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Significance: Lupus nephritis (LuN) is a chronic inflammatory kidney disease. The cellular mechanisms by which LuN progresses to kidney failure are poorly characterized. Automated instance segmentation of immune cells in immunofluorescence images of LuN can probe these cellular interactions. Aim: Our specific goal is to quantify how sample fixation and staining panel design impact automated instance segmentation and characterization of immune cells. Approach: Convolutional neural networks (CNNs) were trained to segment immune cells in fluorescence confocal images of LuN biopsies. Three datasets were used to probe the effects of fixation methods on cell features and the effects of one-marker versus two-marker per cell staining panels on CNN performance. Results: Networks trained for multi-class instance segmentation on fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) samples stained with a two-marker panel had sensitivities of 0.87 and 0.91 and specificities of 0.82 and 0.88, respectively. Training on samples with a one-marker panel reduced sensitivity (0.72). Cell size and intercellular distances were significantly smaller in FFPE samples compared to fresh frozen (Kolmogorov–Smirnov, p≪0.0001). Conclusions: Fixation method significantly reduces cell size and intercellular distances in LuN biopsies. The use of two markers to identify cell subsets showed improved CNN sensitivity relative to using a single marker.

The SARS-CoV-2 virus is known as the causal agent for the current COVID-19 global pandemic. The majority of COVID-19 patients develop acute respiratory distress syndrome (ARDS), while some experience a cytokine storm effect, which is considered as one of the leading causes of patient mortality. Lipids are known to be involved in the various stages of the lifecycle of a virus functioning as receptors or co-receptors that controls viral propagation inside the host cell. Therefore, lipid-related metabolomics aims to provide insight into the immune response of the novel coronavirus. Our study has focused on determination of the potential metabolomic biomarkers utilizing a Teslin® Substrate in paper spray mass spectrometry (PS-MS) for the development of a rapid detection test within 60 seconds of analysis time. In this study, results were correlated with PCR tests to reflect that the systemic responses of the cells were affected by the COVID-19 virus.

Snake antivenom is the only specific treatment for snakebite envenoming, but life-threatening anaphylaxis is a severe side effect and drawback for the use of these typically mammalian serum products. The present study investigates the hypotheses whether serum IgE antibodies against the epitope galactose-alpha-1,3-galactose (α-gal) located on the heavy chain of non-primate mammalian antibodies are a possible cause for hypersensitivity reactions to snake antivenom. Serum samples from 55 patients with snakebite envenoming were obtained before administration of snake antivenom and tested for serum IgE (sIgE) against α-gal and total IgE. Early anaphylactic reactions (EARs) during the first 3 h after antivenom administration were classified into mild, moderate or severe and correlated with the presence of sIgE against α-gal. Fifteen (27%) out of 55 patients (37 male, 18 female, median 34 years, range 9–90 years) developed EARs after antivenom administration. Eleven, three and one patients had mild, moderate and severe EARs, respectively. Serum IgE against α-gal was detected in 17 patients (31%); in five (33%) out of 15 patients with EARs and in 12 (30%) out of 40 patients without EAR (Odds Ratio = 1.2; 95%-confidence interval: 0.3–4.2) with no correlation to severity. Although the prevalence of serum IgE against α-gal was high in the study population, very high levels of total IgE in the majority of patients question their clinical relevance and rather indicate unspecific sIgE binding instead of allergy. Lack of correlation between α-gal sIgE and EARs together with significantly increased total IgE levels suggest that sIgE against α-gal is not the major trigger for hypersensitivity reactions against snake antivenom., Highlights • Prevalence of serum IgE against α-gal is high in Laotian snakebite victims. • Serum IgE against α-gal is not the major trigger for EARs against snake antivenom. • The rate of EARs against snake antivenom from Thailand is 27%.

Objective The role of antiphospholipid antibodies (aPL) during apparently normal pregnancy is still unclear. IgA aPL are prevalent in populations of African origin. Our aim was to measure all isotypes of anticardiolipin (anti-CL) and anti–β2 glycoprotein I (anti-β2GPI) in healthy pregnant and non-pregnant women of different ethnicities. Methods Healthy Sudanese pregnant women ( n = 165; 53 sampled shortly after delivery), 96 age-matched Sudanese female controls and 42 healthy pregnant and 249 non-pregnant Swedish women were included. IgA/G/M anti-CL and anti-β2GPI were tested at one time point only with two independent assays in Sudanese and serially in pregnant Swedes. IgA anti-β2GPI domain 1 and as controls IgA/G/M rheumatoid factor (RF), IgG anti–cyclic citrullinated peptide 2 (anti-CCP2) and anti–thyroid peroxidase (anti-TPO) were investigated in Sudanese females. Results Pregnant Sudanese women had significantly higher median levels of IgA anti-CL, IgA anti-β2GPI ( p 2GPI (both assays; p 2GPI occurrence was increased among Sudanese pregnant women compared with national controls. No corresponding increase during pregnancy was found for IgA anti-β2GPI domain 1 antibodies. Both IgG anti-CL and IgG control autoantibodies decreased during and directly after pregnancy among Sudanese. Serially followed Swedish women showed no changes in IgA aPL, whereas IgG/M anti-CL decreased. Conclusions IgA aPL are increased in Sudanese but not in Swedish women, without corresponding increase in IgA domain 1. Whether due to ethnicity and/or environmental influences the occurrence of IgA aPL during Sudanese pregnancies, and its clinical significance, is yet to be determined.

Caused by the tick-borne spirochete Borrelia burgdorferi, Lyme disease (LD) is the most common vector-borne infectious disease in North America and Europe. Though timely diagnosis and treatment are effective in preventing disease progression, current tests are insensitive in early stage LD, with a sensitivity of $400/test) and extended sample-to-answer timelines (>24 h). To address these challenges, we created a cost-effective and rapid point-of-care (POC) test for early-stage LD that assays for antibodies specific to seven Borrelia antigens and a synthetic peptide in a paper-based multiplexed vertical flow assay (xVFA). We trained a deep-learning-based diagnostic algorithm to select an optimal subset of antigen/peptide targets and then blindly tested our xVFA using human samples (N(+) = 42, N(-) = 54), achieving an area-under-the-curve (AUC), sensitivity, and specificity of 0.950, 90.5%, and 87.0%, respectively, outperforming previous LD POC tests. With batch-specific standardization and threshold tuning, the specificity of our blind-testing performance improved to 96.3%, with an AUC and sensitivity of 0.963 and 85.7%, respectively.

ABO blood group is the most important blood type system for transfusion medicine. A paper-based analytical device (PAD) for ABO blood typing has been proposed. The device was composed of Whatman No. 113 paper, an absorbent gel pad, and a 3D-printing cassette. The 3D-printing cassette contained two circular holes for display of letters “A” and “B” on the PAD. Whole blood was dropped onto hydrophilic letters A and B on the PAD, in which the anti-A and anti-B were pre-immobilized, respectively. An absorbent gel pad was used to adsorb excess blood sample and washing solution during the washing step. The particle size of agglutinated red blood cells (RBCs) could not be eluted out of the paper by the elution solution. In contrast, non-agglutinated RBCs were washed out by means of elution solution. The devices could be used for real blood samples in a wide range of hematocrit levels, 21–59%. Unknown blood group samples (n = 500) were identified by the developed device and the results were compared with the conventional method, revealing 100% accuracy. Because of its compact size with low-cost fabrication, the portable ABO blood typing device has great potential for point-of-care testing, particularly in developing countries.

Respiratory pathogens have been detected in forensic investigations using multiple techniques; however, no study has examined the use of automated, nested, multiplex polymerase chain reaction (ANM‐PCR), commonly used in living patients, in the forensic setting. This retrospective study assessed the utility of ANM‐PCR in detecting respiratory pathogens in the pediatric forensic setting. Respiratory samples from 35 cases were tested for up to 20 respiratory pathogens. 51.4% of these cases yielded a positive ANM‐PCR result, 20% of which were considered the cause of or contributory to death. The most commonly detected pathogens were rhinovirus/enterovirus and respiratory syncytial virus, and these were the only pathogens determined to play a significant role in cause of death. The sampled sites and postmortem intervals tested did not affect the likelihood of a positive or negative test. ANM‐PCR panels are effective, affordable, and rapid ancillary tools in evaluating cause of death in the forensic pediatric population.

The fast and precise detection of potential allergen-specific immunoglobulin E (sIgE) is imperative for the diagnosis and appropriate treatment of allergic diseases. In this study, we have successfully fabricated a novel paper-based immunoassay device for the detection of sIgE in allergic diseases. We used Can f 1, one of the main dog allergens, as a model allergen to detect sIgE in human sera. To achieve excellent performance, the experimental parameters were optimized. Further, we extended this device for potential applications in the clinical diagnosis of allergic diseases: worthwhile clinical performance in the detection of allergens was achieved as compared to that achieved by commercial enzyme-linked immunosorbent assay (ELISA) kit. Therefore, it was proven that this strategy has the advantages of high-throughput, rapid, sensitive, and highly accurate detection of trace amounts of sIgEs. Furthermore, by simply changing the antigen and antibody, this device could be used for the high-throughput detection of other allergens, so as to achieve multiallergen detection and appropriate desensitization therapy, thereby making it promising in the determination of allergic diseases in clinics.

To study the relationship between exposure to airborne particles in a pulp and paper mill and markers of inflammation and coagulation in blood. Personal sampling of inhalable dust was performed for 72 subjects working in a Swedish pulp and paper mill. Stationary measurements were used to study concentrations of total dust, respirable dust, PM10 and PM2.5, the particle surface area and the particle number concentrations. Markers of inflammation, interleukins (IL-1b, IL-6, IL-8, and IL-10), C-reactive protein (CRP), serum amyloid A (SAA), and fibrinogen and markers of coagulation factor VIII, von Willebrand, plasminogen activator inhibitor, and D-dimer were measured in plasma or serum. Sampling was performed on the last day of the work free period of 5 days, before and after the shift the first day of work and after the shifts the second and third day. In a mixed model analysis, the relationship between particulate exposures and inflammatory markers was determined. Sex, age, smoking, and BMI were included as covariates. The average 8-h time-weighted average (TWA) air concentration levels of inhalable dust were 0.30 mg/m(3), range 0.005-3.3 mg/m(3). The proxies for average 8-h TWAs of respirable dust were 0.045 mg/m(3). Significant and consistent positive relations were found between several exposure metrics (PM 10, total and inhalable dust) and CRP, SAA and fibrinogen taken post-shift, suggesting a dose-effect relationship. This study supports a relationship between occupational particle exposure and established inflammatory markers, which may indicate an increased risk of cardiovascular disease. Funding Agencies|Swedish Research Council for Health, Working Life and Welfare [2006-0152]

Investigation of cell signaling pathways is important for the study of pathogenesis of cancer. However, the related operations used in these studies are time consuming and labor intensive. Thus, the development of effective therapeutic strategies may be hampered. In this work, gel-free cell culture and subsequent immunoassay has been successfully integrated and conducted in a paper-based microreactor array. Study of the activation level of different kinases of cells stimulated by different conditions, i.e., IL-6 stimulation, starvation, and hypoxia, was demonstrated. Moreover, rapid screening of cell signaling cascades after the stimulations of HGF, doxorubicin, and UVB irradiation was respectively conducted to simultaneously screen 40 kinases and transcription factors. Activation of multi-signaling pathways could be identified and the correlation between signaling pathways was discussed to provide further information to investigate the entire signaling network. The present technique integrates most of the tedious operations using a single paper substrate, reduces sample and reagent consumption, and shortens the time required by the entire process. Therefore, it provides a first-tier rapid screening tool for the study of complicated signaling cascades. It is expected that the technique can be developed for routine protocol in conventional biological research laboratories.

Objectives Innovative techniques are urgently required to remove pathogenic bacterial contamination of drinking water. This study aimed to evaluate the biocidal activity of calcium hypochlorite [Ca(OCl)2], silver nanoparticles (AgNPs) and Ca(OCl)2/AgNPs composite against bacteria isolated from drinking water supplies (tap and hand pump water). A field trial was subsequently performed to evaluate the efficacy of a biocidal filter paper containing Ca(OCl)2/AgNPs composite against existing pathogenic bacteria and indicator coliform bacteria. Methods A total of 100 water samples were collected from the main source and water troughs used for cattle drinking and were examined for the presence of pathogenic bacteria. The susceptibility of 60 isolated strains to Ca(OCl)2, AgNPs and Ca(OCl)2/AgNPs was evaluated by the broth macrodilution method. The field trial examined different water samples collected from water supplies (pre- and post-treatment) using filter paper impregnated with Ca(OCl)2/AgNPs. Results Ca(OCl)2 loaded on AgNPs at a concentration of 1.5 mg/L showed a lethal effect (100%) on Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae following 180 min of exposure. Furthermore, filter paper impregnated with Ca(OCl)2/AgNPs exhibited a biocidal effect (100%) against existing pathogenic bacteria, bacterial total viable count, total coliform count and faecal coliform count. Conclusions Use of the disinfectant Ca(OCl)2 against isolated bacteria revealed the existence of bacterial resistance. Enhancement of the biocidal effect of Ca(OCl)2 using AgNPs was reliable, proving that Ca(OCl)2/AgNPs composite had a biocidal effect against isolated bacteria at 1.5 mg/L as well as inactivation of coliforms and pathogenic bacteria during percolation through bactericidal filter paper.

White blood cell count is an important indicator of each individual's health condition. An abnormal white blood cell count usually results from an infection, cancer, or other conditions that trigger systemic inflammation responses. White blood cell count also provides predictive information on the incidence of cardiovascular diseases and Type 2 diabetes. Therefore, monitoring white blood cell count on a regular basis can potentially help individuals to take preventive measures and improve healthcare outcomes. Currently, white blood cell count is primarily conducted in centralized laboratories, and it requires specialized equipment and dedicated personnel to perform the test and interpret the results. So far there has been no rapid test that allows white blood cell count in low-resource settings. In this study, we have demonstrated a vertical flow platform that quantifies white blood cells by trapping them in the paper. White blood cells were tagged with gold nanoparticles, and flowed through the paper via a small orifice. The white blood cell count was determined by measuring the colorimetric intensity of gold nanoparticles on the surface of white blood cells that were trapped in the paper mesh. Using this platform, we were able to quantify white blood cells in 15 μL of blood, and visually differentiate the abnormal count of white blood cells from the normal count. The proposed platform enabled rapid white blood cell count in low resource settings with a small sample volume requirement. Its low-cost, instrument-free operations would be attractive for point-of-care applications.

In the pulp and paper industry, during the manufacturing process, the agglomeration of pitch particles (composed of triglycerides, fatty acids, and esters) leads to the formation of black pitch deposits in the pulp and on machinery, which impacts on the process and pulp quality. Traditional methods of pitch prevention and treatment are no longer feasible due to environmental impact and cost. Consequently, there is a need for more efficient and environmentally friendly approaches. The application of lipolytic enzymes, such as lipases and esterases, could be the sustainable solution to this problem. Therefore, an understanding of their structure, mechanism, and sources are essential. In this report, we review the microbial sources for the different groups of lipolytic enzymes, the differences between lipases and esterases, and their potential applications in the pulping industry.

The lack of molecular diagnosis in the field of cancer in Iraq has motivated us to perform a genetic analysis of pediatric acute myelogenous leukemia (AML), including class I and II aberrations. Peripheral blood or bone marrow cells were collected from 134 AML children aged ≤15 years. Flinders Technology Associates (FTA) filter paper cards were used to transfer dried blood samples from five Iraqi hospitals to Japan. DNA sequencing was performed to identify class I mutations. Nested RT-PCR was used to detect class II aberrations, except that MLL rearrangement was detected according to long distance inverse-PCR. NPM1 and FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations were analyzed by GeneScan using DNA template. Among 134 Iraqi pediatric AML samples, the most prevalent FAB subtype was M2 (33.6 %) followed by M3 (17.9 %). Class I mutations: 20 (14.9 %), 8 (6.0 %), and 8 (6.0 %) patients had FLT3-ITD, FLT3-TKD, and KIT mutations, respectively. Class II mutations: 24 (17.9 %), 19 (14.2 %), and 9 (6.7 %) children had PML-RARA, RUNX1-RUNX1T1, and CBFB-MYH11 transcripts, respectively. MLL rearrangements were detected in 25 (18.7 %) patients. NPM1 mutation was detected in seven (5.2 %) cases. Collectively, approximately 30 % of AML children were proved to carry favorable prognostic genetic abnormalities, whereas approximately 10 % had high FLT3-ITD allelic burden and needed a special treatment plan including allogeneic hematopoietic stem cell transplantation. Acute promyelocytic leukemia (APL) was frequent among Iraqi pediatric AML. It is likely that molecular diagnosis using FTA cards in underdeveloped countries could guide doctors towards an appropriate treatment strategy.

Background Lysosomal storage diseases (LSD) are a group of genetic conditions which could present a vast spectrum of abnormalities that may include skeletal abnormalities, organ dysfunction, neuronal involvement, and tissue accumulation of complex molecules, among other manifestations. Definitive diagnosis of LSD is generally obtained by specific enzyme assays performed in leukocytes, fibroblasts, or more recently, dried-blood filter paper (DBFP) samples. Methods We recently introduced dried-leukocytes filter paper (DLFP) as an alternative source of enzyme to assay heparan sulfamidase and galactocerebrosidase activities, which could not be measured in DBFP samples using fluorometric methods. We present a new fluorometric methods on DLFP samples, for evaluation of α-glucosidase (GAA), β-glucosidase (GBA), and N-acetylgalactosamine-6-sulfatase (GALNS) activities, key enzyme assays for the identification of patients with Pompe disease (PD), Gaucher disease (GD), and Morquio A disease (MD), respectively. Results We show a clear discrimination between confirmed PD, GD, and MD patients and healthy controls. Conclusions We conclude that the assays of GAA, GBA, and GALNS on DLFP are reliable and useful methods for the identification of PD, GD, and MD diseases, respectively. As sample preparation is feasible in standard biochemical laboratories and transportation is very simple, it could enable patients living in remote areas to be investigated, diagnosed and eventually treated with the specific therapies available for these diseases.

Objectives Recombinant feline interferon-ω therapy is an immunomodulator currently used in the treatment of different retroviral diseases including feline immune deficiency virus and feline leukaemia virus. Although its mechanism of action remains unclear, this drug appears to potentiate the innate response. Acute phase proteins are one of the key components of innate immunity and studies describing their use as a monitoring tool for the immune system in animals undergoing interferon-ω therapy are lacking. This study aimed to determine whether interferon-ω therapy influences acute phase protein concentrations namely serum amyloid-A, α-1-glycoprotein and C-reactive protein. Methods A single-arm study was performed using 16 cats, living in an animal shelter, naturally infected with retroviruses and subjected to the interferon-ω therapy licensed protocol. Samples were collected before (D0), during (D10 and D30) and after therapy (D65). Serum amyloid-A and C-reactive protein were measured by specific enzyme-linked immunosorbent assay kits and α-1-glycoprotein by single radial immunodiffusion. Results All the acute phase proteins significantly increased in cats undergoing interferon-ω therapy (D0/D65: P

Interferon (IFN)-γ release assays (IGRAs) are probably the most accurate tests for the detection of latent Mycobacterium tuberculosis infection, but IGRAs are labour intensive and the transport of samples over longer distances is difficult. IFN-γ-induced protein (IP)-10 is expressed at 100-fold higher levels than IFN-γ, and IP-10 release assays have comparable performance to IGRAs. The aim of this study was to explore the diagnostic potential of a novel IP-10 release assay based on dried plasma spots (DPS). The presence of IP-10 and IFN-γ was determined in plasma and in DPS by ELISA. Diagnostic algorithms for plasma and DPS tests for IP-10 were developed on a training cohort comprising 60 tuberculosis (TB) patients and 59 healthy controls. Diagnostic accuracy was assessed in a validation cohort comprising 78 TB patients and 98 healthy controls. Plasma was measured in Spain and DPS samples were sent to Denmark using the conventional postal service for analysis. IP-10 was readily detectable in both plasma and DPS, and correlation was excellent (r2 = 0.95). QuantiFERON-TB Gold In-Tube (QFT-TB) and IP-10 in DPS and plasma rendered comparable sensitivity (78%, 82% and 84%, respectively), specificity (100%, 97% and 97%, respectively) and indeterminate rates (p>0.55). The DPS-based IP-10 test has comparable diagnostic accuracy to the QFT-TB and samples can be sent via conventional mail over long distances for analysis without affecting the results.

Background: Adult opsoclonus-myoclonus (OM), a disorder of eye movements accompanied by myoclonus affecting the trunk, limbs, or head, is commonly associated with an underlying malignancy or precipitated by viral infection. Methods: We present the first two reports of post-streptococcal OM associated with antibodies against a 56 kDa protein. Two young girls presented with opsoclonus and myoclonus following a febrile illness and pharyngitis. Protein purification techniques were employed. Amino acid sequences of human neuroleukin (NLK) and streptococcal proteins were compared using the protein-protein BLAST application. Results: The antigen was identified as NLK (glucose-6-phosphate isomerase, GPI). GPI is present on the cell surface of streptococcus making the protein a candidate target for molecular mimicry. Conclusions: We have identified NLK as an antigenic target in two patients with post-streptococcal OM. The pathogenicity of the antibodies is uncertain. The potential role of anti-neuroleukin antibodies in the pathogenesis of OM is discussed. We propose that OM may represent a further syndrome in the growing spectrum of post-streptococcal neurological disorders. The role of streptococcus in OM and the frequency with which anti-NLK responses occur in both post-infectious and paraneoplastic OM should be investigated further.

Robustness of comprehensive DNA- and RNA-based assays at diagnosis of acute myeloid leukemia using blood and bone marrow stored on filter cards

Background The use of filter paper as a simple, inexpensive tool for storage and transportation of blood, ‘Dried Blood Spots’ or Guthrie cards, for diagnostic assays is well-established. In contrast, there are a paucity of diagnostic evaluations of dried cerebrospinal fluid (CSF) spots. These have potential applications in low-resource settings, such as Laos, where laboratory facilities for central nervous system (CNS) diagnostics are only available in Vientiane. In Laos, a major cause of CNS infection is Japanese encephalitis virus (JEV). We aimed to develop a dried CSF spot protocol and to evaluate its diagnostic performance using the World Health Organisation recommended anti-JEV IgM antibody capture enzyme-linked immunosorbent assay (JEV MAC-ELISA). Methodology and Principal Findings Sample volumes, spotting techniques and filter paper type were evaluated using a CSF-substitute of anti-JEV IgM positive serum diluted in Phosphate Buffer Solution (PBS) to end-limits of detection by JEV MAC-ELISA. A conventional protocol, involving eluting one paper punch in 200μl PBS, did not detect the end-dilution, nor did multiple punches utilising diverse spotting techniques. However, pre-cut filter paper enabled saturation with five times the volume of CSF-substitute, sufficiently improving sensitivity to detect the end-dilution. The diagnostic accuracy of this optimised protocol was compared with routine, neat CSF in a pilot, retrospective study of JEV MAC-ELISA on consecutive CSF samples, collected 2009–15, from three Lao hospitals. In comparison to neat CSF, 132 CSF samples stored as dried CSF spots for one month at 25–30°C showed 81.6% (65.7–92.3 95%CI) positive agreement, 96.8% (91.0–99.3 95%CI) negative agreement, with a kappa coefficient of 0.81 (0.70–0.92 95%CI). Conclusions/Significance The novel design of pre-cut filter paper saturated with CSF could provide a useful tool for JEV diagnostics in settings with limited laboratory access. It has the potential to improve national JEV surveillance and inform vaccination policies. The saturation of filter paper has potential use in the wider context of pathogen detection, including dried spots for detecting other analytes in CSF, and other body fluids., Author Summary Japanese encephalitis virus infection is widespread in Asia, and primarily affects children in poor, rural areas. The virus spreads to the brain and spinal cord resulting in significant death and disability. Diagnosis requires testing for the immune response ‘antibody’ specific to Japanese encephalitis virus in the cerebrospinal fluid that surrounds the brain and spinal cord. However, in most areas where the infection occurs there are no laboratory facilities. In the absence of laboratory diagnosis, we have poor knowledge of the extent of the problem in these areas. Dried blood spots on filter paper have been used widely for many years as simple, cost-effective tools for transporting samples for testing. However, there have been few attempts at using dried cerebrospinal fluid spots. We developed a technique for using dried spots to store and test cerebrospinal fluid for antibodies to Japanese encephalitis virus. We compared the results of dried spots with routine neat cerebrospinal fluid in 132 patient samples, and demonstrated excellent agreement between the two tests. This novel method for saturating dried cerebrospinal fluid spots has the potential to enhance our knowledge of Japanese encephalitis virus epidemiology, and inform health policies where they are most needed. It could also be transferred for use in diagnosing other infectious diseases, including using other body fluid samples.

Research was carried out to determine whether the likelihood of obtaining a positive Acid Phosphatase (AP) test result is affected by the make and type of paper used. Also, we aimed to investigate the frequency of AP positive reactions occurring after 2 min using a series of known semen dilutions and to determine whether spermatozoa transfer onto the paper during the act of AP screening. In this research, most brands of paper tested were able to detect a 1 in 40 semen dilution within 2 min. Leaving AP test papers for longer will allow the detection of greater dilutions of semen and as the amount of ejaculation is not reliably known in most casework situations and levels of AP activity can vary in different men, this will increase the seminal detection rate in sexual offence allegations.

Background: Induction of a humoral response against amyloid-β peptide may be beneficial for Alzheimer’s disease (AD) patients and may alleviate the onset and progression of AD. DNA-based vaccination provides a unique alternative method of immunization for treatment and prevention of AD. Currently, the two major delivery methods used for enhancing DNA uptake and immune responses to DNA vaccines in humans are electroporation (EP) and gene gun (GG). Objective: The goal of this translational study was to evaluate the efficacy of an AD DNA epitope vaccine (DepVac) delivered intramuscularly by EP or intradermally by GG. Methods: Humoral and cellular immune responses to immunization with DepVac were evaluated by ELISA and ELISPOT, respectively. Functional activity of the antibodies was also assessed. Results: EP- and GG-mediated immunizations with DepVac induced similar anti-amyloid-β (Aβ) antibody and T cell responses. Anti-Aβ antibodies bound to amyloid plaques in AD brain tissue and to toxic forms of Aβ42 peptide. Conclusion: Both delivery methods are effective at promoting potent antibodies specific for Aβ.

Background: The vascular deposition of amyloid known as cerebral amyloid angiopathy (CAA) – an age-associated condition and a common finding in Alzheimer’s disease – compromises cerebral blood flow, causing macro/microhemorrhages and/or cognitive impairment. Very little is known about the mechanisms causing CAA-related degeneration of cerebral vascular cells. The Dutch E22Q familial amyloid-β (Aβ) variant is primarily associated with CAA, and manifests clinically with severe cerebral hemorrhages. Objective: We aimed to determine the molecular mechanisms causing apoptosis of cerebral endothelial cells in the presence of wild-type Aβ40 or its vasculotropic E22Q variant. Methods: We challenged human brain microvascular endothelial cells with both Aβ variants, and studied the apoptotic pathways triggered by these peptides. Results: Caspase-mediated apoptotic pathways were elicited by both peptides within time frames correlating with their aggregation properties and formation of oligomeric/protofibrillar assemblies. Our data revealed a primary activation of caspase-8 (typically triggered by death receptors) with secondary engagement of caspase-9, with cytochrome C and apoptosis-inducing factor release from the mitochondria, suggesting the independent or synergistic engagement of extrinsic and intrinsic apoptotic mechanisms. Conclusion: Our data demonstrate the induction of caspase-8- and caspase-9-dependent mitochondrial-mediated apoptotic pathways by Aβ oligomers/protofibrils in vascular cells, likely implicating a primary activation of death receptors.

Purpose: To ensure the efficiency and safety of transplanted human embryonic stem cell (hESC)-derived osteoblast-like cells (hESC-OS) for bone regeneration, this study was designed to determine the effects of continuous cell expansion on the osteoblastic differentiation stability, pluripotency, and tumorigenic potential of long-term expanded hESC-OS. Methods: hESCs manually harvested as cell aggregates or enzymatically dissociated as single cells were directly incubated in osteogenic medium and serially passaged to passage 25. Expression of osteoblast-related genes, pluripotent regulator genes, and genes related to tumorigenesis were examined at the primary passage and every 5 passages thereafter. hESC-OS were subcutaneously transplanted into nude mice for 4–24 weeks to test for teratoma formation. hESC-OS were recultivated in hESC culture conditions to evaluate the extent to which reverse differentiation back to the undifferentiated stage may occur. Results: hESC-OS derived from hESC aggregates and dissociated cells exhibited comparable osteoblast differentiation patterns. Expression levels of osteoblast-related genes reached plateau levels at passages 5–10 before declining in higher passages. Expression of tumor-associated genes was not significantly increased. Only hESC-OS at primary and first passages formed teratomas after 4 weeks in vivo. The hESC-OS were not able to revert to hESCs. Conclusions: Expanded hESC-OS demonstrated lineage-specific differentiation stability, did not maintain the pluripotency of hES cells, and were genetically stable. Thus, hESC-OS may be considered for large animal preclinical studies.

Venous thromboembolism (VTE) is a common complication in patients with malignant disease. First recognised by Bouillard in 1823 and later described by Trousseau in 1844, multiple studies have since provided considerable evidence for a clinical association between VTE and cancer. Across all cancers, the risk for VTE is elevated 7-fold; in certain malignancies, the risk for VTE may be increased up to 28-fold. Venous thromboembolism is the second leading cause of death in patients with cancer; among survivors, complications commonly include recurrent VTE and post-thrombotic syndrome, and (more rarely) chronic thromboembolic pulmonary hypertension, which are costly, and have a profound impact on the patient's quality of life. Tumour cells can activate blood coagulation through multiple mechanisms, including production of procoagulant, fibrinolytic, and proaggregating activities, release of proinflammatory and proangiogenic cytokines, and interacting directly with host vascular and blood cells (e.g., endothelial cells, leukocytes, and platelets) through adhesion molecules. Increasing evidence suggests that elements of the haemostatic system also have a direct role in eliciting or enhancing angiogenesis, cell survival, and metastasis. Despite the problem posed by VTE in the setting of cancer, it is evident that a significant number of oncologists do not recognise the link between cancer, its treatment, and thrombogenesis. On 22 May 2009, a group of UK-based physicians met in London, UK, to evaluate recent data on cancer thrombosis. This article (1 of 4) briefly reviews key data on the epidemiology and pathophysiology of VTE as a context for a discussion and consensus statement developed by meeting attendees, on the implications of this information for UK clinical practice.

The two main arms of the stress system include the autonomic nervous system (ANS) and the hypothalamic-pituitary-adrenal (HPA) axis. These two neural stress systems coordinate the response of many other physiological systems to a stressor, including the immune and cardiovascular systems, bringing the body back to homeostasis. The nervous and immune systems communicate with each other in a bidirectional manner. In this review, we will discuss the use of noninvasive methods to evaluate the immune system, ANS and HPA axis. Collection of sweat and saliva, and measurement of heart rate variability are noninvasive methods that can be applied to evaluate neuroimmune interactions. Recently, we validated a new methodology to simultaneously evaluate a large array of neural and immune biomarkers in sweat, collected through cutaneous sweat patches and measured by recycling immunoaffinity chromatography. Noninvasive and ambulatory methodologies of biomarker collection can overcome several limitations intrinsic to invasive methods, such as reducing the stress triggered by collection itself and allowing a wider application to field and community-based settings. Ultimately, simultaneous evaluation of neural and immune systems with noninvasive techniques will help elucidate the underlying interactions of these systems and their role in disease susceptibility and progression of stress-related disorders.

An emerging area of research in autism spectrum disorder (ASD) is the role of prenatal exposure to inflammatory mediators during critical developmental periods. Epidemiological data has highlighted this relationship showing significant correlations between prenatal exposure to pathogens, including influenza, and the occurrence of ASD. Although there has not been a definitive molecular mechanism established, researchers have begun to investigate this relationship as animal models of maternal infection have support- ed epidemiological findings. Several groups utilizing these animal models have found that activation of the maternal immune system, termed maternal immune activation (MIA), and more specifically the exposure of the developing fetus to maternal cytokines precipitate the neurological, immunological and behavioral abnormalities observed in the offspring of these animals. These abnormalities have correlated with clinical findings of immune dysregulation, neurological and behavioral abnormalities in some autistic individuals. Additionally, researchers have observed genetic variations in these models in genes which regulate neurological and immunological development, similar to what is observed clinically in ASD. Altogether, the role of MIA and cytokine dysregulation, as a key mediator in the neuropathological, behavioral and possibly genetic irregularities observed clinically in autism are important factors that warrant further investigation.

Psychological stress is an important factor in susceptibility to many diseases. Our laboratory has been investigating the impact of stress on the susceptibility to Theiler’s virus-induced demyelination (TVID), a mouse model of multiple sclerosis. Using immunodominant viral peptides specific for identification of either CD4+ or CD8+ T cells, stress reduced IFN-γ-producing virus-specific CD4+ and CD8+ T cells in the spleen and CD8+ T cells in the CNS. Expression of mRNA for the Th1 transcription factor T-bet and the Th2 transcription factor GATA-3 were decreased in spleen cells isolated from stressed mice. Cytokine production by cells isolated from the CNS or spleens following stimulation with virus indicated that stress decreased both type 1 and type 2 responses. The adverse effects of stress were partially reversed by concurrent RU486 administration but mimicked by dexamethasone, indicating a major role for glucocorticoids. Global stress-induced immunosuppression resulted in higher levels of virus replication and dissemination. The higher viral load subsequently led to an earlier disease onset and more severe clinical and histological signs of demyelinating disease. Our results have important implications for understanding the pathogenesis of MS, and suggest that stressful events during early infection with an agent capable of inducing demyelination may result in immunosuppression and failure to eliminate the pathogen, which in turn may lead to the development of MS.

Stress is known to suppress immune function and increase susceptibility to infections and cancer. Paradoxically, stress is also known to exacerbate asthma, and allergic, autoimmune and inflammatory diseases, although such diseases should be ameliorated by immunosuppression. Moreover, the short-term fight-or-flight stress response is one of nature’s fundamental defense mechanisms that enables the cardiovascular and musculoskeletal systems to promote survival, and it is unlikely that this response would suppress immune function at a time when it is most required for survival (e.g. in response to wounding and infection by a predator or aggressor). These observations suggest that stress may suppress immune function under some conditions while enhancing it under others. The effects of stress are likely to be beneficial or harmful depending on the type (immunoprotective, immunoregulatory/inhibitory, or immunopathological) of immune response that is affected. Studies have shown that several critical factors influence the direction (enhancing vs. suppressive) of the effects of stress or stress hormones on immune function: (1) Duration (acute vs. chronic) of stress: Acute or short-term stress experienced at the time of immune activation can enhance innate and adaptive immune responses. Chronic or long-term stress can suppress immunity by decreasing immune cell numbers and function and/or increasing active immunosuppressive mechanisms (e.g. regulatory T cells). Chronic stress can also dysregulate immune function by promoting proinflammatory and type-2 cytokine-driven responses. (2) Effects of stress on leukocyte distribution: Compartments that are enriched with immune cells during acute stress show immunoenhancement, while those that are depleted of leukocytes, show immunosuppression. (3) The differential effects of physiologic versus pharmacologic concentrations of glucocorticoids, and the differential effects of endogenous versus synthetic glucocorticoids: Endogenous hormones in physiological concentrations can have immunoenhancing effects. Endogenous hormones at pharmacologic concentrations, and synthetic hormones, are immunosuppressive. (4) The timing of stressor or stress hormone exposure relative to the time of activation and time course of the immune response: Immunoenhancement is observed when acute stress is experienced at early stages of immune activation, while immunosuppression may be observed at late stages of the immune response. We propose that it is important to study and, if possible, to clinically harness the immunoenhancing effects of the acute stress response, that evolution has finely sculpted as a survival mechanism, just as we study its maladaptive ramifications (chronic stress) that evolution has yet to resolve. In view of the ubiquitous nature of stress and its significant effects on immunoprotection as well as immunopathology, it is important to further elucidate the mechanisms mediating stress-immune interactions and to meaningfully translate findings from bench to bedside.

HLA-D/DR alleles as defined by cellular and serological typing can also be identified by biochemical methods. The Southern blot technique provides an additional typing facility which can be applied to DNA obtained from any source of nucleated cells. The polymorphism revealed by Southern blot analyses, the so-called restriction fragment length polymorphism (RFLP), depends upon the restriction enzyme and cDNA probes used. To identify HLA-DR specificities a protocol was developed based on the use of the results of southern blot analyses with several restriction enzymes and cDNA probes within a panel of HLA-D/DR homozygous cells representing the DR1 to DRw8 alleles. First, hybridizations with the 3' untranslated sequence of the DR beta cDNA probe, after digestion of the DNA with PvuII (PvuII-DR beta 3') allows the selective identification of DR1, DR2 and DRw8; DR3, DR5 and DRw6 are found as one group as well as DR4 and DR7 as another. Second, TaqI-DQ alpha hybridization allows the splitting of DRw6-Dw18, DRw6-Dw19 and DRw6-Dw9 from the DR3, DR5 and DRw6 group. The other alleles DR3, DR4, DR5, DRw6-Dw16 and DR7 are revealed by dehybridization and rehybridization of the blot with a DR beta cDNA probe. This protocol was used to test whether in a panel of 30 randomly chosen individuals the HLA-DR typing could be performed. The results were highly concordant to the serotyping. Furthermore by adding the Pst-DR beta and TaqI-DQ alpha RFLPs, most of the MLC defined Dw specificities could also be identified. An overview of the specific fragments described here has been summarized in matrices which can be used as references for DNA-typing in computerized analyses.

Paper mill workers are exposed to culturable microorganisms (MOs). We hypothesized that inflammatory airway response could be detected in sputum of nonsymptomatic workers. From four paper mills, we included 29 healthy nonsmoking men. Workers exposed to high levels of MOs (HMOE, n = 17) were compared with workers exposed to low levels of MO (LMOE, n = 12). A reference group of 22 healthy, nonsmoking, nonexposed (NE) men were also included. We performed differential cell counts of induced sputum, studied gene expressions of isolated sputum macrophages and analyzed inflammatory parameters, including matrix metalloproteinases. Sputum from HMOE workers had a significantly higher percentage of neutrophils than that from LMOE workers (P < 0.05) and NE controls (P < 0.001). There was also an increased gene expression of different pro-inflammatory cytokines, interleukin-6, tumor necrosis factor-alpha, and macrophage inflammatory protein-1beta in isolated airway macrophages and increased levels of total matrix metalloprotease-9 activity in induced sputum from the HMOE group. Our findings indicate that paper industry workers exposed to MOs develop subclinical airway inflammation.

Contains fulltext : 51936.pdf (Publisher’s version ) (Closed access) OBJECTIVE: To evaluate cognitive outcome in adult survivors of bacterial meningitis. METHODS: Data from three prospective multicentre studies were pooled and reanalysed, involving 155 adults surviving bacterial meningitis (79 after pneumococcal and 76 after meningococcal meningitis) and 72 healthy controls. RESULTS: Cognitive impairment was found in 32% of patients and this proportion was similar for survivors of pneumococcal and meningococcal meningitis. Survivors of pneumococcal meningitis performed worse on memory tasks (p

Compliance and acceptance for the finger-prick method of blood collection is generally better than for venipuncture. A finger-prick method of blood collection with quantitative antibody recovery is even more important for seroepidemiological surveys. Finger-prick blood collected and dried onto filter paper has been used; but, unfortunately, this method has several disadvantages, including loss of antibody activity, possible contact contamination from blood spots on adjacent filter papers, and difficulties in extracting antibodies, justifying the search for other methods of collecting and transporting blood samples. We report on a simple method of collecting a measured amount of finger-prick blood onto a sample pad, which is immediately transferred to storage/extraction buffer. The diluted blood sample is never dried, and because of the storage buffer, can be transported and stored without refrigeration. Furthermore, the diluted blood samples can then be tested directly without further preparation. We systematically compared several storage/extraction buffers and commercially available filter papers. We showed that antibody recovery was not significantly affected by the type of filter papers used but was significantly affected by the storage/extraction buffer used. The best such buffer is StabilZyme Select.

This work demonstrates the application of a 3D culture system-Cells-in-Gels-in-Paper (CiGiP)-in evaluating the metabolic response of lung cancer cells to ionizing radiation. The 3D tissue-like construct-prepared by stacking multiple sheets of paper containing cell-embedded hydrogels-generates a gradient of oxygen and nutrients that decreases monotonically in the stack. Separating the layers of the stack after exposure enabled analysis of the cellular response to radiation as a function of oxygen and nutrient availability; this availability is dictated by the distance between the cells and the source of oxygenated medium. As the distance between the cells and source of oxygenated media increased, cells show increased levels of hypoxia-inducible factor 1-alpha, decreased proliferation, and reduced sensitivity to ionizing radiation. Each of these cellular responses are characteristic of cancer cells observed in solid tumors. With this setup we were able to differentiate three isogenic variants of A549 cells based on their metabolic radiosensitivity; these three variants have known differences in their metastatic behavior in vivo. This system can, therefore, capture some aspects of radiosensitivity of populations of cancer cells related to mass-transport phenomenon, carry out systematic studies of radiation response in vitro that decouple effects from migration and proliferation of cells, and regulate the exposure of oxygen to subpopulations of cells in a tissue-like construct either before or after irradiation.

Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely. Eight healthy smokers aged 40–65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1β), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis. A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL. The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression. NHS Health Research Authority ( 13/LO/0256 ).

Alkalophilic Cellulosimicrobium cellulans CKMX1 isolated from mushroom compost is an actinomycete that produces industrially important and environmentally safer thermostable cellulase-free xylanase, which is used in the pulp and paper industry as an alternative to the use of toxic chlorinated compounds. Strain CKMX1 was previously characterized by metabolic fingerprinting, whole-cell fatty acids methyl ester analysis, and 16S rDNA and was found to be C. cellulans CKMX1. Crude enzyme (1027.65 U/g DBP) produced by C. cellulans CKMX1, having pH and temperature optima of 8.0 and 60 °C, respectively, in solid state fermentation of apple pomace, was used in the production of bleached wheat straw pulp. Pretreatment with xylanase at a dose of 5 U/g after pulping decreased pulp kappa points by 1.4 as compared with the control. Prebleaching with a xylanase dose of 5 U/g pulp reduced the chlorine charge by 12.5%, increased the final brightness points by approximately 1.42% ISO, and improved the pulp strength properties. Xylanase could be substituted for alkali extraction in C–Ep–D sequence and used for treating chemically bleached pulp, resulting in bleached pulp with higher strength properties. Modification of bleached pulp with 5 U of enzyme/g increased pulp whiteness and breaking length by 1.03% and 60 m, respectively; decreased tear factor of pulp by 7.29%; increased bulk weight by 3.99%, as compared with the original pulp. Reducing sugars and UV-absorbing lignin-derived compound values were considerably higher in xylanase-treated samples. Cellulosimicrobium cellulans CKMX1 has a potential application in the pulp and paper industries.

To compare data based on computerized and paper versions of health status questionnaires (HSQs) for sampling patient-reported outcomes (PROs) in patients with fibromyalgia (FM). In addition, to examine associations between patient characteristics (age, education, computer experience) and differences between versions. Finally, to evaluate the acceptability of computer-based questionnaires among patients with FM.Method: The study population comprised female patients diagnosed with FM. All patients completed six HSQs: the Fibromyalgia Impact Questionnaire (FIQ), the Major Depression Inventory (MDI), the 36-item Short Form Health Survey (SF-36), the painDETECT questionnaire (PDQ), the Coping Strategies Questionnaire (CSQ), and the Generalized Anxiety Disorder Self-Assessment Questionnaire (GAD-10), both on paper and using a touch screen. One HSQ was tested at a time in a repeated randomized cross-over design. The two versions were completed with a 5-min interval and between each HSQ the participants had a 5-min break. Means, mean differences with 95% confidence intervals (CIs), medians, median differences, and intraclass correlation coefficients (ICCs) were calculated for all HSQs, including relevant subscales. Associations between patient characteristics and differences between versions were explored using Spearmans correlation coefficients.Results: Twenty women, mean age 48.4 years, participated in the study. Except for one item, ICCs between touch-screen and paper versions of the HSQs examined indicated acceptable agreement (ICC = 0.71-0.99). Overall, mean and median differences revealed no differences between versions. No significant associations were observed for patient characteristics. None of the participants preferred paper questionnaires over computerized versions.Conclusions: The computerized HSQs using a touch screen gave comparable results to answers given on paper and were generally preferred by the participants.

Background: About 40% of patients with limbic encephalitis do not have detectable CNS antibodies. Some of these patients have immune-mediated limbic encephalitis, but their frequency is unknown. Aims: (1) To determine the spectrum of limbic encephalitis identified on clinical grounds in a single institution, and compare it with that in patients referred for antibody analysis. (2) To correlate clinical outcomes with the cellular location of the autoantigens. Methods: Prospective clinical case studies. Immunohistochemistry with rat brain, live hippocampal neurones, HeLa cells expressing Kv potassium channels and immunoblot. Results: In 4 years, 17 patients were identified in the Hospital of the University of Pennsylvania, Philadelphia, USA, and the serum or CSF samples of 22 patients diagnosed elsewhere were also studied. 9 of our 17 (53%) patients had antibodies to known neuronal antigens (paraneoplastic or voltage gated potassium channels (VGKCs)) and 5 (29%) to novel cell-membrane antigens (nCMAg) typically expressed in the hippocampus and sometimes in the cerebellum. Considering the entire series, 19 of 39 (49%) patients had antibodies to known antigens, and 17 (44%) to nCMAg. Follow-up (2–48 months, median 19 months) was available for 35 patients. When compared with patients with antibodies to intraneuronal antigens, a significant association with response to treatment was found in those with antibodies to cell-membrane antigens in general (VGKC or nCMAg, p = 0.003) or to nCMAg (p = 0.006). Conclusions: (1) 82% of patients with limbic encephalitis prospectively identified on clinical grounds had CNS antibodies; (2) responsiveness to treatment is not limited to patients with VGKC antibodies; (3) in many patients (29% from a single institution), the autoantigens were unknown but were found to be highly enriched in neuronal cell membranes of the hippocampus; and (4) these antibodies are associated with a favourable outcome.

Background: Some ganglioside complexes (GSCs) are target antigens for serum antibodies in patients with Guillain–Barre syndrome (GBS). Anti-GSC antibodies may be associated with particular clinical features of GBS. Objective: To investigate antibodies to GSCs in the sera of patients with Miller Fisher syndrome (MFS) characterised by elevation of the IgG anti-GQ1b antibody. Results: In all, 7 of 12 (58%) consecutive patients with MFS were found to have IgG antibodies to GSCs containing GQ1b, of whom 5 had IgG antibodies to GQ1b-GM1 complex (GQ1b/GM1) and 2 had antibodies to GQ1b/GD1a; 4 of 5 patients without sensory symptoms had anti-GQ1b/GM1 antibodies. Conclusions: At least three different specificities in MFS-associated antibodies, GQ1b-specific, anti-GQ1b/GM1-positive and anti-GQ1b/GD1a-positive, were observed. In patients with MFS not only GQ1b itself but also clustered epitopes of GSCs, including GQ1b, may be considered to be prime target antigens for serum antibodies. A tendency to escape sensory disturbances is shown by anti-GQ1b/GM1-positive MFS.

Background: Fatigue is a major complaint of multiple sclerosis (MS) patients. However, little is known about its pathophysiological mechanisms. Evidence from chronic fatigue syndrome and studies on sickness behaviour suggest that immune and neuroendocrine factors may play a causative role in the development of fatigue. Methods: We compared whole blood stimulatory capacity for pro- (TNFα, IFNγ) and anti-inflammatory cytokines (IL-10) as well as hypothalamo-pituitary-adrenal (HPA) axis function in 15 MS patients with marked fatigue and 15 patients without fatigue as determined by the Fatigue Severity Scale (FSS). Results: Proinflammatory cytokines were significantly higher (TNFα: 478.9 v 228.2 pg/ml, p = 0.01; IFNγ: 57.6 v 27.8 pg/ml; p = 0.01) in MS patients with fatigue. Furthermore, TNFα values significantly correlated with daytime sleepiness as measured by the Epworth Sleepiness Scale (r = 0.64, p = 0.001). Controlling for disease activity (as measured by the Cambridge Multiple Sclerosis Basic Score), disease duration, Expanded Disability Status Scale, and depression further increased the correlation of cytokine production and fatigue. HPA axis activity was not related to fatigue but was modestly correlated with cognitive impairment. Conclusion: Our data suggest that fatigue in MS is at least partially mediated through activation of proinflammatory cytokines. In line with earlier findings, HPA axis dysfunction seems not to be relevant in MS fatigue pathogenesis but appears to be linked to cognitive impairment. Our findings suggest that increased levels of inflammatory cytokines may be involved in MS fatigue. Investigation of cytokine profiles may increase the understanding of fatigue pathogenesis in MS.

Objective: To investigate the relation of endothelial cell activation with delayed cerebral ischaemia (DCI) and outcome after subarachnoid haemorrhage (SAH).Methods: Concentrations of soluble (s) intercellular adhesion molecule-1, sE-selectin, sP-selectin, ED1-fibronectin, von Willebrand Factor (vWf), and vWf propeptide were measured within three days of SAH onset. The associations with poor outcome were investigated at three months in 106 patients. In 90 patients in whom the occurrence of cerebral ischaemia could be dated accurately, two analyses were undertaken: one for all ischaemic events (n = 32), including those related to treatment, and another for spontaneous DCI ( n = 11). Concentrations of markers were dichotomised at their medians. The associations of endothelial cell activation markers with outcome were expressed as odds ratios ( OR) from logistic regression and those with ischaemic events as hazard ratios (HR) derived from Cox regression.Results: Early vWf concentrations were associated with poor outcome ( crude OR = 4.6 (95% CI, 2.0 to 10.9; adjusted OR = 3.3 (1.1 to 9.8). Early levels of vWf were also positively related to occurrence of all ischaemic events ( crude HR = 2.3 ( 1.1 to 4.9); adjusted HR = 1.8 ( 0.8 to 3.9) and with occurrence of spontaneous DCI ( crude HR = 3.5 (0.9 to 13.1); adjusted HR = 2.2 (0.5 to 9.8). None of the other markers showed any associations.Conclusions: Concentrations of sICAM-1, sP-selectin, sE- selectin, and ED1-fibronectin do not predict the occurrence of DCI or outcome. The positive associations of raised early vWf concentrations with ischaemic events and poor outcome after SAH may reflect a predisposition to further ischaemic injury through formation of microthrombi in the cerebral circulation.

Paraneoplastic neuropathy is a clinical and immunological heterogeneous disorder and attempts have been made to classify subgroups of this disease. Only 30-50% of the clinical defined cases have antineuronal antibodies.The clinical and immunological features of 36 patients with paraneoplastic neuropathy from the authors' database were analysed including the type and course of the neuropathy, associated tumours, and the presence of antineuronal and other autoantibodies.Antineuronal antibodies were detected in 17/36 patients (47%) and anti-Hu was the most frequent antineuronal antibody. Nine patients had high titre antinuclear antibodies (ANA, median titre 1/1000) without antineuronal antibodies. ANA reactivities were different in most patients. Comparison of the ANA positive and ANA negative patients revealed that ANA positive paraneoplastic neuropathy is more frequently associated with breast cancer but is not associated with lung cancer (p0.05). The main clinical type in these patients was sensorimotor neuropathy. No ANA positive patient had central nervous system involvement. Although the Rankin score at the time of diagnosis was not different, the functional outcome in ANA positive patients was better than in ANA negative patients (p0.05).Paraneoplastic neuropathy is a heterogeneous disorder. ANA may define a subgroup of paraneoplastic neuropathy with different clinical and immunological features and may be related to better prognosis of the neuropathic symptoms.

This study investigated the effects of cyanobacteria from pulp-and-paper waste-treatment systems on biological toxicity removal and biodegradation of certain wastewater contaminants. In field and batch studies, using the Microtox®assay, cyanobacterial biomass and final wastewater toxicity were significantly correlated. In softwood-based wastewater, a decrease in toxicity was negatively correlated with cyanobacterial biomass, but the correlation was positive in hardwood-based wastewater. In the softwood-based wastewater, toxicity remained higher in the light than it was in the dark, whereas in hardwood-based wastewater, toxicity was lower in the light than it was in the dark. All of these results were light-dependent, suggesting that the photosynthetic growth of cyanobacteria is required to induce significant effects. When grown in mixed cultures with bacterial degraders, cyanobacteria from pulp-and-paper waste-treatment systems generally impeded the biodegradation of the wastewater contaminants phenol and dichloroacetate (DCA). However, there was one case where the cyanobacterium Phormidium insigne improved the bacterial degradation of DCA. Doubling inorganic nutrient concentrations did not improve phenol or DCA biodegradation in the majority of cases, indicating that nutrient competition is not a major factor. These data suggest that cyanobacteria play an important role during the biological treatment of contaminants, and, hence, toxicity removal in pulp-and-paper waste-treatment systems.Key words: cyanobacteria, heterotrophic bacteria, biodegradation, pulp and paper waste-treatment, wastewater toxicity.

Background: Human immunodeficiency virus (HIV) leucoencephalopathy (HIVL) is an uncommon and rapidly progressive form of AIDS dementia complex (ADC) that has remained poorly understood. Tumour necrosis factor α (TNFα), which has been implicated in the pathogenesis of ADC, is predominantly localised in macrophages in the HIV infected brain, although in vitro studies indicate that neurones can express this cytokine. Objective: To examine the clinical/neuroradiological features of HIVL and the expression of TNFα in HIVL. Methods: Six patients who presented with rapidly progressive dementia within four to 12 weeks of the primary manifestation of their HIV infection were evaluated. Clinical history, treatment regimens, and imaging studies were reviewed, and brain samples from three of the patients were studied by means of immunohistochemistry. Results: Imaging studies showed diffuse bilateral deep white matter changes in all six patients. Clinical and imaging abnormalities improved in five of the six patients within weeks after initiation of antiretroviral treatment. Brain biopsies of two showed pronounced microglia/macrophage activation, but only scant viral protein (gp41) expression. Staining for TNFα was found in microglia/macrophages, and surprisingly, in neurones also. Postmortem analysis of a third patient also showed TNFα expression in neurones of the frontal cortex and basal ganglia. Conclusion: This study provides the first demonstration of staining for TNFα in the neurones of the HIV infected brain, and suggests that the process underlying this rapidly progressive form of ADC may reflect indirect mechanisms mediated by host factors, particularly TNFα.

Background: It has been proposed that, independent of the e4 allele, APOE promoter polymorphisms (−491 A/T and −219 G/T) may be risks factor for Alzheimer’s disease by modulating APOE expression. Objective: To measure the level of APOE expression in Alzheimer’s disease. Methods: Brains were obtained at necropsy from 114 patients with early and late onset sporadic Alzheimer’s disease in Greater Manchester (UK) during years 1986 to 2001. Total RNA was extracted from 84 brains. Purified lymphocytes were obtained from fresh blood from 16 probable Alzheimer cases from Lille (France). APOE and β-actin gene expression was determined by reverse transcriptase polymerase chain reaction in brain and lymphocytes. Results: An inverse correlation between APOE expression level and Aβ loads was observed. As previously described and extended to 114 cases here, an association between the −219 TT genotype and a higher level of parenchymal Aβ deposition was found, irrespective of APOE e4 allele status. This effect was more pronounced in older individuals, whereas higher Aβ load appeared more closely related to e4 in the younger age group (cut off point at the median age at death (72.5 years)). The −219 TT genotype was associated with a decrease in APOE expression. There was a 60% decrease in APOE expression in lymphocytes from probable Alzheimer cases v controls (p = 0.01). Conclusions: In the oldest individuals, reduced APOE expression, modulated in part by −219 G/T polymorphism, may influence risk and constitute a determinant Aβ load in Alzheimer’s disease.

Background: Polymerase chain reaction (PCR) is used to detect viruses in the cerebrospinal fluid (CSF) of patients with neurological disease. However, data to assist its use or interpretation are limited. Objective: We investigated factors possibly influencing viral detection in CSF by PCR, which will also help clinicians interpret positive and negative results. Methods: CSF from patients with was tested for human herpesviruses types 1–6, JC virus, enteroviruses, and Toxoplasma gondii . The likelihood of central nervous system (CNS) infection was classified as likely, possible, or unlikely. PCR findings in these categories were compared using single variable and logistic regression analysis. Results: Of 787 samples tested, 97 (12%) were PCR positive for one or more viruses. Of episodes likely to be CNS viral infections, 30% were PCR positive compared to 5% categorised as unlikely. The most frequent positive findings were Epstein Barr virus (EBV), enteroviruses, and herpes simplex virus (HSV). Enteroviruses and HSV were found predominantly in the likely CNS viral infection group, whereas EBV was found mainly in the unlikely group. Positive PCR results were more likely when there were 3–14 days between symptom onset and lumbar puncture, and when CSF white cell count was abnormal, although a normal CSF did not exclude a viral infection. Conclusions: The diagnostic yield of PCR can be maximised by using sensitive assays to detect a range of pathogens in appropriately timed CSF samples. PCR results, in particular EBV, should be interpreted cautiously when symptoms cannot readily be attributed to the virus detected.

Objectives: Migraine has been identified as an independent risk factor for ischemic stroke. Both neurogenic inflammation and platelet activation have been linked to the pathophysiology of migraine. Increased platelet activation results in up-regulation of specific binding to leukocytes which promotes pro-inflammatory leukocyte secretion and their tethering to endothelium, a mechanism that has been demonstrated in stroke and which could provide a link to migraine. We aimed to determine whether platelet–leukocyte aggregation is increased in migraine patients outside an acute attack. Methods: Seventy two patients with migraine according to IHS criteria were compared to a control group (n = 72). Whole blood flow cytometry was used to quantify the activation dependent P selectin on the platelet, and to assess the fraction of platelets bound to the different leukocyte subsets. Results: Migraine patients showed significantly more platelet–leukocyte aggregates compared to the control subjects (p = 0.003). This effect was driven by an increased polymorphonuclear cell–platelet aggregation (p = 0.003) whereas platelet aggregation with monocytes and lymphocytes was not. Platelet activation was also increased (p = 0.001). Conclusions: In migraine pro-inflammatory platelet adhesion to leukocytes occurs during the headache free interval similar to that seen in acute coronary and cerebrovascular syndromes. This may suggest a link between migraine and stroke on a cellular level.

There is an imperative need to develop a rapid antibody test that can be used for diagnosis of clinical cases in travelers and expatriates, primary surveillance in areas of unknown endemicity, detection of early infection in childhood and for monitoring chemotherapeutic programs. A rapid-format, simple and qualitative flow through immuno filtration test has been developed for the identification of total IgG antibodies to recombinant filarial antigen WbSXP-1. This test system employs colloidal gold-protein A reagent as the antibody capture reagent. The sensitivity and specificity of the test was evaluated in a total of 1,230 serum samples. The sensitivity of the test was found to be 90.8% with brugian (n = 70) and 91.4% with bancroftian (n = 140) microfilaraemic subjects. The test showed minimum reactivity (4/10) with Loa loa microfilaria (MF) positive sera and no reactivity (0/20) with Onchocerca MF positive sera. This rapid diagnosis is found to be non-reactive with individuals having other parasitic diseases including schistosomiasis (n = 10), soil-transmitted helminthiases (n = 34) and protozoan infections (n = 33) indicating the potential of this test as a prospective method of diagnosis for both brugian and bancroftian lymphatic filariasis. Stability kinetics was studied at different temperatures and different time periods. The rapid flow-through immuno filtration test is advantageous since it can be stored at room temperature, is user friendly and is particularly applicable in the field as an initial screening method, for epidemiological monitoring of filarial infections in bancroftian and brugian endemic regions of the world.

Background: The NOTCH4 gene is located at 6p21.3, a site shown in several studies to have significant linkage with Alzheimer’s disease. Objective: To investigate the potential impact of two polymorphisms within this gene on the risk of developing Alzheimer’s disease. Methods: Genotyping of promoter and 5′-UTR polymorphisms was done in Scottish, English, and French populations. The potential functionality of the 5′-UTR polymorphism was assessed by testing its impact on Aβ load in Alzheimer brains and also by undertaking electrophoretic mobility shift assays and transfection experiments. Results: No association of the Notch4 polymorphisms alone with the disease was observed in any of the populations. However, an interaction of the 5′-UTR C/T polymorphism with the e4 allele of the APOE gene was detected in United Kingdom populations but not in the French. No relation between the 5′-UTR polymorphism and Aβ loads was detected overall or in the presence or absence of the e4 allele. No DNA protein specific binding was found with proteins from neuroblastoma, glioma, or astrocytoma cells, and no allele dependent transcriptional activity was detected. Conclusions: No association between two NOTCH4 polymorphisms alone and Alzheimer’s disease was observed in the three populations, but there was evidence of an increased risk associated with the 5′-UTR CC genotype in e4 bearers in the United Kingdom. As no functionality for this polymorphism could be determined, it is likely that the interaction is spurious or results from a linkage disequilibrium of this 5′-UTR polymorphism with another marker elsewhere in the 6p21.3 locus.