EGFR-targeted prodrug activation using bioorthogonal alkene-azide click-and-release chemistry.

Epidermal growth factor receptor (EGFR) is overexpressed in many cancers and therefore serves as an excellent target for prodrug activation. Functionalised trans-cyclooctenes (TCO) were conjugated to an EGFR antibody (cetuximab), providing a reagent for pre-targeting and localisation of the bioorthogonal reagent. The TCOs react with a 4-azidobenzyl carbamate doxorubicin prodrug via a [3 + 2]-cycloaddition and subsequent self-immolation leads to release of doxorubicin (click-and-release). In vitro cell-based assays demonstrated proof-of-concept, that cetuximab conjugated to highly strained TCO (AB-d-TCO) could bind to the EGFR in a melanoma cell line, and selectively activate the doxorubicin prodrug. In a non-EGFR expressing melanoma cell line, no significant prodrug activation was observed. In vivo experiments using this combination of AB-d-TCO and the azido-doxorubicin prodrug in a murine melanoma model revealed no significant anti-tumour activity or increased survival, suggesting there was insufficient prodrug activation and drug release at the tumour site.

Poloxamer 407-chitosan grafted thermoresponsive hydrogels achieve synchronous and sustained release of antigen and adjuvant from single-shot vaccines.

Sustained-release vaccine delivery systems may enhance the immunogenicity of subunit vaccines and reduce the need for multiple vaccinations. The aim of this study was to develop a thermoresponsive hydrogel using poloxamer 407-chitosan (CP) grafted copolymer as a delivery system for single-shot sustained-release vaccines. The CP copolymer was synthesized using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide chemistry. The CP copolymer was a free flowing solution at ambient temperature and transformed rapidly into a gel at body temperature. The hydrogels were loaded with vaccine antigen and adjuvants or the vaccine components were encapsulated in poly (lactic-co-glycolic acid) nanoparticles in order to ensure synchronous release. The CP hydrogels were stable for up to 18 days in vitro. Release of both nanoparticles and the individual components was complete, with release of the individual components being modulated by incorporation into nanoparticles. In vivo, a single dose of CP hydrogel vaccine induced strong, long lasting, cellular and humoral responses that could protect against the development of tumors in a murine melanoma model.

Defining Molecular Details of the Chemistry of Biofilm Formation by Raman Microspectroscopy.

Two protocols that allow for the comparison of Raman spectra of planktonic cells and biofilm formed from these cells in their growth phase have been developed. Planktonic cells are washed and flash-frozen in <1 min to reduce the time for metabolic changes during processing, prior to freeze-drying. Biofilm is formed by standing cells in 50 µL indentations in aluminum foil in an atmosphere of saturated water vapor for 24-48 h. The results for Escherichia coli type K12 cells, which do not readily form biofilm, are compared to those for Staphylococcus epidermidis cells, which prolifically synthesize biofilm. For E. coli, the Raman spectra of the planktonic and biofilm samples are similar with the exception that the spectral signature of RNA, present in planktonic cells, could not be detected in biofilm. For S. epidermidis, major changes occur upon biofilm formation. In addition to the absence of the RNA features, new bands occur near 950 cm-1 and between 1350 and 1420 cm-1 that are associated with an increase in carbohydrate content. Unlike the case in E. coli biofilm, the intensity of G base ring modes is reduced in but A and T base ring signatures become more prominent. For S. epidermis in the biofilm's amide III region, there is evidence of an increase in the level of ß-sheet structure accompanied by a decrease in α-helical content. The presence of biofilm is confirmed by microscope-aided photography and, separately, by staining with methyl violet.

Fetal inflammatory response and risk for psychiatric disorders.

Inflammation contributes to numerous neuropsychiatric disorders, especially those that first appear in childhood. Maternal intrauterine environment, including the placenta, has a role in brain development and risk for neuropsychiatric disorders. This study examines the link between fetal inflammatory syndrome (FIRS), which is placental inflammation in the peri-partem period, and neuropsychiatric disorders during childhood.This is a retrospective cohort study using data from electronic medical records over a 19-year period at one women's hospital. The study includes 4851 children born with placentas meeting criteria for and 31,927 controls identified with normal placentas born during the same period. To be diagnosed with FIRS placenta must contain chorionic vasculitis and/or funisitis. Children had to be in study period for at least 5 years. The primary outcome of the study is incidence of neuropsychiatric disorders during childhood. The secondary outcomes were psychiatric medications prescribed, and psychiatric hospitalizations and treatment. Children born to placentas meeting criteria for FIRS were more likely to be diagnosed with neuropsychiatric disorders (OR = 1.21, CI 95% [1.09,1.35]). Specifically, they were more likely to be diagnosed with autism spectrum disorder (OR = 1.35, CI 95% [1.08, 1.67]), ADHD (OR = 1.27, CI 95% [1.07, 1.49]), conduct disorder (OR = 1.50, CI 95% [1.24, 1.81]), PTSD (OR = 2.46. CI 95% [1.21, 5.04]), adjusting for maternal history of psychiatric disorders, intra-partem substance use, and prescriptions of anti-inflammatory drugs. Children born with placental inflammation are at an increased risk to develop neuropsychiatric disorders. This has profound implications for future research, and early detection, monitoring, and treatment in these children.

Parent-in-training: Resident and fellow experiences from pregnancy to parenthood.

BACKGROUND: Residents and fellows with children face distinct challenges; however, knowledge of factors associated with increased parental stress is limited. OBJECTIVE: This study aimed to investigate experiences and concerns of physician trainees and identify factors associated with higher parental stress. METHODS: An anonymous survey was distributed to all resident and fellow trainees in June 2021 to assess experiences regarding parental leave, breastfeeding, and childcare. We used the Parental Stress Scale (PSS) to identify the factors associated with stress and analyzed the results using descriptive statistics, linear regression, and thematic analysis. RESULTS: Of 1719 trainees, 509 participated (62% women, 30% response rate); half were parents. One-third of the respondents (152/470) said that childcare costs affected the number of children they plan to have; One-third of respondents (152/470) said that childcare costs affected the number of children they plan to have; 45% (210/470) said childcare costs affected when they plan to have children. Among parents, the mean PSS score was 44.3 ± 12.3, with no significant gender differences. More women identified as primary or coprimary caregivers (97% [113/117] vs. 79% [60/76], p < .001) and anticipated training extensions due to parental leave (36% vs. 13% men, p = .009). Breastfeeding was associated with significantly higher PSS scores (p = .017). Twenty-four percent of breastfeeding parents (22/93) felt that their program/institution did not support their breastfeeding goals; lack of perceived support was associated with significantly higher PSS scores (63.6 ± 13.1 vs. 38.6 ± 8.7, p < .001). Trainees experiencing unreliable childcare had significantly higher PSS scores (p = .005). Forty percent (64/159) changed their career plans after becoming parents. CONCLUSIONS: Physician trainee parents experience high stress, with women bearing disproportionate burdens in the domains of parental leave and breastfeeding. These results should inform policies promoting trainee wellness and gender equity.

Communicating Certainty in Pathology Reports.

CONTEXT.­: Pathology reports are the main modality in which results are communicated to other physicians. For various reasons, the diagnosis may be qualified on a spectrum of uncertainty. OBJECTIVE.­: To examine how communication of uncertainty is an unexamined source of possible medical error. No study to our knowledge has examined pathology reports across multiple institutions. This study seeks to identify commonly used phrases of diagnostic uncertainty and their interpreted meanings by surgical pathologists and clinicians. DESIGN.­: Anonymous surveys were completed at 3 major US academic institutions by 18 practicing staff pathologists, 12 pathology residents, 53 staff clinicians, and 50 resident/allied health professional clinicians at 5 standard tumor boards. All participants rated percentage certainty associated with 7 diagnostic terms. Pathologists answered 2 questions related to the ability to clarify a diagnosis using a comment and comfort wording pathology reports. Clinicians answered questions on how often they read a pathology report comment, if they found the comment helpful, and how comfortable they were in reading pathology reports. RESULTS.­: A wide range in percentage certainty was found for each of the 7 diagnostic phrases. Both staff and resident clinicians and residents showed wide variability in interpreting the phrases. Twenty-five of 50 staff clinicians (52%) were very comfortable reading a pathology report, whereas only 4 of 53 resident clinicians (8%) were very comfortable reading a pathology report. Twenty-four of 53 staff clinicians (63%) reported always reading the comment, yet only 20 of 53 (27%) always found the comment helpful. The phrases "diagnostic of" and "consistent with" had the strongest agreement in meaning. The weakest agreement was between "suspicious for" and "compatible with." CONCLUSIONS.­: Efforts to standardize diagnostic terms may improve communication.

Patients Who Take Home Their Surgical Pathology Specimens: A Preliminary Study.

Patients regularly request to take possession of their human tissues after they have become surgical pathology specimens. To date, few formal research studies have examined the prevalence of this practice or the reasoning patients' request that their specimens to be returned to them. This study interviews patients from 2015 to 2017 at one US academic medical center who requested their surgical pathology specimens. Of the 22 eligible patients, 8 patients agreed to be interviewed. Interviews lasted 10 to 30 minutes and included 5 questions. The questions were: (1) What motivated your decision to obtain your surgical pathology specimen, (2) What, if anything, did you do with your specimen, (3) What were positive aspects of your experience, (4) What were negative aspects of your experience, (5) What can the pathology department change to better support patients who request their surgical pathology specimens? Verbatim transcripts were generated and a mixed-methods analysis was performed. The type of specimens included products of conception, placenta and cord, costal cartilage and ribs, loop explant recorder, pacemaker, below knee amputation, and cervix, uterus, Fallopian tubes, and ovaries. The dominant themes included adversity, medical interest, souvenir, cultural beliefs, and curiosity. Subthemes included becoming whole in the afterlife, preservation, my body, restoration, honoring, and regret. In conclusion, pathologists can expand their role as patient advocates and advance patient-centered pathology by supporting patient's individual needs, motivations, and goals, when they request their surgical pathology specimens.

Microcontainers for protection of oral vaccines, in vitro and in vivo evaluation.

Oral vaccines are highly desirable due to simple logistics, mass vaccination potential and for mucosal immunity. Subunit vaccines are preferred due to high safety, but are inherently difficult to deliver orally, thus providing motivation for the use of advanced oral delivery systems. Polymeric devices in micrometer size (microcontainers) were tested here for this purpose. Microcontainers were loaded with a vaccine consisting of spray dried cubosomes with OVA and Quil-A, and coated with a pH-sensitive lid for oral delivery to C57Bl/6 mice. The microcontainers were explored in vitro and in vivo for their potential as oral vaccine delivery system in an oral prime-boost setting and as an oral booster after a subcutaneously injected prime. The residence time of microcontainers in the small intestine was less than one hour. Eudragit® L100-55 was therefore chosen as lid material on the microcontainers as it remained stable in vitro at pH 4.7, which simulated the maximal pH of the stomach, and allowed release of the cubosomes within 30-60 min at pH 6.6, which simulated the mean pH of the distal half of the small intestine. In vitro small angle X-ray scattering showed that cubosomes dissolved in small intestinal fluid when not confined in microcontainers but when loaded into microcontainers they were released as hexosomes. However, while microcontainers could protect and release particles with OVA and Quil-A within relevant time frames in vitro, an immune response was not elicited in vivo after oral administration. Nonetheless, some effect was observed when the microcontainers were used to deliver oral boosters following a subcutaneous prime. This work indicates that oral vaccination with subunit vaccines has potential when combined with a parenteral prime and that oral delivery systems like microcontainers may be used to increase the potency of vaccines with low oral immunogenicity.

A "Pathology Explanation Clinic (PEC)" for Patient-Centered Laboratory Medicine Test Results.

This concept paper addresses communication issues arising between physicians and their patients. To facilitate the communication of essential diagnostic pathology information to patients, and address their questions and concerns, we propose that "Pathology Explanation Clinics" be created. The Pathology Explanation Clinics would provide a channel for direct communications between pathologists and patients. Pathologists would receive special training as "Certified Pathologist Navigators" in preparation for this role. The goal of Pathology Explanation Clinics would be to help fill gaps in communication of information contained in laboratory reports to patients, further explain its relevance, and improve patient understanding of the meaning of such information and its impact on their health and health-care choices. Effort would be made to ensure that Certified Pathologist Navigators work within the overall coordination of care by the health-care team.

Spray dried cubosomes with ovalbumin and Quil-A as a nanoparticulate dry powder vaccine formulation.

Subunit vaccine formulations are often produced as liquid dispersions through complicated processes. It is desirable, however, to have simple, cheap and up-scalable methods to produce nanoparticulate subunit vaccines in powder form. Here, a simple single-step spray drying process for production of powder cubosome precursors with the model antigen ovalbumin (OVA) and the adjuvant Quil-A is presented. The cubosomes were characterized in vitro and evaluated in vivo by subcutaneous and oral administration for their potential as a vaccine formulation. Hydrated cubosomes had average particle size of 257 ±â€¯8 nm and zeta potential of -18.0 ±â€¯0.6 mV. The powder contained 10.6 ±â€¯0.7% w/w OVA prior to hydration, of which 65 ±â€¯1% was released within the first 20 min in 9.5 mM PBS at pH 7.3, with the remaining OVA gradually released over the following 24 h. Immunization with cubosomes resulted in significantly stronger antigen-specific serum IgG responses (p < 0.01), CD8+ T cell expansion (p < 0.0001) and target T cell killing compared to controls when given s.c., and was ineffective orally. This study shows that spray drying is a suitable method for producing nanoparticulate vaccine formulations in dry powder form.

Bioactivity and prostate tissue distribution of metformin in a preprostatectomy prostate cancer cohort.

Metformin has recently been shown to have potential to reduce prostate cancer risk. We conducted a randomized, double-blind, placebo-controlled trial to determine the modulating effects of metformin on tissue and systemic biomarkers of drug activity and its distribution into the prostate tissue. Twenty patients with prostate cancer scheduled to undergo prostatectomy were randomly assigned to receive either extended-release metformin or placebo for a median of 34 days before surgery. Prostatectomy and serum samples were analyzed for metformin concentrations, serum biomarkers of drug activity (prostate-specific antigen, insulin, insulin-like growth factor-1, insulin-like growth factor binding protein 3, sex hormone-binding globulin, and testosterone) and tissue biomarkers of proliferation, apoptosis, cell cycle regulation, and mTOR inhibition. For participants in the metformin arm, the prostate tissue and serum metformin concentrations ranged from 0.88 to 51.2 µg/g tissue and from not detectable to 3.6 µg/ml, respectively. There were no differences between the two groups in either the postintervention tissue biomarker expression in the prostatectomy tissue or pre to postintervention changes in serum biomarkers. We conclude that metformin distributes to human prostate tissue, suggesting that metformin could exert its effects directly on tissue targets. However, there was no difference in tissue and systemic drug effect biomarkers between the two treatment arms. Future studies with longer intervention duration and larger sample size should be considered in order to evaluate the potential of metformin for prostate cancer prevention.

Preliminary evaluation of a thermosensitive chitosan hydrogel for Echinococcus granulosus vaccine delivery.

The EG95 vaccine is effective in protecting grazing animals from infection with Echinococcus granulosus. Six male lambs were used in the study, two were each vaccinated subcutaneously with 50µg EG95/1mg Quil-A, two animals were each vaccinated with 50µg EG95/1mg Quil-A in 1% chitosan thermolabile gel subcutaneously, and two animals served as non-vaccinated controls. Two vaccinations were given at a 7 week interval. Two vaccinations induced a significantly higher antibody titre in the chitosan group compared with the Quil-A only group. The chitosan vaccine group also had a significantly higher antibody titre compared with a positive control sera from vaccinated and challenged sheep. Incorporating the EG95/Quil-A vaccine in a thermo-responsive chitosan sol-gel stimulated, after the second injection, a high level of antibody absorbance which remained high for at least one year. This response was significantly greater than the response to vaccine without the gel.

Twin-screw extruded lipid implants containing TRP2 peptide for tumour therapy.

Much effort has been put in the development of specific anti-tumour immunotherapies over the last few years, and several studies report on the use of liposomal carriers for tumour-associated antigens. In this work, the use of lipid implants, prepared using two different extruders, was investigated for sustained delivery in tumour therapy. The implants consisted of cholesterol, soybean lecithin, Dynasan 114, trehalose, ovalbumin (OVA) or a TRP2 peptide, and Quil-A. Implants were first produced on a Haake Minilab extruder, and then a scale-down to minimal quantities of material on a small scale ZE mini extruder was performed. All formulations were characterised in terms of extrudability, implant properties and in vitro release behaviour of the model antigen ovalbumin. The type of extruder used to produce the implants had a major influence on implant properties and the release behaviour, demonstrating that extrusion parameters and lipid formulations have to be individually adapted to each extrusion device. Subsequently, lipid implants containing TRP-2 peptide were extruded on the ZE mini extruder and investigated in vitro and in vivo. The in vivo study showed that mice having received TRP2 loaded implants had delayed tumour growth for 3days compared to groups having received no TRP2.

Communicating Uncertainty in Surgical Pathology Reports: A Survey of Staff Physicians and Residents at an Academic Medical Center.

In order to document perceptions of text comments appearing in surgical pathology reports, questionnaires were distributed to 4 groups of caregivers: university staff pathologists, resident pathologists, faculty clinicians (other than pathologists), and resident clinicians at a teaching hospital. Results of this pilot study showed a wide degree of variability existed within each group of surgical pathology report users, with respect to percent confidence assigned to various phrases, commonly used to express diagnostic uncertainty, appearing often as free-text comments in surgical pathology reports. The unavailability of immunohistochemistry tests, or ambiguous immunohistochemistry test results, was especially problematic. With respect to modes of communication between the surgical pathology laboratory and its service users, clinicians indicated they preferred to use tumor boards/interdisciplinary conferences, face-to-face meetings, and phone calls to clarify their interpretations of a pathologist's diagnoses, as compared with simply reading free-text comments. On the other hand, surgical pathologists rely heavily on their use of the comment portion of a surgical pathology report to clarify, modify, or expand on the diagnoses they render. The majority of clinicians stated that they "always" read the free-text comment portion of a surgical pathology report, whereas some acknowledged they do not always read it. Pathology residents had significantly less confidence in the ability of a free-text comment on a surgical pathology report to clarify a diagnosis (χ2 = 46.36, P < .0001). Pathology departments should consider standardizing definitions and weighting the words and phrases they use in their free-text comment sections of surgical pathology reports.

Segmental arterial mediolysis and fibromuscular dysplasia: what comes first, the chicken or the egg?

Segmental arterial mediolysis (SAM) is a rare vasculopathy characterized by lysis of the outer media in splanchnic arteries and formation of dissecting pseudoaneurysms that may spontaneously rupture, leading to massive and often fatal intraabdominal hemorrhage. The pathogenesis of SAM is poorly understood. Healed SAM lesions closely resemble fibromuscular dysplasia (FMD), leading some authors to postulate that SAM represents a precursor to FMD despite distinct clinical differences between these two disorders. Herein, we present a 61-year-old woman with fatal SAM who showed histologic features in her aorta suggesting the opposite pathogenetic relationship, with an unclassified "FMD-like" arteriopathy preceding development of SAM.

Clinical Study of Ursodeoxycholic Acid in Barrett's Esophagus Patients.

Prior research strongly implicates gastric acid and bile acids, two major components of the gastroesophageal refluxate, in the development of Barrett's esophagus and its pathogenesis. Ursodeoxycholic acid (UDCA), a hydrophilic bile acid, has been shown to protect esophageal cells against oxidative stress induced by cytotoxic bile acids. We conducted a pilot clinical study to evaluate the clinical activity of UDCA in patients with Barrett's esophagus. Twenty-nine patients with Barrett's esophagus received UDCA treatment at a daily dose of 13 to 15 mg/kg/day for 6 months. The clinical activity of UDCA was assessed by evaluating changes in gastric bile acid composition and markers of oxidative DNA damage (8-hydroxydeoxyguanosine), cell proliferation (Ki67), and apoptosis (cleaved caspase-3) in Barrett's esophagus epithelium. The bile acid concentrations in gastric fluid were measured by liquid chromatography/mass spectrometry. At baseline, UDCA (sum of unchanged and glycine/taurine conjugates) accounted for 18.2% of total gastric bile acids. After UDCA intervention, UDCA increased significantly to account for 93.4% of total gastric bile acids (P < 0.0001). The expression of markers of oxidative DNA damage, cell proliferation, and apoptosis was assessed in the Barrett's esophagus biopsies by IHC. The selected tissue biomarkers were unchanged after 6 months of UDCA intervention. We conclude that high-dose UDCA supplementation for 6 months resulted in favorable changes in gastric bile acid composition but did not modulate selected markers of oxidative DNA damage, cell proliferation, and apoptosis in the Barrett's esophagus epithelium. Cancer Prev Res; 9(7); 528-33. ©2016 AACRSee related article by Brian J. Reid, p. 512.

Assessment of stroke risk in southern Arizona, the pairing of acculturation and stroke risk factor development.

Stroke is a leading cause of mortality in the United States. Hispanics have the same incidence of stroke, but are more likely to have subsequent strokes than non-Hispanic whites. This difference in outcome may be attributable to differences in stroke risk factor awareness. Patients at a community health center in Tucson, AZ completed an anonymous survey regarding existing and perceived health issues. Patient responses were compared in terms of ethnicity and acculturation, as indicated by language preference. Patient responses (n = 301, Spanish: 150, English: 151) indicated that proportionately fewer non-acculturated Hispanics than acculturated Hispanic and non-Hispanic patients indicated that they were at risk for stroke. Acculturated Hispanics and non-Hispanics displayed similar morbidity trends, including increased obesity, hypertension, diabetes, heart problems, depression, and previous stroke. These findings suggest that Hispanics become less healthy and more at risk for stroke and stroke risk factors as they become acculturated.