T1 bacteriophage as an indicator for decontamination of laminar-flow biological safety cabinets.

Type 1 coliphage dried onto a glass surface was used as an indicator to monitor decontamination of biological safety cabinets. When desiccated virus was treated with formaldehyde vapor (5,000 or 10,000 ppm) adjusted to 70 to 90% relative humidity immediately before testing, viral inactivation was slow for the first 50 min but then accelerated, being complete in the next 10 min. However, when virus was incubated in an atmosphere containing 70% humidity for 1 h before formaldehyde was added, inactivation was complete within 3 min, indicating that careful attention must be paid to relative humidity in decontamination of safety cabinets.     

Yersiniae in pond water and snails

A Yersinia enterocolitica serotype 9 was isolated from pond water; Y. enterocolitica-like bacteria were also isolated from pond water and from three species of snails (Lymnaea palustris elodes, Helisoma sp., Oxyloma retusa) from the Edwin S. George Reserve in southeastern Michigan. There was evidence for biochemical stability among some of the organisms over a period of years. There also was evidence of transmission of these organisms to snails from the water.     

In Vivo and In Vitro Effects of Some Plant Hormones on Rat Erythrocyte Carbonic Anhydrase and Glucose-6-Phosphate Dehydrogenase Activities

The present study was undertaken to determine in vivo and in vitro effects of some plant growth regulators on rat erythrocyte carbonic anhydrase (CA) and glucose-6-phosphate dehydrogenase (G6PD) activities. Both in vivo and in vitro, spermidine and kinetin did not affect enzymatic activities of CA and G6PD, whereas putrescine decreased these activities, and abscisic acid increased them. Since plants use such growth regulators, their effects should be considered on mammals consuming them since they may possess important biological effects.     

The Helmholtz Network for Bioinformatics: an integrative web portal for bioinformatics resources

SUMMARY: The Helmholtz Network for Bioinformatics (HNB) is a joint venture of eleven German bioinformatics research groups that offers convenient access to numerous bioinformatics resources through a single web portal. The 'Guided Solution Finder' which is available through the HNB portal helps users to locate the appropriate resources to answer their queries by employing a detailed, tree-like questionnaire. Furthermore, automated complex tool cascades ('tasks'), involving resources located on different servers, have been implemented, allowing users to perform comprehensive data analyses without the requirement of further manual intervention for data transfer and re-formatting. Currently, automated cascades for the analysis of regulatory DNA segments as well as for the prediction of protein functional properties are provided. AVAILABILITY: The HNB portal is available at http://www.hnbioinfo.de     

Cutting edge: Immune stimulation by neisserial porins is toll-like receptor 2 and MyD88 dependent

The immunopotentiating activity of neisserial porins, the major outer membrane protein of the pathogenic Neisseria, is mediated by its ability to stimulate B cells and up-regulate the surface expression of B7-2. This ability is dependent on MyD88 and Toll-like receptor (TLR)2 expression, as demonstrated by a lack of a response by B cells from MyD88 or TLR2 knockout mice to the porins. Using previously described TLR2-dependent reporter constructs, these results were confirmed and were shown to be due to induction of NF-kappaB nuclear translocation. This is the first demonstration of known vaccine adjuvant to stimulate immune cells via TLR2.     

Neisseria meningitidis PorB, a TLR2 ligand, induces an antigen-specific eosinophil recall response: potential adjuvant for helminth vaccines?

Efficacious adjuvants are important components of new vaccines. The neisserial outer membrane protein, PorB, is a TLR2 ligand with unique adjuvant activity. We demonstrate that PorB promotes Th2-skewed cellular immune response to the model Ag, OVA, in mice, including Ag-specific recall eosinophil recruitment to the peritoneum. PorB induces chemokine secretion by myeloid cells using both TLR2-dependent and -independent mechanisms, suggesting that anatomical distribution of TLR2(+) cells may not be a limiting factor for potential vaccine strategies. The results from this study suggest that PorB, and other TLR2 ligands, may be ideal for use against pathogens where eosinophilia may be protective, such as parasitic helminths.     

Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

Background

Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures.

Results

We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting.

Conclusions

Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.     

Alimentary prion infections: Touchdown in the intestine

Neurodegenerative diseases are caused by proteinaceous aggregates, usually consisting of misfolded proteins which are often typified by a high proportion of β-sheets that accumulate in the central nervous system. These diseases, including Morbus Alzheimer, Parkinson disease and Transmissible Spongiform Encephalopathies (TSEs)—also termed prion disorders—afflict a substantial proportion of the human population and, as such, the etiology and pathogenesis of these diseases has been the focus of mounting research. Although many of these diseases arise from genetic mutations or are sporadic in nature, the possible horizontal transmissibility of neurodegenerative diseases poses a great threat to population health. In this article we discuss recent studies that suggest that the “non-transmissible” status bestowed upon Alzheimer and Parkinson diseases may need to be revised as these diseases have been successfully induced through tissue transplants. Furthermore, we highlight the importance of investigating the “natural” mechanism of prion transmission including peroral and perenteral transmission, proposed routes of gastrointestinal uptake and neuroinvasion of ingested infectious prion proteins. We examine the multitude of factors which may influence oral transmissibility and discuss the zoonotic threats that Chronic Wasting disease (CWD), Bovine Spongiform Encephalopathy (BSE) and Scrapie may pose resulting in vCJD or related disorders. In addition, we suggest that the 37 kDa/67 kDa laminin receptor on the cell surface of enterocytes, a major cell population in the intestine, may play an important role in the intestinal pathophysiology of alimentary prion infections.Key words: prion, 37 kDa/67 kDa laminin receptor, CJD, BSE, CWD, scrapie, Alzheimer disease, Parkinson disease, intestine, enterocytesMany different mechanisms exist which underlie the etiology of the numerous neurodegenerative diseases affecting the human population. Amongst the most prominent are Morbus Alzheimer, prion disorders, Parkinson disease, Chorea Huntington, frontotemporal dementia and amylotrophic lateral sclerosis. The molecular mechanisms underlying these diseases vary; however, all neurodegenerative diseases share a common feature: they are caused by protein aggregation. The only neurodegenerative diseases proven to be transmissible are prion disorders. In contrast to frontotemporal dementia, recent evidence suggests that Alzheimer and Parkinson diseases may also be transmissible. Pre-symptomatic Alzheimer disease (APP23) mice exhibited an increase in the Alzheimer phenotype when brain homogenate of autopsied human Alzheimer disease patients and older, amyloid beta- (Aβ-) laden APP23 mice was injected into their hippocampi.¹ These findings suggest that the Aβ-abundant brain homogenate of Alzheimer disease patients may possess the ability to induce or supplement the overproduction of Aβ, possibly leading to the onset of Alzheimer disease.The pathological feature associated with Parkinson disease is the formation of Lewy bodies in cell bodies and neuronal processes in the brain.² The main component of these protein aggregates is α-synuclein (reviewed in ref. ²). Autopsies of Parkinson disease patients revealed that Lewy bodies had formed on healthy embryonic neurons that had been grafted onto the brain tissue of the patients several years before (prior to said examination).^3–5 It may thus be proposed that α-synuclein transmission is possible from diseased to healthy neurons, suggesting that Parkinson disease may be transmissible from a Parkinson disease patient to a healthy individual. These findings imply that Alzheimer and Parkinson diseases may be transmissible through tissue transplants and the use of contaminated surgical tools.⁶Prion disorders, also termed Transmissible Spongiform Encephalopathies (TSEs), are fatal neurodegenerative diseases that affect the central nervous system (CNS) of multiple animal species. In lieu of the social, economic and political ramifications of such infections, as well as the possible intra- and interspecies transmissibility of such disorders, various routes of experimental transmission have been investigated including intracerebral, intraperitoneal, intraventricular, intraocular, intraspinal and subcutaneous injections (reviewed in ref. ^7–9). However, such routes of transmission are not representative of the “natural” mechanism as the majority of prion disorders are contracted through ingestion of infectious prion (PrP^Sc) containing material. Thus, the peroral and perenteral prion transmission is of greatest consequence with respect to TSE disease establishment. Moreover, the presence of PrP^Sc in the buccal cavity of scrapie-infected sheep¹⁰ (reviewed in ref. ¹¹) and the possible horizontal transfer as a result hereof, as may be similarly proposed for animals suffering from other TSEs, may further contribute to the oral transmissibility of TSEs.A number of model systems have been employed to study TSE transmissibility. Owing to ethical constraints, TSE transmissibility to humans via the oral route may not be directly investigated and as a result hereof, alternative model systems are needed. These may include the use of transgenic mice, cell lines which are permissive to infection¹² and experimental animals such as sheep, calves, goats, minks, ferrets and non-human primates (reviewed in ref. ⁹).Intestinal entry of PrP^Sc has been proposed to occur via two pathways, the membranous (M) cell-dependent and M cell-independent pathways (Fig. 1).^13,14 The former involves endocytic M (microfold)-cells, which cover the intestinal lymphoid follicles (Peyer''s patches)¹⁴ and may take up prions and thereby facilitate the translocation of these proteins across the intestinal epithelium into the lymphoid tissues (reviewed in ref. ⁹) as has been demonstrated in a cellular model.¹³ Following such uptake by the M cells, the prions may subsequently pass to the dendritic cells and follicular dendritic cells (FDCs) (Fig. 1), which allow for prion transport to the mesenteric lymph nodes and replication, respectively.¹⁵ The prion proteins may subsequently gain access to the enteric nervous system (ENS) and ultimately the central nervous system (CNS).¹⁵Open in a separate windowFigure 1Proposed routes of gastrointestinal entry of ingested infectious prions (PrP^Sc) as well as possible pathways of amplification and transport to the central nervous system.However, prion intestinal translocation has been observed in the absence of M cells and has been demonstrated to be as a result of the action of polar, 37 kDa/67 kDa LRP/LR (non-integrin laminin receptor; reviewed in ref. ^16–18) expressing enterocytes. Enterocytes are the major cell population of the intestinal epithelium and due to their ability to endocytose pathogens, nutrients and macromolecules,¹⁹ it has been proposed that these cells may represent a major entry site for alimentary prions (Fig. 1).Since enterocyte prion uptake has been demonstrated to be dependent on the presence of LRP/LR on the apical brush border of the cells,^14,20 the interaction between varying prion protein strains and the receptor^21–23 may be employed as a model system to study possible oral transmissibility of prion disorders across species as well as the intestinal pathophysiology of alimentary prion infections.²⁴ Moreover, the blockage of such interactions through the use of anti-LRP/LR specific antibodies has been reported to reduce PrP^Sc endocytosis¹⁹ and thus these antibodies may serve as potential therapeutics to prevent infectious prion internalization and thereby prevent prion infections. It must be emphasized that the adhesion of prion proteins to cells is not solely dependent on the LRP/LR-PrP^Sc interactions;²⁴ however, this interaction is of importance with regards to internalization and subsequent pathogenesis.We applied the aforementioned cell model to study the possible oral transmission of PrP^BSE, PrP^CWD and ovine PrP^Sc to cervids, cattle, swine and humans.²⁴ The direct transmission of the aforementioned animal prion disorders to humans as a result of dietary exposure and the possible establishment of zoonotic diseases is of great public concern. It must however be emphasized that the study investigated the co-localization of LRP/LR and various prion strains and not the actual internalization process.PrP^BSE was shown to co-localize with LRP/LR on human enterocytes²⁴, thereby suggesting that PrP^BSE is transmissible to humans via the oral route which is widely accepted as the manner by which variant CJD originated. This suspicion was previously investigated using a macaque model, which was successfully perorally infected by BSE-contaminated material and subsequently lead to the development of a prion disorder that resembles vCJD.²⁵ These results, due to the evolutionary relatedness between macaques and humans, allowed researchers to confirm the oral transmissibility of PrP^BSE to humans. PrP^BSE may also potentially lead to prion disorder establishment in swine,²⁴ livestock of great economic and social importance.The prion disorder affecting elk, mule deer and white-tailed deer is termed CWD. Cases of the disease are most prevalent in the US but are also evident in Canada and South Korea.^26,27 As the infectious prion isoform is reported to be present in the blood²⁸ and skeletal muscle,²⁹ hunting, consumption of wild venison and contact with other animal products derived from CWD-infected elk and deer may thereby pose a public health risk. Our studies demonstrate that PrP^CWD co-localizes with LRP/LR on human enterocytes²⁴ thereby suggesting a possible oral transmissibilty of this TSE to humans. This is, however, inconsistent with results obtained during intra-cerebral inoculation of the brains and spinal cords of transgenic mice overexpressing the human cellular prion protein (PrP^c),^26,27 which is essential for TSE disease establishment and progression. Further, discrepancies have also been reported with respect to non-human primates, as squirrel monkeys have been successfully intracerebrally inoculated with mule-deer prion homogenates,³⁰ while cynolmolgus macaques were resistant to infection.³¹ CWD has been transmitted to ferrets, minks and goats³² and as these animals may serve as domestic animals or livestock, secondary transmission from such animals to humans, through direct contact or ingestion of infected material, may be an additional risk factor that merits further scientific investigation.Ovine PrP^Sc co-localization with LRP/LR on human and bovine enterocytes may be indicative of the infectious agents'' ability to effect cross-species infections. The oral transmissibility of Scrapie has been confirmed in hamsters fed with sheep-scrapie-infected material.³³The discrepancies with regards to the transmissibility of certain infectious prion proteins when assessed by different model systems may be due to the experimental transmission route employed. Oral exposure often results in significantly prolonged incubation times when compared to intracerebral inoculation techniques and thus failure of transgenic mice and normal experimental animals to develop disease phenotypes after being fed TSE-contaminated material may not necessarily indicate that the infection process failed.¹⁴ Apart from the route of infection, numerous other factors may influence transmission between species, including dose, PrP polymorphisms and genetic factors, the prion strain employed as well as the efficacy of prion transport to the CNS.³⁴ The degree of homology between the PrP^c protein in the animals serving as the infectious prion source and recipient has also been described as a feature limiting cross-species transmission.³⁴ The negative results, as referred to above, obtained upon prion-protein inoculation of animal models may have resulted due to the slow rate at which the infectious prion induces conformational conversion of the endogenous PrP^c in the animal cells and this in turn results in low levels of infectious prion replication and symptom development.²⁷Furthermore, even in the event that certain prion disorders are not directly transmissible to humans, most are transmissible to at least a single species of domestic animal or livestock. The infectious agents properties may be altered in the secondary host such that it becomes transmissible to humans (reviewed in ref. ³⁵). Thus, interspecies transmission between animals may indirectly influence human health.It is noteworthy to add that although the oral route of PrP^Sc transmission may result in prolonged incubation times, it may broaden the range of susceptible hosts. A common constituent of food is ferritin, a protein that is resistant to digestive enzyme hydrolysis and, due to its homology across species, it may serve as co-transporter of PrP^Sc and facilitate enterocyte internalization of the infectious prion.³⁶ It may thus be proposed that prion internalization may occur via a ferritin-PrP^Sc complex even in the absence of co-localization between the infectious agent and LRP/LR such that many more cross-species infections (provided that the other infection factors are favorable) may be probable.³⁷ In addition, digestive enzymes in the gastrointestinal tract facilitate PrP^Sc binding to the intestinal epithelium and subsequent intestinal uptake³⁶ and thus depending on the individuals'' digestive processes, the susceptibility to infection and the rate of disease development may vary accordingly. As a result hereof, though laboratory experiments in cell-culture and animal models may render a particular prion disorder non-infectious to humans, this may not be true for all individuals.In lieu of the above statements, with particular reference to inconsistencies in reported results and the multiple factors influencing oral transmissibility of TSEs, further transmission studies are required to evaluate the zoonotic threat which CWD, BSE and Scrapie may pose through ingestion.     

T-Cell activation by t(9;22) acute lymphoblastic leukemia-derived dendritic-like cells is associated with increased tapasin expression

Dendritic-like cells from t(9;22) acute lymphoblastic leukemia (ALL) blasts can activate T cells, while the original unmodified leukemic blasts cannot. To determine whether these functional differences were associated with differences in antigen-processing machinery (APM) component expression, we have measured the level of APM component expression in unmodified blasts and ALL-derived dendritic-like cells. Seven t(9;22) ALL patient samples and one cell line were studied for APM component expression utilizing a unique panel of recently developed monoclonal antibodies and a recently developed intracellular staining technique. In addition, the HLA class I antigen cell surface expression was measured. HLA class I antigens were similarly expressed on the unmodified blasts and on the autologous dendritic-like cells. Intracellular HLA class I antigen and tapasin expression (P=0.03 for both) were upregulated in all t(9;22) ALL-derived dendritic-like cells, in comparison to the unmodified blasts. These results provide a potential mechanism for the ability of t(9;22) ALL-derived dendritic-like cells to induce T-cell activation and, suggest that tapasin upregulation may serve as a marker to standardize and monitor the quality of the dendritic-like cells used in immunotherapy. Supported partially by The Heidi Leukemia Research Fund, Buffalo, NY, and by PHS grants CA 67108 and CA 16056 awarded by the National Cancer Institute, DHHS.     

Pharmacokinetic and -dynamic modelling of G-CSF derivatives in humans

ABSTRACT: BACKGROUND: The human granulocyte colony-stimulating factor (G-CSF) is routinely applied to support recovery of granulopoiesis during the course of cytotoxic chemotherapies. However, optimal use of the drug is largely unknown. We showed in the past that a biomathematical compartment model of human granulopoiesis can be used to make clinically relevant predictions regarding new, yet untested chemotherapy regimen. In the present paper, we aim to extend this model by a detailed pharmacokinetic and -dynamic modelling of two commonly used G-CSF derivatives Filgrastim and Pegfilgrastim. RESULTS: Model equations are based on our physiological understanding of the drugs which are delayed absorption of G-CSF when applied to the subcutaneous tissue, dose-dependent bioavailability, unspecific first order elimination, specific elimination in dependence on granulocyte counts and reversible protein binding. Pharmacokinetic differences between Filgrastim and Pegfilgrastim were modelled as different parameter sets. Our former cell-kinetic model of granulopoiesis was essentially preserved, except for a few additional assumptions and simplifications. We assumed a delayed action of G-CSF on the bone marrow, a delayed action of chemotherapy and differences between Filgrastim and Pegfilgrastim with respect to stimulation potency of the bone marrow. Additionally, we incorporated a model of combined action of Pegfilgrastim and Filgrastim or endogenous G-CSF which interact via concurrent receptor binding. Unknown pharmacokinetic or cell-kinetic parameters were determined by fitting the predictions of the model to available datasets of G-CSF applications, chemotherapy applications or combinations of it. Data were either extracted from the literature or were received from cooperating clinical study groups. Model predictions fitted well to both, datasets used for parameter estimation and validation scenarios as well. A unique set of parameters was identified which is valid for all scenarios considered. Differences in pharmacokinetic parameter estimates between Filgrastim and Pegfilgrastim were biologically plausible throughout. CONCLUSION: We conclude that we established a comprehensive biomathematical model to explain the dynamics of granulopoiesis under chemotherapy and applications of two different G-CSF derivatives. We aim to apply the model to a large variety of chemotherapy regimen in the future in order to optimize corresponding G-CSF schedules or to individualize G-CSF treatment according to the granulotoxic risk of a patient.