Effect of Bioactivation on Traditional Surfaces and Zirconium Nitride: Adhesion and Proliferation of Preosteoblastic Cells and Bacteria.

PURPOSE: The aim of this vitro study was to reproduce and evaluate the response of bone and bacteria to traditional and innovative implant surfaces with difference wettability. MATERIALS AND METHODS: Two hundred fifty-two samples made of grade 4 titanium with different coating (machined [MAC]; double-etched, Ti-AE; zirconium nitride [Ti-ZrN]) were used for this in vitro study. Disks were divided into test (bioactivated using plasma of argon) and control group (untreated). To assess the surface morphology of the specimens, representative images were acquired via scanning electron microscopy (SEM). Murine preosteoblasts (MC3T3-E1) were used to study the biologic response in vitro, while the quantification of protein adsorption was achieved through the incubation of the titanium samples in a 2% solution of fetal bovine serum (FBS) in phosphate-buffered saline (PBS). The sterilized titanium disks were then colonized by bacterial species from a single sputum sample obtained from a healthy volunteer. For every analysis, 24 disks were used (12 for each group). RESULTS: SEM and topographic analyses demonstrated a Sa value of 0.33 (Ti-ZrN), 0.34 (MAC), and 0.62 (Ti-AE). Compared with the control groups, plasma treatment significantly increased the protein adsorption level on all the different titanium surfaces (5.88 ± 0.21 vs 7.85 ± 0.21, 7.13 ± 0.14 vs 9.74 ± 0.65, 4.41 ± 0.62 vs 6.13 ± 0.52, respectively, for MAC, Ti-treated, and Ti-ZrN). Similar behavior was described for cell adhesion (27.67 ± 2.03 vs 58.00 ± 20.13, 116.67 ± 12.02 vs 159.33 ± 8.09, 52.00 ± 4.73 vs 78.33 ± 4.67, respectively, for MAC, Ti-treated, and Ti-ZrN). Plasma treatment significantly augmented the number of CFU only in MAC and ZrN samples. CONCLUSION: With the limitations of this in vitro study, the following conclusions could be drawn: (1) rough implant surfaces present a higher adhesion and proliferation of preosteoblastic cells and bacterial biofilm; (2) rough implant surfaces benefited the most by the plasma of argon treatment.

Inhibition of herpes simplex type 1 and type 2 infections by Oximacro(®), a cranberry extract with a high content of A-type proanthocyanidins (PACs-A).

In the absence of efficient preventive vaccines, topical microbicides offer an attractive alternative in the prevention of Herpes simplex type 1 (HSV-1) and type 2 (HSV-2) infections. Because of their recognized anti-adhesive activity against bacterial pathogens, cranberry (Vaccinium macrocarpon Ait.) extracts may represent a natural source of new antiviral microbicides. However, few studies have addressed the applications of cranberry extract as a direct-acting antiviral agent. Here, we report on the ability of the novel cranberry extract Oximacro(®) and its purified A-type proanthocyanidins (PACs-A), to inhibit HSV-1 and HSV-2 replication in vitro. Analysis of the mode of action revealed that Oximacro(®) prevents adsorption of HSV-1 and HSV-2 to target cells. Further mechanistic studies confirmed that Oximacro(®) and its PACs-A target the viral envelope glycoproteins gD and gB, thus resulting in the loss of infectivity of HSV particles. Moreover, Oximacro(®) completely retained its anti-HSV activity even at acidic pHs (3.0 and 4.0) and in the presence of 10% human serum proteins; conditions that mimic the physiological properties of the vagina - a potential therapeutic location for Oximacro(®). Taken together, these findings indicate Oximacro(®) as an attractive candidate for the development of novel microbicides of natural origin for the prevention of HSV infections.

Escherichia coli Overexpressing a Baeyer-Villiger Monooxygenase from Acinetobacter radioresistens Becomes Resistant to Imipenem.

Antimicrobial resistance is a global issue currently resulting in the deaths of hundreds of thousands of people a year worldwide. Data present in the literature illustrate the emergence of many bacterial species that display resistance to known antibiotics; Acinetobacter spp. are a good example of this. We report here that Acinetobacter radioresistens has a Baeyer-Villiger monooxygenase (Ar-BVMO) with 100% amino acid sequence identity to the ethionamide monooxygenase of multidrug-resistant (MDR) Acinetobacter baumannii. Both enzymes are only distantly phylogenetically related to other canonical bacterial BVMO proteins. Ar-BVMO not only is capable of oxidizing two anticancer drugs metabolized by human FMO3, danusertib and tozasertib, but also can oxidize other synthetic drugs, such as imipenem. The latter is a member of the carbapenems, a clinically important antibiotic family used in the treatment of MDR bacterial infections. Susceptibility tests performed by the Kirby-Bauer disk diffusion method demonstrate that imipenem-sensitive Escherichia coli BL21 cells overexpressing Ar-BVMO become resistant to this antibiotic. An agar disk diffusion assay proved that when imipenem reacts with Ar-BVMO, it loses its antibiotic property. Moreover, an NADPH consumption assay with the purified Ar-BVMO demonstrates that this antibiotic is indeed a substrate, and its product is identified by liquid chromatography-mass spectrometry to be a Baeyer-Villiger (BV) oxidation product of the carbonyl moiety of the ß-lactam ring. This is the first report of an antibiotic-inactivating BVMO enzyme that, while mediating its usual BV oxidation, also operates by an unprecedented mechanism of carbapenem resistance.

The 6-Aminoquinolone WC5 inhibits different functions of the immediate-early 2 (IE2) protein of human cytomegalovirus that are essential for viral replication.

The human cytomegalovirus (HCMV) immediate-early 2 (IE2) protein is a multifunctional factor essential for viral replication. IE2 modulates both viral and host gene expression, deregulates cell cycle progression, acts as an immunomodulator, and antagonizes cellular antiviral responses. Based on these facts, IE2 has been proposed as an important target for the development of innovative antiviral approaches. We previously identified the 6-aminoquinolone WC5 as a promising inhibitor of HCMV replication, and here, we report the dissection of its mechanism of action against the viral IE2 protein. Using glutathione S-transferase (GST) pulldown assays, mutagenesis, cell-based assays, and electrophoretic mobility shift assays, we demonstrated that WC5 does not interfere with IE2 dimerization, its interaction with TATA-binding protein (TBP), and the expression of a set of cellular genes that are stimulated by IE2. On the contrary, WC5 targets the regulatory activity exerted by IE2 on different responsive viral promoters. Indeed, WC5 blocked the IE2-dependent negative regulation of the major immediate-early promoter by preventing IE2 binding to the crs element. Moreover, WC5 reduced the IE2-dependent transactivation of a series of indicator constructs driven by different portions of the early UL54 gene promoter, and it also inhibited the transactivation of the murine CMV early E1 promoter by the IE3 protein, the murine cytomegalovirus (MCMV) IE2 homolog. In conclusion, our results indicate that the overall anti-HCMV activity of WC5 depends on its ability to specifically interfere with the IE2-dependent regulation of viral promoters. Importantly, our results suggest that this mechanism is conserved in murine CMV, thus paving the way for further preclinical evaluation in an animal model.

Detection of Mimivirus in bronchoalveolar lavage of ventilated and nonventilated patients.

BACKGROUND/AIMS: We investigated the prevalence of Mimivirus in bronchoalveolar lavage (BAL) specimens from ventilated versus nonventilated patients. METHODS: The occurrence of Mimivirus DNA was evaluated by two previously developed real-time PCR assays in 69 BAL specimens: 30 from patients on mechanical ventilation for at least 48 h and 39 from nonventilated patients from different clinical settings, including lung transplant recipients. RESULTS: None of the BAL specimens from ventilated and nonventilated patients resulted positive for Mimivirus. CONCLUSION: This study, similarly to other studies that used molecular assays to detect Mimivirus, found no occurrence of the virus in the lower respiratory tract, thus being in contrast to serological investigations which reported a significant association between Mimivirus and the development of pneumonia. Gene polymorphism could explain these results or, alternatively, it could be hypothesized that Mimivirus does not represent a common cause of lower respiratory tract infection in either ventilated or nonventilated patients. Further studies on a larger population of patients from a different clinical setting evaluating both serology and DNA detection in lower respiratory tract specimens, including BAL and possibly tissue samples, could allow a better definition of the epidemiological and pathological role of Mimivirus in the development of pneumonia.

Development of a quantitative real-time nucleic acid sequence-based amplification assay with an internal control using molecular beacon probes for selective and sensitive detection of human rhinovirus serotypes.

Evidence demonstrating that human rhinovirus (HRV) disease is not exclusively limited to the upper airways and may cause lower respiratory complications, together with the frequency of HRV infections and the increasing number of immunocompromised patients underline the need for rapid and accurate diagnosis of HRV infections. In this study, we developed the first quantitative real-time nucleic acid sequence-based amplification assay with an internal control using molecular beacon probes for selective and sensitive detection of human rhinovirus serotypes. We described a simple method to accurately quantify RNA target by computing the time to positivity (TTP) values for HRV RNA. Quantification capacity was assessed by plotting these TTP values against the starting number of target molecules. By using this simple method, we have significantly increased the diagnostic accuracy, precision, and trueness of real-time NASBA assay. Specificity of the method was verified in both in silico and experimental studies. Moreover, for assessment of clinical reactivity of the assay, NASBA has been validated on bronchoalveolar lavage (BAL) specimens. Our quantitative NASBA assay was found to be very specific, accurate, and precise with high repeatability and reproducibility.

Detection of human rhinoviruses in the lower respiratory tract of lung transplant recipients.

The occurrence of human rhinoviruses (HRV) and its relationship to clinical and histopathological findings were investigated in 127 bronchoalveolar lavage specimens from 36 lung transplant recipients by real-time RT-PCR. In addition, 286 samples from 235 other immunocompromised and immunocompetent patients were also studied. HRV was detected in 41.7% of lung transplant recipients vs 14.5% of other patients (p < 0.0001), and no differences in viral load were observed. Acute respiratory insufficiency was found in 15 cases, three of which were HRV positive (viral load, 6.3 x 10(6) RNA copies/ml in one patient with chronic graft dysfunction). A diagnosis of pneumonia was made in 10 out of 127 cases, two of which were HRV positive (viral load, 10(3)-10(4) in cases of co-infection). Acute rejection was diagnosed in 12 cases, three of which were HRV positive (viral load, 10(3) in two cases of co-infection and 10(5) in a single infection). HRV infection may involve the lower respiratory tract, particularly in the presence of an impaired pulmonary background, such as a transplanted lung. Clinical evaluation should take into account the viral load, with a load of >10(5) possibly being associated with clinical symptoms, although lower loads can be detected in both symptomatic and asymptomatic patients.

Human cytomegalovirus glycoprotein B genotyping from bronchoalveolar lavage specimens.

The genes encoding glycoprotein complexes of human cytomegalovirus are often polymorphic; in particular, glycoprotein B (gB), which is essential for both in vivo and in vitro replication, is encoded by the highly polymorphic gene UL55. In this study, the distribution of gB genotypes was investigated in 44 bronchoalveolar lavage specimens from adult patients positive for human cytomegalovirus DNA by a multiplex nested fast PCR able to amplify 5 gB genotypes (gB1-gB5). The distribution of gB genotypes was as follows: 12 (27.3%) gB1, 11 (25%) gB2, 9 (20.4%) gB3, 4 (9.1%) gB4, 0 gB5, and 8 (18.2%) mixed genotypes. No difference in prevalence was found in relation to clinical features, including immunological status, non-transplant or transplant condition, and type of transplanted organ, or in follow-up specimens; while gB4 and gB3 were shown to be significantly more prevalent in patients with respiratory insufficiency, and gB4 and gB2 in those with pneumonia. The prevalence of gB genotypes in the lower respiratory tract was similar to that previously reported using other specimen types and patients, with gB1 found to be the most prevalent. The association of gB genotypes with specific clinical features should be further investigated.

Improvement of HRV quantification using cRNA-based standards for real time RT-PCR.

Real Time RT-PCR developed in recent years represents an useful tool in the diagnosis of RNA viruses. In order to accurately quantify and normalize a RNA target, efficiency of reverse-transcription must be considered. In this study, a cRNA-standard-based quantitative Real Time RT-PCR have been developed for HRV quantification on bronchoalveolar lavage (BAL) specimens. Results has been compared to a quantitative plasmid standard-based Real Time RT-PCR previously developed by us. Large amount of pHRV was linearized and purified. Blunt ends were generated and cRNA production was carried out. Dilutions of cRNA were generated and dynamic range, intra- and inter-test variability, sensitivity, and limit of detection were evaluated. Sixty-seven BAL, previously resulted positive to our plasmid standard-based method, were evaluated using cRNA-standard quantification. cRNA curve showed a broad dynamic range with a good intra- and inter-test variability, with an average of 3.23 threshold cycles more in comparison to plasmid standard-based curve. In terms of specimen quantification, a difference of 1.07 log was found, showing a significant underrate using plasmid standard-based quantification. The method for cRNA-standard construction seems more suitable for quantification of RNA viruses, in order to normalize the quantification in reverse-transcription.

Quantitative detection of the new polyomaviruses KI, WU and Merkel cell virus in transbronchial biopsies from lung transplant recipients.

BACKGROUND: Recently, three new polyomaviruses-KI, WU and Merkel cell (MCV)-have been discovered and their detection has been reported in different types of specimens, including respiratory samples, suggesting their shedding in the airways. In lung graft recipients, viral agents are associated with events that may limit the success of transplantation, including organ infection/disease and allograft rejection. AIMS: To evaluate the prevalence of KI, WU and MCV in transbronchial biopsies from lung transplant recipients and investigate the association with clinical and histopathological features. METHODS: The quantitation of new polyomaviruses DNA by real-time PCR and association with clinical and histopathological findings were evaluated in 66 transbronchial biopsies from lung transplant recipients. Results KI, WU and MCV were detected in 9.2%, 12.3% and 33.8% of specimens, respectively; with mean viral load ranging from 81 copies/10(4) cells for WU to 258 for MCV, thus not differing from that previously reported in native lungs. No significant association with clinical and histopathological findings (including acute respiratory insufficiency, interstitial and organising pneumonia, acute and chronic rejection) was found. CONCLUSIONS: Results showed a relatively high frequency of detection of the novel polyomaviruses in transbronchial biopsies from lung transplant recipients. It is likely that this accounted for the positive results found in some cases with different pathological background, although no significant association with a specific clinical and/or histopathological pattern was found.

Non-organ-specific and anti-endothelial antibodies in relation to CMV infection and acute rejection in renal transplant recipients.

The presence of non-organ-specific (NOSA) and anti-endothelial antibodies (AECAs) and the onset of rejection in relation to cytomegalovirus (CMV) infection was investigated in 96 renal transplant recipients: 48 CMV pp65-antigenemia-negative (group 1) and 48 positive (group 2). The presence of autoantibodies (autoAbs) was evaluated before and following renal transplantation (first three months) by indirect immunofluorescence. Before transplantation, none of the patients was positive to AECAs, while eight (8.3%) were positive to NOSAs. Post-transplantation, AECA were found in none of patients from group 1 vs. 15/48 (31.2%) from group 2 (p<0.05); NOSAs were detected in 9/48 (18.8%) and 9/48 patients from group 1 and 2, respectively. An acute rejection was diagnosed in ten cases: six of interstitial type (antigenemia-, and AECA-negative; two NOSA-positive); four of vascular type (all of them NOSA-negative, 3/4 antigenemia-, and AECA-positive). CMV infection did not seem to be significantly associated with the appearance of NOSAs, while there was a significant correlation with the occurrence of AECAs. No significant correlation was found between acute rejection and the occurrence of NOSAs, while 75% of the cases of vascular rejection was associated to CMV infection and AECA-positivity, suggesting the pathogenic role of CMV-mediated endothelial damage.

Detection of human herpesvirus-7 DNA in bronchoalveolar lavage.

OBJECTIVES: Human herpesvirus-7 (HHV-7) is a highly seroprevalent virus that, following primary infection, establishes latency or persistence in some tissues, including lung. The aim of this study was to investigate the prevalence of HHV-7 in the lower respiratory tract of hospitalized adult patients. METHODS: The prevalence of HHV-7 DNA was determined by quantitative real-time PCR in 212 bronchoalveolar lavage (BAL) samples obtained from 153 patients. The molecular epidemiology and clinical role of HHV-7 were evaluated. RESULTS: HHV-7 DNA was positive in 44 of 212 specimens (20.7%), obtained from 40 of 153 patients (26.1%), in particular 22/68 (32.35%) and 18/86 (20.9%) in transplant and non-transplant patients, respectively (1 patient evaluated both before and after transplantation). No significant difference according to transplant condition or discharge diagnosis was found. Viral load was >100,000 genome equivalents/ml BAL in 6/22 (27.3%) transplant recipients and 4/18 (22.2%) non-transplant patients (p = n.s.). CONCLUSIONS: The evaluation of HHV-7 DNA in BAL may be useful to investigate its potential role in lower respiratory tract infection, alone or in association with other viral and/or non-viral pathogens and to distinguish latency from reactivation.

Polyomavirus-associated nephropathy: critical issues in virological monitoring.

Polyomavirus-associated nephropathy is one of the most common viral complications in renal transplant recipients. Viral replication is the single common feature of all patients at risk of nephropathy. Therefore, screening for virus replication allows for the identification of patients at risk of developing nephropathy and permits earlier intervention, in particular a pre-emptive reduction of immunosuppression, with improvement of outcome. This paper describes the modes of virological monitoring and its role in the clinical management of renal transplant patients.

Real time PCR TaqMan assays for detection of polyomaviruses KIV and WUV in clinical samples.

Recently, polyomaviruses KI and WU were identified in the airways of patients with acute respiratory symptoms. The epidemiology and pathogenesis of these two viruses are not fully understood, and the development of molecular assays, such as Real Time PCR, was useful for examining their biology and role in different clinical syndromes. The evaluation of different target regions for the amplification of polyomaviruses KI and WU, comparing published primer/probe sets and sets designed in the laboratory is described and was used for testing 175 clinical specimens (84 stools and 91 tonsils). The results showed that the laboratory designs were more sensitive for the detection of polyomaviruses KI and WU DNA in clinical samples. The choice of the primer/probe set, and primarily of the region for amplification, may be relevant for understanding the pathogenic role of viruses such as polyomaviruses KI and WU.

Detection of parvovirus B19 in the lower respiratory tract.

BACKGROUND: Human parvovirus B19 infection generally displays a self-limiting course followed by viral clearance; although, in some cases, persistent infection may occur. Few cases of severe pulmonary disease following primary infection in both immunocompetent and immunocompromised patients were reported. OBJECTIVES: To investigate the prevalence and clinical impact of parvovirus B19 in the lower respiratory tract. STUDY DESIGN: The prevalence of parvovirus B19-DNA was evaluated by Real-Time PCR in 264 bronchoalveolar lavages (BAL) from 189 adult patients over a full-year period and related to demographic characteristics, underlying pathologies, immune status, admission to intensive care unit, mortality within 28 days, and discharge diagnosis. RESULTS: Parvovirus B19-DNA was detected in 7/189 (3.7%) patients, without significant association to demographic characteristics, immune status, transplant versus non-transplant status, admission to intensive care unit, presence of haematological conditions. In two lung transplant recipients surveillance specimens were positive to B19. Four of the remaining five patients presented respiratory insufficiency. A significant association to mortality was found, as 3/7 (42.9%) positive patients died within 28 days. No patient presented serological evidence of recent or acute infection and viremia. CONCLUSIONS: Parvovirus B19 may be detected at low frequency in BAL specimens from patients with different pathological backgrounds. This finding could be due to chronic infection with virus persistence in the lower respiratory tract, also in the absence of symptoms unequivocally attributable to B19. The high rate of mortality warrants the need for further studies to evaluate the opportunity to consider parvovirus B19 in the diagnostic work-up of lower respiratory tract infections.

Detection of PARV4, genotypes 1 and 2, in healthy and pathological clinical specimens.

The molecular epidemiology and tissue distribution of Human Parvovirus 4 (PARV4) and its variant PARV5 (Parvoviridae family) are poorly known. The aim of this study was to investigate the epidemiological role and prevalence of PARV4/5 by a nested-PCR on different clinical specimens, including blood samples from healthy donors, healthy and pathological skin samples, and bronchoalveolar lavages (BAL). Among blood specimens, 2/53 were positive; 3/37 and 23/105 of healthy and pathological skin specimens resulted positive, respectively, whereas no BAL was positive. PARV4/5 may be present in different healthy and pathological samples, suggesting the need for further investigating its tissue distribution.

Quantitative RT real time PCR and indirect immunofluorescence for the detection of human parainfluenza virus 1, 2, 3.

Human parainfluenza viruses (HPIVs) are distributed worldwide and are involved mainly in the pathogenesis of respiratory tract infections. The development and optimization of three quantitative reverse transcription real time polymerase chain reactions (RT Real Time Qt-PCRs) and an indirect immunofluorescence (IFA) for the detection and quantitation of HPIV-1, -2 and -3 in clinical samples are described. Efficiency, sensitivity, specificity, inter- and intra-assay variability and turnaround time of the two methods were compared. These assays have been validated on 131 bronchoalveolar lavage specimens. Based on the results obtained, the molecular methods represent a valid and rapid tool for clinical management and should be included in diagnostic panels aimed to evaluate suspected respiratory tract infections.

Development of a LUX real-time PCR for the detection and quantification of human herpesvirus 7.

Human herpesvirus 7 is a highly seroprevalent beta-herpesvirus that, following primary infection, remains latent in CD4+ T cells and determines a persistent rather than a latent infection in various tissues and organs, including the lung and skin. This paper describes the development of an in-house light upon extension real-time PCR assay for quantification of human herpesvirus 7 DNA in clinical samples. The efficiency, sensitivity, specificity, inter- and intra-assay variability, and dynamic range have been determined. Subsequently, the assay has been validated by evaluating the human herpesvirus 7 load in bronchoalveolar lavages and skin specimens, chosen as 2 persistency sites, from healthy and pathological individuals. The real-time PCR assay developed in this study could be a useful tool to detect and quantify human herpesvirus 7 DNA in different clinical specimens to elucidate its epidemiological and pathogenic roles.

What role for human rhinoviruses in the lower respiratory tract?

Human rhinoviruses (HRV) usually cause upper airway infections. However, viral replication in the tracheobronchial tree has been disclosed, although its clinical role is poorly known. We evaluated the prevalence of HRV in 159 bronchoalveolar lavages from 88 patients and describe a lung transplant recipient with a high HRV load in association with acute rejection. HRV was detected in 22/88 patients (25.0%): 7/18 lung transplant recipients, 11/41 immunocompetent, and 4/29 immunocompromised (p = n.s.). No lung disease was significantly associated with HRV positivity. It should be recommended to include HRV in the virological diagnostic work-up of lower respiratory specimens to elucidate their role.