Characterization of three novel β-defensin antimicrobial peptides in rainbow trout (Oncorhynchus mykiss)

An initial bioinformatics investigation followed by cloning and sequencing analysis, has led to the identification of three novel members (omDB-2, omDB-3, omBD-4) of the β-defensin family in rainbow trout (Oncorhynchus mykiss). The contiguous sequences could be translated to give predicted peptides of 62 (omDB-2), 63 (omDB-3) and 68 (omDB-4) amino acids (aa) in length, with mature peptides of 43 (omDB-2), 39 (omDB-3) and 42 (omDB-4) aa, with no obvious proregion present. Analysis of the gene organization found that all three new genes contained three exons divided by two introns, as seen in defensin genes of other fish species. Constitutive expression of all the trout defensins was detected by RT-PCR in a wide range of mucosal and systemic tissues from healthy fish, with omDB-3 and omDB-4 showing the highest expression levels. Following bacterial challenge in vivo, the defensin genes were induced at the three mucosal sites examined (skin, gill, gut), with levels of omDB-2 and omDB-3 increased some 16-fold in gut and gill respectively. Using polyinosinic polycytosinic RNA (polyI:C) as a viral mimic, all of the four trout β-defensin genes were induced in head kidney primary leucocyte cultures at 4 h post-stimulation, with omDB-1 and omDB-3 particularly highly expressed. These data suggest that β-defensins are likely an important component of the innate defences of fish, and reveal an added level of antimicrobial peptide complexity in fish to that known previously.     

Cutaneous distribution of plasmacytoid dendritic cells in lupus erythematosus. Selective tropism at the site of epithelial apoptotic damage

Recent evidences suggest a significant role of Plasmacytoid dendritic cells (PDC) role in the pathogenesis of lupus erythematosus (LE) via production of type I IFN. Taking advantage on the availability of multiple reagents (CD123, BDCA2, and CD2ap) specifically recognizing PDC on fixed tissues, we investigated the occurrence of PDC in a cohort of 74 LE patients. The large majority of LE biopsies (67/74; 90.5%) showed cutaneous infiltration of PDC. PDC were more frequently observed (96.4 vs 72.2) and numerous in cutaneous LE compared to systemic LE (SLE) and correlated with the density of the inflammatory infiltrate (r=0.40; p<0.001). PDC reduction in SLE might be related to a broader tissue distribution of this cellular population, as indicated by their occurrence in kidneys in 11 out of 24 (45.8%) cases studied. The distribution of cutaneous PDC showed two distinct patterns. More commonly, PDC were observed within perivascular inflammatory nodules in the dermis, associated with CD208+ mature DC and T-bet+ cells [D-PDC]. A second component was observed along the dermal-epithelial junction [J-PDC], in association with cytotoxic T-cells in areas of severe epithelial damage. Notably, chemerin reactivity was observed in 64% of LE biopsies on endothelial cells and in the granular layer keratinocytes. Cutaneous PDC in LE strongly produced type I IFN, as indicated by the diffuse MxA expression, and the cytotoxic molecule granzyme B.This study confirms cutaneous PDC infiltration as hallmark of LE. The topographical segregation in D-PDC and J-PDC suggests a novel view of the role of these cells in skin autoimmunity.     

Evaluation of an Array-Based Method for Human Papillomavirus Detection and Genotyping in Comparison with Conventional Methods Used in Cervical Cancer Screening

Cervical cancer is the second-most prevalent cancer in young women around the world. Infection with human papillomavirus (HPV), especially high-risk HPV types (HR-HPV), is necessary for the development of this cancer. HPV-DNA detection is increasingly being used in cervical cancer screening programs, together with the Papanicolau smear test. We evaluated the usefulness of introducing this new array-based HPV genotyping method (i.e., Clinical Arrays Papillomavirus Humano) in the cervical cancer screening algorithm in our center. The results obtained using this method were compared to those obtained by the hybrid capture II high-risk HPV DNA test (HC-II) and Papanicolau in a selected group of 408 women. The array-based assay was performed in women that were HC-II positive or presented cytological alterations. Among 246 array-positive patients, 123 (50%) presented infection with ≥2 types, and HR-HPV types were detected in 206 (83.7%), mainly HPV-16 (24.0%). Up to 132 (33.2%) specimens were classified as ASCUS (for atypical squamous cells of undetermined significance), and only 48 (36.4%) of them were HPV-DNA positive by either assay; however, 78.7% of these cases were caused by HR-HPV types. The agreement between both HPV-DNA detection techniques was fairly good (n = 367). Screening with Papanicolau smear and HC-II tests, followed by HPV detection and genotyping, provided an optimal identification of women at risk for the development of cervical cancer. Furthermore, with the identification of specific genotypes, either in single or multiple infections, a better prediction of disease progression was achieved. The array method also made allowed us to determine the possible contribution of the available vaccines in our setting.Cervical cancer is the second most prevalent type of cancer in women worldwide. A total of 500,000 new cases are diagnosed each year and cause more than 270,000 deaths (15). Since the 1940s, screening programs for cervical cancer prevention, mainly based on the Papanicolau smear test, have been implemented in resource-rich countries, resulting in a remarkable decrease in its incidence and related mortality (16). However, this test has a limited sensitivity, especially for detecting precancerous lesions (1, 6).Genital human papillomavirus (HPV) is a highly common sexually transmitted infection. Although most HPV infections are transient and asymptomatic, epidemiological studies worldwide have demonstrated that persistent infection with certain genotypes is the necessary cause for the development of cervical cancer and its precursor lesions (3, 19, 24). More than 100 HPV types have been described and classified into high-risk types (HR-HPV) and low-risk types (LR-HPV) according to the probability of developing cervical cancer (14). Therefore, in addition to the Papanicolau smear test, HPV detection assays have been implemented in many countries to improve cervical cancer screening. These assays have a higher sensitivity than the Papanicolau smear test for the detection of women at risk of developing precancerous lesions (12).Since HPV cannot be grown in conventional cell cultures and serological assays are unreliable, molecular techniques constitute the best choice to diagnose HPV infection. Currently, the only assay that has been approved by the U.S. Food and Drug Administration for the detection of HPV-DNA is the Hybrid Capture II system (HC-II; Digene Corp., Gaithersburg, MD). This signal amplification assay was designed to detect LR-HPV and HR-HPV genotypes in two different kits but does not provide genotype information.The interest of HPV genotyping has increased in light of the recently licensed HPV bivalent and tetravalent vaccines (9, 23). Genotyping also allows clinicians to monitor patients according to the oncogenic risk of the HPV types identified. Several genotyping assays have been developed over the last years with a variety of amplification and detection strategies (reviewed in references 4 and 13). Methods based on consensus PCR and reverse hybridization of PCR products provide high sensitivity and extensive typing information, including identification of multiple infections. Recently, an assay based on amplification and array hybridization has been commercialized for the detection and genotyping of HPV in routine clinical specimens (Clinical Arrays Papillomavirus Humano [CAPH]; Genomica S.A.U., Madrid, Spain). This assay provides the possibility to detect simple or mixed-type infections with 35 HPV types (20 HR-HPV and 15 LR-HPV).The aim of the present study was to assess the usefulness of introducing this new array-based HPV detection and genotyping method in the cervical cancer screening algorithm in our center, a reference hospital with 600,000-habitant coverage. With this goal, we compared the results obtained using this method with those obtained by HC-II and the cytology findings.     

Clinical, laboratory, diagnostic and prognostic aspects of canine lymphoma: a retrospective study

During the period of 8 years, 120 dogs affected by lymphoma were referred to the veterinary teaching hospital of the University. Canine lymphoma was classified and staged using a standardised diagnostic approach that involved the acquisition of detailed clinical history, physical examination and extended laboratory workup including lymph node cytology. Additionally, immunophenotyping was available in 22 cases. Multicentric lymphoma was the most common form of disease identified clinically, while the cytological classification revealed the B-cell polymorphic centroblastic subtype as the most frequent type of lymphoma in the study. Furthermore, the influence of several prognostic factors in relation to remission rate, relapsing rate and survival time were evaluated for 16 patients treated with a specific chemotherapy protocol and for which a complete follow-up was available. The results of our study suggest that chronic inflammation may represent a negative prognostic factor shortening both the relapsing and the survival times, while pre-treatment with steroids may have a negative effect on the survival time.     

Differential Interferon Responses Enhance Viral Epitope Generation by Myocardial Immunoproteasomes in Murine Enterovirus Myocarditis

Murine models of coxsackievirus B3 (CVB3)-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection, with host-specific outcomes ranging from complete recovery in resistant mice to chronic disease in susceptible hosts. To identify susceptibility factors that modulate the course of viral myocarditis, we show that type-I interferon (IFN) responses are considerably impaired in acute CVB3-induced myocarditis in susceptible mice, which have been linked to immunoproteasome (IP) formation. Here we report that in concurrence with distinctive type-I IFN kinetics, myocardial IP formation peaked early after infection in resistant mice and was postponed with maximum IP expression concomitant to massive inflammation and predominant type-II IFN responses in susceptible mice. IP activity is linked to a strong enhancement of antigenic viral peptide presentation. To investigate the impact of myocardial IPs in CVB3-induced myocarditis, we identified two novel CVB3 T cell epitopes, virus capsid protein 2 [285-293] and polymerase 3D [2170-2177]. Analysis of myocardial IPs in CVB3-induced myocarditis revealed that myocardial IP expression resulted in efficient epitope generation. As opposed to the susceptible host, myocardial IP expression at early stages of disease corresponded to enhanced CVB3 epitope generation in the hearts of resistant mice. We propose that this process may precondition the infected heart for adaptive immune responses. In conclusion, type-I IFN-induced myocardial IP activity at early stages coincides with less severe disease manifestation in CVB3-induced myocarditis.Myocarditis is often induced by cardiotropic viruses: in about 20% of patients, viral myocarditis leads to its sequela dilated cardiomyopathy, which is linked to chronic inflammation and persistence of cardiotropic viruses.^1,2,3,4 Dilated cardiomyopathy is the most common cause of heart failure in young patients and appears to be a major cause of sudden unexpected death in this cohort. Enteroviruses, including group-B coxsackieviruses, have been linked to the development of myocarditis and dilated cardiomyopathy associated with adverse prognosis.^5,6 Well-established murine models of coxsackievirus B3 (CVB3) myocarditis mimic the human disease progress and are valuable in delineating the underlying mechanisms that determine the divergent courses of myocarditis^7,8,9,10: resistant C57BL/6 mice eliminate the virus following mild acute myocarditis; no chronic inflammation is detected. In contrast, major histocompatibility complex (MHC)-matched A.BY/SnJ mice develop severe acute infection and ongoing chronic myocarditis, thus conferring susceptibility to chronic disease.^7,9Host responses to viral infection trigger the release of interferons (IFNs). IFNs of the α/β subtype are assigned to type I IFNs, whereas IFN-γ is the only type II IFN. IFNs exert numerous antiviral effects in innate and adaptive immunity.¹¹ Although type I IFN-receptor-deficiency was not associated with a dramatic effect on early viral replication in the heart, type I IFN signaling was found to be essential for the prevention of early death due to CVB3-infection.¹² The extraordinary impact of type I IFNs was substantiated in a recent study illustrating acute fulminant infection and chronic disease progression in IFN-β deficient mice.¹³ Deletion of type II IFN receptors was not associated with enhanced mortality in CVB3-infection.¹² IFN-γ responses were shown to be protective in cellular immunity in CVB3-infection.⁹ In addition, expression of IFN-γ conferred protection in enterovirus myocarditis, which may be linked to the activation of nitric oxide-mediated antiviral activity of macrophages.^14,15 Thus, both type I and type II IFN are active in CVB3- myocarditis.One downstream effect of IFN signaling is the induction of immunoproteasome (IP) formation in the target organ of the immune response. Particularly IFN-γ was shown to induce IP expression.^16,17,18 Efficient generation of viral epitopes that stimulate CD8⁺ T cells strongly relies on host-cell IP and, in addition, protein degradation by proteasomes is also essential in the regulation of inflammatory and stress responses, cell cyclus, and apoptosis control.¹⁹ The 20S proteasome as the catalytic core of the proteasome resembles a cylinder-shaped structure of stacked heptameric rings formed by either α or β subunits. The proteolytic function of the so-called standard proteasome is restricted to the β1, β2, and β5 subunit.²⁰ Three alternative catalytic subunits, the so-called immunosubunits β1i, β2i, and β5i, which are incorporated into 20S proteasomes, thus forming IP with altered catalytic characteristics, are expressed on cytokine stimulation.^21,22 It is highly notable that IP activity is linked to a strong enhancement of antigenic viral peptide presentation.^{23,24,25,26,27}Cardiac proteasomes contribute to the modulation of cardiac function in health and disease.²⁸ However, apart from the reported observation that IPs are expressed in the myocardium in acute CVB3 myocarditis, their functional impact has not been studied so far.¹⁰ The present study focuses on IFN-induced myocardial IP activity in CVB3 myocarditis.     

Episodic memory for spatial context biases spatial attention

The study explores the bottom-up attentional consequences of episodic memory retrieval. Individuals studied words (Experiment 1) or pictures (Experiment 2) presented on the left or on the right of the screen. They then viewed studied and new stimuli in the centre of the screen. One-second after the appearance of each stimulus, participants had to respond to a dot presented on the left or on the right of the screen. The dot could follow a stimulus that had been presented, during the study phase, on the same side as the dot (congruent condition), a stimulus that had been presented on the opposite side (incongruent condition), or a new stimulus (neutral condition). Subjects were faster to respond to the dot in the congruent compared to the incongruent condition, with an overall right visual field advantage in Experiment 1. The memory-driven facilitation effect correlated with subjects’ re-experiencing of the encoding context (R responses; Experiment 1), but not with their explicit memory for the side of items’ presentation (source memory; Experiment 2). The results indicate that memory contents are attended automatically and can bias the deployment of attention. The degree to which memory and attention interact appears related to subjective but not objective indicators of memory strength.     

Leptin increases growth of primary ossification centers in fetal mice

The effect of peripheral leptin on fetal primary ossification centers during the early phases of bone histogenesis was investigated by administration of leptin to pregnant mice. Fourteen pregnant mice were divided into two groups. The treated pregnant group was subcutaneously injected in the intrascapular region with supraphysiologic doses (2 mg kg⁻¹) of leptin (Vinci Biochem, Firenze, Italy) in a volume of 0.1 mL per 10 g body weight, at the 7th, 9th and 11th day of gestation. The control group was treated with physiological solution in the same manner and same times as the treated group. The new-born mice were killed 1 day after birth and the primary ossification centers were stained with Alizarin Red S after diaphanizing the soft tissues in 1% potassium hydroxide. The development of both endochondral and intramembranous ossification centers was morphometrically analysed in long bones. The results showed that the ossification centers of mice born by mothers treated with leptin grow more rapidly in both length and cross-sectional area compared with mice born by the untreated mothers. As the development of long bones depends on endochondral ossification occurring at proximal and distal epiphyseal plates as well as on intramembranous ossification along the periosteal surface, it appears that leptin activates the differentiation and proliferation of both chondrocytes and osteoblasts. The role of leptin as a growth factor of cartilage and bone is discussed in the light of the data reported in the literature.     

Sublingual zolpidem is more effective than oral zolpidem in initiating early onset of sleep in the post-nap model of transient insomnia: a polysomnographic study

ObjectiveOX22 is zolpidem formulated for sublingual administration. The primary objective of the present study was to evaluate the efficacy of single doses of sublingual zolpidem (5 and 10 mg) versus oral zolpidem (10 mg), with regard to latency to persistent sleep (LPS), in a post-nap model of insomnia.MethodsTwenty-one healthy volunteers included in this study were recorded by polysomnography during 2 consecutive nights and, on the day in between, during a 2 h nap. Eighteen out of these 21 subjects were finally analyzed. Treatment was randomly administered before the second recording night to subjects demonstrating at least 30 min of sleep during the nap recording.ResultsContrast analyses show that 10 mg OX22 significantly shortened LPS compared to oral zolpidem administration of 10 mg (12.8 ± 9.9 and 18.4 ± 11.3 min, respectively; p < .05). No treatment effects could be evidenced on total sleep time, time awake after sleep onset and sleep architecture parameters for OX22 compared to oral zolpidem. All treatments were well tolerated and did not induce next-day residual effects.ConclusionThe present results show that OX22, a sublingual formulation of zolpidem, has a significant earlier sleep initiation as compared to an equivalent dose of oral zolpidem in healthy volunteers in a post-nap model of insomnia.