“Smart” Materials for Biosensing Devices: Cell-Mimicking Supramolecular Assemblies and Colorimetric Detection of Pathogenic Agents

Mimicking cell membrane and the biomolecular recognition associated with membranes represents a great technical challenge, yet it has opened doors to innovative diagnostic and therapeutic methods. Our work has focused on design and synthesis of a class of smart materials exploiting biological principals for use in biosensors: these materials are functional polymeric assemblies that mimic the cell membrane and conveniently report the presence of pathogens with a color change. Biologically active cell membrane components are incorporated into conjugated polymers with desirable optical properties and the binding of the target molecules onto the material triggers conformational and electronic shifts that are reflected in a chromatic change (a so-called biochromic shift) that is conveniently observed and recorded. Langmuir–Blodgett thin films and vesicle bilayers provide ideal configurations for precise delivery of the biological binding entity to the sensing interface, and for control of molecular orientation for effective biomolecular interaction. Polydiacetylenic membrane-mimicking materials containing cell surface receptor gangliosides and sialic acid residues, respectively were formulated into these architectures and used for colorimetric detection of bacterial toxins and influenza virus. One advantage of these biochromic conjugated polymer (BCP) sensors is that their molecular recognition and signal transduction functionalities are resident in a single functional unit, making them amenable to convenient microfabrication and use.     

In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy

One of the major limitations in the current set of techniques available to neuroscientists is a dearth of methods for imaging individual cells deep within the brains of live animals. To overcome this limitation, we developed two forms of minimally invasive fluorescence microendoscopy and tested their abilities to image cells in vivo. Both one- and two-photon fluorescence microendoscopy are based on compound gradient refractive index (GRIN) lenses that are 350-1,000 microm in diameter and provide micron-scale resolution. One-photon microendoscopy allows full-frame images to be viewed by eye or with a camera, and is well suited to fast frame-rate imaging. Two-photon microendoscopy is a laser-scanning modality that provides optical sectioning deep within tissue. Using in vivo microendoscopy we acquired video-rate movies of thalamic and CA1 hippocampal red blood cell dynamics and still-frame images of CA1 neurons and dendrites in anesthetized rats and mice. Microendoscopy will help meet the growing demand for in vivo cellular imaging created by the rapid emergence of new synthetic and genetically encoded fluorophores that can be used to label specific brain areas or cell classes.     

Preexposure of murine macrophages to CpG oligonucleotide results in a biphasic tumor necrosis factor alpha response to subsequent lipopolysaccharide challenge

Bacterial DNA and synthetic oligonucleotides containing CpG sequences (CpG-DNA and CpG-ODN) provoke a proinflammatory cytokine response (tumor necrosis factor alpha [TNF-alpha], interleukin-12 [IL-12], and IL-6) and increased mortality in lipopolysaccharide (LPS)-challenged mice via a TNF-alpha-mediated mechanism. It was hypothesized that preexposure of macrophages to CpG-ODN would result in an increased TNF-alpha response to subsequent LPS challenge in vitro. Using the murine macrophage cell line RAW 264.7, we demonstrated both a rapid proinflammatory cytokine response (TNF-alpha) and a delayed inhibitory cytokine response (IL-10) with CpG-ODN. Preexposure of macrophages to CpG-ODN for brief periods (1 to 3 h) augmented TNF-alpha secretion and mRNA accumulation following subsequent LPS challenge (1 microg/ml). However, prolonged preexposure to CpG-ODN (6 to 9 h) resulted in suppression of the TNF-alpha protein and mRNA response to LPS. The addition of anti-IL-10 antibody to CpG-ODN during preexposure resulted in an increase in the LPS-induced TNF-alpha response over that induced by CpG-ODN preexposure alone. Thus, while brief preexposure of macrophages to CpG-ODN augments the proinflammatory cytokine response to subsequent LPS challenge, prolonged preexposure elicits IL-10 production, which inhibits the TNF-alpha response. Although the initial proinflammatory effects of CpG-DNA are well established, the immune response to CpG-DNA may also include autocrine or paracrine feedback mechanisms, leading to a complex interaction of proinflammatory and inhibitory cytokines.     

Outbreak of serogroup C meningococcal disease caused by a variant of Neisseria meningitidis serotype 2a ET-15 in a community of men who have sex with men

We describe an outbreak, in a community of men who have sex with men, of serogroup C meningococcal disease caused by a genetic variant of the serotype 2a ET-15 Neisseria meningitidis characterized by a point mutation in the gene coding for the serotype 2a antigen. A microbiological characterization of the outbreak strain is presented in this report.     

Role of HPV DNA testing in predicting cervical intraepithelial lesions: comparison of HC HPV and ISH HPV

Human papillomavirus (HPV) is widely accepted as the primary agent involved in the development of squamous intraepithelial neoplasia and cervical carcinoma. Several commercial tests are available for detecting HPV DNA. This study compares the efficacy of INFORM HPV (in situ hybridization [ISH] HPV) and HCII (HC HPV) in predicting cervical lesions. A total of 762 sequential Papanicolaou (Pap) smears determined by cytologic examination to be either atypical squamous cells of undetermined significance (ASC-US) or low-grade squamous intraepithelial lesions (LSIL) were tested by both Hybrid Capture (HC) HPV and ISH HPV; 250 follow-up biopsies were reviewed as the reference standard for presence or absence of a lesion. ISH HPV and HC HPV differed significantly in accurately predicting biopsy findings from ASC-US and LSIL cases. The overall sensitivity and specificity of ISH HPV were 97% (28/29) and 86% (191/221); and HC HPV was 79% (23/29) and 56% (123/221). The positive predictive value (PPV) of ISH HPV was 48% (28/58) vs HC HPV value of 19% (23/121). Negative predictive value (NPV) was also better with ISH HPV at 99% (191/192) and HC HPV at 95% (123/129). Of equal importance, ISH HPV demonstrated a lower false-positive rate compared to HC HPV, 12% (30/250) vs 39% (98/250), as well as having a slightly lower false-negative rate 0.4% (1/250) vs 2.4% (6/250). ISH HPV is more predictive of biopsy histopathology in patients with detectable cervical lesions than is HC HPV. Effective triage of patients by HPV analysis using ISH HPV as compared to HC HPV has the potential of significant public health impact by reducing unnecessary colposcopies, as well as adverse medical, social, and psychological patient consequences.     

Sexual transmission of hepatitis B infection despite the presence of hepatitis B virus immunity in recipients of allogeneic bone marrow transplantation.

BACKGROUND: After hematopoietic cell transplantation (HCT), hepatitis due to hepatitis B virus (HBV) rarely occurred beyond the initial 12 months after transplantation. OBJECTIVES: We investigated the cause of "late" hepatitis due to HBV infection in two recipients after allogeneic HCT. STUDY DESIGN: Two male patients with acute myeloid leukemia and light chain myeloma, respectively, developed HBV-related hepatitis more than 2 years after HCT. All serum samples collected from the recipients, donors and their respective spouses were tested for HBV DNA by nested PCR, and if positive further quantified by Digene Hybrid Capture assay II. The HBV genotype was determined by PCR and sequencing. RESULTS: Genotypic analysis suggested that the cause of "late" hepatitis was due to acute HBV infection transmitted from their respective spouse. CONCLUSION: Our findings suggested that sexual precautions should be taken in these patients after HCT. Alternatively, or even additionally, active vaccination should be delivered to these patients once they have lost their HBV immunity.     

Randomised controlled trial of near-patient testing for glycated haemoglobin in people with type 2 diabetes mellitus

BACKGROUND: Tight glycaemic control in people with type 2 diabetes can lead to a reduction in microvascular and possibly macrovascular complications. The use of near-patient (rapid) testing offers a potential method to improve glycaemic control. AIM: To assess the effect and costs of rapid testing for glycated haemoglobin (HbA1c) in people with type 2 diabetes. DESIGN OF STUDY: Pragmatic open randomised controlled trial. SETTING: Eight practices in Leicestershire, UK. METHOD: Patients were randomised to receive instant results for HbA1c or to routine care. The principal outcome measure was the proportion of patients with an HbA1c <7% at 12 months. We also assessed costs for the two groups. RESULTS: Of the 681 patients recruited to the study 638 (94%) were included in the analysis. The mean age at baseline was 65.7 years (SD = 10.8 years) with a median (interquartile range) duration of diabetes of 4(1-8) years. The proportion of patients with HbA1c < 7% did not differ significantly between the intervention and control groups (37 versus 38%, odds ratio 0.95 [95% confidence interval = 0.69 to 1.31]) at 12 months follow up. The total cost for diabetes-related care was 390 UK pounds per patient for the control group and 370 UK pounds for the intervention group. This difference was not statistically significant. CONCLUSION: Near-patient testing for HbA1c alone does not lead to outcome or cost benefits in managing people with type 2 diabetes in primary care. Further research is required into the use of rapid testing as part of an optimised patient management model including arrangements for patient review and testing.     

Lifestyle Risk Score: handling missingness of individual lifestyle components in meta-analysis of gene-by-lifestyle interactions

Recent studies consider lifestyle risk score (LRS), an aggregation of multiple lifestyle exposures, in identifying association of gene-lifestyle interaction with disease traits. However, not all cohorts have data on all lifestyle factors, leading to increased heterogeneity in the environmental exposure in collaborative meta-analyses. We compared and evaluated four approaches (Naïve, Safe, Complete and Moderator Approaches) to handle the missingness in LRS-stratified meta-analyses under various scenarios. Compared to “benchmark” results with all lifestyle factors available for all cohorts, the Complete Approach, which included only cohorts with all lifestyle components, was underpowered due to lower sample size, and the Naïve Approach, which utilized all available data and ignored the missingness, was slightly inflated. The Safe Approach, which used all data in LRS-exposed group and only included cohorts with all lifestyle factors available in the LRS-unexposed group, and the Moderator Approach, which handled missingness via moderator meta-regression, were both slightly conservative and yielded almost identical p values. We also evaluated the performance of the Safe Approach under different scenarios. We observed that the larger the proportion of cohorts without missingness included, the more accurate the results compared to “benchmark” results. In conclusion, we generally recommend the Safe Approach, a straightforward and non-inflated approach, to handle heterogeneity among cohorts in the LRS based genome-wide interaction meta-analyses.Subject terms: Genetics, Risk factors     

TGF-β2 and PGE2 in Rabbit Blastocoelic Fluid Can Modulate GM-CSF Production by Human Lymphocytes

PROBLEM: During normal pregnancy, major changes occur in the production of Th2/Th1 cytokines at the feto-maternal interface. Th2 cytokines such as interleukin-4 (IL-4) or interleukin-10 (IL-10) are predominantly produced locally in the uterine and placental tissues, whereas the production of Th1 cytokines such as tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) are decreased. Because these modulations might be induced by the embryo, the current study was carried out to test the effect of rabbit blastocoelic fluid on the production of Th2/Th1 cytokines by lymphocytes, and to investiate the possible implication of transforming growth factor β₂ (TGF-β₂) prostaglandin E₂ (PGE₂) as modulators of the production of these cytokines. METHOD OF STUDY: Human peripheral blood lymphocytes (PBL) were cultured along with ConcanavalinA (Con A), and rabbit blastocoelic fluid was collected on day 12 of gestation (BF d-12). Concentrations of cytokines in culture media were determined by enzyme-linked immunoadsorbent assay (ELISA). RESULTS: Addition of BF d-12 in the culture medium induced a strong inhibition of IL-2, TNF-α, IL-10, and granulocyte-macrophage colony-stimulating factor (GM-CSF) production. However, an initial pretreatment of the lymphocytes with BF d-12, followed by a Con A stimulation, led to a marked increase in GM-CSF production, whereas IL-2, TNF-α, and IL-10 secretions were inhibited. It was also demonstrated, for the first time, that a pretreatment of the lymphocytes with TGF-β₂ and PGE₂ increased GM-CSF production to the same level reached after the addition of BF d-12. Furthermore, removal of TGF-β₂ and PGE₂ from BF d-12 by affinity chromatography reduced the effect of BF d-12 on GM-CSF production. CONCLUSIONS: Taken together, these findings suggest that the embryo, in modulating harmful and beneficial cytokine production locally, plays an active role in its protection against maternal immune cellular assault. These results also emphasize the importance of growth factors for successfully maintaining pregnancy.     

Hydrophobic IgG-containing immune complexes in the plasma of autoimmune MRL/lpr mice, lactate dehydrogenase-elevating virus-infected mice, and pigs: association with transforming growth factor-beta and pH-dependent amplification

Persistent infection of mice with lactate dehydrogenase-elevating virus (LDV) is associated with polyclonal B cell activation, autoimmunity, and circulating hydrophobic IgG-containing immune complexes (ICs), which bind to the surfaces of uncoated ELISA plates in the presence of 0.05% Tween 20. We demonstrate here that hydrophobic IgG-containing ICs also appear naturally in the plasma of autoimmune MRL/lpr mice. These and the similar hydrophobic ICs of LDV-infected mice as well as pigs coincide on ELISA plate surfaces with TGF-beta, apparently in the form of an IgG-TGF-beta complex. Circulating hydrophobic IgG-containing ICs are also susceptible to considerable amplification in vitro by exposure to alkaline conditions. By this latter method, the fraction of in vivo hydrophobic IgG, relative to the maximum in vitro chemically inducible IgG, was found to be about 20% in the plasma of LDV-infected mice, 5% in normal mouse plasma, and less than about 2% in pig plasma. These results indicate the potential for both chemically induced and protein-binding contributions to the generation of hydrophobic IgG-containing molecules, and have implications for immunopathological mechanisms in autoimmunity and persistent virus infections.