Low Endotoxin Levels Research Articles

A Phase I Dose Escalation Trial of Donor T Cells Sensitized with Pentadecapeptides of the CMV-pp65 Protein for the Treatment of CMV Infections Following Allogeneic Hematopoietic Stem Cell Transplants.

Abstract 2262Poster Board II-239We have developed a novel alternative method of generating donor-derived T-cell lines specific for CMV-pp65 peptides for use in adoptive immunotherapy. In this approach, 138 pentadecapeptides (15-mers), with 11 amino acid overlaps spanning the entire 561 amino acid sequence of the CMV protein pp65 were synthesized and manufactured under conditions ensuring their purity and sterility for application in clinical trials. A pool of the 138 pentadecapeptides is loaded onto monocyte-derived dendritic cells derived from seropositive donors and the same donor's T cells are sensitized under GMP conditions. In our preclinical studies, we have been able to generate CMV-pp65 specific T-cell lines from each seropositive donor tested, irrespective of HLA genotype. During the culture period of 21-28 days, populations of T cells specific for CMV-pp65 selectively expanded 200-300 fold while T cells reactive against major or minor alloantigens were depleted. Currently, 9 patients (pts) with persistent/refractory CMV antigenemia, one pt with CMV pneumonia diagnosed four weeks after treatment, have been treated using cells manufactured by this method on a clinical trial approved by the FDA. One pt was treated on a single patient IND prior to final FDA approval; 8 pts were enrolled onto the clinical trial - 3 pts at a T cell dose of 5×105/kg; 3 pts at dose level II of 1×106/kg and 2 pts at dose level III of 2×106/kg. We report here the results of the first 7 pts, (the 2 latter pts are too early in treatment to be included at time of abstract admission). CMV specific CTLs were generated from HLA-identical unrelated donors (3 pts) or from HLA-identical siblings (6 pts) for 2 pts who underwent non-myeloablative conventional and 7 pts who received myeloablative T-cell depleted allogeneic transplants. Pts were eligible if they had persistent CMV antigenemia despite treatment with antiviral drugs or had toxicities precluding further treatment with antiviral agents. Prior to infusion, the T cells were tested to be CMV specific by cytotoxicity, intracellular Interferon gamma (IFN-g) production and MHC-tetramer staining (if available). Cells were also assayed to establish lack of alloreactivity, microbiological sterility and low endotoxin levels. The cytotoxic T cells demonstrated cytolytic activity against peptide-loaded autologous PHA blasts, but exhibited no cytotoxicity against non-pulsed HLA-matched or pulsed HLA-mismatched target cells. The HLA-restriction and the specific pp65-derived epitopes of the CMV-specific T-cells were characterized prior to the infusion. CMV-specific frequencies of the CD8+ cells measured by intracellular IFN-g or MHC tetramers ranged from 2 -70%. Post infusion, an increase in the absolute lymphocyte count correlated with an increase in CMV-specific T-cell frequencies to levels as high as 12% of CD8+ cells. These persisted for at least 7 months (10% of CD8+ cells) following the infusion. Notably, the same pp65-derived epitopes and their HLA-restrictions, which characterized the pre-infusion CTLs were detected in the pt specimens post infusion. Freshly isolated T cells from the blood of HLA-A*0201 positive pts obtained 3 months post infusion showed significant lysis of CMV infected and peptide-pulsed MRC-5 fibroblasts, while non infected/pulsed fibroblasts and pulsed HLA-mismatched targets were not lysed. Three of the treated pts exhibited an HLA-A*0201 as well as HLA-B*0701 allele. In all three pts, epitope-specific T cells for the HLA-A*0201 restricted NLVPMVATV peptide and the B*0701 restricted RPHERNGFTV peptide were detected and monitored in pre and post infusion T-cell populations. All 7 pts who were treated for persistent CMV antigenemia tolerated treatment well. None developed early signs or symptoms of GvHD at the dose levels tested. Six of the 7 pts cleared CMV viremia by 2-4 weeks following the T-cell infusions. One of the pts died six weeks after the CTL infusion of respiratory failure despite clearing CMV from blood and bronchial aspirates. The one pt who remained viremic following the CTL infusion continued on oral Valgancyclovir and subsequently became CMV antigen negative. The clearance of CMV antigenemia was preceded by an initial spike of CMV antigenemia and/or CMV PCR in all pts. These results from the first 7 pts on this trial indicate that donor T cells sensitized with this pool of synthetic overlapping CMV pp65 15-mers are safe and clear CMV viremia resistant to standard therapy. Disclosures:No relevant conflicts of interest to declare.

Rapid Culture-Independent Microbial Analysis Aboard the International Space Station (ISS)

A new culture-independent system for microbial monitoring, called the Lab-On-a-Chip Application Development Portable Test System (LOCAD-PTS), was operated aboard the International Space Station (ISS). LOCAD-PTS was launched to the ISS aboard Space Shuttle STS-116 on December 9, 2006, and has since been used by ISS crews to monitor endotoxin on cabin surfaces. Quantitative analysis was performed within 15 minutes, and sample return to Earth was not required. Endotoxin (a marker of Gram-negative bacteria) was distributed throughout the ISS, despite previous indications that mostbacteria on ISS surfaces were Gram-positive [corrected].Endotoxin was detected at 24 out of 42 surface areas tested and at every surface site where colony-forming units (cfu) were observed, even at levels of 4-120 bacterial cfu per 100 cm(2), which is below NASA in-flight requirements (<10,000 bacterial cfu per 100 cm(2)). Absent to low levels of endotoxin (<0.24 to 1.0 EU per 100 cm(2); defined in endotoxin units, or EU) were found on 31 surface areas, including on most panels in Node 1 and the US Lab. High to moderate levels (1.01 to 14.7 EU per 100 cm(2)) were found on 11 surface areas, including at exercise, hygiene, sleeping, and dining facilities. Endotoxin was absent from airlock surfaces, except the Extravehicular Hatch Handle (>3.78 EU per 100 cm(2)). Based upon data collected from the ISS so far, new culture-independent requirements (defined in EU) are suggested, which are verifiable in flight with LOCAD-PTS yet high enough to avoid false alarms. The suggested requirements are intended to supplement current ISS requirements (defined in cfu) and would serve a dual purpose of safeguarding crew health (internal spacecraft surfaces <20 EU per 100 cm(2)) and monitoring forward contamination during Constellation missions (surfaces periodically exposed to the external environment, including the airlock and space suits, <0.24 EU per 100 cm(2)).

Comparison of Endotoxin and Particle Bounce in Marple Cascade Samplers With and Without Impaction Grease

ABSTRACT The health of persons engaged in agricultural activities are often related or associated with environmental exposures in their workplace. Accurately measuring, analyzing, and reporting these exposures is paramount to outcomes interpretation. This paper describes issues related to sampling air in poultry barns with a cascade impactor. Specifically, the authors describe how particle bounce can affect measurement outcomes and how the use of impaction grease can impact particle bounce and laboratory analyses such as endotoxin measurements. This project was designed to (1) study the effect of particle bounce in Marple cascade impactors that use polyvinyl chloride (PVC) filters; (2) to determine the effect of impaction grease on endotoxin assays when sampling poultry barn dust. A pilot study was undertaken utilizing six-stage Marple cascade impactors with PVC filters. Distortion of particulate size distributions and the effects of impaction grease on endotoxin analysis in samples of poultry dust distributed into a wind tunnel were studied. Although there was no significant difference in the overall dust concentration between utilizing impaction grease and not, there was a greater than 50% decrease in the mass median aerodynamic diameter (MMAD) values when impaction grease was not utilized. There was no difference in airborne endotoxin concentration or endotoxin MMAD between filters treated with impaction grease and those not treated. The results indicate that particle bounce should be a consideration when sampling poultry barn dust with Marple samplers containing PVC filters with no impaction grease. Careful consideration should be given to the utilization of impaction grease on PVC filters, which will undergo endotoxin analysis, as there is potential for interference, particularly if high or low levels of endotoxin are anticipated.

Extracellular Hsp72, an endogenous DAMP, is released by virally infected airway epithelial cells and activates neutrophils via Toll-like receptor (TLR)-4

BackgroundNeutrophils play an important role in the pathophysiology of RSV, though RSV does not appear to directly activate neutrophils in the lower airways. Therefore locally produced cytokines or other molecules released by virally-infected airway epithelial cells are likely responsible for recruiting and activating neutrophils. Heat shock proteins (HSPs) are generally regarded as intracellular proteins acting as molecular chaperones; however, HSP72 can also be released from cells, and the implications of this release are not fully understood.MethodsHuman bronchial epithelial cells (16HBE14o-) were infected with RSV and Hsp72 levels were measured by Western blot and ELISA. Tracheal aspirates were obtained from critically ill children infected with RSV and analyzed for Hsp72 levels by ELISA. Primary human neutrophils and differentiated HL-60 cells were cultured with Hsp72 and supernatants analyzed for cytokine production. In some cases, cells were pretreated with polymyxin B prior to treatment with Hsp72. IκBα was assessed by Western blot and EMSA's were performed to determine NF-κB activation. HL-60 cells were pretreated with neutralizing antibody against TLR4 prior to Hsp72 treatment. Neutrophils were harvested from the bone marrow of wild type or TLR4-deficient mice prior to treatment with Hsp72.ResultsInfection of 16HBE14o- with RSV showed an induction of intracellular Hsp72 levels as well as extracellular release of Hsp72. Primary human neutrophils from normal donors and differentiated HL-60 cells treated with increasing concentrations of Hsp72 resulted in increased cytokine (IL-8 and TNFα) production. This effect was independent of the low levels of endotoxin in the Hsp72 preparation. Hsp72 mediated cytokine production via activation of NF-κB translocation and DNA binding. Using bone marrow-derived neutrophils from wild type and TLR4-mutant mice, we showed that Hsp72 directly activates neutrophil-derived cytokine production via the activation of TLR4.ConclusionCollectively these data suggest that extracellular Hsp72 is released from virally infected airway epithelial cells resulting in the recruitment and activation of neutrophils.

Development of an Ex Vivo Assay for Use as a Surrogate Model to Assess the Impact of Endotoxin on Cattle

Current guidelines used by the US Food and Drug Administration to establish a safe level of pyrogens in veterinary parenteral products were derived from work designed for products intended for human use, and there is no consensus on what levels of endotoxin or other pyrogens are safe for domestic animals. The accepted biological measures for endotoxins and other pyrogens have been based on measurement of relatively small changes in body temperature in rabbits rather than an assessment of the inflammation caused by the endotoxins. It is difficult to accurately assess the small changes in body temperature caused by low levels of endotoxin in food-producing animals such as dairy cattle. Thus, the true relevance of guidelines to domestic animals is unknown. Accordingly, a whole-blood assay as a measure of endotoxin-induced inflammation was developed to serve as an in vitro surrogate model for either the rabbit pyrogen test or tests in cattle. Induction of interleukin-6 (IL-6) mRNA occurred at concentrations of lipopolysaccharide that ranged from 1 μg/mL through 1 pg/mL. Because there were still differences in IL-6 induction compared with unstimulated cultures, blood from a second set of animals was treated with lipopolysaccharide concentrations ranging from 1 μg/mL to 1 fg/mL. These results demonstrated that induction of IL-6 occurred somewhere between 100 fg/mL and 1 pg/mL. Because the most sensitive chemical assay can detect endotoxin only at a similar range, the whole-blood assay may be a biological assay that could be used in place of the rabbit pyrogen assay.

Process development for the production of an E. coli produced clinical grade recombinant malaria vaccine for Plasmodium vivax

The global eradication of malaria will require the development of vaccines to prevent infection cause by Plasmodium vivax in addition to Plasmodium falciparum. In an attempt to contribute to this effort we have previously reported the cloning and expression of a vaccine based on the circumsporozoite protein of P. vivax. The synthetic vaccine encodes for a full-length molecule encompassing the N-terminal and C-terminal regions flanking a chimeric repeat region representing VK210 and VK247, the two major alleles of P. vivax CSP. The vaccine, designated vivax malaria protein 001 (VMP001), was purified to >95% homogeneity using a three-column purification scheme and had low endotoxin levels and passed the rabbit pyrogenicity assay. The protein is recognized by monoclonal antibodies directed against the two repeat motifs, as well as polyclonal antibodies. Immunization with VMP001 induced high titer antibodies in mice using Montanide ISA 720. We currently have more than 10,000 doses of purified bulk and 1800 vials of formulated bulk vaccine available for clinical testing and VMP001 is currently undergoing further development as a candidate vaccine to prevent malaria in humans.

The Adjuvant Effect of Ambient Particulate Matter Is Closely Reflected by the Particulate Oxidant Potential

The Adjuvant Effect of Ambient Particulate Matter Is Closely Reflected by the Particulate Oxidant Potential

Improved Purification Process for Cholera Toxin and its Application to the Quantification of Residual Toxin in Cholera Vaccines

A simplified method for the purification of cholera toxin was developed. The 569B strain of Vibrio cholerae, a recognized hyper-producer of cholera toxin, was propagated in a bioreactor under conditions that promote the production of the toxin. The toxin was separated from the bacterial cells using 0.2-microm crossflow microfiltration, the clarified toxin was passed through the membrane into the permeate, and the bacterial cells were retained in the retentate. The 0.2-microm permeate was then concentrated 3-fold and diafiltered against 10 mM phosphate buffer, pH 7.6, using 30-kDa crossflow ultrafiltration. The concentrated toxin was loaded onto a cation exchange column, the toxin was bound to the column, and most of the impurities were passed unimpeded through the column. The toxin was eluted with a salt gradient of phosphate buffer, pH7.0, containing 1.0M NaCl. The peak containing the toxin was assayed for cholera toxin and protein and the purity was determined to be 92%. The toxin peak had a low endotoxin level of 3.1 EU/microg of toxin. The purified toxin was used to prepare antiserum against whole toxin, which was used in a G(M1) ganglioside-binding ELISA to determine residual levels of toxin in an oral inactivated whole-cell cholera vaccine. The G(M1) ganglioside-binding ELISA was shown to be very sensitive and capable of detecting as little as 1 ng/ml of cholera toxin.

Tangential flow filtration of hemoglobin

Bovine and human hemoglobin (bHb and hHb, respectively) was purified from bovine and human red blood cells via tangential flow filtration (TFF) in four successive stages. TFF is a fast and simple method to purify Hb from RBCs using filtration through hollow fiber (HF) membranes. Most of the Hb was retained in stage III (100 kDa HF membrane) and displayed methemoglobin levels less than 1%, yielding final concentrations of 318 and 300 mg/mL for bHb and hHb, respectively. Purified Hb exhibited much lower endotoxin levels than their respective RBCs. The purity of Hb was initially assessed via SDS-PAGE, and showed tiny impurity bands for the stage III retentate. The oxygen affinity (P(50)) and cooperativity coefficient (n) were regressed from the measured oxygen-RBC/Hb equilibrium curves of RBCs and purified Hb. These results suggest that TFF yielded oxygen affinities of bHb and hHb that are comparable to values in the literature. LC-MS was used to measure the molecular weight of the alpha (alpha) and beta (beta) globin chains of purified Hb. No impurity peaks were present in the HPLC chromatograms of purified Hb. The mass of the molecular ions corresponding to the alpha and beta globin chains agreed well with the calculated theoretical mass of the alpha- and beta- globin chains. Taken together, our results demonstrate that HPLC-grade Hb can be generated via TFF. In general, this method can be more broadly applied to purify Hb from any source of RBCs. This work is significant, since it outlines a simple method for generating Hb for synthesis and/or formulation of Hb-based oxygen carriers.

Low Levels of Endotoxin Enhance Allergen-Stimulated Proliferation and Reduce the Threshold for Activation in Human Peripheral Blood Cells

Background: Endotoxins, comprised of bacterial cell wall lipopolysaccharides (LPS), have been reported to have both protective and exacerbating effects on the development and maintenance of allergic disease in humans and on markers of allergic inflammation in animal models of allergy. In this study, we investigated the effect of low concentrations of LPS on human peripheral blood mononuclear cells (PBMC) stimulated with the major cat allergen Fel d 1. Methods: Extensive purification of recombinant (r) Fel d 1 yielded essentially endotoxin-free rFel d 1 (0.2 ng LPS /mg protein). PBMCs prepared from 15 subjects having IgE to cat (>0.7 kU_A/l) and 8 subjects IgE negative to cat were stimulated with 2, 10 or 25 µg/ml of rFel d 1 in the presence or absence of 50 pg/ml LPS. Proliferation was measured after 7 days of culture and supernatants were analyzed for IFNγ, IL-5 and IL-10. Results: LPS (50 pg/ml) increased rFel d 1-stimulated proliferation of PBMCs both from subjects IgE-positive and subjects negative to cat allergens. PBMCs from 13 of the subjects did not proliferate in response to stimulation with 2 and 10 µg/ml rFel d 1 alone but did so in the presence of LPS. Moreover, LPS increased the levels of rFel d 1-stimulated IFNγ in cultures from cat-negative subjects, IL-5 from cat-positive subjects and IL-10 from both groups. Conclusion: Very low doses of LPS enhance proliferation and decrease the apparent threshold level for cell activation, prompting careful evaluation of allergen stimulated T cell activation in vitro.

Commercially available recombinant sonic hedgehog up-regulates Ptc and modulates the cytokine and chemokine expression of human macrophages: An effect mediated by endotoxin contamination?

The Sonic hedgehog (Shh) signalling pathway plays an important role in developmental patterning and proliferation. Recent evidence suggests that Shh also plays a role in the development of the immune system. Here, we demonstrate that components of the Shh signalling pathway are expressed in human macrophages and that the receptor for Shh, Ptc, is up-regulated by a commercially available recombinant preparation of Shh (CArShh). Further, we report that the addition of CArShh up-regulates the production of IL-6, IL-8, MCP-1, IP-10, MIG and RANTES by macrophages, an effect enhanced by the presence of fetal calf serum in the culture medium. In contrast, TGF- β, TNF- α, IL-1b, IL-12 and IL-10 production were not modulated by CArShh and VEGF was minimally up-regulated even in the presence of serum. The up-regulation of these cytokines and chemokines was abrogated by CD14 inhibition and polymixin B, but not reliably inhibited by the specific Shh pathway inhibitor cyclopamine. These results suggest that, although components of the Shh signalling pathway are expressed in macrophages, the modulation of macrophage cytokine and chemokine effector function seen in response to commercially available rShh results from low levels of endotoxin contained within the CArShh preparations employed to explore the effects of Shh in vitro.

Development of Low Endotoxin Gelatin for Regenerative Medicine

The basic concept of tissue engineering, an actively applied technology in regenerative medicine, is the coordination between cells, scaffolds, and proliferation factors. Collagen and gelatin are materials that have mostly been used as scaffolds because of their excellent affinity for cells, they are biodegradable, and their ability to form gel. However, very few of the collagen or gelatin preparations used as tissue engineering material have been prepared with low levels of endotoxin, which may trigger fever, shock and a fall in blood pressure even in very minute quantities. Therefore we decided to decrease the content of endotoxin in gelatin so that this biomaterial is not harmful to the body but still maintains its properties, namely, bioaffinity and biodegradation for use in the fields of cell culture and regenerative medicine. In this paper, we describe a method whereby endotoxin could be removed from gelatin solution by using an ultrafiltration membrane with a molecular weight cut-off of 100 kDa and low endotoxin gelatin, endotoxin activity in 0.1% gelatin solution was below 0.03 EU/ml, was obtained as the resulting filtrate solution.

Endotoxin Levels in Rural Thai and Urban Singaporean Homes

Background: Exposure to dust endotoxin and allergens in early childhood may influence the development of allergic diseases. Aims: This study aimed to evaluate dust endotoxin and dust mite allergens in urban Singapore and rural Thai homes of young children and study potential environmental influences. Methods: Mattress dust endotoxin and Der p 1, Der f 1, group 2 (Der f 2 and Der p 2) and Blo t 5 allergen levels were quantified in 101 infant mattress dust samples, 51 from urban Singapore and 50 from rural Thailand. Comprehensive questionnaires on the home environment and cleaning practices were completed. Results: Endotoxin levels in rural Thailand were significantly higher than in urban Singapore (geometric mean 26,334.12 ± 4.60 and 18,377.85 ± 2.52 endotoxin units/g, respectively; p = 0.032). In contrast, higher levels of Der f 1 (p = 0.02), group 2 (p < 0.01) and Blo t 5 (p < 0.01) allergens were found in Singapore homes compared with rural Thai homes. Multiple logistic regression analysis showed that the use of detergents (p = 0.001) and disinfectants (p = 0.024) to clean floors and mattress protectors (p = 0.021) were independently associated with lower endotoxin levels. Conclusion: Endotoxin levels are higher in rural compared with urban homes in South East Asia. The reverse was true for dust mite allergen levels. Certain identifiable home environmental conditions and practices accounted for the differences in endotoxin levels.

Rapid monitoring of bacteria in dialysis fluids by fluorescent vital staining and microcolony methods

Bacterial contamination of dialysis fluids can cause inflammation and pyrogenic reactions in patients. Bacteria can grow more rapidly in dialysis fluids than in the high quality water used for their preparation, because dialysis fluids contain nutrients and electrolytes. Rapid determination of the microbiological quality of dialysis fluids is important, as it may aid in the reduction of instances of endotoxaemia. Microbiological contamination in dialysis systems should be detected as quickly as possible in order to provide dialysis fluid to patients with safety. Microbial contamination in dialysis fluids is usually assessed by culture-dependent methods such as plate counting. Arvanitidou et al. [1] examined dialysis fluids collected from 85 haemodialysis centres in Greece and found a total of 3500 5000 colony-forming units (CFUs)/ml of heterotrophic bacteria in 40% of measured samples [1]. Oie et al. [2] reported that dialysis fluids are more frequently contaminated than the water, acid or bicarbonate concentrates used for dialysis fluid preparation, and that the bacterial count in 42.5% of 40 dialysis fluid samples was >2000CFU/ml. These data indicate that the microbial contamination levels in a considerable proportion of dialysis fluid samples are relatively high. However, culture-dependent methods often underestimate bacterial numbers, because many bacterial species such as mycobacteria cannot be cultivated by conventional means [3], thus potentially placing the patients at a higher risk of inflammation and pyrogenic reactions. In addition, plate counting can require up to several days to detect bacteria. Therefore, microbiological qualities of dialysis fluids are often evaluated by measuring the endotoxin level. The endotoxin assays are simple and require a few hours to obtain results. However, endotoxin levels are often not correlated with numbers of CFU, and high numbers of colonyforming cells are sometimes detected in dialysis fluids with significantly low endotoxin level [4]. Simple, rapid and accurate methods of enumeration without longtime (e.g. 7 days) culture are essential, in order to maintain microbiological quality of dialysis fluids. Fluorescent staining is widely applied to detect bacteria rapidly [5]. Fluorescent dyes stain the nucleic acid and/or protein within the bacterial cells, and these cells can be counted by fluorescent microscopy, laser scanning cytometry and flow cytometry. Fluorescent vital staining such as 40,6-diamidino-2-phenylindole (DAPI) – carboxyfluorescein diacetate (CFDA) double staining can simultaneously determine the total bacterial count and the number of physiologically active bacteria (bacteria with enzymatic activity), not only in environmental samples (river water, pond water, ground water) but also in medical and pharmaceutical samples (pharmaceutical water [6] and herbal medicines [7]). By this technique, numbers of total bacteria (both active and inactive cells) are determined by DAPI staining, and physiologically active cells are selectively enumerated with CFDA staining, without the need for culture. DAPI is used for total direct counting of microbial cells in aquatic environments, because of its high stainability [5]. CFDA is a popular, ultra-sensitive, fluorescent substrate for estimating esterase activity in live micro-organisms (bacteria, yeast and fungi) [5]. Activity of cytoplasmic esterase of micro-organisms is an indicator of their physiological activity. Not only cells with growth potential but also dormant or injured cells can be stained with CFDA. Many bacteria in water with low nutrient concentrations cannot form large colonies which are Correspondence and offprint requests to: Masao Nasu, Environmental Science and Microbiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan. Email: nasu@phs.osaka-u.ac.jp Nephrol Dial Transplant (2007) 22: 612–616

Respiratory response to endotoxin and dust predicts evidence of inflammatory response in volunteers in a swine barn

It has been shown that exposure in intense exposure in swine barn facilities is associated with increased respiratory symptoms and reduction in pulmonary functions. This study investigated if systemic response could be predicted by FEV(1) response following swine barn exposure. Naïve males were tested at baseline, low and high endotoxin and dust levels. Subjects were classified as "more responsive" (n = 9) or "less responsive" (n = 11) based on FEV(1) reduction following high endotoxin exposure. Health measures included pulmonary function testing, blood samples and nasal lavage. Environmental samples were collected from the barn. White blood cells and blood lymphocytes at low exposure were significantly greater in those who were "more responsive" compared to those who were "less responsive". There was a significant increase in blood lymphocytes, serum IL6, total nasal lavage cells and nasal IL8 at high exposure among "more responsive" subjects compared to "less responsive" subjects. Respiratory response to high-level endotoxin and dust exposure predicts evidence of inflammatory response throughout a range of endotoxin and dust exposures.

Vasoactive Intestinal Peptide and PACAP38 Control N-Methyl-D-aspartic Acid-induced Dendrite Motility by Modifying the Activities of Rho GTPases and Phosphatidylinositol 3-Kinases

Dendrite morphogenesis is highly dynamic and characterized by the addition and elongation of processes and also by their selective maintenance, retraction, and elimination. Glutamate can influence these events via N-methyl-d-aspartic acid (NMDA) receptors. The neuropeptides vasoactive intestinal peptide and pituitary adenylyl cyclase-activating polypeptide-38 (PACAP38) affect neurogenesis and differentiation in the developing nervous system. We report here that the peptides and NMDA acted synergistically on dendrite and branch formation. In stage III hippocampal neurons, NMDA increased not only the addition but also the elimination of new dendrites and branches by activating Rac and Cdc42 and phosphatidylinositol 3-kinases, respectively. When applied alone, the neuropeptides did not influence dendrite or branch formation. However, they reduced the elimination of newly formed dendrites and branches caused by NMDA by preventing the NMDA-induced activation of phosphatidylinositol 3-kinases. This led to the formation of persistent dendrites and branches. Additional timelapse studies on the dynamics of dendrite elongation showed alternating periods of elongation and retraction. Phosphatidylinositol 3-kinases increased the velocities of dendrite elongation and retraction, whereas the neuropeptides prolonged the periods of elongation. By modifying NMDA-induced activation of Rho GTPases and phosphatidylinositol 3-kinases, vasoactive intestinal peptide and PACAP38 could play an important role in the control of dendrite growth and branching during development and in response to neuronal activity.

Do Native and Polymeric α1-Antitrypsin Activate Human Neutrophils In Vitro?

alpha(1)-Antitrypsin (AAT)-Z deficiency is a risk factor for the development of COPD. Compared to wild-type M, AAT-Z has an increased tendency to polymerize, rendering it inactive as a serine proteinase inhibitor. It has been demonstrated that wild-type M- and Z-deficiency AAT polymers are chemotactic for human neutrophils. However, our own studies dispute a proinflammatory role for polymerized AAT-M and AAT-Z, suggesting rather that they are predominantly antiinflammatory, exhibiting inhibitory effects on lipopolysaccharide-stimulated human monocyte activation. The discrepancies between these observations prompted us to re-examine the effects of AAT. The effects of native and polymerized AAT-M and AAT-Z with varying levels of endotoxin contamination (0.08 to 2.55 endotoxin units [EU]/mg protein) on human neutrophil chemotaxis and interleukin (IL)-8 release, in vitro, were evaluated. Neither native nor polymerized (M- or Z-deficient) AAT contaminated with low levels of endotoxin (</= 0.08 EU/mg protein) stimulated neutrophil chemotaxis, whereas N-formyl methionyl leucyl phenylalanine (fMLP), a positive control, increased chemotaxis fourfold. A small but nonsignificant increase in neutrophil chemotaxis, however, was observed with AAT preparations containing higher levels of endotoxin (>/= 0.88 EU/mg protein), and significant chemotaxis occurred when AAT was spiked with either endotoxin or zymosan. In support, native and polymeric AAT-M with low endotoxin contamination completely inhibited neutrophil IL-8 release triggered by the zymosan, while AATs with high endotoxin contamination strongly induced IL-8 release and did not inhibit zymosan-stimulated IL-8 release. The proinflammatory effects of native and polymeric AAT may be critically dependent on the presence of other cell activators, bacterial or otherwise, while pure preparations of AAT appear to exert predominantly antiinflammatory activity.

History and results of the two inter‐laboratory round robin endotoxin assay studies on cotton dust

In the US cotton industry, airborne cotton dust levels are regulated, and other countries are moving to specify safety limits for airborne endotoxins. There is concern about potential respiratory health hazards associated with agricultural and other organic dusts. In laboratories, ranking which samples have high and low levels of endotoxin is usually in good agreement between laboratories. When different laboratories assay identical samples, the levels differ. The objective of this research was to evaluate the intra- and inter-laboratory variability for 13 laboratories measuring endotoxin in cotton dust. Two inter-laboratory round robin endotoxin assay studies were conducted using cotton dust. In the first round robin, each laboratory used their normal in-house assay method and then used a common extraction protocol. In the second round robin, a common extraction protocol and endotoxin assay kit was used. The intra-laboratory results had small variations but inter-laboratory results had very high variations. The inter-laboratory results using a common extraction protocol showed reduced differences. Using the same extraction protocol and endotoxin assay kit, the intra-laboratory variation was small and inter-laboratory variation was reduced but not enough for inter-laboratory agreement. Most of the laboratories were able to discern between the high and low endotoxin concentration dusts. Standardization has reduced the differences in results between laboratories and possibly further standardization may bring closer inter-laboratory agreement.

Quantification of endotoxins in necrotic root canals from symptomatic and asymptomatic teeth

The purpose of this investigation was to quantify the concentration of endotoxin in necrotic root canals and investigate the possible relationship between the concentration of endotoxin and endodontic signs and symptoms. Samples were collected from root canals of 50 patients requiring endodontic treatment due to necrosis of the pulpal tissue. Anaerobic techniques were used to determine the number of c.f.u. in each sample. A quantitative chromogenic Limulus amoebocyte lysate assay was used to measure the concentration of endotoxin in each sample. The presence of c.f.u. was detected by culture in all samples (range 10(2)-5x10(6)). In samples from cases of patients with spontaneous pain, the mean c.f.u. was 1.43x10(6) while in asymptomatic cases it was 9.1x10(4). Endotoxin was present in all the samples studied [range 2390.0-22100.0 endotoxin units (EU) ml-1]. The mean concentration of endotoxin in samples from patients with spontaneous pain was 18540.0 EU ml-1 while in asymptomatic cases it was 12030.0 EU ml-1. Asymptomatic cases generally had lower levels of endotoxin (i.e. a negative association). A positive association was found between endotoxin and symptomatic cases (e.g. spontaneous pain, tenderness to percussion, pain on palpation, swelling and purulent exudates). This study showed that endotoxin is present in high concentrations in root canals of symptomatic teeth. There was a positive correlation between the concentration of endotoxin in the root canal and the presence of endodontic signs and symptoms.

Lipopolysaccharide (LPS) Contamination Plays the Real Role in C-Reactive Protein–Induced IL-6 Secretion From Human Endothelial Cells In Vitro

To the Editor, In a recent article, Taylor et al1 have shown that C-Reactive Protein (CRP), per se, does not activate endothelial cells; rather, lipopolysacchride (LPS) and azide were responsible for the observed cellular activation. We would like to provide our data to further support the conclusion by Taylor and colleagues. We have tested >10 batches of commercial human CRP preparations from 3 companies (Calbiochem, TriChem, and Sigma). Human saphenous vein endothelial cells (HUVECs, passages 3 to 5) were grown …