Potential use of serum-derived bovine immunoglobulin/protein isolate for the management of COVID-19.

COVID-19 manifests as a mild disease in most people but can progress to severe disease in nearly 20% of individuals. Disease progression is likely driven by a cytokine storm, either directly stimulated by SARS-CoV-2 or by increased systemic inflammation in which the gut might play an integral role. SARS-CoV-2 replication in the gut may cause increased intestinal permeability, alterations to the fecal microbiome, and increased inflammatory cytokines. Each effect may lead to increased systemic inflammation and the transport of cytokines and inflammatory antigens from the gut to the lung. Few interventions are being studied to treat people with mild disease and prevent the cytokine storm. Serumderived bovine immunoglobulin/protein isolate (SBI) may prevent progression by (1) binding and neutralizing inflammatory antigens, (2) decreasing gut permeability, (3) interfering with ACE2 binding by viral proteins, and (4) improving the fecal microbiome. SBI is therefore a promising intervention to prevent disease progression in COVID-19 patients.

Serum Bovine Immunoglobulins Improve Inflammation and Gut Barrier Function in Persons with HIV and Enteropathy on Suppressive ART.

BACKGROUND: Systemic inflammation persists in chronic HIV infection and is associated with increased rates of non-AIDS events such as cardiovascular and liver disease. Increased gut permeability and systemic exposure to microbial products are key drivers of this inflammation. Serum-derived bovine immunoglobulin/protein isolate (SBI) supports gut healing in other conditions such as inflammatory bowel disease. METHODS: In this randomized, double-blind study, participants receiving suppressive antiretroviral therapy (ART) with chronic diarrhea received placebo or SBI at 2.5 g BID or 5 g BID for 4 weeks, followed by a 20-week placebo-free extension phase with SBI at either 2.5 or 5 g BID. Intestinal fatty acid binding protein (I-FABP), zonulin, flagellin, lipopolysaccharide (LPS) and LPS-binding protein, and inflammatory markers were measured by ELISA or multiplex assays. Non-parametric tests were used for analysis. RESULTS: One hundred three participants completed the study. By week 24 SBI significantly decreased circulating levels of I-FABP (-0.35 ng/µL, P=0.002) and zonulin (-4.90 ng/µL, P=0.003), suggesting improvement in gut damage, and interleukin-6 (IL-6) (-0.40 pg/µL, P=0.002), reflecting improvement in systemic inflammation. In participants with the lowest quartile of CD4+ T-cell counts at baseline (189-418 cells/µL), CD4+ T-cell counts increased significantly (26 cells/µL; P=0.002). CONCLUSIONS: Oral SBI may decrease inflammation and warrants further exploration as a potential strategy to improve gut integrity and decrease systemic inflammation among persons receiving prolonged suppressive ART.

Evaluation of oral serum-derived bovine immunoglobulins in HIV-infected patients with chronic idiopathic diarrhea.

Objectives To evaluate serum-derived bovine immunoglobulin/protein isolate (SBI) for safety and impact on gastrointestinal (GI) symptoms in HIV patients with chronic idiopathic diarrhea. Methods A multi-center trial comprised of a double-blind, placebo (PBO)-controlled lead-in phase, (participants received PBO or SBI at 2.5 or 5.0 g BID for 4 weeks) followed by a 20-week, PBO-free phase (SBI at either 2.5 or 5.0 g BID). Participants included HIV-infected patients who were virologically suppressed with a history of chronic idiopathic diarrhea, defined as > 3 loose stools per day for ≥ 3 months without an identifiable cause. Safety was evaluated by monitoring adverse events (AEs) and clinical laboratory testing. Health status and changes in GI symptoms were assessed using validated questionnaires. Results SBI was well tolerated by the 103 participants with only 2 withdrawals due to AEs potentially associated with SBI. Mean number of daily unformed stools decreased from about 4 at baseline to less than 2 by week 4 for all study groups. Improvements in several other GI symptoms were also reported. Comparison of the PBO group to SBI groups showed no significant differences, although both SBI cohorts reported significantly improved health status scores. GI symptom improvements were maintained throughout the 20-week PBO-free phase. Conclusions Oral SBI is safe and well tolerated at the doses studied in HIV patients with chronic diarrhea. No conclusions could be drawn regarding impact on GI symptoms. Additional studies are ongoing to examine the biological and immunologic effects of SBI in virologically suppressed HIV-infected patients.

Absorption and safety of serum-derived bovine immunoglobulin/protein isolate in healthy adults.

PURPOSE: Previous studies have shown that oral administration of bovine immunoglobulin protein preparations is safe and provides nutritional and intestinal health benefits. The purpose of this study was to evaluate the plasma amino acid response following a single dose of serum-derived bovine immunoglobulin/protein isolate (SBI) and whether bovine immunoglobulin G (IgG) is present in stool or in blood following multiple doses of SBI in healthy volunteers. METHODS: A total of 42 healthy adults were administered a single dose of placebo or SBI at one of three doses (5 g, 10 g, or 20 g) in blinded fashion and then continued on SBI (2.5 g, 5 g, or 10 g) twice daily (BID) for an additional 2 weeks. Serial blood samples were collected for amino acid analysis following a single dose of placebo or SBI. Stool and blood samples were collected to assess bovine IgG levels. RESULTS: The area under the curve from time 0 minute to 180 minutes for essential and total amino acids as well as tryptophan increased following ingestion of 5 g, 10 g, or 20 g of SBI, with a significant difference between placebo and all doses of SBI (p<0.05) for essential amino acids and tryptophan but only the 10 g and 20 g doses for total amino acids. Bovine IgG was detected in the stool following multiple doses of SBI. No quantifiable levels of bovine IgG were determined in plasma samples 90 minutes following administration of a single dose or multiple doses of SBI. CONCLUSION: Oral administration of SBI leads to increases in plasma essential amino acids during transit through the gastrointestinal tract and is safe at levels as high as 20 g/day.

Attenuation of Colitis by Serum-Derived Bovine Immunoglobulin/Protein Isolate in a Defined Microbiota Mouse Model.

BACKGROUND: The pathogenesis of inflammatory bowel disease (IBD) is complex and multifaceted including genetic predisposition, environmental components, microbial dysbiosis, and inappropriate immune activation to microbial components. Pathogenic bacterial provocateurs like adherent and invasive E. coli have been reported to increase susceptibility to Crohn's disease. Serum-derived bovine immunoglobulin/protein isolate (SBI) is comprised primarily of immunoglobulins (Igs) that bind to conserved microbial components and neutralize exotoxins. AIM: To demonstrate that oral administration of SBI may modulate mucosal inflammation following colonization with E. coli, LF82, and exposure to dextran sodium sulfate (DSS). METHODS: Defined microbiota mice harboring the altered Schaedler flora (ASF) were administered SBI or hydrolyzed collagen twice daily starting 7 days prior to challenge with E. coli LF82 and continuing for the remainder of the experiment. Mice were treated with DSS for 7 days and then evaluated for evidence of local and peripheral inflammation. RESULTS: Igs within SBI bound multiple antigens from all eight members of the ASF and E. coli LF82 by western blot analysis. Multiple parameters of LF82/DSS-induced colitis were reduced following administration of SBI, including histological lesion scores, secretion of cytokines and chemokines from cecal biopsies, intestinal fatty acid binding protein (I-FABP) and serum amyloid A from plasma. CONCLUSIONS: Oral administration of SBI attenuated clinical signs of LF82/DSS-induced colitis in mice. The data are consistent with the hypothesis that SBI immunoglobulin binding of bacterial antigens in the intestinal lumen may inhibit the inflammatory cascades that contribute to IBD, thus attenuating DSS-induced colitis.

Bovine immunoglobulin/protein isolate binds pro-inflammatory bacterial compounds and prevents immune activation in an intestinal co-culture model.

Intestinal barrier dysfunction is associated with chronic gastrointestinal tract inflammation and diseases such as IBD and IBS. Serum-derived bovine immunoglobulin/protein isolate (SBI) is a specially formulated protein preparation (>90%) for oral administration. The composition of SBI is greater than 60% immunoglobulin including contributions from IgG, IgA, and IgM. Immunoglobulin within the lumen of the gut has been recognized to have anti-inflammatory properties and is involved in maintaining gut homeostasis. The binding of common intestinal antigens (LPS and Lipid A) and the ligand Pam3CSK4, by IgG, IgA, and IgM in SBI was shown using a modified ELISA technique. Each of these antigens stimulated IL-8 and TNF-α cytokine production by THP-1 monocytes. Immune exclusion occurred as SBI (≤50 mg/mL) bound free antigen in a dose dependent manner that inhibited cytokine production by THP-1 monocytes in response to 10 ng/mL LPS or 200 ng/mL Lipid A. Conversely, Pam3CSK4 stimulation of THP-1 monocytes was unaffected by SBI/antigen binding. A co-culture model of the intestinal epithelium consisted of a C2BBe1 monolayer separating an apical compartment from a basal compartment containing THP-1 monocytes. The C2BBe1 monolayer was permeabilized with dimethyl palmitoyl ammonio propanesulfonate (PPS) to simulate a damaged epithelial barrier. Results indicate that Pam3CSK4 was able to translocate across the PPS-damaged C2BBe1 monolayer. However, binding of Pam3CSK4 by immunoglobulins in SBI prevented Pam3CSK4 translocation across the damaged C2BBe1 barrier. These results demonstrated steric exclusion of antigen by SBI which prevented apical to basal translocation of antigen due to changes in the physical properties of Pam3CSK4, most likely as a result of immunoglobulin binding. This study demonstrates that immunoglobulins in SBI can reduce antigen-associated inflammation through immune and steric exclusion mechanisms and furthers the mechanistic understanding of how SBI might improve immune status and reduce inflammation in various intestinal disease states.

Use of a centrifugal bioreactor for cartilaginous tissue formation from isolated chondrocytes.

Although a centrifugal bioreactor (CCBR) supports high-density mammalian suspension cell cultures by balancing drag, buoyancy, and centrifugal forces, to date anchorage-dependent cultures have not been tried. Also, steady or intermittent hydrostatic pressures of 8 to 500 kPa, and shears of 0.02 to 1.4 N/m(2) can be simultaneously applied in the CCBR. This article demonstrates the use of a CCBR to stimulate chondrogenesis in a high-density culture. At 3 weeks, histological results show even distribution of glycosaminoglycan (GAG) and collagen, with 1,890 ± 270 cells/mm(2) cell densities that exceed those of 1,470 ± 270 in pellet cultures. Analysis of collagen content reveals similar levels for all treatment groups; 6.8 ± 3.5 and 5.0 ± 0.4 µg collagen/µg DNA for 0.07 and 0.26 MPa CCBR cultures, respectively, in contrast to 6.6 ± 1.9 values for control pellet cultures. GAG levels of 5.6 ± 1.5 and 4.1 ± 0.9 µg GAG /µg DNA are present for cultures stressed at 0.07 and 0.26 MPa, respectively, in comparison to control pellet cultures at the 8.4 ± 0.9 level. Although results to date have not revealed mechanical stress combinations that stimulate chondrogenesis over unstressed controls, system advantages include continuous culture at cell densities above those in the pellet, precise medium control, the ability to independently vary multiple mechanical stresses over a broad range, and the flexibility for integration of scaffold features for future chondrogenesis stimulation studies.

Polyelectrolyte multilayers in tissue engineering.

The layer-by-layer assembly of sequentially adsorbed, alternating polyelectrolytes has become increasingly important over the past two decades. The ease and versatility in assembling polyelectrolyte multilayers (PEMs) has resulted in numerous wide ranging applications of these materials. More recently, PEMs are being used in biological applications ranging from biomaterials, tissue engineering, regenerative medicine, and drug delivery. The ability to manipulate the chemical, physical, surface, and topographical properties of these multilayer architectures by simply changing the pH, ionic strength, thickness, and postassembly modifications render them highly suitable to probe the effects of external stimuli on cellular responsiveness. In the field of regenerative medicine, the ability to sequester growth factors and to tether peptides to PEMs has been exploited to direct the lineage of progenitor cells and to subsequently maintain a desired phenotype. Additional novel applications include the use of PEMs in the assembly of three-dimensional layered architectures and as coatings for individual cells to deliver tunable payloads of drugs or bioactive molecules. This review focuses on literature related to the modulation of chemical and physical properties of PEMs for tissue engineering applications and recent research efforts in maintaining and directing cellular phenotype in stem cell differentiation.

Engineered three-dimensional liver mimics recapitulate critical rat-specific bile acid pathways.

A critical hepatic function is the maintenance of optimal bile acid (BA) compositions to achieve cholesterol homeostasis. BAs are rarely quantified to assess hepatic phenotype in vitro since existing analytical techniques have inadequate resolution. We report a detailed investigation into the biosynthesis and homeostasis of eight primary rat BAs in conventional in vitro hepatocyte cultures and in an engineered liver mimic. The three-dimensional (3D) liver mimic was assembled with layers of primary rat hepatocytes and liver sinusoidal endothelial cells. A high-pressure liquid chromatography and mass spectrometry technique was developed with a detection limit of 1 ng/mL for each BA, which is significantly lower than previous approaches. Over a 2-week culture, only 3D liver mimics exhibited the ratio of conjugated cholic acid to chenodeoxycholic acid that has been observed in vivo. This ratio, an important marker of BA homeostasis, was significantly higher in stable collagen sandwich cultures indicating significant deviation from physiological behavior. The biosynthesis of tauro-ß-muricholic acid, a key primary rat BA, doubled only in the engineered liver mimics while decreasing in the other systems. These trends demonstrate that the 3D liver mimics provide a unique platform to study hepatic metabolism.

Fluid flow through a high cell density fluidized-bed during centrifugal bioreactor culture.

An increasing demand for products such as tissues, proteins, and antibodies from mammalian cell suspension cultures is driving interest in increasing production through high-cell density bioreactors. The centrifugal bioreactor (CCBR) retains cells by balancing settling forces with surface drag forces due to medium throughput and is capable of maintaining cell densities above 10(8) cells/mL. This article builds on a previous study where the fluid mechanics of an empty CCBR were investigated showing fluid flow is nonuniform and dominated by Coriolis forces, raising concerns about nutrient and cell distribution. In this article, we demonstrate that the previously reported Coriolis forces are still present in the CCBR, but masked by the presence of cells. Experimental dye injection observations during culture of 15 microm hybridoma cells show a continual uniform darkening of the cell bed, indicating the region of the reactor containing cells is well mixed. Simulation results also indicate the cell bed is well mixed during culture of mammalian cells ranging in size from 10 to 20 microm. However, simulations also allow for a slight concentration gradient to be identified and attributed to Coriolis forces. Experimental results show cell density increases from 0.16 to 0.26 when centrifugal force is doubled by increasing RPM from 650 to 920 at a constant inlet velocity of 6.5 cm/s; an effect also observed in the simulation. Results presented in this article indicate cells maintained in the CCBR behave as a high-density fluidized bed of cells providing a homogeneous environment to ensure optimal growth conditions.

Kinetic simulation of a centrifugal bioreactor for high population density hybridoma culture.

Demand for increasingly complex post-translationally modified proteins, such as monoclonal antibodies (mAbs), necessitates the use of mammalian hosts for production. The focus of this article is a continuous centrifugal bioreactor (CCBR) capable of increasing volumetric productivity for mAb production through high density hybridoma culture, exceeding 10(8) cells/mL. At these extreme densities, environmental conditions such as substrate and inhibitor concentrations rapidly change dramatically affecting the growth rate. The development of a kinetic model predicting glucose, mAb, lactate, and ammonium concentrations based on dilution rate and cell density is shown in this article. Additionally, it is found that pH affects both growth rate and viability, and a range of 6.9-7.4 is needed to maintain growth rate above 90% of the maximum. Modeling shows that operating an 11.4 mL CCBR inoculated with 2.0 x 10(7) cells/mL at a dilution rate of 1.3 h(-1), results in a predicted growth rate 82% of the maximum value. At the same dilution rate increasing density to 6.0 x 10(7) cells/mL decreases the predicted growth rate to 60% of the maximum; however, by increasing dilution rate to 6.1 h(-1) the growth rate can be increased to 86% of the maximum. Using the kinetic model developed in this research, the concentration of glucose, mAb, lactate, and ammonium are all predicted within 13% of experimental results. This model and an understanding of how RPM impacts cell retention serve as valuable tools for maintaining high density CCBR cultures, ensuring maximum growth associated mAb production rates.

A study of the Coriolis effect on the fluid flow profile in a centrifugal bioreactor.

Increasing demand for tissues, proteins, and antibodies derived from cell culture is necessitating the development and implementation of high cell density bioreactors. A system for studying high density culture is the centrifugal bioreactor (CCBR), which retains cells by increasing settling velocities through system rotation, thereby eliminating diffusional limitations associated with mechanical cell retention devices. This article focuses on the fluid mechanics of the CCBR system by considering Coriolis effects. Such considerations for centrifugal bioprocessing have heretofore been ignored; therefore, a simpler analysis of an empty chamber will be performed. Comparisons are made between numerical simulations and bromophenol blue dye injection experiments. For the non-rotating bioreactor with an inlet velocity of 4.3 cm/s, both the numerical and experimental results show the formation of a teardrop shaped plume of dye following streamlines through the reactor. However, as the reactor is rotated, the simulation predicts the development of vortices and a flow profile dominated by Coriolis forces resulting in the majority of flow up the leading wall of the reactor as dye initially enters the chamber, results are confirmed by experimental observations. As the reactor continues to fill with dye, the simulation predicts dye movement up both walls while experimental observations show the reactor fills with dye from the exit to the inlet. Differences between the simulation and experimental observations can be explained by excessive diffusion required for simulation convergence, and a slight density difference between dyed and un-dyed solutions. Implications of the results on practical bioreactor use are also discussed.

Engineering the stem cell microenvironment.

Multipotent stem cells in the body facilitate tissue regeneration, growth, and wound healing throughout life. The microenvironment in which they reside provides signals that direct these progenitors to proliferate, differentiate, or remain dormant; these factors include soluble molecules, the extracellular matrix, neighboring cells, and physical stimuli. Recent advances in the culture of embryonic stem cells and adult progenitors necessitate an increased understanding of these phenomena. Here, we summarize the interactions between stem cells and their local environment, drawing on in vivo observations and tissue culture studies. In addition, we describe novel methods of characterizing the effects of various environmental factors and review new techniques that enable scientists and engineers to more effectively direct stem cell fate.

Model-based optimization of a conductive matrix enzyme electrode.

A mathematical model has been developed to describe the mechanism for internal mass transfer and enzyme reaction kinetics of an amperometric conductive matrix enzyme electrode. The model is simplified and solved analytically to arrive at a representation for the response slope in the linear range as well as for the response time. This is the first time that the response time of an enzyme electrode is described by a mathematical model. Simulations give information on how the design parameters influence the performance of the electrode for a glucose oxidase catalyzed sensing reaction process. Based on this information, several designs were constructed and tested showing suitable agreement with theoretical predictions. Finally, an optimized electrode was designed and validated.