A microfluidic strategy to capture antigen-specific high affinity B cells.

Assessing B cell affinity to pathogen-specific antigens prior to or following exposure could facilitate the assessment of immune status. Current standard tools to assess antigen-specific B cell responses focus on equilibrium binding of the secreted antibody in serum. These methods are costly, time-consuming, and assess antibody affinity under zero-force. Recent findings indicate that force may influence BCR-antigen binding interactions and thus immune status. Here, we designed a simple laminar flow microfluidic chamber in which the antigen (hemagglutinin of influenza A) is bound to the chamber surface to assess antigen-specific BCR binding affinity of five hemagglutinin-specific hybridomas under 65- to 650-pN force range. Our results demonstrate that both increasing shear force and bound lifetime can be used to enrich antigen-specific high affinity B cells. The affinity of the membrane-bound BCR in the flow chamber correlates well with the affinity of the matched antibodies measured in solution. These findings demonstrate that a microfluidic strategy can rapidly assess BCR-antigen binding properties and identify antigen-specific high affinity B cells. This strategy has the potential to both assess functional immune status from peripheral B cells and be a cost-effective way of identifying individual B cells as antibody sources for a range of clinical applications.

Mitigating neutrophil trafficking and cardiotoxicity with DS-IkL in a microphysiological system of a cytokine storm.

A feature of severe COVID-19 is the onset of an acute and intense systemic inflammatory response referred to as the "cytokine storm". The cytokine storm is characterized by high serum levels of inflammatory cytokines and the subsequent transport of inflammatory cells to damaging levels in vital organs (e.g., myocarditis). Immune trafficking and its effect on underlying tissues (e.g., myocardium) are challenging to observe at a high spatial and temporal resolution in mouse models. In this study, we created a vascularized organ-on-a-chip system to mimic cytokine storm-like conditions and tested the effectiveness of a novel multivalent selectin-targeting carbohydrate conjugate (composed of DS - dermatan sulfate and IkL - a selectin-binding peptide, termed DS-IkL) in blocking infiltration of polymorphonuclear leukocytes (PMN). Our data shows that cytokine storm-like conditions induce endothelial cells to produce additional inflammatory cytokines and facilitate infiltration of PMNs into tissue. Treatment of tissues with DS-IkL (60 µM) reduced PMN accumulation in the tissue by >50%. We then created cytokine storm-like conditions in a vascularized cardiac tissue-chip and found that PMN infiltration increases the spontaneous beating rate of the cardiac tissue, and this effect is eliminated by treatment with DS-IkL (60 µM). In summary, we demonstrate the utility of an organ-on-a-chip platform to mimic COVID-19 related cytokine storm and that blocking leukocyte infiltration with DS-IkL could be a viable strategy to mitigate associated cardiac complications.

Convection and extracellular matrix binding control interstitial transport of extracellular vesicles.

Extracellular vesicles (EVs) influence a host of normal and pathophysiological processes in vivo. Compared to soluble mediators, EVs can traffic a wide range of proteins on their surface including extracellular matrix (ECM) binding proteins, and their large size (∼30-150 nm) limits diffusion. We isolated EVs from the MCF10 series-a model human cell line of breast cancer progression-and demonstrated increasing presence of laminin-binding integrins α3ß1 and α6ß1 on the EVs as the malignant potential of the MCF10 cells increased. Transport of the EVs within a microfluidic device under controlled physiological interstitial flow (0.15-0.75 µm/s) demonstrated that convection was the dominant mechanism of transport. Binding of the EVs to the ECM enhanced the spatial concentration and gradient, which was mitigated by blocking integrins α3ß1 and α6ß1. Our studies demonstrate that convection and ECM binding are the dominant mechanisms controlling EV interstitial transport and should be leveraged in nanotherapeutic design.

Considerations on Integrating Prostate-Specific Membrane Antigen Positron Emission Tomography Imaging Into Clinical Prostate Cancer Trials by National Clinical Trials Network Cooperative Groups.

PURPOSE: As prostate-specific membrane antigen (PSMA) positron emission tomography (PET) becomes increasingly available in the United States, the greater sensitivity of the technology in comparison to conventional imaging poses challenges for clinical trials. The NCI Clinical Imaging Steering Committee (CISC) PSMA PET Working Group was convened to coordinate the identification of these challenges in various clinical scenarios and to develop consensus recommendations on how best to integrate PSMA PET into ongoing and upcoming National Clinical Trials Network (NCTN) trials. METHODS: NCI CISC and NCI Genitourinary Steering Committee members and leadership nominated clinicians, biostatisticians, patient advocates, and other imaging experts for inclusion in the PSMA PET Working Group. From April to July 2021, the working group met independently and in conjunction with the CISC to frame challenges, including stage migration, response assessment, trial logistics, and statistical challenges, and to discuss proposed solutions. An anonymous, open-ended survey was distributed to members to collect feedback on challenges faced. Representatives from each NCTN group were invited to present an overview of affected trials. From these discussions, the consensus document was developed and circulated for the inclusion of multiple rounds of feedback from both the Working Group and CISC. RESULTS: The current consensus document outlines the key challenges for clinical prostate cancer trials resulting from the increasing availability of PSMA PET. We discuss implications for patient selection and definition of end points and provide guidance and potential solutions for different clinical scenarios, particularly with regard to best practices in defining eligibility criteria and outcome measures. RECOMMENDATIONS: This article provides guidance regarding clinical trial design and conduct, and the interpretation of trial results.

Organ-on-a-chip model of vascularized human bone marrow niches.

Bone marrow niches (endosteal and perivascular) play important roles in both normal bone marrow function and pathological processes such as cancer cell dormancy. Unraveling the mechanisms underlying these events in humans has been severely limited by models that cannot dissect dynamic events at the niche level. Utilizing microfluidic and stem cell technologies, we present a 3D in vitro model of human bone marrow that contains both the perivascular and endosteal niches, complete with dynamic, perfusable vascular networks. We demonstrate that our model can replicate in vivo bone marrow function, including maintenance and differentiation of CD34+ hematopoietic stem/progenitor cells, egress of neutrophils (CD66b+), and niche-specific responses to doxorubicin and granulocyte-colony stimulating factor. Our platform provides opportunities to accelerate current understanding of human bone marrow function and drug response with high spatial and temporal resolution.

Stem cell therapy: a potential for the perils of pancreatitis.

Acute and chronic pancreatitis carry a significant disease burden and there is no definite treatment that exists for either. They are associated with local and systemic inflammation and lead to numerous complications. Stem cell therapy has been explored for other disease processes and is a topic of research that has gained momentum with regards to implications for acute and chronic pancreatitis. They not only carry the potential to aid in regeneration but also prevent pancreatic injury as well as injury of other organs and hence the resultant complications. Stem cells appear to have immunomodulatory properties and clinical potential as evidenced by numerous studies in animal models. This review article discusses the types of stem cells commonly used and the properties that show promise in the field of pancreatitis.

Critical Issues and Recent Advances in Anticoagulant Therapy: A Review.

As the population is aging, clinicians are coming across more patients with atrial fibrillation and venous thromboembolism requiring anticoagulation to prevent stroke and systemic embolisms. Due to a high prevalence and unfavorable consequences, managing thromboembolic diseases have become areas of clinical concern. Traditional anticoagulants like heparin, low molecular weight heparin and warfarin have been used for the prevention and treatment of venous and arterial thromboses. But, issues of bleeding, parenteral route of administration, or the need for frequent monitoring due to variability in response respectively limit their use. The article gives an overview of coagulation along with existing therapy available for anticoagulation and to present an update on utility and recent advances of new oral anticoagulants (NOACs) beginning from their nomenclature, advantages, disadvantages, precautions and contraindications compared with those of vitamin K antagonists (VKAs) based on a large number of recent studies and clinical trials.

Estimation of the incidence of animal rabies in Punjab, India.

BACKGROUND: Rabies is a devastating zoonotic disease of mammals that causes encephalitis and death. It is endemic in India, with an estimated annual 20,000 human deaths (one-third of the global rabies burden). The magnitude of animal rabies incidence is unknown. METHODS: In four sub-districts of Punjab, India, we monitored canine and livestock populations from August 15, 2016 to August 14, 2017. Demographic, clinical and rabies diagnostic laboratory (RDL) data were collected from suspected cases of rabies. The annual incidence rate / 10,000 animal years at risk (95% CI) in each sub-district was estimated for each species. RESULTS: During 2016-2017, a total of 41 suspected rabies cases were detected in the four selected sub-districts in Punjab. Laboratory confirmed rabies (LCR) incidence was 2.03/10,000 dog years (0.69, 5.96) and 2.71/10,000 dog years (1.14, 6.43) in stray and pet dogs, respectively. The LCR incidence in farmed buffalo and cattle was 0.19/10,000 buffalo years (0.07, 0.57) and 0.23/10,000 cattle years (0.06, 0.88), respectively. The LCR incidence amongst equine was 4.28/10,000 equine years (0.48, 38.10). Stray cattle rabies incidence in the selected sub-districts was 9.49/10,000 cattle years (3.51, 25.67). If similar enhanced surveillance for rabies was conducted state-wide, we estimate that 98 (34-294) buffalo, 18 (2-156) equine, 56 (15-214) farmed cattle, 96 (35-259) stray cattle, 128 (54-303) pet dogs and 62 (21-182) stray dogs would be expected to be confirmed with rabies in Punjab annually. CONCLUSION: These results indicate that rabies incidence in animals, particularly in dogs and stray cattle, is much higher than previously suspected. We recommend that statewide enhanced disease surveillance should be conducted to obtain more accurate estimates of rabies incidence in Punjab to facilitate better control of this important disease.

Characterization of a novel Lytic Polysaccharide Monooxygenase from Malbranchea cinnamomea exhibiting dual catalytic behavior.

A novel Lytic Polysaccharide Monooxygenase (LPMO) family AA9 (PMO9A_MALCI) protein from thermophilic fungus Malbranchea cinnamomea was cloned and expressed in Pichia pastoris. The expressed protein was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. SDS-PAGE analysis showed PMO9A_MALCI to be ~27 kDa protein. High performance anion exchange chromatography and mass spectrometry confirmed that purified protein was active against an array of cellulosic (avicel, carboxy methyl cellulose) and hemicellulosic (birch wood xylan, wheat arabinoxylan and rye arabinoxylan) substrates, releasing both oxidized and unoxidized cello-oligosaccharide and xylo-oligosaccharide products respectively. Presence of double oxidized products during mass spectrometric analysis as well as in-silico analysis confirmed that the expressed protein belongs to Type 3 LPMO family. Molecular dynamic simulations further confirmed the sharing of common amino acid residues conserved for catalysis of both cellulosic and hemicellulosic substrates which further indicates that both substrates are equally preferred. Enzymatic cocktails constituted by replacing a part of commercial cellulase CellicCTec2 with PMO9A_MALCI (9:1/8:2) led to synergistic improvement in saccharification of acid and alkali pretreated biomass. This is the first report on heterologous expression of LPMO from M. cinnamomea, exhibiting catalysis of cellulose and pure xylan.

Clostridium Difficile Infection in Inflammatory Bowel Disease Patients.

BACKGROUND: The rising incidence of Clostridium difficile infection (CDI) in the general population has been recognized by health care organizations worldwide. The emergence of hypervirulent strains has made CDI more challenging to understand and treat. Inflammatory bowel disease (IBD) patients are at higher risk of infection, including CDI. OBJECTIVE: A diagnostic approach for recurrent CDI has yet to be validated, particularly for IBD patients. Enzyme immunoassay (EIA) for toxins A and B, as well as glutamate dehydrogenase EIA, are both rapid testing options for the identification of CDI. Without a high index of suspicion, it is challenging to initially differentiate CDI from an IBD flare based on clinical evaluation alone. METHODS: Here, we provide an up-to-date review on CDI in IBD patients. When caring for an IBD patient with suspected CDI, it is appropriate to empirically treat the presumed infection while awaiting further test results. RESULTS: Treatment with vancomycin or fidaxomicin, but not oral metronidazole, has been advocated by an expert review from the clinical practice update committee of the American Gastroenterology Association. Recurrent CDI is more common in IBD patients compared to non-IBD patients (32% versus 24%), thus more aggressive treatment is recommended for IBD patients along with early consideration of fecal microbiota transplant. CONCLUSION: Although the use of infliximab during CDI has been debated, clinical experience exists supporting its use in an IBD flare, even with active CDI when needed.

Challenges to human rabies elimination highlighted following a rabies outbreak in bovines and a human in Punjab, India.

In August 2015, a rabies outbreak occurred in bovines in a Punjab village, India; subsequently, a farmer in the same village died of rabies in October 2015. We surveyed farmers to describe the outbreak, the demographics and rabies prophylaxis administered to householders on case farms, and farmers' knowledge of rabies prevention and treatment. We used multiple correspondence analysis to guide investigation of associations between demographics, farm status and rabies knowledge, attitudes and practices. The number of affected bovines was unusually high; 15 cattle and buffalo died on 13 smallholder farms (attack rate 4%). Post-exposure vaccinations were administered to 24 people (median 2 doses). Affected farms had significantly larger households and were more likely to keep their livestock outside (therefore, accessible by stray dogs), suggesting that the impact of the outbreak was disproportionally borne by households of lower socio-economic status. Primary sources of rabies information were friends and neighbours, not health authorities or media. Women who had not received formal education were less likely to have heard of rabies. Although case farm participants were more likely to have heard about rabies from a veterinarian, their knowledge and practices to prevent rabies did not reflect the level expected considering their contact with a health professional; they were more likely to believe that traditional remedies prevent rabies and less likely to tell their children to avoid playing with stray dogs than participants from other farms. This study highlights knowledge dissemination disparities that, if typical of rural locations, could obstruct attempts to eliminate canine-mediated human rabies in India. Therefore, understanding the pervasiveness and influence of traditional medical beliefs on treatment-seeking behaviour, communication structures within villages and the impact of local practices such as carcass disposal on dog populations will be essential to ensure that rabies control strategies are effective in rural India.

Non-Alcoholic Fatty Pancreas Disease (NAFPD): A Silent Spectator or the Fifth Component of Metabolic Syndrome? A Literature Review.

BACKGROUND AND OBJECTIVE: Fat accumulation in the pancreas has remained a relatively unknown disease since it was initially described in 1926. However, it has gained increasing attention in the past two decades with the emergence of the obesity epidemic. Pancreatic steatosis is a general term used for fat accumulation in the pancreas. It is further classified into fatty replacement, fatty infiltration, lipomatous pseudo-hypertrophy, non-alcoholic fatty pancreas disease (NAFPD) and non-alcoholic fatty steatopancreatitis (NASP). NAFPD is defined as obesity-associated accumulation of fat in the pancreas without significant alcohol consumption. Data on the prevalence of NAFPD are limited due to a lack of standardized screening tests. METHODS: MEDLINE/PubMed was searched to find relevant studies and abstracts on pancreatic steatosis. RESULTS: Pancreatic fat can be quantified by various imaging techniques including ultrasonography, computed tomography, magnetic resonance imaging and magnetic resonance spectroscopy. The pathophysiology of NAFPD has not been completely understood. Chronic exposure of ß-cells to hyperglycemia and higher levels of free fatty acids results in increased intracellular triglyceride accumulation, which ultimately causes reduced insulin secretion, insulin resistance, cell apoptosis and subsequent fatty replacement. This vicious cycle likely is a determining factor in the development of diabetes mellitus and metabolic syndrome. There is no approved pharmacologic therapy for NAFPD. Caloric restriction might have a role in normalization of ß-cell function by reducing pancreatic fat content. Troglitazone (blend of telmisartan and sitagliptin) has demonstrated effectiveness in animal models but is still in experimental stages. CONCLUSION: The cause and effect relationship between the metabolic syndrome and NAFPD has not yet been established. Further studies are required to study the effect of NAFPD on glucose hemostasis.

Producing methane, methanol and electricity from organic waste of fermentation reaction using novel microbes.

Residual solid and liquid streams from the one-pot CRUDE (Conversion of Raw and Untreated Disposal into Ethanol) process were treated with two separate biochemical routes for renewable energy transformation. The solid residual stream was subjected to thermophilic anaerobic digestion (TAD), which produced 95 ±â€¯7 L methane kg-1 volatile solid with an overall energy efficiency of 12.9 ±â€¯1.7%. A methanotroph, Methyloferula sp., was deployed for oxidation of mixed TAD biogas into methanol. The residual liquid stream from CRUDE process was used in a Microbial Fuel Cell (MFC) to produce electricity. Material balance calculations confirmed the integration of biochemical routes (i.e. CRUDE, TAD, and MFC) for developing a sustainable approach of energy regeneration. The current work demonstrates the utilization of different residual streams originated after food waste processing to release minimal organic load to the environment.

Expression of catalytically efficient xylanases from thermophilic fungus Malbranchea cinnamomea for synergistically enhancing hydrolysis of lignocellulosics.

In this study, two xylanase genes (GH10 and GH11) derived from Malbranchea cinnamomea, designated as XYN10A_MALCI and XYN11A_MALCI, respectively, were expressed in Pichia pastoris X33. The maximum level of xylanase expression was found to be 24.3U/ml for rXYN10A_MALCI and 573.32U/ml for rXYN11A_MALCI. The purified recombinant rXYN11A_MALCI was stable at 70°C and catalytically active against a variety of substituted (arabinoxylans) as well as unsubstituted xylans. The hydrolytic potential of recombinant xylanases for enhancing the hydrolysis of acid/alkali pretreated lignocellulosics (rice straw and bagasse) by the commercial cellulase Cellic CTec2 was assessed which revealed that both rXYN10A_MALCI and rXYN11A_MALCI act synergistically with commercial cellulases and resulted in 1.54 and 1.58 folds improved hydrolysis of acid treated rice straw and alkali treated rice straw using cocktail comprising of Cellic CTec2 and XYN11A_MALCI (8:2 ratio) when compared to Cellic CTec2 alone at same protein loading rate of (∼5.7mg/g biomass).

Infliximab use in ulcerative colitis flare with clostridium difficile infection: A report of two cases and literature review.

Clostridium difficile infection (CDI) is a major cause of morbidity and mortality in patients with inflammatory bowel disease (IBD), especially in ulcerative colitis (UC). The incidence and severity of CDI in IBD has shown an increasing trend in the last two decades. Patients with IBD are predisposed to CDI secondary to the recurrent use of antibiotics, corticosteroids, and immunosuppressants and secondary to dysbiosis. It is clinically challenging to distinguish the symptoms of CDI from an IBD flare. The worsening of IBD symptoms demands escalation of steroids or initiation of biologics. However, the management of CDI in IBD, not responding to antibiotics, is not well described beyond a few case reports. We report two cases of CDI with active UC flare. The patients did not respond to antibiotics or intravenous corticosteroids but had rapid resolution of CDI symptoms after receiving infliximab infusion. The optimal dosing and infusion frequency of infliximab in management of CDI in UC/IBD remains to be established.

Vascularization and innervation of slits within polydimethylsiloxane sheets in the subcutaneous space of athymic nude mice.

Success of cell therapy in avascular sites will depend on providing sufficient blood supply to transplanted tissues. A popular strategy of providing blood supply is to embed cells within a functionalized hydrogel implanted within the host to stimulate neovascularization. However, hydrogel systems are not always amenable for removal post-transplantation; thus, it may be advantageous to implant a device that contains cells while also providing access to the circulation so retrieval is possible. Here we investigate one instance of providing access to a vessel network, a thin sheet with through-cut slits, and determine if it can be vascularized from autologous materials. We compared the effect of slit width on vascularization of a thin sheet following subcutaneous implantation into an animal model. Polydimethylsiloxane sheets with varying slit widths (approximately 150, 300, 500, or 1500 µm) were fabricated from three-dimensional printed molds. Subcutaneous implantation of sheets in immunodeficient mice revealed that smaller slit widths have evidence of angiogenesis and new tissue growth, while larger slit widths contain native mature tissue squeezing into the space. Our results show that engineered slit sheets may provide a simple approach to cell transplantation by providing a prevascularized and innervated environment.

Hepatocyte transplantation: Consider infusion before incision.

Human hepatocyte transplantation is undergoing study as a bridge, or even alternative, to orthotopic liver transplantation (OLT). This technique has undergone multiple developments over the past thirty years in terms of mode of delivery, source and preparation of cell cultures, monitoring of graft function, and use of immunosuppression. Further refinements and improvements in these techniques will likely allow improved graft survival and function, granting patients higher yield from this technique and potentially significantly delaying need for OLT.

Elderly donor graft for liver transplantation: Never too late.

The definitive treatment for end stage liver disease remains a liver transplant and hence livers are needed for these patients along with cases of acute fulminant liver failure. Hence livers are a scarce and highly valuable commodity in the current time. By extending the pool of donors to include the elderly livers, it allows for increased availability of donors and reduces the mortality that is associated with the waiting list itself. There is an increasing prevalence of end stage liver disease due to conditions like chronic hepatitis B and C, non-alcoholic steatohepatitis, alcoholic liver disease. Many studies show non-inferior outcomes when elderly livers are used as a vigorous selection process is implemented. The process takes into account the characteristics of the donor, graft and recipient allowing for appropriate donor-recipient coupling. To meet the increasing demands of livers, elderly donors should be utilized for liver transplantation. The aim of this review article is to describe the aging process of the liver and the outcomes associated with use of elderly livers for transplantation.