Trasmissione post-natale del citomegalovirus attraverso il latte materno al neonato VLBW

Introduction Postnatal human cytomegalovirus (CMV) infection is usually asymptomatic in term babies, while preterm infants are more susceptible to symptomatic CMV infection. Breastfeeding plays a dominant role in the epidemiology of transmission of postnatal CMV infection, but the risk factors of symptomatic CMV infection in preterm infants are unknown. Patients and Methods Between December 2003 and August 2006, eighty Very Low Birth Weight (VLBW) preterm infants (gestational age ≤ 32 weeks and birth weight < 1500 g), admitted to the Neonatal Intensive Care Unit of St Orsola-Malpighi General Hospital, Bologna were recruited. All of them were breastfed for at least one month. During the first week of life, serological test for CMV was performed on maternal blood. Furthermore, urinary CMV culture was performed in all the infants in order to exclude a congenital CMV infection. Urine samples from each infant were collected and processed for CMV culture once a week. Once every 15 days a blood sample was taken from each infant to evaluate the complete blood count, the hepatic function and the C reactive protein. In addition, samples of fresh breast milk were processed weekly for CMV culture. A genetic analysis of virus variant was performed in the urine of the infected infants and in their mother’s milk to confirm the origin of infection. Results We evaluated 80 VLBW infants and their 68 mothers. Fifty-three mothers (78%) were positive for CMV IgG antibodies, and 15 (22%) were seronegative. In the seronegative group, CMV was never isolated in breast milk, and none of the 18 infants developed viruria; in the seropositive group, CMV was isolated in 21 out of 53 (40%) mother’s milk. CMV was detected in the urine samples of 9 out of 26 (35%) preterm infants, who were born from 21 virolactia positive mothers. Six of these infants had clinically asymptomatic CMV infection, while 3 showed a sepsis-like illness with bradycardia, tachypnea and repeated desaturations. Eight out of nine infants showed abnormal hematologic values. The detection of neutropenia was strictly related to CMV infection (8/9 infected infants vs 17/53 non infected infants, P<.005), such as the detection of an increase in conjugated bilirubin (3/9 infected infants vs 2/53 non infected infants, P<.05). The degree of neutropenia was not different between the two groups (infected/non infected). The use of hemoderivatives (plasma and/or IgM–enriched immunoglobulin) in order to treat a suspected/certain infection in newborn with GE< 28 ws was seen as protective against CMV infection (1/4 infected infants vs 18/20 non infected infants [GE<28 ws]; P<.05). Furthermore, bronchopulmonary dysplasia (defined both as oxygen-dependency at 30 days of life and 36 ws of postmenstrual age) correlated with symptomatic infection (3/3 symptomatic vs 0/6 asymptomatic: P<.05). Conclusion Our data suggest that CMV infection transmitted to preterm newborn through human milk is always asymptomatic when newborns are clinically stable. Otherwise, the infection can worsen a preexisting disease such as bronchopulmonary dysplasia. Human milk offers many nutritional and psychological advantages to preterm newborns: according to our data, there’s no reason to contraindicate it neither to pasteurize the milk of all the mothers of preterm infants who are CMV seropositive.

Studien zur physiologischen Funktion löslicher Zytokinrezeptoren und zur Wirkungsweise viral kodierter Zytokine

Interleukin-6 (IL-6) aktiviert Zielzellen durch Bindung an den Interleukin-6-Rezeptor (IL-6R) und anschließende Homodimerisierung von gp130. IL-6 alleine kann nur Zellen aktivieren, die IL-6R exprimieren, der Komplex aus IL-6 und löslichem IL-6R (sIL-6R) kann gp130 auf Zellen aktivieren, die keinen IL-6R exprimieren. Von gp130 gibt es eine lösliche Form (sgp130), die in Komplexen mit sIL-6R und IL-6 vorliegen kann.Es wurden rekombinante Versionen von sgp130 konstruiert, exprimiert und aufgereinigt. Die sgp130 Proteine inhibieren die sIL-6R-abhängige Stimulation von Zellen, nicht jedoch über membrangebundenen IL-6R vermittelte IL-6-Aktivitäten. sgp130 inhibiert also selektiv sIL-6R-abhängige Antworten und hat keinen Einfluß auf IL-6-Antworten über membrangebundenen IL-6R.Das Genom von Humanem Herpesvirus-8 kodiert für ein virales IL-6 (vIL-6). Um zu klären, ob vIL-6 direkt an IL-6R oder gp130 bindet, wurden Immunpräzipitationen mit radioaktiv markiertem vIL-6 durchgeführt. Dabei zeigte vIL-6 eine direkte Interaktion mit gp130, nicht jedoch mit IL-6R.Die biologische Aktivität von vIL-6 ist IL-6R-unabhängig. Es gibt keinen Unterschied in der Effektivität von vIL-6 bei der Stimulation von Zellen die nur gp130 oder gp130 und IL-6R exprimieren. Die Ergebnisse demonstrieren, daß vIL-6 das erste bekannte Zytokin ist, welches direkt gp130 binden und aktivieren kann.

Trascriptome analysis and functional genomics of Neisseria meningitidis in human blood

Neisseria meningitidis (Nm) is the major cause of septicemia and meningococcal meningitis. During the course of infection, it must adapt to different host environments as a crucial factor for survival. Despite the severity of meningococcal sepsis, little is known about how Nm adapts to permit survival and growth in human blood. A previous time-course transcriptome analysis, using an ex vivo model of human whole blood infection, showed that Nm alters the expression of nearly 30% of ORFs of the genome: major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. Starting from these data, mutagenesis studies of a subset of up-regulated genes were performed and the mutants were tested for the ability to survive in human whole blood; Nm mutant strains lacking the genes encoding NMB1483, NalP, Mip, NspA, Fur, TbpB, and LctP were sensitive to killing by human blood. Then, the analysis was extended to the whole Nm transcriptome in human blood, using a customized 60-mer oligonucleotide tiling microarray. The application of specifically developed software combined with this new tiling array allowed the identification of different types of regulated transcripts: small intergenic RNAs, antisense RNAs, 5’ and 3’ untranslated regions and operons. The expression of these RNA molecules was confirmed by 5’-3’RACE protocol and specific RT-PCR. Here we describe the complete transcriptome of Nm during incubation in human blood; we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. In addition the tiling array analysis demonstrated that Nm expresses a set of new transcripts, not previously identified, and suggests the presence of a circuit of regulatory RNA elements used by Nm to adapt to proliferate in human blood.

Cathepsin D primes caspase-8 activation by multiple intra-chain proteolysis

During the resolution of inflammatory responses, neutrophils rapidly undergo apoptosis. A direct and fast activation of caspase-8 by cathepsin D was shown to be crucial in the initial steps of neutrophil apoptosis. Nevertheless, the activation mechanism of caspase-8 remains unclear. Here, by using site-specific mutants of caspase-8, we show that both cathepsin D-mediated proteolysis and homodimerization of caspase-8 are necessary to generate an active caspase-8. At acidic pH, cathepsin D specifically cleaved caspase-8 but not the initiator caspase-9 or -10 and significantly increased caspase-8 activity in dimerizing conditions. These events were completely abolished by pepstatin A, a pharmacological inhibitor of cathepsin D. The cathepsin D intra-chain proteolysis greatly stabilized the active site of caspase-8. Moreover, the main caspase-8 fragment generated by cathepsin D cleavage could be affinity-labeled with the active site probe biotin-VAD-fluoromethyl ketone, suggesting that this fragment is enzymatically active. Importantly, in an in vitro cell-free assay, the addition of recombinant human caspase-8 protein, pre-cleaved by cathepsin D, was followed by caspase-3 activation. Our data therefore indicate that cathepsin D is able to initiate the caspase cascade by direct activation of caspase-8. As cathepsin D is ubiquitously expressed, this may represent a general mechanism to induce apoptosis in a variety of immune and nonimmune cells.

Virological and pathological findings in Bluetongue virus serotype 8 infected sheep

Twenty-seven sheep of the four most common Swiss breeds and the English breed Poll Dorset were experimentally infected with a northern European field strain of bluetongue virus serotype 8 (BTV-8). Animals of all breeds developed clinical signs, viremia and pathological lesions, demonstrating that BTV-8 is fully capable of replicating and inducing bluetongue disease (BT) in the investigated sheep. Necropsy performed between 10 and 16 days post-infectionem (d.p.i.) revealed BT-typical hemorrhages, effusions, edema, erosions and activation of lymphatic tissues. Hemorrhages on the base of the Arteria pulmonalis and the left Musculus papillaris subauricularis were frequently present. Histology confirmed the macroscopical findings. Using a score system, clinical manifestation and pathology were found to be significantly related. Furthermore, clinical signs and fever were shown to be indicative for the concurrent presence of high amounts of viral ribonucleic acid (RNA) in blood. Spleen, lung, lymph nodes and tonsils from all animals were analyzed regarding viral RNA loads and infectivity using real-time reverse transcriptase PCR (rRT-PCR) and virus isolation in cell culture, respectively. The highest amount of viral RNA was detected in spleen and lung and rRT-PCR revealed to be a more sensitive method for virus detection compared to virus isolation. A long-term follow-up was performed with three sheep showing that BTV-8 viral RNA in blood was present up to 133 d.p.i. and in certain tissues even on 151 d.p.i. No significant breed-related differences were observed concerning clinicopathological picture and viremia, and the Swiss sheep were as susceptible to BTV-8 infection as Poll Dorset sheep, demonstrating a remarkably high virulence of BTV-8 for indigenous sheep breeds.

Functional expression of CCR1, CCR3, CCR4, and CXCR4 chemokine receptors on human platelets

Platelets are known to contain platelet factor 4 and beta-thromboglobulin, alpha-chemokines containing the CXC motif, but recent studies extended the range to the beta-family characterized by the CC motif, including RANTES and Gro-alpha. There is also evidence for expression of chemokine receptors CCR4 and CXCR4 in platelets. This study shows that platelets have functional CCR1, CCR3, CCR4, and CXCR4 chemokine receptors. Polymerase chain reaction detected chemokine receptor messenger RNA in platelet RNA. CCR1, CCR3, and especially CCR4 gave strong signals; CXCR1 and CXCR4 were weakly positive. Flow cytometry with specific antibodies showed the presence of a clear signal for CXCR4 and weak signals for CCR1 and CCR3, whereas CXCR1, CXCR2, CXCR3, and CCR5 were all negative. Immunoprecipitation and Western blotting with polyclonal antibodies to cytoplasmic peptides clearly showed the presence of CCR1 and CCR4 in platelets in amounts comparable to monocytes and CCR4 transfected cells, respectively. Chemokines specific for these receptors, including monocyte chemotactic protein 1, macrophage inflammatory peptide 1alpha, eotaxin, RANTES, TARC, macrophage-derived chemokine, and stromal cell-derived factor 1, activate platelets to give Ca(++) signals, aggregation, and release of granule contents. Platelet aggregation was dependent on release of adenosine diphosphate (ADP) and its interaction with platelet ADP receptors. Part, but not all, of the Ca(++) signal was due to ADP release feeding back to its receptors. Platelet activation also involved heparan or chondroitin sulfate associated with the platelet surface and was inhibited by cleavage of these glycosaminoglycans or by heparin or low molecular weight heparin. These platelet receptors may be involved in inflammatory or allergic responses or in platelet activation in human immunodeficiency virus infection.

Type I IFN induction in response to Listeria monocytogenes in human macrophages: evidence for a differential activation of IFN regulatory factor 3 (IRF3)

Listeria monocytogenes is a prototypic bacterium for studying innate and adaptive cellular immunity as well as host defense. Using human monocyte-derived macrophages, we report that an infection with a wild-type strain, but not a listeriolysin O-deficient strain, of the Gram-positive bacterium L. monocytogenes induces expression of IFN-beta and a bioactive type I IFN response. Investigating the activation of signaling pathways in human macrophages after infection revealed that a wild-type strain and a hemolysin-deficient strain of L. monocytogenes activated the NF-kappaB pathway and induced a comparable TNF response. p38 MAPK and activating transcription factor 2 were phosphorylated following infection with either strain, and IFN-beta gene expression induced by wild-type L. monocytogenes was reduced when p38 was inhibited. However, neither IFN regulatory factor (IRF) 3 translocation to the nucleus nor posttranslational modifications and dimerizations were observed after L. monocytogenes infection. In contrast, vesicular stomatitis virus and LPS triggered IRF3 activation and signaling. When IRF3 was knocked down using small interfering RNA, a L. monocytogenes-induced IFN-beta response remained unaffected whereas a vesicular stomatitis virus-triggered response was reduced. Evidence against the possibility that IRF7 acts in place of IRF3 is provided. Thus, we show that wild-type L. monocytogenes induced an IFN-beta response in human macrophages and propose that this response involves p38 MAPK and activating transcription factor 2. Using various stimuli, we show that IRF3 is differentially activated during type I IFN responses in human macrophages.

Gene duplication: a drive for phenotypic diversity and cause of human disease

Gene duplication is one of the key factors driving genetic innovation, i.e., producing novel genetic variants. Although the contribution of whole-genome and segmental duplications to phenotypic diversity across species is widely appreciated, the phenotypic spectrum and potential pathogenicity of small-scale duplications in individual genomes are less well explored. This review discusses the nature of small-scale duplications and the phenotypes produced by such duplications. Phenotypic variation and disease phenotypes induced by duplications are more diverse and widespread than previously anticipated, and duplications are a major class of disease-related genomic variation. Pathogenic duplications particularly involve dosage-sensitive genes with both similar and dissimilar over- and underexpression phenotypes, and genes encoding proteins with a propensity to aggregate. Phenotypes related to human-specific copy number variation in genes regulating environmental responses and immunity are increasingly recognized. Small genomic duplications containing defense-related genes also contribute to complex common phenotypes.

Survey of bluetongue virus infection in free-ranging wild ruminants in Switzerland.

BACKGROUND In 2006, bluetongue virus serotype 8 (BTV-8) was detected for the first time in central Europe. Measures to control the infection in livestock were implemented in Switzerland but the question was raised whether free-ranging wildlife could be a maintenance host for BTV-8. Furthermore Toggenburg orbivirus (TOV), considered as a potential 25th BTV serotype, was detected in 2007 in domestic goats in Switzerland and wild ruminants were considered a potential source of infection. To assess prevalences of BTV-8 and TOV infections in wildlife, we conducted a serological and virological survey in red deer, roe deer, Alpine chamois and Alpine ibex between 2009 and 2011. Because samples originating from wildlife carcasses are often of poor quality, we also documented the influence of hemolysis on test results, and evaluated the usefulness of confirmatory tests. RESULTS Ten out of 1,898 animals (0.5%, 95% confidence interval 0.3-1.0%) had detectable antibodies against BTV-8 and BTV-8 RNA was found in two chamois and one roe deer (0.3%, 0.1-0.8%). Seroprevalence was highest among red deer, and the majority of positive wild animals were sampled close to areas where outbreaks had been reported in livestock. Most samples were hemolytic and the range of the optical density percentage values obtained in the screening test increased with increasing hemolysis. Confirmatory tests significantly increased specificity of the testing procedure and proved to be applicable even on poor quality samples. Nearly all samples confirmed as positive had an optical density percentage value greater than 50% in the ELISA screening. CONCLUSIONS Prevalence of BTV-8 infection was low, and none of the tested animals were positive for TOV. Currently, wild ruminants are apparently not a reservoir for these viruses in Switzerland. However, we report for the first time BTV-8 RNA in Alpine chamois. This animal was found at high altitude and far from a domestic outbreak, which suggests that the virus could spread into/through the Alps. Regarding testing procedures, hemolysis did not significantly affect test results but confirmatory tests proved to be necessary to obtain reliable prevalence estimates. The cut-off value recommended by the manufacturer for the screening test was applicable for wildlife samples.

Identification of Trypanosoma brucei components involved in trypanolysis by normal human serum

Normal human serum (NHS) confers human resistance to infection by the parasite Trypanosoma brucei owing to the trypanolytic activity of apolipoprotein L1 (APOL1), present in two serum complexes termed Trypanolytic Factors (TLF-1 and -2). In order to identify parasite components involved in the intracellular trafficking and activity of TLFs, an inducible RNA interference (RNAi) genomic DNA library constructed in bloodstream form T. brucei was subjected to RNAi induction and selection for resistant parasites under NHS conditions favouring either TLF-1 or TLF-2 uptake. While TLF-1 conditions readily selected the haptoglobin-haemoglobin (HP-HB) surface receptor TbHpHbR as expected, given its known ability to bind TLF-1, under TLF-2 conditions no specific receptor for TLF-2 was identified. Instead, the screen allowed the identification of five distinct factors expected to be involved in the assembly of the vacuolar proton pump V-ATPase and consecutive endosomal acidification. These data confirm that lowering the pH during endocytosis is required for APOL1 toxic activity.

Multibranched acquired periungual fibrokeratomas with confounding histopathologic findings resembling papillomavirus infection: a report of two cases

Acquired periungual fibrokeratomas are benign fibrous tissue tumors and are considered as the topographical variant of acquired digital fibrokeratoma. They usually present as solitary tumors. In some instance, the entity may appear in multibranched fashion. The main histopathologic features consist of acanthosis, thick collagen bundles mainly oriented in a vertical axis forming a central core, and numerous proliferating fibroblasts. In this article, we present two cases of acquired multibranched periungual fibrokeratoma and depict their varying clinical features over time. Binucleation and perinuclear halos of keratinocytes mimicking human papillomavirus (HPV) infection were detected microscopically, but there was no reactivity with HPV immunostaining. In context, anti-HPV immunostaining may be helpful in the differentiation of fibrokeratomas from HPV infection. On the other hand, it should be kept in mind that these histopathologic findings may be found in acral biopsies independent of viral effects.

A viral gene that activates lytic cycle expression of Kaposi’s sarcoma-associated herpesvirus

Herpesviruses exist in two states, latency and a lytic productive cycle. Here we identify an immediate-early gene encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus eight (HHV8) that activates lytic cycle gene expression from the latent viral genome. The gene is a homologue of Rta, a transcriptional activator encoded by Epstein–Barr virus (EBV). KSHV/Rta activated KSHV early lytic genes, including virus-encoded interleukin 6 and polyadenylated nuclear RNA, and a late gene, small viral capsid antigen. In cells dually infected with Epstein–Barr virus and KSHV, each Rta activated only autologous lytic cycle genes. Expression of viral cytokines under control of the KSHV/Rta gene is likely to contribute to the pathogenesis of KSHV-associated diseases.

CD147 facilitates HIV-1 infection by interacting with virus-associated cyclophilin A

Cyclophilin A (CyPA) is specifically incorporated into the virions of HIV-1 and has been shown to enhance significantly an early step of cellular HIV-1 infection. Our preliminary studies implicated CD147 as a receptor for extracellular CyPA. Here, we demonstrate a role for CyPA–CD147 interaction during the early steps of HIV-1 infection. Expression of human CD147 increased infection by HIV-1 under one-cycle conditions. However, susceptibility to infection by viruses lacking CyPA (simian immunodeficiency virus or HIV-1 produced in the presence of cyclosporin A) was unaffected by CD147. Virus-associated CyPA coimmunoprecipitated with CD147 from infected cells. Antibody to CD147 inhibited HIV-1 entry as evidenced by the delay in translocation of the HIV-1 core proteins from the membrane and inhibition of viral reverse transcription. Viruses whose replication did not require CyPA (SIV or mutant HIV-1) were resistant to the inhibitory effect of anti-CD147 antibody. These results suggest that HIV-1 entry depends on an interaction between virus-associated CyPA and CD147 on a target cell.

Global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein B

Human cytomegalovirus (HCMV) infection alters the expression of many cellular genes, including IFN-stimulated genes (ISGs) [Zhu, H., Cong, J.-P., Mamtora, G., Gingeras, T. & Shenk, T. (1998) Proc. Natl. Acad. Sci. USA 95, 14470–14475]. By using high-density cDNA microarrays, we show that the HCMV-regulated gene expression profile in fibroblasts does not differ substantially from the response generated by IFN. Furthermore, we identified the specific viral component triggering this response as the envelope glycoprotein B (gB). Cells treated with gB, but not other herpesviral glycoproteins, exhibited the same transcriptional profile as HCMV-infected cells. Thus, the interaction of gB with its as yet unidentified cellular receptor is the principal mechanism by which HCMV alters cellular gene expression early during infection. These findings highlight a pioneering paradigm for the consequences of virus–receptor interactions.

Epstein-Barr viral latency is disrupted by the immediate-early BRLF1 protein through a cell-specific mechanism.

Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, is a human herpesvirus associated with epithelial cell malignancies (nasopharyngeal carcinoma) as well as B-cell malignancies. Understanding how viral latency is disrupted is a central issue in herpesvirus biology. Epithelial cells are the major site of lytic EBV replication within the human host, and viral reactivation occurs in EBV-associated nasopharyngeal carcinomas. It is known that expression of a single viral immediate-early protein, BZLF1, is sufficient to initiate the switch from latent to lytic infection in B cells. Cellular regulation of BZLF1 transcription is therefore thought to play a key role in regulating the stringency of viral latency. Here we show that, unexpectedly, expression of another viral immediate-early protein, BRLF1, can disrupt viral latency in an epithelial cell-specific fashion. Therefore, the mechanisms leading to disruption of EBV latency appear to be cell-type specific.