Semi-preparative purification and validation of monoclonal antibodies for immunotherapy in mice

A number of rat hybridomas were adapted to grow in RPMI containing either 5% IgG-depleted FCS of 1% serum-free Nutridoma. Alternatively, protein-free Ultradoma PF was used. Growth in these media allowed purification procedures to be used that are based on tangential ultrafiltration in combination with affinity chromatography on gels linked to protein G or anti-rat L chain coupled antibodies. The isolated antibody preparations were found to be pure and to consist of monomeric intact IgG. The yield and recovery of mAb using this procedure were found to be consistently high. These antibody preparations were analyzed for endotoxin contamination. Whereas during isolation endotoxin contamination increased, the endotoxin content per mg purified protein did not. Affinity chromatography on Detoxi-gel resulted in the efficient removal of this contamination and using this protocol the antibody preparations obtained were found to be of sufficient purity, activity and low endotoxin content to permit their in vivo use in animal models of immunotherapy

Potential causes of mortality for horseshoe crabs (Limulus polyphemus) during the biomedical bleeding process

Biomedical companies catch and bleed horseshoe crabs for the production of Limulus amebocyte lysate (LAL), a product used for protecting public health (Berkson and Shuster, 1999). LAL is a clotting agent, derived solely from horseshoe crab blood cells, which is used to detect the presence of pathogenic gramnegative bacteria in injectable drugs and implantable medical and dental devices (Mikkelsen, 1988; Novitsky, 1991). In addition, LAL is used in many diagnostic tests for such illnesses as gram-negative bacterial meningitis and typhoid fever (Ding and Ho, 2001). Because the LAL test allows one to detect femtogram levels of endotoxin (Ding and Ho, 2001), it is the most effective test for detecting endotoxin contamination, and its increasing use in medical and pharmaceutical laboratories makes it a highly valued product

Influence of Immune Status on the Airborne Colonization of Piglets with Methicillin-Resistant Staphylococcus aureus (MRSA) Clonal Complex (CC) 398

Colonized vertebrates including humans and pigs are to date the main reservoirs of livestock-associated Methicillin-resistant Staphylococcus aureus (LA-MRSA). Currently, the mechanisms underlying colonization of pigs are not fully understood. We investigated the influence of piglet pre-immune status on airborne MRSA colonization. Three groups of MRSA-negative piglets were primed and exposed to airborne LA-MRSA (104 colony forming units (cfu)/m3) in an aerosol chamber for 24 h. One group was treated intramuscularly with dexamethasone (1 mg/kg body weight) to imitate weaning stress. The second group was exposed to bacterial endotoxin containing MRSA aerosol. Both conditions play a role in the development of multifactorial diseases and may promote MRSA colonization success. The third group served as control. The piglets' MRSA status was monitored for 21 days via swab samples. At necropsy, specific tissues and organs were analyzed. Blood was collected to examine specific immunological parameters. The duration of MRSA colonization was not extended in both treated groups compared to the control group, indicating the two immune-status influencing factors do not promote MRSA colonization. Blood sample analysis confirmed a mild dexamethasone-induced immune suppression and typical endotoxin-related changes in peripheral blood. Of note, the low-dose dexamethasone treatment showed a trend of increased MRSA clearance

High throughput genomic sequencing of bioaerosols in broiler chicken production facilities

Chronic inhalation exposure to agricultural dust promotes the development of chronic respiratory diseases among poultry workers. Poultry dust is composed of dander, chicken feed, litter bedding and microbes. However, the microbial composition and abundance has not been fully elucidated. Genomic DNA was extracted from settled dust and personal inhalable dust collected while performing litter sampling or mortality collection tasks. DNA libraries were sequenced using a paired-end sequencing-by-synthesis approach on an Illumina HiSeq 2500. Sequencing data showed that poultry dust is predominantly composed of bacteria (64–67%) with a small quantity of avian, human and feed DNA (\u3c 2% of total reads). Staphylococcus sp. AL1, Salinicoccus carnicancri and Lactobacillus crispatus were the most abundant bacterial species in personal exposure samples of inhalable dust. Settled dust had a moderate relative abundance of these species as well as Staphylococcus lentus and Lactobacillus salivarius. There was a statistical difference between the microbial composition of aerosolized and settled dust. Unlike settled dust composition, aerosolized dust composition had little variance between samples. These data provide an extensive analysis of the microbial composition and relative abundance in personal inhalable poultry dust and settled poultry dust

Dinucleoside polyphosphates : newly detected uraemic compounds with an impact on leucocyte oxidative burst

Background. Dinucleoside polyphosphates (NpnN) have pathophysiologic roles in cardiovascular disease and are newly detected uraemic retention solutes. They were retrieved in human plasma, tissues and cells. Although their impact on several cell systems involved in vascular damage (endothelium, smooth muscle cells and thrombocytes) has been evaluated, their effect on different types of leucocytes has never been studied. Methods. This study evaluates, for the first time, the impact of NpnN on monocyte, granulocyte and lymphocyte oxidative burst activity at baseline and after stimulation with N-formyl-methionine-leucine-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA) in whole blood. Diadenosine triphosphate (Ap(3)A) to diadenosine hexaphosphate (Ap(6)A) were tested to investigate the effect of the number of phosphate groups on reactive oxygen species (ROS) production. The effect of the type of nucleoside was evaluated by comparing adenosine guanosine tetraphosphate, diguanosine tetraphosphate, uridine adenosine tetraphosphate (Up(4)A) and diadenosine tetraphosphate (Ap(4)A). Results. This study demonstrated that lymphocytes are especially susceptible to intracellular diadenosine polyphosphates. Depending on the phosphate chain length, different effects were observed. At baseline and with fMLP Ap(4)A, Ap(5)A and Ap(6)A enhanced lymphocyted-free radical production. In addition, Ap(3)A, Ap(4)A and Ap5A increased PMA-stimulated ROS production in lymphocytes. Monocytes and granulocytes parallel the lymphocyte response albeit with an inhibition of Ap(6)A on granulocytes. Considering NpnN with four phosphate groups, Up(4)A showed the most important stimulatory effects on monocytes and Ap(4)A on lymphocytes. Conclusions. NpnN mainly have a leucocyte-activating impact, most significant for Ap(4)A, considering phosphate chain length, and for Up(4)A, considering the type of nucleosides. These results suggest that the pro-inflammatory effects of NpnN can contribute to the development of atherosclerosis, probably in the early stages of chronic kidney disease, but their chemical composition affects their activity

Downregulation of Tumor Necrosis Factor Expression in the Human Mono-Mac-6 Cell Line

Mono-Mac-6 cells, but not U937 cells, can be Induced to rapidly express tumor necrosis factor (TNF) mRNA and protein when triggered with Ilpopolysaccharlde (LPS) at 1 pg/mI. Preincubatlon of the cells for 3 d with low amounts of LPS (10 ng/mI) results In nearly complete suppression of TNF secretion. This downreguiatlon appears to occur at the pretranslational level since specIfIc mRNA is virtually undetectable under these conditions. By contrast, the same prelncubatlon with 10 ng/mI LPS results in enhanced phagocytosls (28.6-67.2% for Staphylococcus aureus), demonstrating that not all monocyte functions are suppressed. While these results show that only stringent exclusion of LPS from culture media allows for Induction of TNF In the Mono-Mac-6 cell line, the pronounced effect of LPS preincubatlon may also provide a suitable model with which to study the mechanisms of LPS-lnduced desensitizatIon

Shwartzman reaction after human renal homotransplantation.

In three human recipients, five renal homografts were destroyed within a few minutes to hours after their revascularization in the new host. The kidneys, removed one to 54 days later, had cortical necrosis. The major vessels were patent, but the arterioles and glomeruli were the site of fibrin deposition. There was little or no fixation of host immunoglobulins in the homografts. The findings were characteristic of a generalized Shwartzman reaction. Although the cause (or causes) of the Shwartzman reaction in our patients is not known, they may have been conditioned by the bacterial contamination and hemolysis that often attend hemodialysis, by immunosuppression and by the transplantation itself. Some of the patients have preformed lymphocytotoxic antibodies. Thus, certain patients may be predisposed. High-risk patients should be recognized and treated prophylactically with anticoagulants

Analysis of black holes in our knowledge concerning animal health in the organic food production chain

Although a large body of research on health aspects can be found via the web or in conference proceedings, only few papers reach the peer reviewed literature. This aspect makes it difficult to judge the quality of available data that relate to health aspects associated with organic livestock production. Many data deal with "on farm case type studies" which often do not lead to a direct relation between certain management procedures and incidence of certain health aspects. The principle that organic held animals are in better condition concerning their health as compared to conventionally held animals has not yet been proven. Well-controlled studies are needed to show the effects of organically held animals on innate and adaptive immune responses and disease susceptibility. The conditions in organic livestock production lead to novel challenges concerning social interactions, physical requirements, climatic conditions and infectious burden which requires certain breeds of animals that differ considerably from the conventionally held animals. More research is needed to identify these breeds whereby breeding selection criteria specific to organic conditions and principles should be used. A number of health problems such as infestation with parasites is an important problem that has been noted in organic livestock (poultry and pig) production and more research is needed to both prevent and treat these infections. Although largely recommended, phytotherapy and homeopathy do not have any demonstrated efficacy in managing helminths. A multidisciplinary approach is needed to address these questions. Investigations should be concerned with aspects such as rotation, stocking density, disinfections strategies, genetic predisposition and novel (plant derived or biological) antihelminthics. A major drawback in the use of phytotherapeuticals is the lack of good clinical trials, most claims are based on traditional use, anecdotal evidence or extrapolation of human data. Another problem is the consistency/standardisation of the product; variations exist in the levels of "active" ingredients between lots. Extensive studies have shown that garlic has a beneficial effect on human health and therefore would possibly be a suitable candidate to increase the health of animals in organic production. Further research is needed to substantiate this claim. Although mastitis is not seen more often in organic dairy production compared to conventional farms, it is still frequently encountered and both basic and applied research concerning prevention and cure without the traditional use of antibiotics is needed. More knowledge is needed concerning effects of alternative treatments in organic livestock production. As long as alternative treatments have not formally been proven to be effective, allopathic treatments should be preferred. Studies are needed concerning the welfare aspects of disease and insufficient or postponed treatment in organic animal production systems. It is generally accepted that feather picking and cannibalism is a major problem in organic poultry and more research is needed to counter this unwanted behaviour of the animals. The following aspects including genetic influences, housing environment, feed composition and the presence of external parasites may be involved. Vaccination has been one of the largest success factors in the prevention of disease in production animals. Most vaccines used nowadays are genetically engineered and should thus carry the GMO label. GMO based vaccines produced by incorporating genetic material from bacteria or viruses into plants is an attractive approach but does not comply with the principles of organic farming. Investigations concerning various vaccination strategies with or without GMO approaches should be evaluated to prevent various infectious diseases in organic livestock production systems. Organic livestock production leads to an increase or re-emergence of certain zoonotic diseases ( Campylobacter, toxoplasma). This is a serious drawback concerning claims that organic products are healthier as compared to conventional products and research into the prevalence of certain zoonotic infections, risk factors, farm management, post slaughter decontamination and consumer perception/education is badly needed. Several micro-organisms are described to be present in feeds and could be sources of infections of farm animals. Such pathogens can be either present on the outside of the plant (epiphytes) or on the inside of the plant (endophytes). The incidence of pathogenic bacteria in/on plants used for organic animal feed is not clear and therefore research on the incidence of these pathogens is needed. Biological cycles such as the utilisation of manure from organic farms may potentially lead to the creation of infectious reservoirs. Research is needed to provide evidence for this hypothesis and strategies should be devised to prevent this problem. Little is known about the role of pests in the occurrence of disease in organic livestock production. Research is needed into the efficacy of alternative pest control strategies since current pest control methods are not in agreement with organic production principles (use of anticoagulants for rodent control). The obligatory use of straw bedding in swine household management leads to higher dust and bioareosol (endotoxin) levels in stables. This may affect susceptibility to lung disease (farmer and animal) and investigations concerning measures to reduce dust and bioaerosol levels are needed. Current literature on this subject is not concordant and true differences may exist between management systems from different countries. Piglet mortality is an important problem in organic production systems and research concerning genetic and housing aspects is currently performed but not yet reported in the peer-reviewed literature. Amino acid imbalance and research into novel feed sources with high protein content is needed. More knowledge is known concerning possible anti-nutritive factors in these feeds and the effects of these new nutrients on gut health of the animals. Amino acid imbalance may lead to higher excretion of excess amino acids into the environment. On farm research should be performed to substantiate whether these claims also apply to organic production systems. It has been suggested that organic feeds for animals in organic production systems may contain higher mycotoxin levels thereby adversely affecting their health status. As yet no conclusive data concerning this issue has been published and research into this area is needed to prove or reject this myth. As yet it is not known whether phytase levels in organic feed for pigs are sufficient to allow optimal utilisation of organic phosphor thereby also leading to a lower environmental pollution; further investigations are needed in this area

Challenging the Clostridium botulinum toxin type A (BoNT/A) with a selection of microorganisms by culture methods and extended storage of used vials to assess the loss of sterility

In 2002, botulinum toxin type A (BoNT/A) was approved by the US Food and Drug Administration (FDA) for cosmetic use. However, there may be procedural differences between the ways in which a clinician handles, applies and stores the product compared to the suggested guidelines of the manufacturer for handling and storage. To this end vials (N = 12) of BoNT/A were tested for the incidence of microbial contamination followed by challenging the product with a selection of microorganisms by culture methods and by using a calcein release assay to contaminate multi-dose vials at the single concentration used for facial aesthetics. A culture, droplet method was used to count microorganisms challenged with the therapeutic product and to compare viability levels in appropriate controls as well as measuring their lytic properties via an existing cell-free system involving calcein release. Counts of test organisms within the droplets, with the product and the controls without the product were undertaken using Image J software. The result from the incidence of in-vial contamination was inconclusive. Bacterial levels between controls and product challenged groups demonstrated no differences in the growth of viable microorganisms following immediate contact (p = ≥ 0.05). The cell-free calcein release assay demonstrated differences at all time points for low levels of lysis in each case with bacterial lipid extract and were statistically significant (p = 0.011). Although these data appear to correlate with the minimum inhibitory concentration, the additives and vial integrity are also likely to contribute to the maintenance of BoNT/A sterility

Enhancement of platelet response to immune complexes and IgG aggregates by lipid A-rich bacterial lipopolysaccharides.

The effect of the common lipid moiety of bacterial LPS on secretion from washed human platelets has been studied. The lipid A-rich LPS of S. minnesota R595 and a lipid A preparation both potentiated platelet serotonin secretion in response to IgG aggregates or immune complexes up to 50-fold but had little effect in the absence of IgG. Lipid A has been shown to bind immune aggregates, raising the possibility that its mechanism of action involved effective enlargement or insolubilization of the aggregates. IgG aggregates of dimer to tetramer size were shown to be platelet simuli, equivalent on a weight basis to larger soluble aggregates. The effect of both sizes of aggregates on platelets were equally enhanced by the LPS, indicating that increased size of aggregates alone could not account for the effect of LPS. Similarly, because lipid A-rich LPS enhanced platelet response to already insoluble immune complexes, its mechanism of action cannot simply be insolubilization of immune aggregates. These LPS did not enhance platelet stimulation by antiplatelet antibody, monosodium urate crystals, or thrombin and only slightly enhanced stimulation by insoluble human skin collagen. This indicates some stimulus specificity in the ability of LPS to increase platelet secretion. The enhancement of cell response to immune complexes by the common lipid region of LPS may represent a mechanism for the diverse effects of LPS in vivo and in vitro