SARC-F Is Better Correlated with Muscle Function Indicators than Muscle Mass in Older Hemodialysis Patients.

BACKGROUND: Sarcopenia can be characterized by European Consensus for Sarcopenia (EWGSOP2) criteria, but it methods are not easily accessible. Likewise, the Strength, Assistance with walking, Rise from a Chair, Climb stairs and Falls (SARC-F) has been proposed. OBJECTIVE: The aim of this study was i) to evaluate the prevalence for risk sarcopenia and ii) to correlate the SARC-F with components of the EWGSOP2 consensus in hemodialysis (HD) patients. MEASUREMENTS: This cross-sectional study enrolled ninety-five (male n= 59; 62%) HD older patients. Sarcopenia risk was assessed using the SARC-F, which ≥4 score indicates sarcopenia risk. Sarcopenia was confirmed through of the EWGSOP2 consensus, including the handgrip strength (HGS <27kg for men and <16kg for women) using the dynamometer, muscle mass through appendicular muscle mass (ASMI/m2 <7.0 kg/m2 for men and 5.5 kg/m2 for women) using the bioimpedance electrical, and physical performance through of gait speed (GS <0.8 m/s). RESULTS: From 95 patients, n=21(22%) presented sarcopenia risk. SARC-F ≥4 group are older (64.9±13.9 vs. 56.9±14.6 y, p= 0.028), presented lower ASMI (7.4±1.2 vs. 8.3±1.8 kg/m2, p=0.033), HGS (20.5±5.7 vs. 27.2±10.2 kg, p=0.005), and GS (0.5±0.1 vs. 0.7±0.1 m/s, p=0.001) than SARC-F<4 group. SARC-F score was negatively correlated with EWGSOP2 components: ASMI x SARC-F (r=-0.27, p=0.007), HGS x SARC-F (r=-0.35, p=0.0005), and GS x SARC-F (r=-0.47, p<0.0001). Although, no difference of number of patients with low or normal ASMI values was found, 62% and 95% of SARC-F≥4 group patients presented low HGS and gait speed, respectively. CONCLUSIONS: In older HD patients, 22% presented sarcopenia risk. In addition, SARC-F is better correlated with muscle function indicators (HGS and gait speed) than muscle mass.

Age-dependent pathogenesis of clade 2.3.4.4A H5N2 HPAIV in experimentally infected Broad Breasted White turkeys.

Highly pathogenic avian influenza (HPAI) is a viral disease with devastating consequences to the poultry industry as it results in high morbidity, mortality and international trade restrictions. In the present study, we characterized age-related differences in terms of pathology in commercial white broad breasted turkeys inoculated with A/turkey/Minnesota/12582/2015 (H5N2) HPAIV clade 2.3.4.4A, a virus from the largest HPAI poultry outbreak that affected the Unites States in 2014-2015. Turkeys infected at 6-weeks of age showed inapparent to little clinical signs with rapid disease progression, reaching 100% mortality at 3 days post infection (dpi). In contrast, turkeys infected at 16-weeks of age developed ataxia and lethargy and reached 100% mortality by 5 dpi. Infection in the 6-weeks old turkeys resulted in peracute lesions consistent of extensive hemorrhages, edema and necrosis, but inflammation was not prominent. In the 16-weeks old turkeys, necrosis and hemorrhages in tissues were accompanied by a more prominent subacute inflammatory infiltrate. Both age groups showed presence of avian influenza virus (AIV) nucleoprotein (NP) in multiple cell types including neurons, glial cells, ependymal cells, respiratory epithelial cells, air capillary epithelium and pulmonary macrophages, cardiac myocytes, smooth muscle fibers, pancreatic acini and ductal cells. Cells of the vascular walls stained strongly positive for viral antigens, but no positivity was found in the endothelial cells of any organs. These findings indicate that age is a determinant factor in the progression of the disease and delay of mortality during infection with the H5N2 clade 2.3.4.4A HPAI virus in naïve white broad breasted turkeys.

Robot-Assisted Oesophagectomy: Recommendations Towards a Standardised Ivor Lewis Procedure.

A considerable number of reports have been published on the feasibility, techniques, and early postoperative results of robotic-assisted oesophageal surgery. However, these are mostly smaller case series, suggesting that the robot-assisted Ivor Lewis procedure is still in the implementation phase and far from being standardised. Oesophageal surgeons from seven robotic university centres in Germany, experienced in both minimally invasive and robot-assisted minimally invasive surgery, took part in a workshop on robot-assisted surgery. An intensive exchange of opinions and experiences, followed by a step-by-step re-enactment of the operation in a cadaver lab, enabled us to develop a standardised robot-assisted Ivor Lewis surgical workflow, which is presented here. Systematic and objective comparison of experiences and results using a robot-assisted Ivor Lewis procedure has made it possible to develop a standardised surgical workflow that is now clinically applied in our centres. It is hoped that standardisation of this procedure will help to maintain patient safety, prevent medical errors, and facilitate the learning curve, while introducing robotic surgery into a centre.

Evaluation of the robotic approach concerning pitfalls in rectal surgery.

INTRODUCTION: The feasibility and advantages of robotic rectal surgery (RRS) in comparison to conventional open or laparoscopic rectal resections have been postulated in several reports. But well-known challenges and pitfalls of minimal invasive rectal surgery have not been evaluated by a prospective, multicenter setting so far. Aim of this study was to analyze the perioperative outcome of patients following RRS especially in regard to the pitfalls such as obesity, male patients and low tumors by a European multicenter setting. METHODS: This prospective study included 348 patients undergoing robotic surgery due to rectal cancer in six major European centers. Clinicopathological parameters, morbidity, perioperative recovery and short-term outcome were analyzed. RESULTS: A total of 283 restorative surgeries and 65 abdominoperineal resections were carried out. The conversion rate was 4.3%, mean blood loss was 191 ml, and mean operative time was 315 min. Postoperative complications with a Clavien-Dindo score >2 were observed in 13.5%. Obesity and low rectal tumors showed no significant higher rates of major complications or impaired oncological parameters. Male patients had significant higher rates of major complications and anastomotic leakage (p = 0.048 and p = 0.007, respectively). DISCUSSION: RRS is a promising tool for improvement of rectal resections. The well-known pitfalls of minimal-invasive rectal surgery like obesity and low tumors were sufficiently managed by RRS. However, RRS showed significantly higher rates of major complications and anastomotic leakage in male patients, which has to be evaluated by future randomized trials.

Expression of cancer testis antigens CT10 (MAGE-C2) and GAGE in gastrointestinal stromal tumors.

INTRODUCTION: Expression of cancer testis antigens (CTAs) has been associated with prognosis in gastrointestinal stromal tumors (GIST) and other malignancies. CTAs are currently being investigated for cancer immunotherapy. MATERIALS AND METHODS: We analyzed two CTAs, CT10/MAGE-C2 and GAGE, in 51 GIST by immunohistochemistry and correlated it with established histopathological criteria for malignancy. RESULTS: GAGE expression was found in 6/51 (12%) patients, whereas 5/51 (10%) patients expressed CT10/MAGE-C2. 7/51(14%) patients expressed at least one of both CTAs, in 4/51 (8%) patients both CTAs were positive. High-grade GIST are more likely to express GAGE (p = 0.002) and CT10/MAGE-C2 (p = 0.007) compared to less aggressive tumors. All patients with GAGE or CT10/MAGE-C2 expression had moderate- or high-risk of recurrence according to the established risk criteria. The presence of GAGE correlates with mitotic rate (p = 0.001) and tumor size (p = 0.02), but not with tumor location (p = 0.60). CT10/MAGE-C2 also significantly correlates with mitotic rate (p = 0.004) and tumor size (p = 0.002), whereas no correlation could be found with tumor location (p = 0.36). DISCUSSION: CT10/MAGE-C2 and GAGE should be explored together with other previously described CTAs as targets for immunotherapy of GIST in cases, which are refractory to conventional therapy.

Genesis of pandemic influenza.

During the last decade the number of reported outbreaks caused by highly pathogenic avian influenza (HPAI) in domestic poultry has drastically increased. At the same time, low pathogenic avian influenza (LPAI) strains, such as H9N2 in many parts of the Middle East and Asia and H6N2 in live bird markets in California, have become endemic. Each AI outbreak brings the concomitant possibility of poultry-to-human transmission. Indeed, human illness and death have resulted from such occasional transmissions with highly pathogenic avian H7N7 and H5N1 viruses while avian H9N2 viruses have been isolated from individuals with mild influenza. The transmission of avian influenza directly from poultry to humans has brought a sense of urgency in terms of understanding the mechanisms that lead to interspecies transmission of influenza. Domestic poultry species have been previously overlooked as potential intermediate hosts in the generation of influenza viruses with the capacity to infect humans. In this review, we will discuss molecular and epidemiological aspects that have led to the recurrent emergence of avian influenza strains with pandemic potential, with a particular emphasis on the current Asian H5N1 viruses.

Adaptation of influenza A/Mallard/Potsdam/178-4/83 H2N2 virus in Japanese quail leads to infection and transmission in chickens.

To assess the potential of quail as an intermediate host of avian influenza, we tested the influenza A/Mallard/ Potsdam/178-4/83 (H2N2) virus to determine whether through adaptation a mallard strain can replicate and transmit in quail, as well as other terrestrial birds. After five serial passages of lung homogenate a virus arose that replicated and transmitted directly to contact cage mates. To test whether adaptation in quail led to interspecies transmission, white leghorn chickens were infected with the wild-type (mall/178) and quail-adapted (qa-mall/178) viruses. The results show that mall/178 H2N2 does not establish an infection in chickens nor does it transmit, while qa-mall/178 H2N2 infects and transmits to contact chickens causing clinical signs like depression and diarrhea. Completed sequences indicate six amino acid changes spanning four genes, PB2, PB1, HA, and NP, suggesting that the internal genes play a role in host adaptation. Further adaptation of qa-mall/178 in white leghorn chickens created a virus that replicated more efficiently in the upper and lower respiratory tract. Sequence analysis of the chicken-adapted virus points to a deletion in the neuraminidase stalk region.

Pandemic influenza: preventing the emergence of novel strains and countermeasures to ameliorate its effects.

Influenza is a seasonal disease that peaks every year in the winter months. Antigenic drift of the viral surface proteins, particularly the hemagglutinin (HA), is responsible for the virus's ability to evading the host's immune system, and for the severity of the disease. Pandemic influenza arises when an influenza virus carrying a novel HA gene enters into the naive human population, resulting in excess morbidity and mortality. Three major influenza pandemics were experienced in the last century and the emergence of a new pandemic strain is considered a matter of time. Our current understanding suggests that pandemic influenza strains arise from influenza viruses circulating in the natural reservoir, although the presence of intermediate hosts is considered essential in this process. Pigs and land-based birds have been shown to play a major role in the ecology of influenza viruses by providing an environment in which influenza viruses can change their phenotype, expand their host range, and eventually transmit to humans. In recent years, a great detail of attention has been placed on understanding the epidemiological and molecular factors that can lead to interspecies transmission of influenza viruses. In this review we will discuss the ecological and molecular aspects that lead to pandemic influenza as well as the intervention strategies at our disposal that can reduce the emergence of pandemic influenza strains and/or minimize their effects.

Isolation and characterization of H3N2 influenza A virus from turkeys.

Five 34-wk-old turkey breeder layer flocks in separate houses of 2550 birds each in a single farm in Ohio experienced a drop in egg production from late January to early February 2004. Tracheal swabs (n = 60), cloacal swabs (n = 50), and convalescent sera (n = 110) from the flocks were submitted to the laboratory for diagnostics. Virus isolation was attempted in specific-pathogen free embryonating chicken eggs and Vero and MDCK cells. Virus characterization was performed using agar gel immunodiffusion, the hemagglutination test, the hemagglutination inhibition test, the virus neutralization test, reverse transcription-polymerase chain reaction, sequencing, and phylogenetic analysis. A presumptive influenza virus was successfully propagated and isolated on the first passage in MDCK cells, but initially not in Vero cells or specific-pathogen free chicken embryos. After two passages in MDCK cells, it was possible to propagate the isolate in specific-pathogen free chicken embryos. Preliminary sequence analysis of the isolated virus confirmed that it was influenza A virus with almost 100% (235/236) identity with the matrix gene of a swine influenza A virus, A/Swine/Illinois/100084/01 (H1N2). However, it was not possible to subtype the virus using conventional serotyping methods. The results of genetic characterization of the isolated virus showed that it was the H3N2 subtype and was designated as A/Turkey/OH/313053/04 (H3N2). Phylogenetic analysis of the eight gene segments of the virus showed that A/Turkey/OH/313053/04 (H3N2) isolate was most closely related to the triple-reassortant H3N2 swine viruses [A/Swine/WI/14094/99 (H3N2)] that have been circulating among pigs in the United States since 1998, which contains gene segments from avian, swine, and human viruses. The A/Turkey/OH/313053/04 (H3N2) isolated from turkeys in this study was classified as a low pathogenic avian influenza A virus because it only caused a drop in egg production with minor other clinical signs and no mortality.

Responsiveness to a pandemic alert: use of reverse genetics for rapid development of influenza vaccines.

BACKGROUND: In response to the emergence of severe infection capable of rapid global spread, WHO will issue a pandemic alert. Such alerts are rare; however, on Feb 19, 2003, a pandemic alert was issued in response to human infections caused by an avian H5N1 influenza virus, A/Hong Kong/213/03. H5N1 had been noted once before in human beings in 1997 and killed a third (6/18) of infected people. The 2003 variant seemed to have been transmitted directly from birds to human beings and caused fatal pneumonia in one of two infected individuals. Candidate vaccines were sought, but no avirulent viruses antigenically similar to the pathogen were available, and the isolate killed embryonated chicken eggs. Since traditional strategies of vaccine production were not viable, we sought to produce a candidate reference virus using reverse genetics. METHODS: We removed the polybasic aminoacids that are associated with high virulence from the haemagglutinin cleavage site of A/Hong Kong/213/03 using influenza reverse genetics techniques. A reference vaccine virus was then produced on an A/Puerto Rico/8/34 (PR8) backbone on WHO-approved Vero cells. We assessed this reference virus for pathogenicity in in-vivo and in-vitro assays. FINDINGS: A reference vaccine virus was produced in Good Manufacturing Practice (GMP)-grade facilities in less than 4 weeks from the time of virus isolation. This virus proved to be non-pathogenic in chickens and ferrets and was shown to be stable after multiple passages in embryonated chicken eggs. INTERPRETATION: The ability to produce a candidate reference virus in such a short period of time sets a new standard for rapid response to emerging infectious disease threats and clearly shows the usefulness of reverse genetics for influenza vaccine development. The same technologies and procedures are currently being used to create reference vaccine viruses against the 2004 H5N1 viruses circulating in Asia.

Land-based birds as potential disseminators of avian mammalian reassortant influenza A viruses.

Chickens, quail, and other land-based birds are extensively farmed around the world. They have been recently implicated in zoonotic outbreaks of avian influenza in Hong Kong. The possibility that land-based birds could act as mixing vessels or disseminators of avian/mammalian reassortant influenza A viruses with pandemic potential has not been evaluated. In this report, we investigated whether chickens and Japanese quail are susceptible to a mammalian influenza virus (A/swine/Texas/4199-2/98 [H3N2]). This virus did not grow in chickens and replicated to low levels in Japanese quail but did not transmit. Replacing the H3 gene of this virus for one of the avian H9 viruses resulted in transmission of the avian/swine reassortant virus among quail but not among chickens. Our findings demonstrated that Japanese quail could provide an environment in which viruses like the A/swine/Texas/4199-2/98 [H3N2] virus could further reassort and generate influenza viruses with pandemic potential.

Functional analysis of PA binding by influenza a virus PB1: effects on polymerase activity and viral infectivity.

Influenza A virus expresses three viral polymerase (P) subunits-PB1, PB2, and PA-all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain alpha, contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain alpha. The role of the minimal PB1 domain alpha in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.

Universal primer set for the full-length amplification of all influenza A viruses.

To systematically identify and analyze the 15 HA and 9 NA subtypes of influenza A virus, we need reliable, simple methods that not only characterize partial sequences but analyze the entire influenza A genome. We designed primers based on the fact that the 15 and 21 terminal segment specific nucleotides of the genomic viral RNA are conserved between all influenza A viruses and unique for each segment. The primers designed for each segment contain influenza virus specific nucleotides at their 3'-end and non-influenza virus nucleotides at the 5'-end. With this set of primers, we were able to amplify all eight segments of N1, N2, N4, N5, and N8 subtypes. For N3, N6, N7, and N9 subtypes, the segment specific sequences of the neuraminidase genes are different. Therefore, we optimized the primer design to allow the amplification of those neuraminidase genes as well. The resultant primer set is suitable for all influenza A viruses to generate full-length cDNAs, to subtype viruses, to sequence their DNA, and to construct expression plasmids for reverse genetics systems.

Generation of influenza A viruses entirely from cloned cDNAs.

We describe a new reverse-genetics system that allows one to efficiently generate influenza A viruses entirely from cloned cDNAs. Human embryonic kidney cells (293T) were transfected with eight plasmids, each encoding a viral RNA of the A/WSN/33 (H1N1) or A/PR/8/34 (H1N1) virus, flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator-together with plasmids encoding viral nucleoprotein and the PB2, PB1, and PA viral polymerases. This strategy yielded >1 x 10(3) plaque-forming units (pfu) of virus per ml of supernatant at 48 hr posttransfection. The addition of plasmids expressing all of the remaining viral structural proteins led to a substantial increase in virus production, 3 x 10(4)-5 x 10(7) pfu/ml. We also used reverse genetics to generate a reassortant virus containing the PB1 gene of the A/PR/8/34 virus, with all other genes representing A/WSN/33. Additional viruses produced by this method had mutations in the PA gene or possessed a foreign epitope in the head of the neuraminidase protein. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and in the production of vaccines and gene therapy vectors.

Genetic analysis of the internal ribosome entry segment of bovine viral diarrhea virus.

Pestiviruses and hepaciviruses are atypical members of the Flaviviridae due to their unique biological properties, including the utilization of internal ribosome entry for translation initiation. In contrast to internal initiation in picornaviruses, which depends on numerous canonical initiation factors, the mode of internal ribosome entry in pestiviruses and hepaciviruses resembles prokaryotic translation initiation. To identify functionally important elements within the bovine viral diarrhea virus (BVDV) internal ribosome entry segment (IRES), we carried out a mutational analysis of the 5' untranslated region (5' UTR) of BVDV cloned in the intercistronic region of a bicistronic reporter plasmid. IRES function was assessed in a bicistronic transcript by inserting the 5' 901 nucleotides of BVDV genome, which correspond to the 385 nucleotides of the 5' UTR and 515 nucleotides of the open reading frame (ORF) encoding for Npro and 4 amino acids from the capsid protein. The resulting Npro-luciferase fusion encoded by the 3' cistron was cleaved efficiently to release the luciferase reporter. In vivo translation analyses showed that stem-loops Ia and Ib in the 5' UTR were completely dispensable for efficient translation, whereas stem-loop IIIe and the hairpin end of IIIb were only partially required. In contrast, deletions or insertions in any of other four stem-loop structures, including domains II, IIIa, IIIc, and IIId, caused nearly 10-fold reductions of in vivo IRES activity. The tolerance of structural modifications within the distal portion of domain IIIb and IIIe correlated with a low level of sequence conservation in these regions among pestiviruses. The 5' boundary of the IRES resides at the 5' end of stem-loop II near nucleotide 75. The 3' of the IRES extends into the 5' end of the polyprotein ORF because removal of the Npro coding region reduced translation by 21%.

The matrix 1 protein of influenza A virus inhibits the transcriptase activity of a model influenza reporter genome in vivo.

The M1 protein of influenza virus inhibits the in vitro transcriptase activity of ribonucleoprotein cores from virions. This inhibitory activity is thought to be relevant in vivo because accumulation of M1 at the late stages of viral replication may be the cue to halt viral mRNA production. A model influenza reporter genome was used to explore the effect of M1 on the activity of the influenza virus transcriptase complex within cultured cells. Expression of M1 in cells bearing the model influenza virus reporter genome was accompanied by a reduction of CAT gene expression to 12% of control levels. Quantification of RNA by ribonuclease protection assay revealed that the influenza reporter genome mRNA levels in M1-expressing cells were reduced by approximately 74% compared with those of cells expressing a control protein. These findings are consistent with the proposed model in which M1 is responsible for limiting viral transcription during late stages of infection. By expressing truncated forms of M1, the inhibitory activity was found to reside within the amino-terminal half of the M1 protein. Two independent inhibitory domains were identified in this region: one between amino acid residues 1-90 and the other spanning residues 91-127.

A monoclonal antibody against acetylcholinesterase inhibits the formation of amyloid fibrils induced by the enzyme.

A monoclonal antibody (mAb) 25B1 directed against fetal bovine-serum acetylcholinesterase (FBS AChE) was used to examine the ability of the cholinergic enzyme to promote the assembly of amyloid-beta peptides (A beta) into Alzheimers fibrils. This mAb binds to the peripheral anionic site of the enzyme and allosterically inhibits catalytic activity of FBS AChE. Several techniques, including thioflavine-T fluorescence, turbidity, and negative-staining at the electron microscopy level, were used to assess amyloid formation. Inhibition of amyloid formation was dependent on the molar ratio AChE:mAb 25B1, and at least 50% of the inhibition of the AChE promoting effect occurs at a molar ratio similar to that required for inhibition of the esterase activity. Our results suggest that mAb 25B1 inhibits the promotion of the amyloid fibril formation triggered by AChE by affecting the lag period of the A beta aggregation process.

A 48-amino-acid region of influenza A virus PB1 protein is sufficient for complex formation with PA.

The concerted activity of four influenza virus proteins, PB1, PB2, PA, and NP is necessary and sufficient for transcription and replication of the viral genome in the nucleus of the cell. The three P proteins form a heterotrimeric complex in virions and the nuclei of infected cells. Biochemical analyses have shown specific interactions between PB1 and PA as well as PB1 and PB2, indicating that PB1 is the backbone of the complex. To identify domains of PB1 involved in binding PA, a two-hybrid system adapted for mammalian cells (CV-1) was implemented. First, we demonstrate the ability of PB1 and PA to interact efficiently and specifically in reciprocal combinations of two-hybrid reporter moieties, suggesting that transcription factor module fusion did not interfere sterically or allosterically with interaction between PB1 and PA. Subsequent analyses with a set of chimeric proteins with truncations of the PB1 C termini, N termini, or internal sequences led to the identification of a region at the N terminus of PB1 responsible for binding PA. Forty-eight amino acids at the N terminus of PB1 were sufficient for binding PA in vivo with the same efficiency as the complete PB1 protein. This region of PB1 responsible for binding PA does not overlap with other previously described PB1 functional domains involved in nuclear transport and RNA polymerization. We propose to name this region of interaction with PA domain alpha, to differentiate it from other functional domains described for PB1.