Isolation of HLA-DR-naturally presented peptides identifies T-cell epitopes for rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) immunopathogenesis revolves around the presentation of poorly characterised self-peptides by human leucocyte antigen (HLA)-class II molecules on the surface of antigen-presenting cells to autoreactive CD4 +T cells. Here, we analysed the HLA-DR-associated peptidome of synovial tissue (ST) and of dendritic cells (DCs) pulsed with synovial fluid (SF) or ST, to identify potential T-cell epitopes for RA. METHODS: HLA-DR/peptide complexes were isolated from RA ST samples (n=3) and monocyte-derived DCs, generated from healthy donors carrying RA-associated shared epitope positive HLA-DR molecules and pulsed with RA SF (n=7) or ST (n=2). Peptide sequencing was performed by high-resolution mass spectrometry. The immunostimulatory capacity of selected peptides was evaluated on peripheral blood mononuclear cells from patients with RA (n=29) and healthy subjects (n=12) by flow cytometry. RESULTS: We identified between 103 and 888 HLA-DR-naturally presented peptides per sample. We selected 37 native and six citrullinated (cit)-peptides for stimulation assays. Six of these peptides increased the expression of CD40L on CD4 +T cells patients with RA, and specifically triggered IFN-γ expression on RA CD4 +T cells compared with healthy subjects. Finally, the frequency of IFN-γ-producing CD4 +T cells specific for a myeloperoxidase-derived peptide showed a positive correlation with disease activity. CONCLUSIONS: We significantly expanded the peptide repertoire presented by HLA-DR molecules in a physiologically relevant context, identifying six new epitopes recognised by CD4 +T cells from patients with RA. This information is important for a better understanding of the disease immunopathology, as well as for designing tolerising antigen-specific immunotherapies.

[IGG4-related disease. Report of 52 patients]. / Enfermedad relacionada a IgG4. Serie clínica de pacientes chilenos.

BACKGROUND: IgG4-related disease (IgG4 RD) is an immune-mediated fibro-inflammatory disorder, with tissue infiltration of IgG4+ plasma cells. It causes pseudotumors, tumors, and a wide spectrum of clinical manifestations. AIM: To report the clinical, laboratory, histopathological and treatment characteristics of a group of Chilean patients with IgG4 RD. MATERIAL AND METHODS: Review of medical records of 52 patients aged 18 to 76 years with IgG4 RD seen at six medical centers. RESULTS: Elevated IgG4 serum levels (> 135 mg/dl) were found in 18 of 44 (41%) patients. There was histological confirmation of the disease in 46 patients. The most common sites of involvement were lungs, eyes and kidneys. Eighteen (35%) patients had only one organ involved, 34 (65%) patients had two organs and 13 (25%) patients had three or more organs. The involvement of two organs was significantly more common in men (p < 0.05). In patients with only one organ involvement, the most frequent location was orbital and meningeal. All patients with kidney or lung disease had multiorgan involvement. All patients received corticosteroid therapy, 67% synthetic immunosuppressants, and 16% rituximab. CONCLUSIONS: ER-IgG4 can affect any tissue. Multiorgan involvement was more common in this series, with preference for lungs, eyes and kidneys. An excellent response to steroids is characteristic of the disease, but with a high relapse rate that requires additional immunosuppression.

A Mimic of Ankylosing Spondylitis, Ochronosis: Case Report and Review of the Literature.

PURPOSE OF REVIEW: Ochronosis and alkaptonuria are manifestations of the same condition-a rare autosomal recessive disorder resulting from a constitutional lack of homogentisate 1,2-dioxygenase (HGD) with the consequent accumulation of homogentisic acid (HGA). In ochronosis, HGA undergoes autoxidation as well as enzymatic oxidation to form an ochronotic pigment that accumulates in cartilage and connective tissues. In the beginning, there is homogentisic aciduria and pigmentation of cartilages and other connective tissues. In later years, generalized osteoarthritis of the spine and large joints, termed ochronotic arthropathy, develops. RECENT FINDINGS: The diagnosis is confirmed by quantitative measurement of HGA in urine and mutation analysis of the HGD gene. One of the differential diagnoses for the skin findings is exogenous ochronosis, a limited hyperpigmentation of skin caused by some chemicals. As for the lumbar spine findings, there can be radiographic similarities with ankylosing spondylitis (AS) including reduced intervertebral disc spaces and loss of lumbar lordosis; however, ochronosis will spare the sacroiliac joint, and the lumbar spine will show dense, wafer-like disk calcification with a vacuum disc phenomenon and broad syndesmophytes. Here, we present a case of a patient with probable ochronosis that was treated many years as ankylosing spondylitis without response, and we provide a review of the current literature on ochronosis pathogenesis, diagnosis, and treatment.

Paget's Disease of Bone: Osteoimmunology and Osteoclast Pathology.

PURPOSE OF REVIEW: The purpose of this review is to recognize clinical features of Paget's disease of bone and to describe how the osteoclast, a myeloid-derived cell responsible for bone resorption, contributes to the disease. RECENT FINDINGS: Recent studies have identified several variants in SQSTM1, OPTN, and other genes that may predispose individuals to Paget's disease of bone; studies of these genes and their protein products have elucidated new roles for these proteins in bone physiology. Understanding the pathologic mechanisms in the Pagetic osteoclast may lead to the identification of future treatment targets for other inflammatory and autoimmune diseases characterized by abnormal bone erosion and/or osteoclast activation.

Hepatitis C virus-specific cellular immune responses in sustained virological responders with viral persistence in peripheral blood mononuclear cells.

BACKGROUND & AIMS: Hepatitis C virus (HCV)-RNA detection in peripheral blood mononuclear cells (PBMCs) after recovery from HCV infection, is a type of occult HCV infection although is unclear how the viral persistence in PBMCs affects HCV-specific T-cell responses. The aim of this study was to investigate if cellular immune responses are modified by HCV persistence in PBMCs. METHODS: HCV-specific CD4(+) and CD8(+) T-cell responses against six HCV peptides, situated within the non-structural (NS) proteins NS3, NS4b and NS5b, were measured by flow cytometry-through intracellular detection of gamma interferon (IFN-γ) or interleukin 4 (IL-4) and CD69 expression- in 27 sustained virological responders (SVR): 13 with and 14 without occult HCV infection in PBMCs, detected by strand-specific real-time PCR. Ten healthy individuals and 14 chronically infected patients with viraemia, were included as controls. RESULTS: SVR without occult infection showed a higher percentage of activated CD4(+) cells against peptides belonging to NS3 (p124, p153) and NS5b (p257, p294), activated CD8(+) cells against NS3 (p124, p153, p158) and NS5b-p294, as well as an elevated percentage of CD4(+) cells releasing IFN-γ + IL-4 against NS3-p153, and by CD8(+) cells against NS3 (p124, p153). SVR without occult infection showed a higher percentage of activation and release of IFN-γ + IL-4 by both cell subpopulations than the two group of controls, in contrast to SVR with occult infection. CONCLUSION: The lower HCV-specific T-cell response found in SVR with occult infection indicates that the immune response may be impaired when the virus persists in PBMCs.

Downstream of the bioreactor: advancements in recovering fuels and commodity chemicals.

Downstream processing aims at recovering the target product at the required specifications from the bioreactor effluent. Research and development in this field relies on experimental and mathematical tools at the levels of chemical components, unit operations and processes. Recently, advances have been made in addressing the broth mixture complexity early on, in incorporating high-throughput experimentation for data generation and mechanistic understanding of the separation processes, in improving the materials and scalability of specific unit operations, as well as establishing the potential of process integration concepts. Further developments are expected considering the variety of (non-sugar) feedstocks currently under research, the need to transition to renewable energy sources, and the opportunities for improved scale-up through initiatives as Big Data and digital manufacturing.

Microbial Hydrocarbon Formation from Biomass.

Fossil carbon sources mainly contain hydrocarbons, and these are used on a huge scale as fuel and chemicals. Producing hydrocarbons from biomass instead is receiving increased attention. Achievable yields are modest because oxygen atoms need to be removed from biomass, keeping only the lighter carbon and hydrogen atoms. Microorganisms can perform the required conversions, potentially with high selectivity, using metabolic pathways that often end with decarboxylation. Metabolic and protein engineering are used successfully to achieve hydrocarbon production levels that are relevant in a biorefinery context. This has led to pilot or demo processes for hydrocarbons such as isobutene, isoprene, and farnesene. In addition, some non-hydrocarbon fermentation products are being further converted into hydrocarbons using a final chemical step, for example, ethanol into ethene. The main advantage of direct microbial production of hydrocarbons, however, is their potentially easy recovery because they do not dissolve in fermentation broth.

Integration of Gas Enhanced Oil Recovery in Multiphase Fermentations for the Microbial Production of Fuels and Chemicals.

In multiphase fermentations where the product forms a second liquid phase or where solvents are added for product extraction, turbulent conditions disperse the oil phase as droplets. Surface-active components (SACs) present in the fermentation broth can stabilize the product droplets thus forming an emulsion. Breaking this emulsion increases process complexity and consequently the production cost. In previous works, it has been proposed to promote demulsification of oil/supernatant emulsions in an off-line batch bubble column operating at low gas flow rate. The aim of this study is to test the performance of this recovery method integrated to a fermentation, allowing for continuous removal of the oil phase. A 500 mL bubble column is successfully integrated with a 2 L reactor during 24 h without affecting cell growth or cell viability. However, higher levels of surfactants and emulsion stability are measured in the integrated system compared to a base case, reducing its capacity for oil recovery. This is related to release of SACs due to cellular stress when circulating through the recovery column. Therefore, it is concluded that the gas bubble-induced oil recovery method allows for oil separation and cell recycling without compromising fermentation performance; however, tuning of the column parameters considering increased levels of SACs due to cellular stress is required for improving oil recovery.

Enfermedad relacionada a IgG4. Serie clínica de pacientes chilenos / IGG4-related disease. Report of 52 patients

BACKGROUND: IgG4-related disease (IgG4 RD) is an immune-mediated fibro-inflammatory disorder, with tissue infiltration of IgG4+ plasma cells. It causes pseudotumors, tumors, and a wide spectrum of clinical manifestations. AIM: To report the clinical, laboratory, histopathological and treatment characteristics of a group of Chilean patients with IgG4 RD. MATERIAL AND METHODS: Review of medical records of 52 patients aged 18 to 76 years with IgG4 RD seen at six medical centers. RESULTS: Elevated IgG4 serum levels (> 135 mg/dl) were found in 18 of 44 (41%) patients. There was histological confirmation of the disease in 46 patients. The most common sites of involvement were lungs, eyes and kidneys. Eighteen (35%) patients had only one organ involved, 34 (65%) patients had two organs and 13 (25%) patients had three or more organs. The involvement of two organs was significantly more common in men (p < 0.05). In patients with only one organ involvement, the most frequent location was orbital and meningeal. All patients with kidney or lung disease had multiorgan involvement. All patients received corticosteroid therapy, 67% synthetic immunosuppressants, and 16% rituximab. CONCLUSIONS: ER-IgG4 can affect any tissue. Multiorgan involvement was more common in this series, with preference for lungs, eyes and kidneys. An excellent response to steroids is characteristic of the disease, but with a high relapse rate that requires additional immunosuppression.

Recent advances in the microbial production and recovery of apolar molecules.

Several apolar molecules of interest for the production of fuels and chemicals can nowadays be produced by fermentation. Those secreted from the microbial cell are of particular interest for large scale bioprocessing, since they allow for cell reuse, in situ product recovery and competitive production levels. So far, however, bioprocess strategies for fermentation and product recovery have been developed for addressing needs at the laboratory scale, rather than the process scale. Most commonly used strategies include extractive fermentations, product stripping in the gas phase, and off-line de-emulsification followed by intensive centrifugation. At the same time, current techno-economic studies at process scale have demonstrated the absolute need for significant improvements in both microorganism and process technology, for these processes to become competitive.

Feasibility study of an alkaline-based chemical treatment for the purification of polyhydroxybutyrate produced by a mixed enriched culture.

This study focused on investigating the feasibility of purifying polyhydroxybutyrate (PHB) from mixed culture biomass by alkaline-based chemical treatment. The PHB-containing biomass was enriched on acetate under non-sterile conditions. Alkaline treatment (0.2 M NaOH) together with surfactant SDS (0.2 w/v% SDS) could reach 99% purity, with more than 90% recovery. The lost PHB could be mostly attributed to PHB hydrolysis during the alkaline treatment. PHB hydrolysis could be moderated by increasing the crystallinity of the PHB granules, for example, by biomass pretreatment (e.g. freezing or lyophilization) or by effective cell lysis (e.g. adjusting alkali concentration). The suitability of the purified PHB by alkaline treatment for polymer applications was evaluated by molecular weight and thermal stability. A solvent based purification method was also performed for comparison purposes. As result, PHB produced by mixed enriched cultures was found suitable for thermoplastic applications when purified by the solvent method. While the alkaline method resulted in purity, recovery yield and molecular weight comparable to values reported in literature for PHB produced by pure cultures, it was found unsuitable for thermoplastic applications. Given the potential low cost and favorable environmental impact of this method, it is expected that PHB purified by alkaline method may be suitable for other non-thermal polymer applications, and as a platform chemical.

Breaking oil-in-water emulsions stabilized by yeast.

Several biotechnological processes can show an undesirable formation of emulsions making difficult phase separation and product recovery. The breakup of oil-in-water emulsions stabilized by yeast was studied using different physical and chemical methods. These emulsions were composed by deionized water, hexadecane and commercial yeast (Saccharomyces cerevisiae). The stability of the emulsions was evaluated varying the yeast concentration from 7.47 to 22.11% (w/w) and the phases obtained after gravity separation were evaluated on chemical composition, droplet size distribution, rheological behavior and optical microscopy. The cream phase showed kinetic stability attributed to mechanisms as electrostatic repulsion between the droplets, a possible Pickering-type stabilization and the viscoelastic properties of the concentrated emulsion. Oil recovery from cream phase was performed using gravity separation, centrifugation, heating and addition of demulsifier agents (alcohols and magnetic nanoparticles). Long centrifugation time and high centrifugal forces (2 h/150,000×g) were necessary to obtain a complete oil recovery. The heat treatment (60°C) was not enough to promote a satisfactory oil separation. Addition of alcohols followed by centrifugation enhanced oil recovery: butanol addition allowed almost complete phase separation of the emulsion while ethanol addition resulted in 84% of oil recovery. Implementation of this method, however, would require additional steps for solvent separation. Addition of charged magnetic nanoparticles was effective by interacting electrostatically with the interface, resulting in emulsion destabilization under a magnetic field. This method reached almost 96% of oil recovery and it was potentially advantageous since no additional steps might be necessary for further purifying the recovered oil.

Microbial community-based polyhydroxyalkanoates (PHAs) production from wastewater: Techno-economic analysis and ex-ante environmental assessment.

This work investigates the potential for polyhydroxybutyrate (PHB) production from wastewater, from a techno-economic and an environmental perspective, examining scale-up opportunities and bottlenecks prior to commercialisation. Conceptual process design, economic, environmental impacts and sensitivity analysis are developed for one fermentation process and three downstream processing routes, based on alkali, surfactant-hypochlorite and solvent treatments. Environmentally and cost-wise, the alkali treatment is the most favourable with production costs of 1.40€/kg PHB, global warming potential of 2.4kgCO2-eq/kg PHB and non-renewable energy use of 106MJ/kg PHB. The solvent-based process yields the highest costs and environmental burdens: 1.95€/kg PHB, 4.30kgCO2-eq/kg PHB and 156MJ/kg PHB. The production of PHB from wastewater is identified as an interesting alternative to pure culture-polyhydroxyalkanoates production from sugars. However, these results are not yet competitive with those for the petrochemical counterparts. Additional performance improvements may be possible, through process integration and optimisation.

Fermentation broth components influence droplet coalescence and hinder advanced biofuel recovery during fermentation.

Developments in synthetic biology enabled the microbial production of long chain hydrocarbons, which can be used as advanced biofuels in aviation or transportation. Currently, these fuels are not economically competitive due to their production costs. The current process offers room for improvement: by utilizing lignocellulosic feedstock, increasing microbial yields, and using cheaper process technology. Gravity separation is an example of the latter, for which droplet growth by coalescence is crucial. The aim of this study was to study the effect of fermentation broth components on droplet coalescence. Droplet coalescence was measured using two setups: a microfluidic chip and regular laboratory scale stirred vessel (2 L). Some fermentation broth components had a large impact on droplet coalescence. Especially components present in hydrolysed cellulosic biomass and mannoproteins from the yeast cell wall retard coalescence. To achieve a technically feasible gravity separation that can be integrated with the fermentation, the negative effects of these components on coalescence should be minimized. This could be achieved by redesign of the fermentation medium or adjusting the fermentation conditions, aiming to minimize the release of surface active components by the microorganisms. This way, another step can be made towards economically feasible advanced biofuel production.

Microbial advanced biofuels production: overcoming emulsification challenges for large-scale operation.

Isoprenoids and alkanes produced and secreted by microorganisms are emerging as an alternative biofuel for diesel and jet fuel replacements. In a similar way as for other bioprocesses comprising an organic liquid phase, the presence of microorganisms, medium composition, and process conditions may result in emulsion formation during fermentation, hindering product recovery. At the same time, a low-cost production process overcoming this challenge is required to make these advanced biofuels a feasible alternative. We review the main mechanisms and causes of emulsion formation during fermentation, because a better understanding on the microscale can give insights into how to improve large-scale processes and the process technology options that can address these challenges.

Large-scale production of diesel-like biofuels - process design as an inherent part of microorganism development.

Industrial biotechnology is playing an important role in the transition to a bio-based economy. Currently, however, industrial implementation is still modest, despite the advances made in microorganism development. Given that the fuels and commodity chemicals sectors are characterized by tight economic margins, we propose to address overall process design and efficiency at the start of bioprocess development. While current microorganism development is targeted at product formation and product yield, addressing process design at the start of bioprocess development means that microorganism selection can also be extended to other critical targets for process technology and process scale implementation, such as enhancing cell separation or increasing cell robustness at operating conditions that favor the overall process. In this paper we follow this approach for the microbial production of diesel-like biofuels. We review current microbial routes with both oleaginous and engineered microorganisms. For the routes leading to extracellular production, we identify the process conditions for large scale operation. The process conditions identified are finally translated to microorganism development targets. We show that microorganism development should be directed at anaerobic production, increasing robustness at extreme process conditions and tailoring cell surface properties. All the same time, novel process configurations integrating fermentation and product recovery, cell reuse and low-cost technologies for product separation are mandatory. This review provides a state-of-the-art summary of the latest challenges in large-scale production of diesel-like biofuels.