulations poorly represented in peripheral blood, particularly when accurate detection and characterization of highly unstable and poorly expressed molecules is required.

Critical role of multidimensional flow cytometry in detecting occult leptomeningeal disease in newly diagnosed aggressive B-cell lymphomas.

Among histological aggressive non-Hodgkin lymphomas (NHL), the overall risk of central nervous system (CNS) relapse is approximately 5%, a figure which is too low to offer prophylaxis to all patients. The aim of this work is to demonstrate the utility of flow cytometry (FCM) in detecting occult leptomeningeal disease in this subtype of NHL. We studied cerebrospinal fluid (CSF) involvement in 42 newly diagnosed aggressive NHL patients at risk for CNS involvement. We used multicolour FCM to detect CSF infiltrating neoplastic cells. Among the 42 patients studied, 11 had CSF involvement as detected by FCM. Of these, only four were also positive for conventional morphology (p=0.046). These results designate that FCM as the first choice technique in NHL CSF clinical cell analysis.

Immunomodulatory properties of cefaclor: in vivo effect on cytokine release and lymphoproliferative response in rats.

The proper and coordinated response of the host immune system to bacterial infections is known to play a central role in the eradication of an infection. Therefore, the impact of antibiotics on both innate and acquired host immunity may be involved in the therapeutic outcome. The aim of this study was to evaluate the effects of the widely used cephalosporin cefaclor on some parameters of the immune system in ex vivo conditions. The results demonstrated that short-term (3 to 6 days) treatment with this antibiotic induced pleiotropic modification of rat spleen cells upon ex vivo stimulation with the polyclonal mitogen PHA, entailing increased lymphoproliferative responses, augmented IFN-gamma, IL-2 and IL-10 synthesis and decreased production of IL-4 and IL-6 in comparison to spleen cells from control rats. The mononuclear spleen cells of healthy rats released larger amounts of IFN-gamma and IL-2 in culture supernatants in response to polyclonal mitogenic stimulation with PHA compared to the spleens of the control rats receiving vehicle only. Simultaneously, the treatment with cefaclor augmented PHA-induced lymphoproliferative responses and reduced the synthesis of IL-4 and IL-6. These data depict a type 1 cytokine inducing and immunostimulatory pharmacological profile that, by activating the innate and acquired immune system, would be synergistic with cefaclor antibacterial activity.