Rab35 is required for embryonic development and kidney and ureter homeostasis through regulation of epithelial cell junctions.

Background: Rab35 is a member of a GTPase family of endocytic trafficking proteins. Studies in cell lines have indicated that Rab35 participates in cell adhesion, polarity, cytokinesis, and primary cilia length and composition. Additionally, sea urchin Rab35 regulates actin organization and is required for gastrulation. In mice, loss of Rab35 in the CNS disrupts hippocampal development and neuronal organization. Outside of the CNS, the functions of mammalian Rab35 in vivo are unknown. Methods: We generated and analyzed the consequences of both congenital and conditional null Rab35 mutations in mice. Using a LacZ reporter allele, we assessed Rab35 expression during development and postnatally. We assessed Rab35 loss in the kidney and ureter using histology, immunofluorescence microscopy, and western blotting. Results: Congenital Rab35 loss of function caused embryonic lethality: homozygous mutants arrested at E7.5 with cardiac edema. Conditional loss of Rab35, either during gestation or postnatally, caused hydronephrosis. The kidney and ureter phenotype were associated with disrupted actin cytoskeletal architecture, altered Arf6 epithelial polarity, reduced adherens junctions, loss of tight junction formation, defects in EGFR expression and localization, disrupted cell differentiation, and shortened primary cilia. Conclusion: Rab35 is essential for mammalian development and the maintenance of kidney and ureter architecture. Loss of Rab35 leads to non-obstructive hydronephrosis, making the Rab35 mutant mouse a novel mammalian model to study mechanisms underlying this disease.

Immortalized Bovine Satellite Cells for Cultured Meat Applications.

For cultured meat to succeed at scale, muscle cells from food-relevant species must be expanded in vitro in a rapid and reliable manner to produce millions of metric tons of biomass annually. Toward this goal, genetically immortalized cells offer substantial benefits over primary cells, including rapid growth, escape from cellular senescence, and consistent starting cell populations for production. Here, we develop genetically immortalized bovine satellite cells (iBSCs) via constitutive expression of bovine Telomerase reverse transcriptase (TERT) and Cyclin-dependent kinase 4 (CDK4). These cells achieve over 120 doublings at the time of publication and maintain their capacity for myogenic differentiation. They therefore offer a valuable tool to the field, enabling further research and development to advance cultured meat.

A Beefy-R culture medium: Replacing albumin with rapeseed protein isolates.

The development of cost-effective serum-free media is essential for the economic viability of cultured meat. A key challenge facing this goal is the high-cost of recombinant albumin which is necessary in many serum-free media formulations, including a recently developed serum-free medium for bovine satellite cell (BSC) culture termed Beefy-9. Here we alter Beefy-9 by replacing recombinant albumin with rapeseed protein isolate (RPI), a bulk-protein solution obtained from agricultural waste through alkali extraction (pH 12.5), isoelectric protein precipitation (pH 4.5), dissolution of physiologically soluble proteins (pH 7.2), and concentration of proteins through 3 kDa ultrafiltration. This new medium, termed Beefy-R, was then used to culture BSCs over four passages, during which cells grew with an average doubling time of 26.6 h, showing improved growth compared with Beefy-9. In Beefy-R, BSCs maintained cell phenotype and myogenicity. Together, these results offer an effective, low-cost, and sustainable alternative to albumin for serum-free culture of muscle stem cells, thereby addressing a key hurdle facing cultured meat production.

General practice-based undergraduate pharmacy longitudinal clerkship: a theoretically underpinned qualitative evaluation.

BACKGROUND: A Pharmacy Longitudinal Clerkship (PLC) was designed to develop student pharmacists' (SPs) competence in a general practice setting. AIM: The aim was to carry out a theoretically underpinned qualitative evaluation of stakeholder perceptions of influences of behavioural determinants on SP development for clinical practice in general practice. METHOD: General practice-based PLCs were delivered in 2019/20 and 2020/21 for two cohorts of SPs in NHS Highland, Scotland. Qualitative semi-structured interviews were used to explore stakeholder perceptions of influences of behavioural determinants on SP development. Informed written consent was obtained. An interview schedule was developed and piloted using the Theoretical Domains Framework (TDF). Interviews were recorded, transcribed verbatim and analysed using thematic methodology. Ethics approval was granted. RESULTS: Seven SPs and five general practitioner (GP) tutors were interviewed. Key themes were identified mapped to TDF domains and included: knowledge-utilisation and practical application of knowledge; skills-triangulation of skills under clinical supervision; beliefs about capabilities-confidence building with clinical and patient contact; professional role and identity-elucidation of professional roles within general practice. CONCLUSION: This evaluation shows benefits of embedding SPs within clinical teams and immersing them in a clinical environment over a prolonged period in a general practice Pharmacy Longitudinal Clerkship. It is expected this will translate into a more confident transition to postgraduate professional clinical practice. Funding should be sought to test alternative PLC arrangements including: multiple full-time longitudinal placement blocks; or ultimately a year-long longitudinal clerkship programme with an IPE element.

Simple and effective serum-free medium for sustained expansion of bovine satellite cells for cell cultured meat.

Cell-cultured meat offers the potential for a more sustainable, ethical, resilient, and healthy food system. However, research and development has been hindered by the lack of serum-free media that enable the robust expansion of relevant cells (e.g., muscle satellite cells) over multiple passages. Recently, a low-cost serum-free media (B8) was described for pluripotent stem cells. Here, B8 is adapted for bovine satellite cells through the addition of a single component, recombinant albumin, which renders it suitable for long-term satellite cell expansion without sacrificing myogenicity. This new media (Beefy-9) maintains cell growth over the entire period tested (seven passages), with an average doubling time of 39 h. Along with demonstrated efficacy for bovine cells, Beefy-9 offers a promising starting-point for developing serum-free media for other meat-relevant species. Ultimately, this work offers a foundation for escaping cultured meat research's reliance on serum, thereby accelerating the field.

Reducing anticholinergic medication exposure among older adults using consumer technology: Protocol for a randomized clinical trial.

INTRODUCTION: A growing body of scientific evidence points to the potentially harmful cognitive effects of anticholinergic medications among older adults. Most interventions designed to promote deprescribing of anticholinergics have directly targeted healthcare professionals and have had mixed results. Consumer-facing technologies may provide a unique benefit by empowering patients and can complement existing healthcare professional-centric efforts. METHODS: We initiated a randomized clinical trial to evaluate the effectiveness of a patient-facing mobile application (Brain Safe app) compared to an attention control medication list app in reducing anticholinergic exposure among community-dwelling older adults. Study participants are adults aged 60 years and above, currently using at least one prescribed strong anticholinergic, and receiving primary care. The trial plans to enroll a total of 700 participants, randomly allocated in 1:1 proportion to the two study arms. Participants will have the Brain Safe app (intervention arm) or attention control medication list app (control arm) loaded onto a smartphone (study provided or personal device). All participants will be followed for 12 months and will have data collected at baseline, at 6 months, and 12 months by blinded outcome assessors. The primary outcome of the study is anticholinergic exposure measured as total standard daily dose (TSDD) computed from medication prescription electronic records. Secondary outcomes of the study are cognitive function and health-related quality of life. DISCUSSION: A consumer-facing intervention to promote deprescribing of potentially high-risk medications can be part of a multi-pronged approach to reduce inappropriate medication use among older adult patients. Delivering a deprescribing intervention via a mobile app is a novel approach and may hold great promise to accelerate deployment of medication safety initiatives across diverse patient populations. CLINICAL TRIAL REGISTRATION: Registered at ClinicalTrials.gov on October 10, 2019. Identifier number: NCT04121858.

Going Remote-Demonstration and Evaluation of Remote Technology Delivery and Usability Assessment With Older Adults: Survey Study.

BACKGROUND: The COVID-19 pandemic necessitated "going remote" with the delivery, support, and assessment of a study intervention targeting older adults enrolled in a clinical trial. While remotely delivering and assessing technology is not new, there are few methods available in the literature that are proven to be effective with diverse populations, and none for older adults specifically. Older adults comprise a diverse population, including in terms of their experience with and access to technology, making this a challenging endeavor. OBJECTIVE: Our objective was to remotely deliver and conduct usability testing for a mobile health (mHealth) technology intervention for older adult participants enrolled in a clinical trial of the technology. This paper describes the methodology used, its successes, and its limitations. METHODS: We developed a conceptual model for remote operations, called the Framework for Agile and Remote Operations (FAR Ops), that combined the general requirements for spaceflight operations with Agile project management processes to quickly respond to this challenge. Using this framework, we iteratively created care packages that differed in their contents based on participant needs and were sent to study participants to deliver the study intervention-a medication management app-and assess its usability. Usability data were collected using the System Usability Scale (SUS) and a novel usability questionnaire developed to collect more in-depth data. RESULTS: In the first 6 months of the project, we successfully delivered 21 care packages. We successfully designed and deployed a minimum viable product in less than 6 weeks, generally maintained a 2-week sprint cycle, and achieved a 40% to 50% return rate for both usability assessment instruments. We hypothesize that lack of engagement due to the pandemic and our use of asynchronous communication channels contributed to the return rate of usability assessments being lower than desired. We also provide general recommendations for performing remote usability testing with diverse populations based on the results of our work, including implementing screen sharing capabilities when possible, and determining participant preference for phone or email communications. CONCLUSIONS: The FAR Ops model allowed our team to adopt remote operations for our mHealth trial in response to interruptions from the COVID-19 pandemic. This approach can be useful for other research or practice-based projects under similar circumstances or to improve efficiency, cost, effectiveness, and participant diversity in general. In addition to offering a replicable approach, this paper tells the often-untold story of practical challenges faced by mHealth projects and practical strategies used to address them. TRIAL REGISTRATION: ClinicalTrials.gov NCT04121858; https://clinicaltrials.gov/ct2/show/NCT04121858.

A mouse model of BBS identifies developmental and homeostatic effects of BBS5 mutation and identifies novel pituitary abnormalities.

Primary cilia are critical sensory and signaling compartments present on most mammalian cell types. These specialized structures require a unique signaling protein composition relative to the rest of the cell to carry out their functions. Defects in ciliary structure and signaling result in a broad group of disorders collectively known as ciliopathies. One ciliopathy, Bardet-Biedl syndrome (BBS; OMIM 209900), presents with diverse clinical features, many of which are attributed to defects in ciliary signaling during both embryonic development and postnatal life. For example, patients exhibit obesity, polydactyly, hypogonadism, developmental delay and skeletal abnormalities along with sensory and cognitive deficits, but for many of these phenotypes it is uncertain, which are developmental in origin. A subset of BBS proteins assembles into the core BBSome complex, which is responsible for mediating transport of membrane proteins into and out of the cilium, establishing it as a sensory and signaling hub. Here, we describe two new mouse models for BBS resulting from a targeted LacZ gene trap allele (Bbs5-/-) that is a predicted congenital null mutation and conditional (Bbs5flox/flox) allele of Bbs5. Bbs5-/- mice develop a complex phenotype consisting of increased pre-weaning lethality craniofacial and skeletal defects, ventriculomegaly, infertility and pituitary anomalies. Utilizing the conditional allele, we show that the male fertility defects, ventriculomegaly and pituitary abnormalities are only present when Bbs5 is disrupted prior to postnatal day 7, indicating a developmental origin. In contrast, mutation of Bbs5 results in obesity, independent of the age of Bbs5 loss.

Engineering carotenoid production in mammalian cells for nutritionally enhanced cell-cultured foods.

Metabolic engineering of mammalian cells has to-date focused primarily on biopharmaceutical protein production or the manipulation of native metabolic processes towards therapeutic aims. However, significant potential exists for expanding these techniques to diverse applications by looking across the taxonomic tree to bioactive metabolites not synthesized in animals. Namely, cross-taxa metabolic engineering of mammalian cells could offer value in applications ranging fromfood and nutrition to regenerative medicine and gene therapy. Towards the former, recent advances in meat production through cell culture suggest the potential to produce meat with fine cellular control, where tuning composition through cross-taxa metabolic engineering could enhance nutrition and food-functionality. Here we demonstrate this possibility by engineering primary bovine and immortalized murine muscle cells with prokaryotic enzymes to endogenously produce the antioxidant carotenoids phytoene, lycopene and ß-carotene. These phytonutrients offer general nutritive value and protective effects against diseases associated with red and processed meat consumption, and so offer a promising proof-of-concept for nutritional engineering in cultured meat. We demonstrate the phenotypic integrity of engineered cells, the ability to tune carotenoid yields, and the antioxidant functionality of these compounds in vitro towards both nutrition and food-quality objectives. Our results demonstrate the potential for tailoring the nutritional profile of cultured meats. They further lay a foundation for heterologous metabolic engineering of mammalian cells for applications outside of the clinical realm.

Is SBRT Boost Feasible for PET Positive Lymph Nodes for Cervical Cancer? Evaluation using Tumor Control Probability and QUANTEC Criteria.

PURPOSE: This study aimed to examine the feasibility of stereotactic body radiation therapy (SBRT) as an external beam radiation therapy boost to positron emission tomography (PET) positive lymph nodes (LN) in patients with cervical cancer and to evaluate overall tumor control probability (TCP) increase. METHODS AND MATERIALS: Ten patients with cervical cancer and PET positive LN metastasis who received external beam radiation therapy (45 Gy), followed by a 3-dimensional conformal radiation therapy boost (5.4-9 Gy) and tandem-and-ovoid high-dose-rate brachytherapy (16-30 Gy) were retrospectively enrolled in this study. SBRT plans were generated using 21 Gy, 24 Gy, or 30 Gy as a replacement for 3-dimensional conformal radiation therapy boost. The 2 Gy-per-fraction equivalent dose maps were made using an α/ß value of 10 for PET positive LNs and 3 for organs at risk (OARs). TCP values were calculated using a logistic TCP model, where 2 input parameters (D50 and Gamma50 = 2) were modeled by 2 clinical outcomes: our institution and the literature. OAR sparing was evaluated using Quantitative Analyses of Normal Tissue Effects in the Clinic (QUANTEC) dose limits. RESULTS: Thirty percent of 10 patients receiving conventional boost experienced recurrence. The TCP of the SBRT schemes was 88% ± 7% (97% ± 2%; 21 Gy), 96% ± 1% (99% ± 0%; 24 Gy), and 99% ± 1% (100% ± 0%; 30 Gy), and the conventional LN-boost TCP value was 25% ± 11% (58% ± 15%) when TCP input parameters were based on published clinical outcome data for LN SBRT treatments (institutional outcome data). The tumor coverage doses (D90) of the SBRT boost plans were on average 32.34 Gyαß=10 (21 Gy), 37.78 Gyαß=10 (24 Gy), and 55.54Gyαß=10 (30 Gy) higher than the conventional LN boost plan. The QUANTEC OAR dose constraints were met for the bladder, rectum, and bowel in all cases for the SBRT LN 21 Gy group, and in 90% and 70% of cases in the SBRT LN 24 Gy and SBRT LN 30 Gy groups, respectively. CONCLUSIONS: An SBRT boost dose of 30 Gy can be delivered without compromising QUANTEC OAR limits. The use of SBRT increases TCP values, regardless of the input parameters.

Spider prey-wrapping silk is an α-helical coiled-coil/ß-sheet hybrid nanofiber.

Solid-State NMR results on 13C-Ala/Ser and 13C-Val enriched Argiope argentata prey-wrapping silk show that native, freshly spun aciniform silk nanofibers are dominated by α-helical (∼50% total) and random-coil (∼35% total) secondary structures, with minor ß-sheet nanocrystalline domains (∼15% total). This is the most in-depth study to date characterizing the protein structural conformation of the toughest natural biopolymer: aciniform prey-wrapping silks.

Cost analysis of osteoradionecrosis.

OBJECTIVE: Osteoradionecrosis is a significant complication of head and neck cancer treatment, and its most severe form (grade III) necessitates radical surgery. This study aimed to compare the cost of free-flap reconstructive surgery for grade III osteoradionecrosis and similar non-osteoradionecrosis cases in order to assess the cost burden of osteoradionecrosis treatment. METHODS: All patients who underwent free-flap reconstructive surgery for osteoradionecrosis between July 2004 and July 2010 at Auckland City Hospital (19 patients) were identified, and relevant data were collected retrospectively. These patients were matched in terms of age and sex with patients who underwent free-flap reconstructive surgery. RESULTS: The treatment cost was 44 per cent higher in osteoradionecrosis patients when compared to non-osteoradionecrosis patients. CONCLUSION: The significant financial burden on the health system, and the growing evidence for the effectiveness of pentoxifylline, tocopherol and clodronate, should prompt us to explore this alternative treatment further.

Risk prediction scores for postoperative mortality after esophagectomy: validation of different models.

BACKGROUND: Different prediction models for operative mortality after esophagectomy have been developed. The aim of this study is to independently validate prediction models from Philadelphia, Rotterdam, Munich, and the ASA. METHODS: The scores were validated using logistic regression models in two cohorts of patients undergoing esophagectomy for cancer from Switzerland (n = 170) and Australia (n = 176). RESULTS: All scores except ASA were significantly higher in the Australian cohort. There was no significant difference in 30-day mortality or in-hospital death between groups. The Philadelphia and Rotterdam scores had a significant predictive value for 30-day mortality (p = 0.001) and in-hospital death (p = 0.003) in the pooled cohort, but only the Philadelphia score had a significant prediction value for 30-day mortality in both cohorts. Neither score showed any predictive value for in-hospital death in Australians but were highly significant in the Swiss cohort. ASA showed only a significant predictive value for 30-day mortality in the Swiss. For in-hospital death, ASA was a significant predictor in the pooled and Swiss cohorts. The Munich score did not have any significant predictive value whatsoever. CONCLUSION: None of the scores can be applied generally. A better overall predictive score or specific prediction scores for each country should be developed.

Reactive oxygen species regulate caspase activation in tumor necrosis factor-related apoptosis-inducing ligand-resistant human colon carcinoma cell lines.

The effects of reactive oxygen species (ROS) on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in solid cancers have yet to be clearly defined. In this study, we found that the classic uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), induced a reduction in DeltaPsim and generation of ROS. This uncoupling effect enhanced TRAIL-induced apoptosis in TRAIL-resistant human colon carcinoma cell lines (RKO, HT29, and HCT8). Sensitization was inhibited by benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone, indicating the requirement for caspase activation. CCCP per se did not induce apoptosis or release of proapoptotic factors from mitochondria. Generation of ROS by CCCP was responsible for TRAIL-induced Bax and caspase activation because scavenging ROS completely abrogated apical caspase-8 activation and further downstream events leading to cell death. Overexpression of Bcl-2 did not prevent the initial loss of DeltaPsim and ROS generation following CCCP treatment, but did prevent cell death following TRAIL and CCCP exposure. Uncoupling of mitochondria also facilitated TRAIL-induced release of proapoptotic factors. X-linked inhibitor of apoptosis overexpression abrogated TRAIL-induced apoptosis in the presence of CCCP and decreased initiator procaspase-8 processing, indicating that additional processing of caspase-8 required initiation of a mitochondrial amplification loop via effector caspases. Of interest, depletion of caspase-9 in RKO cells did not protect cells from TRAIL/CCCP-induced apoptosis, indicating that apoptosis occurred via a caspase-9-independent pathway. Data suggest that in the presence of mitochondrial-derived ROS, TRAIL induced mitochondrial release of Smac/DIABLO and inactivation of X-linked inhibitor of apoptosis through caspase-9-independent activation of caspase 3.

Casein kinase I attenuates tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by regulating the recruitment of fas-associated death domain and procaspase-8 to the death-inducing signaling complex.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a wide variety of malignant cell lines, in contrast to normal cells, but with considerable heterogeneity in response. Death receptor-mediated apoptosis may be attenuated by a variety of different mechanisms, including phosphorylation-based signaling pathways. We have demonstrated that casein kinase I can attenuate TRAIL-induced apoptosis in human cell lines derived from colon adenocarcinoma (HT29 and HCT8) and pediatric rhabdomyosarcoma (JR1). Inhibition of casein kinase I (CKI) phosphorylation events in HT29, HCT8, and JR1 cells by CKI-7 dramatically increased apoptosis after exposure to TRAIL, in the absence of apoptosis induced by TRAIL treatment alone. CKI inhibition enhanced the recruitment of Fas-associated death domain and procaspase-8 to the death-inducing signaling complex after TRAIL treatment and enhanced cleavage of procaspase-8 at the death-inducing signaling complex. In HT29 cells studied further, rapid cleavage of caspase-8, caspase-3, Bid, and the caspase substrate poly(ADP-ribose) polymerase occurred when CKI-7 and TRAIL were combined. Overexpression of Bcl-2, Bcl-xL, or mutant DN-Fas-associated death domain protected HT29 cells from TRAIL-induced apoptosis in the presence of the CKI inhibitor. In addition, TRAIL combined with CKI-7 promoted the release of cytochrome c, Smac/DIABLO, HtrA2/Omi, and AIF from the mitochondria and down-regulated the expression of XIAP and c-IAP1. Small hairpin RNAs directed against CKI revealed that the CKIalpha isoform contributed significantly to the inhibition of TRAIL-induced apoptosis. These findings suggest that CKIalpha plays an antiapoptotic role through the generation of phosphorylated sites at the level of the death-inducing signaling complex, thereby conferring resistance to caspase cleavage mediated by TRAIL.

Natural history of Hymenoptera venom sensitivity in adults.

BACKGROUND: Epidemiologic studies of Hymenoptera venom allergy in adults show a prevalence of positive venom skin test results, RASTs of 15% to 25%, or both, but most such individuals have had no systemic reactions to stings. The clinical significance and natural history of this apparently common sensitivity is uncertain. OBJECTIVE: We sought to determine the natural history of venom sensitization by observing the rate of decrease or increase in sensitivity in normal adults over 5 to 10 years. The clinical significance of these findings is related to the frequency of systemic reactions to stings during the period of observation. METHODS: Serial observations were planned in 520 volunteers and randomly selected subjects. Two follow-up visits were attempted, once after 2 to 3 years and again after 5 to 9 years, to perform repeat venom skin tests and RASTs and to review any history of interim stings and their outcomes. RESULTS: Follow-up visits were conducted with 398 subjects (375 early visits and 205 late visits). Overall, in the 398 subjects with one or more visits after a mean of 4 years, skin test responses changed from positive to negative in 44 of 98 (45%) and from negative to positive in 27 of 309 (8.7%) of the subjects. Skin test responses changed from positive to negative in 29 of 87 (33%) subjects after 2.5 years and in 43 of 54 (80%) after 6.8 years. Even when the skin test response became negative, venom-specific IgE remained positive in 11 of 29 (38%) subjects after 2.5 years and in 13 of 43 (30%) after 6.8 years. The rate of loss of sensitivity was 12% per year, similar to retrospective estimates. Skin test sensitivity to venoms disappears more rapidly in these subjects without symptoms (half-life, 4 years) than in patients receiving venom immunotherapy (half-life, 7 years). Skin test responses changed from negative to positive in 23 of 288 (8%) subjects after 2.5 years and in 9 of 151 (6%) after 6.8 years. Insect stings caused no reaction in 120 subjects with a negative skin test response, but 17% (11 of 65) of subjects with a positive skin test response (but with a negative history) had systemic reactions when stung. There was no difference between the early and late visits in the frequency of systemic reactions reported. The risk may be higher than 17% for the specific individuals (67% after 2.5 years and 20% after 6.8 years) whose positive skin test responses persist for years. This risk is lower than that of patients with a positive history (50%) but higher than that of "normal" adults or venom-treated patients (<2%). It is still not clear whether any subset of adults with a positive skin test response but a negative history can be identified, in whom the risk of systemic sting reaction would justify venom immunotherapy even before any reaction occurs. CONCLUSION: Asymptomatic venom sensitization in adults is common but transient, disappearing at the rate of 12% per year. However, the risk of a systemic reaction to a subsequent sting is significant in adults without symptoms but with positive venom skin test responses (17%) and may be higher when skin test sensitivity does persist for years.

Prospective observations on stopping prolonged venom immunotherapy.

After a decade spent establishing the safety, efficacy, and optimal techniques for venom immunotherapy, we have begun a series of studies to determine how long venom immunotherapy must be continued. In retrospective surveys, patients who had stopped venom immunotherapy after 1 to 2 years had a substantial risk (25%) of systemic sting reactions, but this was less than 50% of the risk in untreated patients. In this first prospective study, 30 patients elected to stop venom immunotherapy after at least 5 years of therapy. Skin test sensitivity had decreased significantly during therapy in 18/30 patients but remained clearly positive in 23/30 (seven patients became equivocal or negative). Serum venom-specific IgE antibodies were at the lower limit of detection (1 ng/ml) in 11/30 patients. After stopping treatment, the mean serum venom-specific IgG antibody level declined from 5.5 +/- 0.6 micrograms/ml to 2.4 +/- 0.3 micrograms/ml by 9 months, which is the same as the mean venom IgG in untreated patients. After 12 months without therapy, live sting challenge caused no systemic reaction in 29 patients. The mean venom IgG level 1 month after the sting had risen significantly to 4.1 +/- 0.5 micrograms/ml, but there was no significant increase of venom IgE. These results suggest that prolonged venom immunotherapy leads to isotype-specific suppression of the venom IgE antibody response and may provide persistent clinical protection by mechanisms other than IgG blocking antibodies. The observations are to be interpreted very cautiously. Further investigations are needed to extend these observations in additional patients and for longer periods of time, and to examine possible mechanisms for this apparent loss of clinical reactivity.

Clinical and immunologic observations in patients who stop venom immunotherapy.

It is currently recommended that venom immunotherapy (VIT) be continued as long as the sensitivity persists (indicated by positive venom skin tests or RAST). In this pilot study, we performed a retrospective survey of the clinical and immunologic effects of stopping VIT. The 82 patients studied had received maintenance VIT for a mean of 14 months and had stopped VIT a mean of 43 months before evaluation. Subsequent "field" stings in 28 patients caused systemic reactions in six cases (22%), which is significantly higher than the 1% to 3% systemic reaction rate in patients who remain on maintenance VIT. The 22% reaction rate is a minimal estimate caused by loss of venom sensitivity in some patients and residual venom-specific IgG antibody levels in others. Reevaluation of venom skin tests and IgG levels was possible in 43 patients. A tenfold decline from before VIT skin test results was observed in 27 patients (63%). Skin tests remained clearly positive in 32/43 (74%), became weakly positive in 9/43 (21%), and 2/43 (5%) became negative. The IgG level declined from typical maintenance levels before stopping VIT (mean 7.2 +/- 1.2 micrograms/ml) to levels typical of untreated patients at the time of retesting (mean 1.95 +/- 0.3 micrograms/ml). Despite the marked fall of IgG antibody, one third of the patients still had levels in the average range observed in patients receiving maintenance VIT. We conclude that there is a substantial risk of anaphylactic sting reaction if VIT is stopped while venom sensitivity persists.

Diethylstilbestrol on urinary catecholamines and on food intake in castrated male rats.

Moderate changes in food intake produced by diethylstilbestrol in rats were not well correlated with changes in urinary norepinephrine, vanillylmandelic acid or epinephrine. Apparently moderate dietary restrictions are not capable of decreasing adrenergic activity, and the decreased urinary norepinephrine produced by diethylstilbestrol is not associated with decreased availability of dietary precursors.