Preparing Patients for Oral Immunotherapy (PPOINT): International Delphi consensus for procedural preparation and consent.

BACKGROUND: Despite the promise of oral immunotherapy (OIT) to treat food allergies, this procedure is associated with potential risk. There is no current agreement about what elements should be included in the preparatory or consent process. OBJECTIVE: We developed consensus recommendations about the OIT process considerations and patient-specific factors that should be addressed before initiating OIT and developed a consensus OIT consent process and information form. METHODS: We convened a 36-member Preparing Patients for Oral Immunotherapy (PPOINT) panel of allergy experts to develop a consensus OIT patient preparation, informed consent process, and framework form. Consensus for themes and statements was reached using Delphi methodology, and the consent information form was developed. RESULTS: The expert panel reached consensus for 4 themes and 103 statements specific to OIT preparatory procedures, of which 76 statements reached consensus for inclusion specific to the following themes: general considerations for counseling patients about OIT; patient- and family-specific factors that should be addressed before initiating OIT and during OIT; indications for initiating OIT; and potential contraindications and precautions for OIT. The panel reached consensus on 9 OIT consent form themes: benefits, risks, outcomes, alternatives, risk mitigation, difficulties/challenges, discontinuation, office policies, and long-term management. From these themes, 219 statements were proposed, of which 189 reached consensus, and 71 were included on the consent information form. CONCLUSION: We developed consensus recommendations to prepare and counsel patients for safe and effective OIT in clinical practice with evidence-based risk mitigation. Adoption of these recommendations may help standardize clinical care and improve patient outcomes and quality of life.

Investigation of the Role of Interleukin 6 in Vitiligo Pathogenesis.

Interleukin-6 (IL6) is involved in pathogenesis of several autoimmune disorders including vitiligo. Hence, we aimed to investigate the association of IL6 -174 G/C and -572 G/C polymorphisms and its transcript levels with vitiligo; to evaluate the effect of IL-6 on normal human melanocyte (NHM) viability and expression of IL6R, MITF and TYR. IL6 -174 G/C and -572 G/C polymorphisms were genotyped by ARMS-PCR and PCR-RFLP respectively in 343 controls and 322 vitiligo patients. IL6 transcript levels were estimated from PBMCs (96 controls and 77 patients) and skin samples (15 controls and 15 patients) by qPCR. NHM viability was assessed by MTT; IL6R, MITF and TYR transcript and protein levels were monitored by qPCR and ICC respectively. Genetic analyses revealed no association of IL6 -174 G/C polymorphism (p> .05) with vitiligo. Analysis of IL6 -572 G/C revealed reduced risk of vitiligo in individuals with GC/CC genotypes compared to GG genotype (p = .010). IL6 expression was significantly increased (p = .0197) in PBMCs of patients. Further, IL6 expression was significantly higher in non-lesional skin compared to controls (p = .009). In-vitro NHM viability was decreased upon IL-6 exposure (10-50 ng/ml; p< .05), with significantly increased IL6R transcript (p = .042) and protein levels (p = .003) however, MITF transcript (p = .0003) and protein levels (p = .016), and TYR transcript levels (p = .001) were significantly decreased. The results suggest that IL6 -572 G/C polymorphism might be associated with vitiligo susceptibility in Gujarat population. Moreover, increased IL6 expression in vitiligo patients and its effect on NHM suggest a potential role in melanocyte biology. CONCLUSION: The results suggest that IL6 - 572 G/C polymorphism might be associated with vitiligo susceptibility in Gujarat population. Moreover, increased IL6 expression in vitiligo patients and its effect on NHM suggest a potential role in melanocyte biology.

Towards an FDA-cleared basophil activation test.

Food allergy is a global health problem affecting up to 10% of the world population. Accurate diagnosis of food allergies, however, is still a major challenge in medical offices and for patients seeking alternative avenues of diagnosis. A flawless test to confirm or rule out a food allergy does not exist. The lack of optimum testing methods to establish precise clinical correlations remains a major obstacle to effective treatment. Certain IgE measurement methods, including component testing, have received FDA clearance, but they have been used primarily as an analytical tool and not to establish clinical correlations. Most allergy tests are still carried out within the laboratory, and skin tests outside a laboratory setting that are used for food allergy diagnosis rely on non-standardized allergens, according to the FDA definition. Epitope mapping and basophil activation test (BAT) have recently been proposed as a means of establishing better clinical correlations. Yet neither have received FDA clearance for widespread distribution. Of the two methods, the BAT has the advantage of being a functional assay. Over the past few years, several large private practice groups in the United States, have developed BAT as a clinical assay and have started using it in patient care. Given this clinical experience, the vast number of papers published on BAT (more than 1,400 as of 2022) and the trend toward increasing FDA regulation, it is essential to understand the roadmap for regulatory clearance of this assay.

Validation of inducible basophil biomarkers: Time, temperature and transportation.

BACKGROUND: The short stability window of several hours from blood collection to measuring basophil activation has limited the use of flow cytometry-based basophil activation assays in clinical settings. We examine if it is possible to extend this window to 1 day allowing for shipment of samples between laboratories. Several options exist for reporting the results including reporting all the measured values directly, calculating ratios and reporting a single value covering all measured results. Each of these options have different stability and value to the physician. METHODS: Whole blood samples from peanut allergic patients were stimulated with four different peanut concentrations at Day 0, Day 1, and Day 2. Samples were stored under temperature-controlled conditions. Flow cytometry was used to analyze the samples. The basophil activation and degranulation were measured as percentage of positive CD63 basophils and CD203c MFI fold change. Shipped samples were transported under ambient conditions. RESULTS: The results show that CD63 is a stable marker at Day 1. The CD203c ratio decreases significantly at Day 1. Calculating the CD63/IgE ratio proves to be more stable than CD63 alone. The most stable readouts are the semi-quantitative results and the trajectory of the dose response curve. Finally, we confirmed that the stability can be extended to samples shipped overnight to the laboratory. CONCLUSIONS: It is possible to extend the stability of the basophil activation assay to 1 day for samples stored at 18-25°C as well as samples shipped under ambient conditions as long as the temperature is within the 2-37°C range.

Adipose tissue: A natural resource for multipotent mesenchymal stem cells with potential translation to trigerminal layers.

BACKGROUND: The article reports basic science research that establishes that adipose tissue (AT)-derived mesenchymal stem cells (MSCs) have a potential to transgerminal translation. STUDY DESIGN: MSC confirmation was obtained by phenotypic spindle-shaped cells as well as with four positive and three negative markers. The translineage translation of adipose-derived MSCs (ADMSCs) was established. MATERIALS AND METHODS: The lipoaspirate was subjected to enzymatic digestion with collagenase. Stromal vascular factor (SVF) was isolated. With two passages, pure culture of ADMSCs was obtained. They were translated to all the three germinal layers. RESULTS: AT-derived SVF contains ~30% MSCs. They are capable of being translated into endoderm, mesoderm and ectoderm. CONCLUSION: AT is a rich source for MSCs, with immense research possibilities for regeneration and rejuvenation.

The use of in vivo, ex vivo, in vitro, computational models and volunteer studies in vision research and therapy, and their contribution to the Three Rs.

Much is known about mammalian vision, and considerable progress has been achieved in treating many vision disorders, especially those due to changes in the eye, by using various therapeutic methods, including stem cell and gene therapy. While cells and tissues from the main parts of the eye and the visual cortex (VC) can be maintained in culture, and many computer models exist, the current non-animal approaches are severely limiting in the study of visual perception and retinotopic imaging. Some of the early studies with cats and non-human primates (NHPs) are controversial for animal welfare reasons and are of questionable clinical relevance, particularly with respect to the treatment of amblyopia. More recently, the UK Home Office records have shown that attention is now more focused on rodents, especially the mouse. This is likely to be due to the perceived need for genetically-altered animals, rather than to knowledge of the similarities and differences of vision in cats, NHPs and rodents, and the fact that the same techniques can be used for all of the species. We discuss the advantages and limitations of animal and non-animal methods for vision research, and assess their relative contributions to basic knowledge and clinical practice, as well as outlining the opportunities they offer for implementing the principles of the Three Rs (Replacement, Reduction and Refinement).

Laparoscopic restorative proctocolectomy ileal pouch anal anastomosis: How I do it?

Surgery for ulcerative colitis is a major and complex colorectal surgery. Laparoscopy benefits these patients with better outcomes in context of cosmesis, pain and early recovery, especially in young patients. For surgeons, it is a better tool for improving vision and magnification in deep cavities. This is not the simple extension of the laparoscopy training. Starting from preoperative preparation to post operative care there are wide variations as compared to open surgery. There are also many variations in steps of laparoscopic surgery. It involves left colon, right colon and rectal mobilisation, low division of rectum, pouch creation and anastomosis of pouch to rectum. Over many years after standardisation of this technique, it takes same operative time as open surgery at our centre. So we present our standardized technique of laparoscopic assisted restorative proctocolectomy and ileal pouch anal anastomosis (IPAA).

Novel Trocarless, Scarless Technique for Left Lobe Liver Retraction in Laparoscopic Upper Gastrointestinal Surgeries: Simple, Cost-effective and with Better Cosmesis.

Surgeons always look for ways to reduce the size and number of ports in laparoscopy, where in today's era, we have single-incision laparoscopic surgery (SILS). While doing so, principal 'adequate exposure' should not be compromised. For upper gastrointestinal laparoscopic surgeries, we have adopted a novel technique for retraction of the left lobe of liver, which is described here. Device can be made both single sling and double sling, with help of an infant feeding tube and any routinely used suture material. Placement of device does not require any incision, special energy source, or instrument. It can help in SILS. Detailed technique is described in the text. Operative times did not change significantly. Exposure was excellent. No special instruments or energy devices are required; thus, it is cost-effective. Reducing one port for liver retraction gives better cosmetic results. No liver injury due to the device was noticed in any of the cases. This technique is simpler and cheaper and also gives reasonable cosmetic results compared to other techniques described in the literature.

Thoracoscopic part of minimal invasive oesophagectomy in semiprone position: our initial experience.

INTRODUCTION: Surgical resection with curative intent is till the mainstay of treatment for resectable esophageal cancer. Minimal invasive oesophagectomy has the potential to lower morbidity and mortality. In all likelihood, thoracoscopic oesophagectomy in semiprone position gives all the benefits of prone position and can be converted to thoracotomy without change in patient position if needed. The aim of this study is to analyze the feasibility of thoracoscopic oesophagectomy in semiprone position. MATERIALS AND METHODS: This is a retrospective analysis of the data of thoracoscopic oesophgeactomy in semiprone position at Kaizen Hospital, a tertiary care center for gastroenterology during the period of December 2011 to December 2012. All surgeries were performed under general anesthesia with a single-lumen endotracheal tube. Esophageal mobilization was done by thoracoscopic approach in a semiprone position and an end-to-end hand-sewn cervical anastomosis was done. Abdominal part was performed by laparoscopic method in 3 patients and by laparotomy in rest of the patients. RESULTS: Total of 12 patients were included in this study. There was 1 conversion to thoracotomy and 1 surgical mortality. Mean operating time for the thoracoscopic part was 103 minutes, mean estimated blood loss for the thoracoscopic part was 110 mL, mean maximum end-tidal CO2 38.5 mm Hg, mean lymph nodes retrieved 14, and all patients had R0 resection. The median intensive care unit stay was 1 day and hospital stay was 8 days. CONCLUSIONS: Thoracoscopic part of thoracolaparoscopic oesophagectomy in semiprone position is a feasible option. It gives all advantages of prone position and thoracotomy is possible without change in patient position. However, further large-scale studies are required.

Legal scenario in burn care in India.

Physicians engaged in management of burn patients in India need to keep themselves abreast with the legal requirements. Clinical burn management and liaison with local authorities go almost parallel. Concept of the legal rights of Burn Survivor and the family are emerging now in India. Demarcation between physical impairment status and disability to sustain are discussed. Burn Physicians can help their patients by imparting this information. Pertinent details about Workmen's compensation act, Persons with disabilities act and guidelines for calculation of physical impairments are listed.

A randomized, double-blind, phase 3 study of fospropofol disodium for sedation during colonoscopy.

GOALS: This double-blind, multicenter study evaluated the safety and efficacy of intravenous fospropofol (6.5 mg/kg vs. 2 mg/kg) for moderate sedation in patients undergoing colonoscopy. METHODS: In all, 314 patients >or=18 years (American Society of Anesthesiologists PS1 to PS3) were randomized to receive fospropofol 2 mg/kg, fospropofol 6.5- mg/kg, or midazolam 0.02 mg/kg, after pretreatment with intravenous fentanyl 50 mcg. Supplemental doses of study medication were permitted to achieve a Modified Observer's Assessment of Alertness/Sedation scale score

The role of freestyle perforator-based pedicled flaps in reconstruction of delayed traumatic defects.

The use of perforator-based flaps as freestyle pedicled flaps for traumatic defects has been limited. We explored this possible application in small to moderate sized traumatic defects presenting in the delayed phase, with distinct oedema and induration in the potential flap donor area and posttraumatic vessel disease. Attempts to skeletonize perforator vessels are likely to compromise the flap perfusion, and inadequate dissection is likely to limit mobility of the indurated tissues in the flap. Conventionally, an axial pattern pedicled or a free flap would be needed in such cases, thus increasing its magnitude. We used the freestyle technique to cover traumatic defects by retrograde dissection of pedicled perforator-based flaps. As the surgery was performed in the delayed phase, the tissues were indurated and a larger tissue cuff was preserved around the pedicle than would be our practice in elective surgery. In addition, flap dimensions were planned larger than the defect to be closed. The donor defect was either skin grafted or closed primarily. Our study included 11 cases at various sites over the body. All flaps survived, though 3 flaps encountered major complications, 2 of which needed reoperation. None of the flaps failed completely. The pedicled perforator-based flap provides the surgeon with additional reconstructive options in the setting of trauma. These flaps can be safely harvested using indurated tissue; thus in selected cases, a free flap can be avoided, and reliable cover can be provided with a pedicled flap. Nevertheless, clinical judgment is essential to assess the potential vascular territory of the flap.

Microsatellites as EWS/FLI response elements in Ewing's sarcoma.

The ETS gene family is frequently involved in chromosome translocations that cause human cancer, including prostate cancer, leukemia, and sarcoma. However, the mechanisms by which oncogenic ETS proteins, which are DNA-binding transcription factors, target genes necessary for tumorigenesis is not well understood. Ewing's sarcoma serves as a paradigm for the entire class of ETS-associated tumors because nearly all cases harbor recurrent chromosomal translocations involving ETS genes. The most common translocation in Ewing's sarcoma encodes the EWS/FLI oncogenic transcription factor. We used whole genome localization (ChIP-chip) to identify target genes that are directly bound by EWS/FLI. Analysis of the promoters of these genes demonstrated a significant over-representation of highly repetitive GGAA-containing elements (microsatellites). In a parallel approach, we found that EWS/FLI uses GGAA microsatellites to regulate the expression of some of its target genes including NR0B1, a gene required for Ewing's sarcoma oncogenesis. The microsatellite in the NR0B1 promoter bound EWS/FLI in vitro and in vivo and was both necessary and sufficient to confer EWS/FLI regulation to a reporter gene. Genome wide computational studies demonstrated that GGAA microsatellites were enriched close to EWS/FLI-up-regulated genes but not down-regulated genes. Mechanistic studies demonstrated that the ability of EWS/FLI to bind DNA and modulate gene expression through these repetitive elements depended on the number of consecutive GGAA motifs. These findings illustrate an unprecedented route to specificity for ETS proteins and use of microsatellites in tumorigenesis.

Evaluation and comparison of portable emissions measurement systems and federal reference methods for emissions from a back-up generator and a diesel truck operated on a chassis dynamometer.

There is considerable interest in portable emissions measurement systems (PEMS) for emission inventory and regulatory applications. For this study, four commercial PEMS were compared with a Federal Reference Method (FRM) for measuring emissions from a back-up generator (BUG) over steady-state loads and a diesel truck on transient and steady-state chassis dynamometer tests. The agreement between the PEMS and the FRM varied depending on the pollutant and the particular PEMS tested for both the BUG and chassis dynamometer testing. The best performing PEMS for both the BUG and chassis testing was within approximately 12% for NOx of the FRM. For the BUG testing, several PEMS showed agreement with the FRM within approximately 5% for CO2. For the chassis dynamometer testing, the best PEMS showed agreement typically within approximately 5% for CO2. PM measurements for the BUG testing were low compared to the FRM, with the best measurements approximately 20% lower. For the chassis testing, two PM PEMS showed a good correlation but a high bias, while the correlation was worse for the other two PEMS. For each emissions component, some PEMS under different test conditions showed considerably larger deviations than those for the best performing PEMS.

Genome-wide analyses reveal properties of redundant and specific promoter occupancy within the ETS gene family.

The conservation of in vitro DNA-binding properties within families of transcription factors presents a challenge for achieving in vivo specificity. To uncover the mechanisms regulating specificity within the ETS gene family, we have used chromatin immunoprecipitation coupled with genome-wide promoter microarrays to query the occupancy of three ETS proteins in a human T-cell line. Unexpectedly, redundant occupancy was frequently detected, while specific occupancy was less likely. Redundant binding correlated with housekeeping classes of genes, whereas specific binding examples represented more specialized genes. Bioinformatics approaches demonstrated that redundant binding correlated with consensus ETS-binding sequences near transcription start sites. In contrast, specific binding sites diverged dramatically from the consensus and were found further from transcription start sites. One route to specificity was found--a highly divergent binding site that facilitates ETS1 and RUNX1 cooperative DNA binding. The specific and redundant DNA-binding modes suggest two distinct roles for members of the ETS transcription factor family.

Retinal and cerebral microembolization during coronary artery bypass surgery: a randomized, controlled trial.

BACKGROUND: We sought to compare the effects on ophthalmic function of coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB) and off-pump (OPCAB) grafting and to investigate whether retinal microvascular damage is associated with markers of cerebral injury. METHODS AND RESULTS: Retinal microvascular damage was assessed by fluorescein angiography and color fundus photography. Ophthalmic function was tested by the logarithm of the minimum angle of resolution visual acuity (VA), and cerebral injury, by transcranial Doppler ultrasound-detected emboli and S100 protein values. Twenty patients were randomized. Fluorescein angiography and postoperative VA could not be obtained for 1 CABG-CPB patient. Retinal microvascular damage was detected in 5 of 9 CABG-CPB but in none of 10 OPCAB patients (risk difference, 55%; 95% confidence interval [CI], 23% to 88%; P=0.01). Color fundus photography detected microvascular damage in 1 CABG-CPB patient but in no OPCAB patients; this lesion was associated with a field defect, which remained after 3 months of follow-up. There was no difference in postoperative VA. Doppler high-intensity transient signals (HITS) were 20.3 times more frequent in the CABG-CPB than in the OPCAB group (95% CI, 9.1 to 45; P<0.0001). Protein S100 levels were higher in the CABG-CPB than in the OPCAB group 1 hour after surgery (P<0.001). HITS were 14.7 times more frequent (95% CI, 3.5 to 62; P=0.001) and S100 level 2.1 times higher (95% CI, 1.3 to 3.5; P=0.005) when retinal microvascular damage was present. CONCLUSIONS: The relative frequency of retinal microvascular damage between groups shows the extent to which the risk of cerebral injury is reduced with OPCAB. Imaging of part of the cerebral circulation provides evidence to validate markers of cerebral injury.

Genome-based peptide fingerprint scanning.

We have implemented a method that identifies the genomic origins of sample proteins by scanning their peptide-mass fingerprint against the theoretical translation and proteolytic digest of an entire genome. Unlike previously reported techniques, this method requires no predefined ORF or protein annotations. Fixed-size windows along the genome sequence are scored by an equation accounting for the number of matching peptides, the number of missed enzymatic cleavages in each peptide, the number of in-frame stop codons within a window, the adjacency between peptides, and duplicate peptide matches. Statistical significance of matching regions is assessed by comparing their scores to scores from windows matching randomly generated mass data. Tests with samples from Saccharomyces cerevisiae mitochondria and Escherichia coli have demonstrated the ability to produce statistically significant identifications, agreeing with two commonly used programs, peptident and mascot, in 86% of samples analyzed. This genome fingerprint scanning method has the potential to aid in genome annotation, identify proteins for which annotation is incorrect or missing, and handle cases where sequencing errors have caused framing mistakes in the databases. It might also aid in the identification of proteins in which recoding events such as frameshifting or stop-codon read-through have occurred, elucidating alternative translation mechanisms. The prototype is implemented as a clientserver pair, allowing the distribution, among a set of cluster nodes, of a single or multiple genomes for concurrent analysis.

Artificial neural network prediction of antisense oligodeoxynucleotide activity.

An mRNA transcript contains many potential antisense oligodeoxynucleotide target sites. Identification of the most efficacious targets remains an important and challenging problem. Building on separate work that revealed a strong correlation between the inclusion of short sequence motifs and the activity level of an oligo, we have developed a predictive artificial neural network system for mapping tetranucleotide motif content to antisense oligo activity. Trained for high-specificity prediction, the system has been cross-validated against a database of 348 oligos from the literature and a larger proprietary database of 908 oligos. In cross- validation tests the system identified effective oligos (i.e. oligos capable of reducing target mRNA expression to <25% that of the control) with 53% accuracy, in contrast to the <10% success rates commonly reported for trial-and-error oligo selection, suggesting a possible 5-fold reduction in the in vivo screening required to find an active oligo. We have implemented a web interface to a trained neural network. Given an RNA transcript as input, the system identifies the most likely oligo targets and provides estimates of the probabilities that oligos targeted against these sites will be effective.

Computational identification of putative programmed translational frameshift sites.

MOTIVATION: In an effort to identify potential programmed frameshift sites by statistical analysis, we explore the hypothesis that selective pressure would have rendered such sites underabundant and underrepresented in protein-coding sequences. We developed a computer program to compare the frequencies of k-length subsequences of nucleotides with the frequencies predicted by a zero order Markov chain determined by the codon bias of the same set of sequences. The program was used to calculate and evaluate the distribution of 7-base oligonucleotides in the 6000+ putative protein-coding sequences of S. cerevisiae preliminary to the laboratory testing of the most highly underrepresented oligos for frameshifting efficiency. RESULTS: Among the most significant results is the finding that the heptanucleotides CUU-AGG-C and CUU-AGU-U, sites of the programmed +1 translational frameshifts required for the production in yeast of actin filament-binding protein ABP140 and telomerase subunit EST3, respectively, rank among the least represented of phase I heptanucleotides in the coding sequences of S. cerevisiae. Laboratory experiments demonstrated that other underrepresented heptanucleotides identified by the program, for example GGU-CAG-A, are also prone to significant translational frameshifting, suggesting the possibility that genes containing other underrepresented heptamers may also encode transframe products. AVAILABILITY: The program is available for download from http://www.gesteland.genetics.utah.edu/freqAnalysis SUPPLEMENTARY INFORMATION: Complete results from the analysis of S. cerevisiae are available on http://www.gesteland.genetics.utah.edu/freqAnalysis