Preclinical development of a bispecific TNFα/IL-23 neutralising domain antibody as a novel oral treatment for inflammatory bowel disease.

Anti-TNFα and anti-IL-23 antibodies are highly effective therapies for Crohn's disease or ulcerative colitis in a proportion of patients. V56B2 is a novel bispecific domain antibody in which a llama-derived IL-23p19-specific domain antibody, humanised and engineered for intestinal protease resistance, V900, was combined with a previously-described TNFα-specific domain antibody, V565. V56B2 contains a central protease-labile linker to create a single molecule for oral administration. Incubation of V56B2 with trypsin or human faecal supernatant resulted in a complete separation of the V565 and V900 monomers without loss of neutralising potency. Following oral administration of V900 and V565 in mice, high levels of each domain antibody were detected in the faeces, demonstrating stability in the intestinal milieu. In ex vivo cultures of colonic biopsies from IBD patients, treatment with V565 or V900 inhibited tissue phosphoprotein levels and with a combination of the two, inhibition was even greater. These results support further development of V56B2 as an oral therapy for IBD with improved safety and efficacy in a greater proportion of patients as well as greater convenience for patients compared with traditional monoclonal antibody therapies

High Yield Production Process for Shigella Outer Membrane Particles

Gram-negative bacteria naturally shed particles that consist of outer membrane lipids, outer membrane proteins, and soluble periplasmic components. These particles have been proposed for use as vaccines but the yield has been problematic. We developed a high yielding production process of genetically derived outer membrane particles from the human pathogen Shigella sonnei. Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30–45 in a 5 L fermenter. Proteomic analysis of the purified particles showed the preparation to primarily contain predicted outer membrane and periplasmic proteins. These were highly immunogenic in mice. The production of these outer membrane particles from high density cultivation of bacteria supports the feasibility of scaling up this approach as an affordable manufacturing process. Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles. This work provides the basis for a large scale manufacturing process of Generalized Modules of Membrane Antigens (GMMA) for production of vaccines from Gram-negative bacteria

Clinical features and outcomes of elderly hospitalised patients with chronic obstructive pulmonary disease, heart failure or both

Background and objective: Chronic obstructive pulmonary disease (COPD) and heart failure (HF) mutually increase the risk of being present in the same patient, especially if older. Whether or not this coexistence may be associated with a worse prognosis is debated. Therefore, employing data derived from the REPOSI register, we evaluated the clinical features and outcomes in a population of elderly patients admitted to internal medicine wards and having COPD, HF or COPD + HF. Methods: We measured socio-demographic and anthropometric characteristics, severity and prevalence of comorbidities, clinical and laboratory features during hospitalization, mood disorders, functional independence, drug prescriptions and discharge destination. The primary study outcome was the risk of death. Results: We considered 2,343 elderly hospitalized patients (median age 81 years), of whom 1,154 (49%) had COPD, 813 (35%) HF, and 376 (16%) COPD + HF. Patients with COPD + HF had different characteristics than those with COPD or HF, such as a higher prevalence of previous hospitalizations, comorbidities (especially chronic kidney disease), higher respiratory rate at admission and number of prescribed drugs. Patients with COPD + HF (hazard ratio HR 1.74, 95% confidence intervals CI 1.16-2.61) and patients with dementia (HR 1.75, 95% CI 1.06-2.90) had a higher risk of death at one year. The Kaplan-Meier curves showed a higher mortality risk in the group of patients with COPD + HF for all causes (p = 0.010), respiratory causes (p = 0.006), cardiovascular causes (p = 0.046) and respiratory plus cardiovascular causes (p = 0.009). Conclusion: In this real-life cohort of hospitalized elderly patients, the coexistence of COPD and HF significantly worsened prognosis at one year. This finding may help to better define the care needs of this population

Outer membrane particles as Shigella vaccine candidate: generation, proteomic and immunoproteomic analysis

Shigellae are Gram-negative enteric bacteria representing the major cause of dysenteric diseases in infants and young children in developing countries. The genus Shigella comprises 50 different serotypes characterized by the carbohydrate composition of the O-antigen of the lipopolysaccharide. In natural infection the immune response to the O-antigen has been shown to be protective but serotype-specific, and no vaccine against Shigella is currently available. Novartis Vaccines Institute for Global Health (NVGH) aims to develop a broadly protective protein-based vaccine using outer membrane particles. These particles, called Generalized Modules for Membrane Antigens (GMMA), also known as outer membrane vesicles (OMV), are naturally shed from the surface of Gram-negative bacteria and expose the host to proteins of the outer membrane in their native orientation. They are different from detergent-extracted OMV which are depleted of lipooligosaccharides and lipoproteins. As Shigella naturally produce low amounts of GMMA, the tolR gene was deleted to induce GMMA overproduction. To avoid immuno-dominant immune responses to the O-antigen, the genes of the O-antigen biosynthesis gene cluster were deleted. In addition, the msbB gene was deleted to reduce the endotoxicity of the lipopolysaccharide (LPS). GMMA were generated under iron-restricted conditions to induce the expression of highly conserved iron siderophore receptors that have been shown to be protective antigens against Salmonella infection. The proteomics analysis of S. sonnei 53G and S. flexneri 2a 2457T GMMA confirmed that GMMA are highly enriched in outer membrane and periplasmic proteins. Approximately 65% and 71% of the total predicted outer membrane proteins of S. flexneri and S. sonnei, respectively, and 50% of the total predicted periplasmic proteins were found to be present in GMMA. In contrast, only 4% of the total predicted cytoplasmic proteins were indentified in GMMA. We identified 50 outer membrane proteins and 2 predicted extracellular proteins that were conserved in S. sonnei and S. flexneri GMMA. Among these proteins we identified 3 proteins encoded on the virulence plasmid (MxiD, MxiM and IcsA/VirG), and other 3 proteins with a virulence-associated function (SepA, SigA and TieB) which are specific for Shigella and pathogenic E. coli. The proteomic results also confirmed the overexpression of 8 iron-regulated proteins in GMMA of which 5 are conserved in S. sonnei and S. flexneri. Mice were immunized using purified GMMA and immune sera were analyzed by ELISA and bi-dimensional Western blots. All GMMA elicited similarly high levels of total GMMA-specific IgG. By 2 dimensional Western blots (2-D WB) analysis mainly periplasmic proteins and only 3 outer membrane proteins were identified, suggesting that a different approach was needed to identify outer membrane proteins in their native conformation. In this work we present a novel approach to identify immunogenic surface proteins based on immunoprecipitation studies with live bacteria using sera raised against GMMA. We identified 30 immunogenic surface proteins and among those we found OmpA, OmpX and YaeT confirming our previous 2-D WB results. In addition, we identified proteins that were not found by 2-D WB but are known to be immunogenic in Shigella, e.g. TolC, supporting the power of this approach. Furthermore, OmpC, FepA, FhuA, and CirA were identified using this approach suggesting that the native conformation of the epitopes is important for antibody binding and that these epitopes are lost by 2-D WB analysis. A quantitative proteomic analysis of GMMA proteomes, based on stable isotope dimethyl labeling followed by GeLC-MS/MS, revealed that the majority of the 30 immunogenic proteins are the most abundant proteins in GMMA. Interestingly, YiaD, YtfM and one of the iron-regulated proteins, FepA, raised a strong antibody response but are of low abundance in GMMA. The majority of the immunogenic proteins identified in S. sonnei are also present and highly conserved in S. flexneri GMMA. Thus, it is likely that these proteins might raise cross-reactive immune responses. The present work shows for the first time an extensive qualitative and quantitative proteomic analysis of Shigella GMMA. In addition, the analysis of immunogenic surface proteins under native conditions identified proteins that are highly conserved among Shigella serotypes. This work provides the basis for further characterization of GMMA as a protein-based vaccine against ShigellaLa Shigella è un batterio enterico Gram-negativo e rappresenta la principale causa di dissenteria nei neonati e nei bambini al di sotto dei 5 anni nei paesi in via di sviluppo. Il genere Shigella include 50 serotipi diversi distinguibili in base alla composizione saccaridica dell’antigene O del lipopolisaccaride (LPS). Nell’infezione naturale è stato osservato che l’antigene O è in grado di indurre una risposta immunitaria protettiva contro l’infezione da Shigella, ma tale risposta risulta essere specifica contro i singoli serotipi. Non ci sono al momento vaccini disponibili per prevenire l’infezione da Shigella. L’istituto “Novartis Vaccines Institute for Global Health” (NVGH) sta lavorando su un progetto per lo sviluppo di un vaccino proteico protettivo e ad ampio spettro per la prevenzione di infezioni da Shigella basato sull’utilizzo di particelle di membrana esterna. Queste particelle, chiamate Moduli Generalizzati per l’espressione di Antigeni di Membrana (GMMA) e comunemente conosciute in letteratura come vescicole di membrana esterna (OMV), sono naturalmente rilasciate dalla superficie dei batteri Gram-negativi e presentano all’ospite le proteine della membrana esterna nella loro conformazione originaria. Queste particelle si distinguono dalle OMV ottenute mediante estrazione con detergenti che sono prive di lipo-oligosaccaridi e lipoproteine. Poiché Shigella rilascia naturalmente piccole quantità di GMMA, è stata eseguita una delezione del gene tolR al fine di indurre un maggior rilascio di GMMA. Per abolire la risposta immunitaria serotipo-specifica dell’antigene O è stata eseguita una delezione del cluster di geni codificanti per l’antigene O. Una successiva delezione del gene msbB è stata effettuata per ridurre la reattogenicità delle molecole di LPS. Le GMMA sono state ottenute crescendo i ceppi produttori in condizioni limitanti di ferro al fine di indurre l’espressione di recettori del ferro molto conservati in Shigella. Tali recettori si sono dimostrati antigeni protettivi contro l’infezione da Salmonella. L’analisi delle GMMA di S. sonnei 53G e S. flexneri 2a 2457T attraverso approccio proteomico ha confermato che le GMMA contengono prevalentemente proteine di membrana esterna e proteine periplasmatiche. Nelle GMMA abbiamo identificato circa il 65% e il 71% delle proteine di membrana esterna predette in totale rispettivamente in S. flexneri e in S. sonnei e il 50% delle proteine periplasmatiche. Le proteine citoplasmatiche identificate nelle GMMA rappresentano, invece, solamente il 4% delle proteine citoplasmatiche totali predette. Abbiamo identificato 50 proteine di membrana esterna e 2 proteine con predizione extracellulare conservate nelle GMMA di S. sonnei e di S. flexneri. Tra le proteine comuni abbiamo identificato 3 proteine codificate dal plasmide di virulenza (MxiD, MxiM and IcsA/VirG) e altre 3 proteine con una funzione associata alla virulenza (SepA, SigA and TieB) che sono specifiche per Shigella e ceppi patogeni di E. coli. I risultati dell’analisi proteomica hanno inoltre confermato l’overespressione di 8 proteine ferro-regolate nelle GMMA, di cui 5 sono conservate in S. sonnei e S. flexneri. Le GMMA sono state utilizzate per l’immunizzazione di topi e i sieri immuni sono stati analizzati mediante saggi ELISA e Western blots bi-dimensionali (2-D WB). Tutte le GMMA testate hanno stimolato in modo equiparabile la produzione di alti livelli di immunoglobuline di tipo G (IgG) GMMA-specifiche. Dalle analisi di 2-D WB abbiamo identificato proteine immunogeniche prevalentemente periplasmatiche e solo 3 proteine di membrana esterna. Tali risultati hanno suggerito la necessità di trovare un approccio alternativo per l’identificazione di proteine di membrana esterna nella loro conformazione nativa. In questo studio presentiamo un approccio per l’identificazione di proteine immunogeniche di superficie basato su studi di immunoprecipitazione su batteri vivi usando sieri immuni specifici contro le GMMA. Abbiamo identificato 30 proteine immunogeniche di superificie e tra queste abbiamo trovato OmpA, OmpX e YaeT confermando i risultati precedentemente ottenuti mediante 2-D WB. Inoltre, abbiamo identificato proteine che sono riportate immunogeniche in Shigella, come ad esempio TolC, ma che non sono state rilevate mediante 2-D WB supportando l’efficacia di questo approccio. L’ulteriore identificazione delle proteine OmpC, FepA, FhuA e CirA mediante immunoprecipitazione suggerisce che la conformazione nativa degli epitopi è importante per il legame con l’anticorpo e che tali epitopi vengono persi nelle analisi di 2-D WB. Un’analisi proteomica di tipo quantitativa delle GMMA, basata sull’utilizzo di marcatura isotopica stabile con legame dimetilico seguita da GeLC-MS/MS, ha rivelato che la maggior parte delle 30 proteine immunogeniche sono anche le proteine più abbondanti nelle GMMA. E’ interessante notare che nonostante YiaD, YtfM e una delle proteine ferro-regolate (FepA) siano tra le proteine meno abbondanti nelle GMMA, tali proteine hanno indotto una forte risposta immunitaria. La maggior parte delle proteine immunogeniche identificate in S. sonnei sono anche presenti e altamente conservate nelle GMMA di S. flexneri. Perciò, è molto probabile che queste proteine siano in grado di indurre una risposta immunitaria cross-reattiva. Il presente studio mostra per la prima volta un’estensiva analisi proteomica di tipo qualitativa e quantitativa delle GMMA ottenute da Shigella. Inoltre, l’analisi delle proteine immunogeniche di superficie condotta in condizioni native ha permesso di identificare proteine altamente conservate tra i serotipi di Shigella. Questo lavoro fornisce le basi per una successiva caratterizzazione delle GMMA come vaccino a base proteica contro Shigell

Quantitative proteomic analysis of Shigella flexneri and Shigella sonnei Generalized Modules for Membrane Antigens (GMMA) reveals highly pure preparations

Outer membrane blebs are naturally shed by Gram-negative bacteria and are candidates of interest for vaccines development. Genetic modification of bacteria to induce hyperblebbing greatly increases the yield of blebs, called Generalized Modules for Membrane Antigens (GMMA). The composition of the GMMA from hyperblebbing mutants of Shigella flexneri 2a and Shigella sonnei were quantitatively analyzed using high-sensitivity mass spectrometry with the label-free iBAQ procedure and compared to the composition of the solubilized cells of the GMMA-producing strains. There were 2306 proteins identified, 659 in GMMA and 2239 in bacteria, of which 290 (GMMA) and 1696 (bacteria) were common to both S. flexneri 2a and S. sonnei. Predicted outer membrane and periplasmic proteins constituted 95.7% and 98.7% of the protein mass of S. flexneri 2a and S. sonnei GMMA, respectively. Among the remaining proteins, small quantities of ribosomal proteins collectively accounted for more than half of the predicted cytoplasmic protein impurities in the GMMA. In GMMA, the outer membrane and periplasmic proteins were enriched 13.3-fold (S. flexneri 2a) and 8.3-fold (S. sonnei) compared to their abundance in the parent bacteria. Both periplasmic and outer membrane proteins were enriched similarly, suggesting that GMMA have a similar surface to volume ratio as the surface to periplasmic volume ratio in these mutant bacteria. Results in S. flexneri 2a and S. sonnei showed high reproducibility indicating a robust GMMA-producing process and the low contamination by cytoplasmic proteins support the use of GMMA for vaccines. Data are available via ProteomeXchange with identifier PXD002517.ISSN:1438-4221ISSN:1618-0607ISSN:1433-112

THE INTEGRATIVE APPROACH TO THE PSYCHO- PHYSICAL REHABILITATION ISSUES OF BREAST CANCER SURVIVORS

Background Survival rates for breast cancer are increasing, but many clinical needs in a larger and larger population of survivors are not properly met. During and beyond cancer treatments, patients tend to reduce their physical activity levels, gain weight and may complain about chronic pain, lymphedema, fatigue, cognitive problems, depression and anxiety, reduced social contacts and difficulties in resuming daily activities. In addition, younger women must also face fertility issues and early menopause, higher levels of distress regarding body image, sexual health, childcare and career management. Beside this, all the survivors may feel a fear for disease recurrence and many of them are not educated to identify and express their needs. Materials and Methods Tailored supportive strategies, including physical and psychological interventions, should be offered since the time of diagnosis and along the entire cancer journey and the follow up period. They should be aimed not only to the prevention and control of psychological and physical impairments (such as anxiety, stress, depression, lymphedema, genitourinary symptoms and sexual dysfunctions), but also to encourage remodulation of personal lifestyles for tertiary prevention and nurture patient’s resources that can promote a personal growth after the traumatic experience of cancer. Conclusions Toward this goal, many cancer centres worldwide including ours offer dedicated programs of psycho-oncological support, nutritional and physical activity counselling and evidence based complementary treatments (such as acupuncture, mindfulness, meditation, reflexology, herbal medicine, music therapy and more)

Electron microscopy of <i>Shigella sonnei</i> Δ<i>tolR</i> Δ<i>galU</i> GMMA.

<p>GMMA were isolated from the culture supernatant of <i>S. sonnei</i> –pSS Δ<i>tolR</i> Δ<i>msbB</i> by TFF, prepared for negative staining, and viewed by electron microscopy revealing the presence of well-organized membrane vesicles with a diameter of about 30–60 nm. Bar length  = 100 nm.</p

2D gel electrophoresis of <i>Shigella sonnei</i> Δ<i>tolR</i> Δ<i>galU</i> GMMA and immunoblot.

<p>A)200 µg of proteins from <i>S. sonnei</i> Δ<i>tolR</i> Δ<i>galU</i> GMMA were separated in the first dimension on a non linear pH 3–11 gradient, and in the second dimension on a 4–12% polyacrylamide gradient. Visible bands were identified by protein mass fingerprint. OmpA and OmpC were quantified with Image master 2D Platinum 6.0. B) Sera from mice immunized with GMMA from <i>S. sonnei</i> Δ<i>tolR</i> Δ<i>galU</i> were used to study the subset of proteins present in GMMA that are able to raise antibodies. A 2D gel containing 20 µg of GMMA protein from <i>S. sonnei</i> Δ<i>tolR</i> Δ<i>galU</i> was blotted and the membrane was incubated with sera from immunized mice with GMMA from <i>S. sonnei</i> Δ<i>tolR</i> Δ<i>galU</i> in combination with Freund’s adjuvant. Several reactive proteins were identified. The numbers behind the names refer to the position of the proteins in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035616#pone-0035616-t002" target="_blank">Table 2</a>. C) To verify that the signal observed in the 2D Western blot was due exclusively to antibody raised upon immunization with GMMA, 10 µg of GMMA were separated by 1D SDS-PAGE (12% PA) and stained with Coomassie (1) or transferred to a membrane. Western blots were developed using (2) sera raised against GMMA from <i>S. sonnei</i> Δ<i>tolR</i> Δ<i>galU</i> as used for the 2D Western blot in B, (3) preimmune serum, (4) sera raised in mice immunized with Freund’s adjuvant or (5) PBS, or (6) secondary antibody only. A signal could only be observed when sera raised against GMMA were used (2).</p