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Mycotoxins present in cereals are a worldwide problem and are a result of the presence of mycotoxin producing fungi. A strategy to reduce these fungi and mycotoxin levels in contaminated grains is with the use of lactic acid bacteria (LAB) or Bacillus spp., which can degrade or bind toxins. In this study, LAB and Bacillus spp. were isolated from mycotoxin contaminated wheat grains and, together with additional plant-derived strains, an antifungal screening against Fusarium graminearum was performed. Furthermore, these strains were screened for their ability to reduce zearalenone (ZEA) and deoxynivalenol (DON). Finally, the mode of action of the most promising microorganisms was investigated by analyzing toxin reduction with viable and dead cells, cell extracts and supernatants. Out of 212 tested strains, 70 showed high antifungal activity and 42 exhibited the ability to detoxify more than 90% ZEA, i.e., Bacillus licheniformis (19), B. megaterium (13), and Levilactobacillus brevis (10). None of the tested strains were able to decrease DON. The mode of action of ZEA reduction could not be fully elucidated. Neither dead cells (

The first set of data refers to PAR-2 gene expression with the target gene rbF2rl1 assessed in tenocytes harvested from New Zealand White Rabbits’ Achilles tendons. These tenocytes were stimulated in vitro with 20 ng/mL platelet-derived growth factor-BB (PDGF-BB) and compared to the corresponding cell culture without growth factor PDGF-BB. In addition, three inhibitors were tested. In the presence or absence of 40 µM inhibitor concentration and 5 % fetal bovine serum, the following inhibitors were applied: SB203580 = inhibitor for MAPK; LY-294002 = inhibitor for PI3K; PD153035 = inhibitor for EGFR. As control, gene expression was assessed under DMSO = dimethyl sulfoxide (solvent of the inhibitors) or in medium = basal culture medium (with 10 % fetal bovine serum).The second set of data represents morphological aspects of cytoskeletal reorganization for rabbit Achilles tenocytes stimulated in vitro with 20 ng/mL PDGF-BB compared to the corresponding cell culture without PDGF-BB. Data on cell size, on F-actin immunohistochemical labeling intensity, α-tubulin immunohistochemical labeling intensity and on cell aspect ratio (length of the cell divided by its width) are presented. Moreover, analogous to the first set of data, cytoskeletal rearrangement in the presence or absence of the inhibitors SB203580, LY-294002 and PD153035 in the presence or absence of PDGF-BB were assessed.

This study investigated the impact of the depulping of cocoa beans after pod opening, as well as the influences of pod storage (PS) and fermentation time on the fermentation dynamics and the overall quality of beans and liquors made thereof. Twelve variations were conducted in three experimental runs (with/without depulping; 1-/3-day PS; and fermentation times of 3, 4, 5, 6 or 7 days). Fermentation dynamics (e.g., temperature and pH) and the quality of dried beans (e.g., cut-test and fermentation index) and liquors (sensory assessment, quantification of cocoa key-odorants and tastants) were investigated. It was demonstrated that 17–20% of cocoa pulp, relative to the total bean-pulp-mass weight, could be mechanically removed without negatively affecting the bean quality. No significant differences were found in the percentages of well-fermented beans after 5–6 days fermentation with 1-day PS, resulting in 49 ± 9% with, and 48 ± 12% without depulping. There were no significant differences in key tastants present in the liquors; however, significantly less volatile acids and esters were found when liquors were produced from 5–6 day-fermented depulped beans, with 1-day PS, without negatively affecting the sensory profiles. This strategy allows producers to maximize the cacao fruit’s value by integrating part of the pulp into the cocoa value chain.

Large bone defects after trauma demand for adequate bone substitutes. Bone void fillers should be antibacterial and pro-angiogenic. One viable option is the use of composite materials like the combination of PLGA and amorphous calcium phosphate (aCaP).Copper stimulates angiogenesis and has antibacterial qualities. Either copper oxide (CuO) nanoparticles (NPs) were therefore added to PLGA/aCaP/CuO in different concentrations (1, 5 and 10 w/w %) or copper-doped tricalcium phosphate NPs (TCP with 2% of copper) were electrospun into PLGA/CuTCP nanocomposites.Bi-layered nanocomposites of PLGA/aCaP with different copper NPs (CuO or TCP) and a second layer of pristine PLGA were fabricated. Two clinical bacterial isolates (Staphylococcus aureus and Staphylococcus epidermidis) were used to assess antibacterial properties of the copper-containing materials. For angiogenesis, the chorioallantoic membrane (CAM) assay of the chicken embryo was performed.The higher the CuO content, the higher were the antibacterial properties, with 10 % CuO reducing bacterial adhesion most effectively. Vessel and cell densities were highest in the 5 % CuO containing scaffolds, while tissue integration was more pronounced at lower CuO content. The PLGA/aCaP/CuO (1 % CuO) behaved similar like PLGA/CuTCP in all angiogenic and antibacterial readouts, based on the same copper fraction.We conclude that CuO NPs or CuTCP NPs are useful components to increase angiogenic properties of nanocomposites and at the same time exhibiting antibacterial characteristics.

The impact of pod storage (PS) and two drying temperatures of fermented cocoa beans was investigated in Ecuador. Therefore, four variations were simultaneously carried out three times at two locations, independently: 0, 3, and 5 days of PS, dried at 60 °C and 0 days of PS, dried at 80 °C. Pod weight during storage, pulp content, pH, temperature, microbial counts, total free amino acids, protein profiles, sugars, organic acids, cut-test, fermentation index, and sensory profiles were analyzed. Minor differences in fermentation dynamics and bean quality were found between variations with and without PS. A rather accelerated fermentation with pod-stored beans was observed (e.g., faster color change, slightly lower pH in cotyledon after 48 h), along with a significantly higher maximal temperature during 24–42 h (43.1 ± 3.2 °C compared to 39.2 ± 2.0 °C without PS). More well-fermented beans were reached with PS (52.3 ± 22.6%) than without (62.7 ± 9.2%). Differences during fermentation were observed between the locations (e.g., pH, acids, sugars), but sensory evaluation indicated that the impact of location was mitigated with PS. Drying at 80 °C showed no adverse effects, as evidenced by the results of the cut-test and fermentation index. However, sensory evaluations revealed significant differences between 80 °C and 60 °C, with the former exhibiting more bitter and astringent cocoa liquor.

The current study addresses the critical issue of Listeria monocytogenes growth in raw sausage/meat products leading to human infections, most commonly listeriosis, which is known for its high fatality rate. This research focuses on the isolation, identification, and screening of lactic acid bacteria from various meat and fish products in Switzerland. In total, 274 lactic acid bacteria strains were isolated from 30 different products and were screened for their ability to inhibit Listeria monocytogenes growth, with 51 isolates demonstrating anti-Listeria activity at 8 °C, 15 °C, 25 °C, and 37 °C. Further experiments, using a meat model and a raw sausage challenge test, demonstrated that Leuconostoc carnosum DH25 significantly inhibited Listeria monocytogenes growth during the ripening and storage of the tested meat/sausage. This inhibitory effect was found to be attributed to the bacteriocins produced by Leuconostoc carnosum DH25 rather than factors like pH or water activity. The stability of the anti-Listeria substances was examined, revealing their resistance to temperature and pH changes, making Leuconostoc carnosum DH25 a promising protective culture for raw sausages. The genome sequencing of this strain confirms its safety, with no antibiotic resistance genes or virulence factors detected, and reveals the presence of the structural genes for the production of the bacteriocin LeucocinB-Ta11a. This study underscores the potential of LAB strains and their bacteriocins as effective tools for enhancing food safety and preventing Listeria monocytogenes growth in meat products, offering valuable insights into biocontrol strategies in the food industry.

In Ecuador, various processes are applied during cocoa post-harvesting. This study, therefore, explored fermentation parameters across two locations with 2–7 independent runs, focusing on temperature, microbial counts, pH during fermentation and drying, and their impact on cocoa bean quality. Factors including fermentation devices (jute bags, plastic bags, and wooden boxes), pre-drying, turning during fermentation, fermentation duration, and drying temperature were investigated. Fermenting in plastic bags without pre-drying or turning and fermenting in jute bags for only 40 ± 2.0 h yielded low maximal fermentation temperatures Tmax (31.1 ± 0.4 °C and 37.6 ± 1.8 °C), leading to bitter, astringent, woody, and earthy cocoa liquor. Longer fermentation (63 ± 6 h) in wooden boxes with turning (Wt) and in jute bags with pre-drying and turning (Jpt) achieved the highest Tmax of 46.5 ± 2.0 °C, and a more acidic cocoa liquor, particularly in Wt (both locations) and Jpt (location E). Therefore, it is recommended to ferment for a minimum duration from day 1 to 4 (63 ± 6 h), whether using plastic bags (with mandatory pre-drying) or jute bags (with or without pre-drying or turning). Furthermore, this study underscores the risks associated with excessively high drying temperatures (up to 95.2 ± 13.7 °C) and specific dryer types, which can falsify cut-tests and introduce unwanted burnt-roasted off-flavors in the cocoa liquor.

This study investigated the impact of the depulping of cocoa beans after pod opening, as well as the influences of pod storage (PS) and fermentation time on the fermentation dynamics and the overall quality of beans and liquors made thereof. Twelve variations were conducted in three experimental runs (with/without depulping; 1-/3-day PS; and fermentation times of 3, 4, 5, 6 or 7 days). Fermentation dynamics (e.g., temperature and pH) and the quality of dried beans (e.g., cut-test and fermentation index) and liquors (sensory assessment, quantification of cocoa key-odorants and tastants) were investigated. It was demonstrated that 17–20% of cocoa pulp, relative to the total bean-pulp-mass weight, could be mechanically removed without negatively affecting the bean quality. No significant differences were found in the percentages of well-fermented beans after 5–6 days fermentation with 1-day PS, resulting in 49 ± 9% with, and 48 ± 12% without depulping. There were no significant differences in key tastants present in the liquors; however, significantly less volatile acids and esters were found when liquors were produced from 5–6 day-fermented depulped beans, with 1-day PS, without negatively affecting the sensory profiles. This strategy allows producers to maximize the cacao fruit's value by integrating part of the pulp into the cocoa value chain. [ABSTRACT FROM AUTHOR]

Abstract One great challenge in surgical tendon repair is the minimization of peritendinous adhesions. An electrospun tube can serve as a physical barrier around a conventionally sutured tendon. Six New Zealand White rabbits had one Achilles tendon fully transsected and sutured by a 4-strand suture. Another six rabbits had the same treatment, but with the additional electrospun DegraPol tube set around the sutured tendon. The adhesion formation to the surrounding tissue was investigated 12 weeks post-operation. Moreover, inflammation-related protease-activated receptor-2 (PAR-2) protein expression was assessed. Finally, rabbit Achilles tenocyte cultures were exposed to platelet-derived growth factor-BB (PDGF-BB), which mimicks the tendon healing environment, where PAR-2 gene expression was assessed as well as immunofluorescent staining intensity for F-actin and α-tubulin, respectively. At 12 weeks post-operation, the partially degraded DegraPol tube exhibited significantly lower adhesion formation (− 20%). PAR-2 protein expression was similar for time points 3 and 6 weeks, but increased at 12 weeks post-operation. In vitro cell culture experiments showed a significantly higher PAR-2 gene expression on day 3 after exposure to PDGF-BB, but not on day 7. The cytoskeleton of the tenocytes changed upon PDGF-BB stimulation, with signs of reorganization, and significantly decreased F-actin intensity. An electrospun DegraPol tube significantly reduces adhesion up to twelve weeks post-operation. At this time point, the tube is partially degraded, and a slight PAR-2 increase was detected in the DP treated tendons, which might however arise from particles of degrading DegraPol that were stained dark brown. PAR-2 gene expression in rabbit tenocytes reveals sensitivity at around day 10 after injury.

Tendon injuries can result in two major drawbacks. Adhesions to the surrounding tissue may limit the range of motion, while fibrovascular scar formation can lead to poor biomechanical outcomes. Prosthetic devices may help to mitigate those problems. Emulsion electrospinning was used to develop a novel three-layer tube based on the polymer DegraPol (DP), with incorporated insulin-like growth factor-1 (IGF-1) in the middle layer. Scanning electron microscopy was utilized to assess the fiber diameter in IGF-1 containing pure DP meshes. Further characterization was performed with Fourier Transformed Infrared Spectroscopy, Differential Scanning Calorimetry, and water contact angle, as well as through the assessment of mechanical properties and release kinetics from ELISA, and the bioactivity of IGF-1 by qPCR of collagen I, ki67, and tenomodulin in rabbit Achilles tenocytes. The IGF-1-containing tubes exhibited a sustained release of the growth factor up to 4 days and showed bioactivity by significantly upregulated ki67 and tenomodulin gene expression. Moreover, they proved to be mechanically superior to pure DP tubes (significantly higher fracture strain, failure stress, and elastic modulus). The novel three-layer tubes intended to be applied over conventionally sutured tendons after a rupture may help accelerate the healing process. The release of IGF-1 stimulates proliferation and matrix synthesis of cells at the repair site. In addition, adhesion formation to surrounding tissue can be reduced due to the physical barrier.

Cocoa post-harvest practices were monitored on a small-farm scale (ca. 50 kg fresh beans) at five intermediaries from four provinces in Ecuador: (A) in Manabí, (B) and (E) in Los Ríos, (C) in Cotopaxi, (D) in Guayas. Temperature, pH (pulp, cotyledon), cell counts (yeasts, lactic acid bacteria, acetic acid bacteria) were recorded daily, and cut-tests and sensory descriptive analysis evaluated end quality. An overall inconsistency and variability in processing were observed with different fermentation devices (jute/plastic bags, wooden boxes), pre-drying, turning during fermentation, fermentation duration, and different drying processes (temperatures, direct/indirect). Key parameters (maximum temperature, pH cotyledon development) revealed a significant impact of the fermentation device on the post-harvest process and, therefore, on the fermentation development. 67–74 h in jute bags without turning was sufficient to reach well-fermented cocoa beans without moldy off-flavors, whereas 133 h in plastic bags without turning resulted in 3 ± 1% moldy beans and cocoa liquor with moldy off-flavor. Drying at high temperatures (80 ± 10 °C) with direct heat contact resulted in beans roasted to burnt off-flavor. Conclusively, the whole post-harvest process was crucial for well-fermented beans without off-flavor. Plastic bags seemed unsuitable, while jute bags could be an alternative to wooden boxes.

Tendon injuries suffer from a slow healing, often ending up in fibrovascular scar formation, leading to inferior mechanical properties and even re-rupture upon resumption of daily work or sports. Strategies including the application of growth factors have been under view for decades. Insulin-like growth factor-1 (IGF-1) is one of the used growth factors and has been applied to tenocyte in vitro cultures as well as in animal preclinical models and to human patients due to its anabolic and matrix stimulating effects. In this narrative review, we cover the current literature on IGF-1, its mechanism of action, in vitro cell cultures (tenocytes and mesenchymal stem cells), as well as in vivo experiments. We conclude from this overview that IGF-1 is a potent stimulus for improving tendon healing due to its inherent support of cell proliferation, DNA and matrix synthesis, particularly collagen I, which is the main component of tendon tissue. Nevertheless, more in vivo studies have to be performed in order to pave the way for an IGF-1 application in orthopedic clinics.

(1) Background: Surgical tendon repair often leads to adhesion formation, leading to joint stiffness and a reduced range of motion. Tubular implants set around sutured tendons might help to reduce peritendinous adhesions. The lubricant hyaluronic acid (HA) is a viable option for optimizing such tubes with the goal of further enhancing the anti-adhesive effect. As the implant degrades over time and diffusion is presumed, the impact of HA on tendon cells is important to know. (2) Methods: A culture medium of rabbit Achilles tenocytes was supplemented with high-molecular-weight (HMW) HA and the growth curves of the cells were assessed. Additionally, after 3, 7 and 14 days, the gene expression of several markers was analyzed for matrix assembly, tendon differentiation, fibrosis, proliferation, matrix remodeling, pro-inflammation and resolution. (3) Results: The addition of HA decreased matrix marker genes, downregulated the fibrosis marker α-SMA for a short time and slightly increased the matrix-remodeling gene MMP-2. Of the pro-inflammatory marker genes, only IL-6 was significantly upregulated. IL-6 has to be kept in check, although IL-6 is also needed for a proper initial inflammation and efficient resolution. (4) Conclusions: The observed effects in vitro support the intended anti-adhesion effect and therefore, the use of HMW HA is promising as a biodegradable implant for tendon repair.

In Ecuador, various processes are applied during cocoa post-harvesting. This study, therefore, explored fermentation parameters across two locations with 2–7 independent runs, focusing on temperature, microbial counts, pH during fermentation and drying, and their impact on cocoa bean quality. Factors including fermentation devices (jute bags, plastic bags, and wooden boxes), pre-drying, turning during fermentation, fermentation duration, and drying temperature were investigated. Fermenting in plastic bags without pre-drying or turning and fermenting in jute bags for only 40 ± 2.0 h yielded low maximal fermentation temperatures Tmax (31.1 ± 0.4 °C and 37.6 ± 1.8 °C), leading to bitter, astringent, woody, and earthy cocoa liquor. Longer fermentation (63 ± 6 h) in wooden boxes with turning (Wt) and in jute bags with pre-drying and turning (Jpt) achieved the highest Tmax of 46.5 ± 2.0 °C, and a more acidic cocoa liquor, particularly in Wt (both locations) and Jpt (location E). Therefore, it is recommended to ferment for a minimum duration from day 1 to 4 (63 ± 6 h), whether using plastic bags (with mandatory pre-drying) or jute bags (with or without pre-drying or turning). Furthermore, this study underscores the risks associated with excessively high drying temperatures (up to 95.2 ± 13.7 °C) and specific dryer types, which can falsify cut-tests and introduce unwanted burnt-roasted off-flavors in the cocoa liquor. [ABSTRACT FROM AUTHOR]

Abstract Background Recurrent and persistent infections are known to affect airways of patients with Primary Immunodeficiency despite appropriate replacement immunoglobulin serum levels. Interestingly, patients with Chronic Obstructive Pulmonary Disease or with non-CF bronchiectasis also show similar susceptibility to such infections. This may be due to the limited availability of immunoglobulins from the systemic circulation in the conductive airways, resulting in local immunodeficiency. Topical application of nebulized plasma-derived immunoglobulins may represent a means to address this deficiency. In this study, we assessed the feasibility of nebulizing plasma-derived immunoglobulins and delivering them into the airways of rats and non-human primates. Methods Distinct human plasma-derived immunoglobulin isotype preparations were nebulized with an investigational eFlow® nebulizer and analyzed in vitro or deposited into animals. Biochemical and immunohistological analysis of nebulized immunoglobulins were then performed. Lastly, efficacy of topically applied human plasma-derived immunoglobulins was assessed in an acute Streptococcus pneumoniae respiratory infection in mice. Results Characteristics of the resulting aerosols were comparable between preparations, even when using solutions with elevated viscosity. Neither the structural integrity nor the biological function of nebulized immunoglobulins were compromised by the nebulization process. In animal studies, immunoglobulins levels were assessed in plasma, broncho-alveolar lavages (BAL) and on lung sections of rats and non-human primates in samples collected up to 72 h following application. Nebulized immunoglobulins were detectable over 48 h in the BAL samples and up to 72 h on lung sections. Immunoglobulins recovered from BAL fluid up to 24 h after inhalation remained structurally and functionally intact. Importantly, topical application of human plasma-derived immunoglobulin G into the airways of mice offered significant protection against acute pneumococcal pneumonia. Conclusion Taken together our data demonstrate the feasibility of topically applying plasma-derived immunoglobulins into the lungs using a nebulized liquid formulation. Moreover, topically administered human plasma-derived immunoglobulins prevented acute respiratory infection.

Functional lactic acid bacteria (LAB) as starter cultures used in sourdough fermentation have been researched for years. This study evaluated the LAB strains Leuconostoc citreum DCM65 (mannitol, exopolysaccharide producing, antifungal activity) and Lactiplantibacillus plantarum subsp. plantarum MA418 (amylolytic activity) and their potential as single or co-culture starters in sourdough fermented buns containing different levels of sugar (control 9% and reduced 0, 3, 6%). Cell counts, pH development, and organic acids were determined before and after sourdough fermentation (30 °C, 24 h) and physical properties (color, volume, pore structure, and texture) of buns produced thereof were determined after baking. Sourdoughs started with DCM65 and/or MA418 developed up to log 9.2 CFU/g presumptive LAB after 24 h, assertiveness of the added starter cultures species was confirmed by MALDI-TOF MS. Acetic acid and mannitol were only detected in sourdough fermented with DCM65 (single or co-culture) up to 2.5 mg/g and 9.8 mg/g, respectively. The starter cultures applied influenced physical properties of buns. Sourdough buns started with MA418 had higher volume and slice area, and softer crumb; in contrast, buns fermented with DCM65 had a finer pore structure. In summary, both starter cultures showed high potential in sourdough buns with reduced sugar content.

The growth of filamentous fungi during the spontaneous cocoa bean fermentation leads to inferior cocoa bean quality and poses a health risk for consumers due to the potential accumulation of mycotoxins. We recently developed anti-fungal cultures with the capacity to inhibit the growth of mycotoxigenic filamentous fungi on cocoa beans. However, it is not clear how these anti-fungal cultures affect the fermentation process and cocoa bean quality. For that, the anti-fungal co-cultures, Lactobacillus fermentum M017-Saccharomyces cerevisiae H290 (A) and Lb. fermentum 223-S. cerevisiae H290 (B), were applied to 180-kg box fermentations in Honduras in three time-independent replications each including a spontaneous control fermentation. The comparison of inoculated and spontaneous fermentation processes revealed that the co-cultures only marginally affected the fermentation process and cocoa bean quality. Microorganisms reached maximal levels of 6.2-7.6 log CFU/g of yeasts and acetic acid bacteria and 7.9-9.5 log CFU/g of lactic acid bacteria during all fermentations and led to maximal metabolite concentrations in bean cotyledons of 4-12 mg/g ethanol, 2-6 mg/g lactic acid and 6-14 mg/g acetic acid. The fermentation and drying processes resulted in 38-90 mg epicatechin equivalents/g in the cotyledons of dried beans. However, the co-cultures led to up to ten times higher mannitol levels in cotyledons of inoculated beans compared to beans during spontaneous fermentation, and caused a slower fermentation process, detectable as up to 8-12 °C lower temperatures in the centre of the fermenting pulp-bean mass and up to 22% lower proportions of well-fermented beans after drying. Co-culture B-with Lb. fermentum 223 -led to improved cocoa bean quality compared to co-culture A-with Lb. fermentum M017 -, i.e. cocoa beans with 0.5-1.9 mg/g less acetic acid, 4-17% higher shares of well-fermented beans and, on a scale from 0 to 10, to 0.2-0.6 units lower astringency, up to 1.1 units lower off-flavours, and 0.2-0.9 units higher cocoa notes. Therefore, the anti-fungal co-culture B is recommended for future applications and its capacity to limit fungal growth and mycotoxin production during industrial-scale cocoa bean fermentation should be investigated in further studies.

In this study, the potential of Leuconostoc as non-conventional sourdough starter cultures was investigated. A screening for antifungal activities of 99 lactic acid bacteria (LAB) strains revealed high suppression of bakery-relevant moulds in nine strains of Leuconostoc with activities against Penicillium sp., Aspergillus sp., and Cladosporium sp. Mannitol production was determined in 49 Leuconostoc strains with >30 g/L mannitol in fructose (50 g/L)-enriched MRS. Further, exopolysaccharides (EPS) production was qualitatively determined on sucrose (40 g/L)-enriched MRS agar and revealed 59 EPS positive Leuconostoc strains that harboured dextransucrase genes, as confirmed by PCR. Four multifunctional Lc. citreum strains (DCM49, DCM65, MA079, and MA113) were finally applied in lab-scale sourdough fermentations (30 °C, 24 h). Lc. citreum was confirmed by MALDI-TOF MS up to 9 log CFU/g and pH dropped to 4.0 and TTA increased to 12.4. Antifungal compounds such as acetic acid, phenyllactic and hydroxyphenyllactic acids were determined up to 1.7 mg/g, 2.1 µg/g, and 1.3 µg/g, respectively, mannitol up to 8.6 mg/g, and EPS up to 0.62 g/100 g. Due to the observed multifunctionalities and the competitiveness in the natural flour microbiota present in sourdoughs, non-conventional LAB genera such as Leuconostoc seem promising for application in sourdough-based bakery products.

Influenza is a highly contagious, acute, febrile respiratory infection that can have fatal consequences particularly in individuals with chronic illnesses. Sporadic reports suggest that intravenous immunoglobulin (IVIg) may be efficacious in the influenza setting. We investigated the potential of human IVIg to ameliorate influenza infection in ferrets exposed to either the pandemic H1N1/09 virus (pH1N1) or highly pathogenic avian influenza (H5N1). IVIg administered at the time of influenza virus exposure led to a significant reduction in lung viral load following pH1N1 challenge. In the lethal H5N1 model, the majority of animals given IVIg survived challenge in a dose dependent manner. Protection was also afforded by purified F(ab′)2 but not Fc fragments derived from IVIg, supporting a specific antibody-mediated mechanism of protection. We conclude that pre-pandemic IVIg can modulate serious influenza infection-associated mortality and morbidity. IVIg could be useful prophylactically in the event of a pandemic to protect vulnerable population groups and in the critical care setting as a first stage intervention.

Intravascular erythrocyte destruction, accompanied by the release of pro-oxidative and pro-inflammatory components hemoglobin and heme, is a common event in the pathogenesis of numerous diseases with heterogeneous etiology and clinical features. A frequent adverse effect related to massive hemolysis is the renal injury and inflammation. Nevertheless, it is still unclear whether heme––a danger-associated molecular pattern––and ligand for TLR4 or upstream hemolysis-derived products are responsible for these effects. Well-characterized animal models of hemolysis with kidney impairment are needed to investigate how hemolysis drives kidney injury and to test novel therapeutic strategies. Here, we characterized the pathological processes leading to acute kidney injury and inflammation during massive intravascular hemolysis, using a mouse model of phenylhydrazine (PHZ)-triggered erythrocyte destruction. We observed profound changes in mRNA levels for markers of tubular damage (Kim-1, NGAL) and regeneration (indirect marker of tubular injury, Ki-67), and tissue and vascular inflammation (IL-6, E-selectin, P-selectin, ICAM-1) in kidneys of PHZ-treated mice, associated with ultrastructural signs of tubular injury. Moreover, mass spectrometry revealed presence of markers of tubular damage in urine, including meprin-α, cytoskeletal keratins, α-1-antitrypsin, and α-1-microglobulin. Signs of renal injury and inflammation rapidly resolved and the renal function was preserved, despite major changes in metabolic parameters of PHZ-injected animals. Mechanistically, renal alterations were largely heme-independent, since injection of free heme could not reproduce them, and scavenging heme with hemopexin in PHZ-administered mice could not prevent them. Reduced overall health status of the mice suggested multiorgan involvement. We detected amylasemia and amylasuria, two markers of acute pancreatitis. We also provide detailed characterization of renal manifestations associated with acute intravascular hemolysis, which may be mediated by hemolysis-derived products upstream of heme release. This analysis provides a platform for further investigations of hemolytic diseases and associated renal injury and the evaluation of novel therapeutic strategies that target intravascular hemolysis.

During hemolysis, hemoglobin and heme released from red blood cells promote oxidative stress, inflammation and thrombosis. Plasma haptoglobin and hemopexin scavenge free hemoglobin and heme, respectively, but can be depleted in hemolytic states. Haptoglobin and hemopexin supplementation protect tissues, including the vasculature, liver and kidneys. It is widely assumed that these protective effects are due primarily to hemoglobin and heme clearance from the vasculature. However, this simple assumption does not account for the consequent cytoprotective adaptation seen in cells and organs. To further address the mechanism, we used a hyperhemolytic murine model (Townes-SS) of sickle cell disease to examine cellular responses to haptoglobin and hemopexin supplementation. A single infusion of haptoglobin or hemopexin (± equimolar hemoglobin) in SS-mice increased heme oxygenase-1 (HO-1) in the liver, kidney and skin several fold within 1 hour and decreased nuclear NF-ĸB phospho-p65, and vaso-occlusion for 48 hours after infusion. Plasma hemoglobin and heme levels were not significantly changed 1 hour after infusion of haptoglobin or hemopexin. Haptoglobin and hemopexin also inhibited hypoxia/reoxygenation and lipopolysaccharide-induced vaso-occlusion in SS-mice. Inhibition of HO-1 activity with tin protoporphyrin blocked the protections afforded by haptoglobin and hemopexin in SS-mice. The HO-1 reaction product carbon monoxide, fully restored the protection, in part by inhibiting Weibel-Palade body mobilization of P-selectin and von Willebrand factor to endothelial cell surfaces. Thus, the mechanism by which haptoglobin and hemopexin supplementation in hyperhemolytic SS-mice induces cytoprotective cellular responses is linked to increased HO-1 activity.

In relation to the recent trials of Intravenous Immunoglobulin (IVIG) in Alzheimer's Disease (AD) it was demonstrated that different IgG preparations contain varying amounts of natural anti-amyloid β (Aβ) antibodies as measured by ELISA. We therefore investigated the relevance of ELISA data for measuring low-affinity antibodies, such as anti-Aβ. We analysed the binding of different commercial Immunoglobulin G (IgG) preparations to Aβ, actin and tetanus toxoid in different binding assays to further investigate the possible cause for observed differences in binding to Aβ and actin between different IgG preparations. We show that the differences of commercial IgG preparations in binding to Aβ and actin in ELISA assays are artefactual and only evident in in vitro binding assays. In functional assays and in vivo animal studies the different IVIG preparations exhibited very similar potency. ELISA data alone are not appropriate to analyse and rank the binding capacity of low-affinity antibodies to Aβ or other endogenous self-antigens contained in IgG preparations. Additional analytical methods should be adopted to complement ELISA data.