Universal and specific quantitative detection of botulinum neurotoxin genes

<p>Abstract</p> <p>Background</p> <p><it>Clostridium botulinum</it>, an obligate anaerobic spore-forming bacterium, produces seven antigenic variants of botulinum toxin that are distinguished serologically and termed "serotypes". Botulinum toxin blocks the release of acetylcholine at neuromuscular junctions resulting in flaccid paralysis. The potential lethality of the disease warrants a fast and accurate means of diagnosing suspected instances of food contamination or human intoxication. Currently, the Food and Drug Administration (FDA)-accepted assay to detect and type botulinum neurotoxins (BoNTs) is the mouse protection bioassay. While specific and sensitive, this assay requires the use of laboratory animals, may take up to four days to achieve a diagnosis, and is unsuitable for high-throughput analysis. We report here a two-step PCR assay that identifies all toxin types, that achieves the specificity of the mouse bioassay while surpassing it in equivalent sensitivity, that has capability for high-throughput analysis, and that provides quantitative results within hours. The first step of our assay consists of a conventional PCR that detects the presence of <it>C. botulinum </it>regardless of the neurotoxin type. The second step uses quantitative PCR (qPCR) technology to determine the specific serotype of the neurotoxin.</p> <p>Results</p> <p>We assayed purified <it>C. botulinum </it>DNA and crude toxin preparations, as well as food and stool from healthy individuals spiked with purified BoNT DNA, and one stool sample from a case of infant botulism for the presence of the NTNH gene, which is part of the BoNT gene cluster, and for the presence of serotype-specific BoNT genes. The PCR surpassed the mouse bioassay both in specificity and sensitivity, detecting positive signals in BoNT preparations containing well below the 1 LD₅₀required for detection via the mouse bioassay. These results were type-specific and we were reliably able to quantify as few as 10 genomic copies.</p> <p>Conclusions</p> <p>While other studies have reported conventional or quantitative PCR-based assays for the detection of <it>C. botulinum </it>genes, our procedure's high-throughput capability and its portability allows most laboratories to quickly assess the possible presence of BoNTs either in food processing samples or in suspected cases of botulism. Thus, this assay provides rapid and specific detection of BoNT and toxin complex genes and would enable the targeting of appropriate therapeutics to infected individuals in a timely manner.</p

Monitoring the response to neoadjuvant hormone therapy for locally advanced breast cancer using three-dimensional time-resolved optical mammography.

Optical mammography is a functional imaging technique that uses near-infrared light to produce three-dimensional breast images of tissue oxygen saturation and hemoglobin concentration. It has been used to monitor the response to neoadjuvant chemotherapy in breast cancer patients. We present the first results on monitoring tumor response to hormone therapy using optical mammography. We present three case studies from postmenopausal women treated with neoadjuvant hormone therapy for locally advanced breast cancer. The women were scanned before starting treatment, once during treatment, and then before surgery. Changes in physiological and optical properties within the tumor and in the rest of the breast were evaluated. At the time of surgery, two patients partially responded to treatment and one did not respond. The patients that partially responded on ultrasound revealed a corresponding recovery to normal in the hemoglobin concentration images, whereas the nonresponder indicated an increase in hemoglobin concentration in the tumor compared to her pretreatment images. These case studies suggest that optical imaging of the breast during neoadjuvant hormone treatment can provide potentially valuable information, and that physiological changes within the tumor can be seen in response to treatment

The clonal composition of human CD4+CD25+Foxp3+ cells determined by a comprehensive DNA-based multiplex PCR for TCRB gene rearrangements

The characterization of the T-cell receptor (TCR) repertoire of CD4+ regulatory T cells (TR) have been limited due to the RNA degradation that results following permeabilization and fixation as routinely used for intracellular staining of Foxp3. In the present study the clonal composition of human umbilical cord blood (UCB) and adult peripheral blood mononuclear cells (PBMC) CD4+ TR and non-TR was characterized by a DNA-based multiplex PCR which allowed for the consistent clonotypic characterization of cells that have undergone fixation and permeabilization. To validate this method, CD8+ T cells from two HLA A*0201 individuals were sorted and compared clonotypically based upon their ability either to secrete interferon-γ in response to a CMV pp65 epitope or to bind to the corresponding pMHC I tetramer. In the UCB and PBMCs clonotypes shared between the CD4+CD25+Foxp3+ and CD4+CD25+Foxp3− was observed in all 3 UCB and in one adult PBMCs, suggesting that naïve and memory CD4+ TR can share the same clonotypes as CD4+ non-TR in humans

Oral serum-derived bovine immunoglobulin improves duodenal immune reconstitution and absorption function in patients with HIV enteropathy.

ObjectivesTo examine the impact of serum-derived bovine immunoglobulin, an oral medical food known to neutralize bacterial antigen and reduce intestinal inflammation, on restoration of mucosal immunity and gastrointestinal function in individuals with HIV enteropathy.DesignOpen-label trial with intensive 8-week phase of bovine serum immunoglobulin (SBI) 2.5 g twice daily with a 4-week washout period and an optional 9-month extension study.MethodsHIV enteropathy was defined as chronic gastrointestinal symptoms including frequent loose or watery stools despite no identifiable, reversible cause. Upper endoscopy for tissue immunofluorescent antibody assay and disaccharide gut permeability/absorption studies were performed before and after 8 weeks of SBI to test mucosal immunity and gastrointestinal function. Blood was collected for markers of microbial translocation, inflammation, and collagen kinetics. A validated gastrointestinal questionnaire assessed changes in symptoms.ResultsAll eight participants experienced profound improvement in symptoms with reduced bowel movements/day (P = 0.008) and improvements in stool consistency (P = 0.008). Gut permeability was normal before and after the intervention, but D-xylose absorption increased in seven of eight participants. Mucosal CD4 lymphocyte densities increased by a median of 139.5 cells/mm2 from 213 to 322 cells/mm2 (P = 0.016). Intestinal-fatty acid binding protein (I-FABP), a marker of enterocyte damage, initially rose in seven of eight participants after 8 weeks (P = 0.039), and then fell below baseline in four of five who continued receiving SBI (P = 0.12). Baseline serum I-FABP levels were negatively correlated with subsequent rise in mucosal CD4 lymphocyte densities (r = -0.74, P = 0.046).ConclusionSBI significantly increases intestinal mucosal CD4 lymphocyte counts, improves duodenal function, and showed evidence of promoting intestinal repair in the setting of HIV enteropathy

High intensity focused ultrasound in the treatment of breast fibroadenomata (HIFU-F trial)

Background: High intensity focused ultrasound (HIFU) is a non-invasive ablative technique utilising the application of high frequency ultrasound (US) pressure waves to cause tissue necrosis. This emerging technology is currently limited by prolonged treatment times. The aim of the HIFU-F trial was to perform circumferential HIFU treatment as a means of shortening treatment times. Methods: A prospective trial was set up to treat 50 consecutive patients ≥18 years of age. Eligible patients possessed symptomatic fibroadenomata, visible on US. Patients ≥25 years of age required histological confirmation of the diagnosis. Primary outcome measures were reduction in treatment time, reduction in volume on US after 12 months and complication rates. Results: HIFU treatment was performed in 51 patients (53 treatments) with a mean age of 29.8 years (SD 7.2 years) and diameter of 2.6 cm (SD 1.4 cm). Circumferential ablation reduced treatment times by an estimated 19.9 minutes (SD 25.1 minutes), which is a 29.4% (SD 15.2%) reduction compared to whole lesion ablation. Volume reduction of 43.2% (SD 35.4%; p<0.005, paired t-test) was observed on US at 12 months post-treatment. Local complications completely resolved at one month apart from skin hyper-pigmentation, which persisted in nine cases at three months, six cases at six months and six at 12 months. Conclusion: Circumferential HIFU treatment for breast fibroadenomata is feasible to reduce both lesion size and treatment time. HIFU is a non-invasive alternative technique for the treatment of breast fibroadenomata

Accelerated in vivo proliferation of memory phenotype CD4+ T-cells in human HIV-1 infection irrespective of viral chemokine co-receptor tropism.

CD4(+) T-cell loss is the hallmark of HIV-1 infection. CD4 counts fall more rapidly in advanced disease when CCR5-tropic viral strains tend to be replaced by X4-tropic viruses. We hypothesized: (i) that the early dominance of CCR5-tropic viruses results from faster turnover rates of CCR5(+) cells, and (ii) that X4-tropic strains exert greater pathogenicity by preferentially increasing turnover rates within the CXCR4(+) compartment. To test these hypotheses we measured in vivo turnover rates of CD4(+) T-cell subpopulations sorted by chemokine receptor expression, using in vivo deuterium-glucose labeling. Deuterium enrichment was modeled to derive in vivo proliferation (p) and disappearance (d*) rates which were related to viral tropism data. 13 healthy controls and 13 treatment-naive HIV-1-infected subjects (CD4 143-569 cells/ul) participated. CCR5-expression defined a CD4(+) subpopulation of predominantly CD45R0(+) memory cells with accelerated in vivo proliferation (p = 2.50 vs 1.60%/d, CCR5(+) vs CCR5(-); healthy controls; P<0.01). Conversely, CXCR4 expression defined CD4(+) T-cells (predominantly CD45RA(+) naive cells) with low turnover rates. The dominant effect of HIV infection was accelerated turnover of CCR5(+)CD45R0(+)CD4(+) memory T-cells (p = 5.16 vs 2.50%/d, HIV vs controls; P<0.05), naïve cells being relatively unaffected. Similar patterns were observed whether the dominant circulating HIV-1 strain was R5-tropic (n = 9) or X4-tropic (n = 4). Although numbers were small, X4-tropic viruses did not appear to specifically drive turnover of CXCR4-expressing cells (p = 0.54 vs 0.72 vs 0.44%/d in control, R5-tropic, and X4-tropic groups respectively). Our data are most consistent with models in which CD4(+) T-cell loss is primarily driven by non-specific immune activation

Reduced-iodine-dose dual-energy coronary CT angiography: qualitative and quantitative comparison between virtual monochromatic and polychromatic CT images.

To quantitatively evaluate the impact of virtual monochromatic images (VMI) on reduced-iodine-dose dual-energy coronary computed tomography angiography (CCTA) in terms of coronary lumen segmentation in vitro, and secondly to assess the image quality in vivo, compared with conventional CT obtained with regular iodine dose. A phantom simulating regular and reduced iodine injection was used to determine the accuracy and precision of lumen area segmentation for various VMI energy levels. We retrospectively included 203 patients from December 2017 to August 2018 (mean age, 51.7 ± 16.8 years) who underwent CCTA using either standard (group A, n = 103) or reduced (group B, n = 100) iodine doses. Conventional images (group A) were qualitatively and quantitatively compared with 55-keV VMI (group B). We recorded the location of venous catheters. In vitro, VMI outperformed conventional CT, with a segmentation accuracy of 0.998 vs. 1.684 mm &lt;sup&gt;2&lt;/sup&gt; , respectively (p &lt; 0.001), and a precision of 0.982 vs. 1.229 mm &lt;sup&gt;2&lt;/sup&gt; , respectively (p &lt; 0.001), in simulated overweight adult subjects. In vivo, the rate of diagnostic CCTA in groups A and B was 88.4% (n = 91/103) vs. 89% (n = 89/100), respectively, and noninferiority of protocol B was inferred. Contrast-to-noise ratios (CNR) of lumen versus fat and muscle were higher in group B (p &lt; 0.001) and comparable for lumen versus calcium (p = 0.423). Venous catheters were more often placed on the forearm or hand in group B (p &lt; 0.001). In vitro, low-keV VMI improve vessel area segmentation. In vivo, low-keV VMI allows for a 40% iodine dose and injection rate reduction while maintaining diagnostic image quality and improves the CNR between lumen versus fat and muscle. • Dual-energy coronary CT angiography is becoming increasingly available and might help improve patient management. • Compared with regular-iodine-dose coronary CT angiography, reduced-iodine-dose dual-energy CT with low-keV monochromatic image reconstructions performed better in phantom-based vessel cross-sectional segmentation and proved to be noninferior in vivo. • Patients receiving reduced-iodine-dose dual-energy coronary CT angiography often had the venous catheter placed on the forearm or wrist without compromising image quality