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Chamcheu JC, Siddiqui IA, Adhami VM, et al. Int J Nanomedicine. 2018;13:4189–4206. An error during the preparation of Figures 1 and 5 for publishing led to the inadvertent creation of duplicate regions in images from these figures on pages 4194 and 4199, respectively. The journal wishes to apologise for this error. The correct versions of Figures 1 and 5 are as follows: Figure 1 Size characterization and encapsulation and loading efficiencies of chitosan-based nanoEGCG. (A) Size measurement and distribution of nanoEGCGusing dynamic light scattering. (B) Zeta potential measurement of nanoEGCG. (C) Representative transmission electron microscopy photomicrographs showing the relative homogeneous size and morphology of (i) diluted nanoEGCGand (ii) undiluted nanoEGCG, and (iii) CHI-Void-NPs. Scale bar=200 nm; the insets represent higher magnification. (D) Encapsulation and loading efficiency of EGCG on to chitosan nanoparticles as monitored with UV-vis spectra for free EGCG (not encapsulated) and total EGCG (encapsulated + free). (E) UV-vis spectra used to construct the standard curve, with EGCGconcentrations of 25, 12.5, 6.25, 3.12, and 1.6 μg/mL.Abbreviations: EGCG, (–)-epigallocatechin-3-gallate; nanoEGCG, CHI-EGCG-NPs, chitosan-based polymeric nanoparticle formulation of EGCG; CHI-Void-NPs, chitosan-based void (without EGCG) nanoparticles; UV-vis, ultraviolet–visible. Figure 5 Effect of topically applied free EGCG and nanoEGCG on infiltrating immune cells and expression of differentiation markers in IMQ-treated mouse skin lesions: Mice were treated in 4 groups as described in the legends to Figure 4 and Figure S5. (A–D, F–I, K–N, P–S) Photomicrographs showing immunohistological features of: (A–D) mast cells (toluidine blue staining); (F–I) epidermis/dermis (NE, brown staining), and microabscesses (arrow); (K–N) macrophages (F4/80, red staining); and (P–S) double immunofluorescence staining for loricrin (green) and T-lymphocytes (CD4+, red staining). Nuclei were counterstained blue with DAPI. Magnification for all panels ×200. (E, J, O, T, U) Quantitative analyses of changes in immune cells: (E) mast cells; (J) NE+ cells; (O) F4/80+ cells; and (U) loricrin in the 4 treatment groups. Each data point represents the mean±SD of 4 random fields/mouse from 5 mice/group. *p, 0.05, **p, 0.01, ***p, 0.001, and ****p, 0.0001 for the indicated 2-way comparisons.Abbreviations: EGCG, (–)-epigallocatechin-3-gallate; nanoEGCG, chitosan-based polymeric nanoparticle formulation of EGCG; IMQ, imiquimod; NE, neutrophil elastase; LPV, low-power view.

Jean Christopher Chamcheu,1,2,* Imtiaz A Siddiqui,1,* Vaqar M Adhami,1,* Stephane Esnault,3 Dhruba J Bharali,4 Abiola S Babatunde,1,5 Stephanie Adame,1 Randall J Massey,6 Gary S Wood,1 B Jack Longley,1 Shaker A Mousa,4 Hasan Mukhtar11Department of Dermatology, School of Medicine and Public Health, University of Wisconsin, and the Middleton VA Medical Center, Madison, WI, USA; 2Department of Basic Pharmaceutical Sciences, School of Pharmacy, College of Health and Pharmaceutic Sciences, University of Louisiana at Monroe, Monroe, LA, USA; 3Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, The University of Wisconsin–Madison School of Medicine and Public Health, Madison, WI, USA; 4The Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Albany, NY, USA; 5Department of Hematology, University of Ilorin, Ilorin, Nigeria; 6Electron Microscope Facility, Medical School Research Support Progs, School of Medicine and Public Health, University of Wisconsin, and the Middleton VAMedical Center, Madison, WI, USA *These authors contributed equally to this work Background: Psoriasis is a chronic and currently incurable inflammatory skin disease characterized by hyperproliferation, aberrant differentiation, and inflammation, leading to disrupted skin barrier function. The use of natural agents that can abrogate these effects could be useful for the treatment of psoriasis. Earlier studies have shown that treatment of keratinocytes and mouse skin with the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) mitigated inflammation and increased the expression of caspase-14 while promoting epidermal differentiation and cornification. However, bioavailability issues have restricted the development of EGCG for the treatment of psoriasis.Materials and methods: To overcome these limitations, we employed a chitosan-based polymeric nanoparticle formulation of EGCG (CHI-EGCG-NPs, hereafter termed nanoEGCG) suitable for topical delivery for treating psoriasis. We investigated and compared the efficacy of nanoEGCG versus native or free EGCG in vitro and in an in vivo imiquimod (IMQ)-induced murine psoriasis-like dermatitis model. The in vivo relevance and efficacy of nanoEGCG formulation (48 µg/mouse) were assessed in an IMQ-induced mouse psoriasis-like skin lesion model compared to free EGCG (1 mg/mouse).Results: Like free EGCG, nanoEGCG treatment induced differentiation, and decreased proliferation and inflammatory responses in cultured keratinocytes, but with a 4-fold dose advantage. Topically applied nanoEGCG elicited a significant (p20-fold dose advantage over free EGCG.Conclusion: Based on these observations, our nanoEGCG formulation represents a promising drug-delivery strategy for treating psoriasis and possibly other inflammatory skin diseases. Keywords: chitosan nanoparticles, topical delivery of chitosan nanoformulated EGCG, psoriasis-like skin inflammation, phytochemical treatment of psoriasis, normal human epidermal keratinocytes, differentiation, anti-inflammatory action

Background: Obstructive sleep apnoea (OSA) can cause left atrial (LA) and left ventricular (LV) remodelling, which is linked to atrial fibrillation (AF). Whether continuous positive airway pressure (CPAP) can reverse LA and LV remodelling in patients with OSA and paroxysmal AF (PAF) has yet to be studied. We assessed the impact of CPAP treatment on LA and LV size and function in patients with OSA and PAF before and after catheter ablation., Methods: In a randomised controlled trial, we screened patients with PAF for OSA. We enrolled patients with an Apnoea-Hypopnoea Index ≥15/hour. The burden of AF was monitored by an implantable loop recorder in all patients. Patients were then randomised to CPAP treatment or standard care. Transthoracic echocardiography was performed at baseline and after 6 and 12 months to assess LV and LA function and remodelling with advanced echocardiographic imaging techniques., Results: We enrolled 109 patients (63±7 years, body mass index 29.6±4.3, 76% men). 83 patients were scheduled for pulmonary vein isolation (PVI) and 26 for clinical follow-up only. 55 patients were randomised to CPAP and 54 to standard care. The burden of AF decreased significantly in patients who underwent PVI irrespective of treatment with CPAP (p for difference ≤0.001). Patients in the study group had LV ejection fraction (LVEF) and LV global longitudinal strain (GLS) within the normal range, increased LA Volume Index (LAVI), LA volume (by speckle tracking) and decreased LA reservoir strain at baseline. We did not observe any improvement in LVEF, GLS, LAVI, LA volume or LA reservoir strain in either group during the 12 months of follow-up., Conclusions: In patients with PAF and OSA, treatment with CPAP was not associated with reverse LA remodelling within 12 months of follow-up., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: BMI variability has been associated with increased cardiovascular disease risk in individuals with type 2 diabetes, however comparison between clinical studies and real-world observational evidence has been lacking. Furthermore, it is not known whether BMI variability has an effect independent of HbA1c variability., Methods: We investigated the association between BMI variability and 3P-MACE risk in the Harmony Outcomes trial (n = 9198), and further analysed placebo arms of REWIND (n = 4440) and EMPA-REG OUTCOME (n = 2333) trials, followed by real-world data from the Tayside Bioresource (n = 6980) using Cox regression modelling. BMI variability was determined using average successive variability (ASV), with first major adverse cardiovascular event of non-fatal stroke, non-fatal myocardial infarction, and cardiovascular death (3P-MACE) as the primary outcome., Results: After adjusting for cardiovascular risk factors, a + 1 SD increase in BMI variability was associated with increased 3P-MACE risk in Harmony Outcomes (HR 1.12, 95% CI 1.08-1.17, P < 0.001). The most variable quartile of participants experienced an 87% higher risk of 3P-MACE (P < 0.001) relative to the least variable. Similar associations were found in REWIND and Tayside Bioresource. Further analyses in the EMPA-REG OUTCOME trial did not replicate this association. BMI variability's impact on 3P-MACE risk was independent of HbA1c variability., Conclusions: In individuals with type 2 diabetes, increased BMI variability was found to be an independent risk factor for 3P-MACE across cardiovascular outcome trials and real-world datasets. Future research should attempt to establish a causal relationship between BMI variability and cardiovascular outcomes., (© 2024. The Author(s).)

Aims: The non-invasive myocardial work index (MWI) has been validated in patients without aortic stenosis (AS). A thorough assessment of methodological limitations is warranted before this index can be applied to patients with AS., Methods and Results: We simultaneously measured left ventricular pressure (LVP) by using a micromanometer-tipped catheter and obtained echocardiograms in 20 patients with severe AS. We estimated LVP curves and calculated pressure-strain loops using three different models: (i) the model validated in patients without AS; (ii) the same model, but with pressure at the aortic valve opening (AVO) adjusted to diastolic cuff pressure; and (iii) a new model based on the invasive measurements from patients with AS. Valvular events were determined by echocardiography. Peak LVP was estimated as the sum of the mean aortic transvalvular gradient and systolic cuff pressure. In same-beat comparisons between invasive and estimated LVP curves, Model 1 significantly overestimated early systolic pressure by 61 ± 5 mmHg at AVO compared with Models 2 and 3. However, the average correlation coefficients between estimated and invasive LVP traces were excellent for all models, and the overestimation had limited influence on MWI, with excellent correlation (r = 0.98, P < 0.001) and good agreement between the MWI calculated with estimated (all models) and invasive LVP., Conclusion: This study confirms the validity of the non-invasive MWI in patients with AS. The accuracy of estimated LVP curves improved when matching AVO to the diastolic pressure in the original model, mirroring that of the AS-specific model. This may sequentially enhance the accuracy of regional MWI assessment., Competing Interests: Conflict of interest: K.R. is co-inventor of the ‘Method for myocardial work analysis’, currently licenced by General Electrics. O.A.S. is also a co-inventor of the method for myocardial work analysis and has filed a patent on ‘Estimation of blood pressure in the heart’. He has received lecture fees from GE Healthcare. The remaining authors have nothing to disclose., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background: A precision medicine approach in type 2 diabetes requires the identification of clinical and biological features that are reproducibly associated with differences in clinical outcomes with specific anti-hyperglycaemic therapies. Robust evidence of such treatment effect heterogeneity could support more individualized clinical decisions on optimal type 2 diabetes therapy., Methods: We performed a pre-registered systematic review of meta-analysis studies, randomized control trials, and observational studies evaluating clinical and biological features associated with heterogenous treatment effects for SGLT2-inhibitor and GLP1-receptor agonist therapies, considering glycaemic, cardiovascular, and renal outcomes. After screening 5,686 studies, we included 101 studies of SGLT2-inhibitors and 75 studies of GLP1-receptor agonists in the final systematic review., Results: Here we show that the majority of included papers have methodological limitations precluding robust assessment of treatment effect heterogeneity. For SGLT2-inhibitors, multiple observational studies suggest lower renal function as a predictor of lesser glycaemic response, while markers of reduced insulin secretion predict lesser glycaemic response with GLP1-receptor agonists. For both therapies, multiple post-hoc analyses of randomized control trials (including trial meta-analysis) identify minimal clinically relevant treatment effect heterogeneity for cardiovascular and renal outcomes., Conclusions: Current evidence on treatment effect heterogeneity for SGLT2-inhibitor and GLP1-receptor agonist therapies is limited, likely reflecting the methodological limitations of published studies. Robust and appropriately powered studies are required to understand type 2 diabetes treatment effect heterogeneity and evaluate the potential for precision medicine to inform future clinical care., (© 2023. Springer Nature Limited.)

Purpose: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an established treatment predominantly for malignancies. Chronic graft-versus-host disease (cGVHD) is the leading long-term complication after allo-HSCT, but knowledge on cGVHD and health-related quality of life (HRQOL) in long-term survivors of allo-HSCT performed in childhood, adolescence, and young adulthood (CAYA) is scarce. Therefore, we aimed to (1) assess prevalence and risk factors of active cGVHD using the 2014 National Institutes of Health-Consensus criteria, (2) investigate associations between cGVHD severity, patient-reported symptom burden, and HRQOL, and (3) compare HRQOL of survivors to population norms. Methods: We conducted a nationwide cross-sectional study in long-term survivors of CAYA allo-HSCT combining clinical examinations and patient-reported outcome measures. Results: We included 103 survivors, 55 (53%) females, median age of 19.6 years [range 0.3-29.9] at HSCT, 16.8 years [6.0-32.0] from HSCT, and 77 (75%) with underlying malignancy. Overall, 32 (31%) survivors were diagnosed with active cGVHD. The risk of active cGVHD was increased with prior acute GVHD and reduced with in vivo T cell depletion. cGVHD severity was associated with increased symptom burden, but not with adverse HRQOL. Compared to Norwegian population norms, allo-HSCT survivors reported significantly lower HRQOL. Conclusion: These results indicate a high prevalence of cGVHD in long-term survivors of CAYA allo-HSCT. Although we did not find an association between cGVHD severity and HRQOL, survivors reported significantly poorer HRQOL compared to population norms. Knowledge on the long-term consequences of cGVHD will be important for optimizing treatment and long-term follow-up care after CAYA allo-HSCT.

The association between body weight variability and the risk of cardiovascular disease (CVD) has been investigated previously with mixed findings. However, there has been no extensive study which systematically evaluates the current evidence. Furthermore, the impact of ethnicity and type 2 diabetes on this phenomena has not yet been investigated. Therefore, the aim of this study was to comprehensively evaluate the effect of weight variability on risk of CVD (any cardiovascular (CV) event, composite CV outcome, CV death, Stroke, Myocardial Infarction) and the influence of ethnicity and type 2 diabetes status on the observed association. A systematic review and meta-analysis was performed according to the meta-analyses of observational studies in epidemiology (MOOSE) guidelines. The electronic databases PubMed, Web of Science, and the Cochrane Library were searched for studies that investigated the relationship between body weight or BMI variability and CV diseases using Medical Subject Headings (MeSH) terms and keywords. The relative risks (RRs) for the outcomes were collected from studies, pooled, and analysed using a random-effects model to estimate the overall relative risk. Of 5645 articles screened, 23 studies with a total population of 15,382,537 fulfilled the prespecified criteria and were included. Individuals in the highest strata of body weight variability were found to have significantly increased risk of any CV event (RR = 1.27; 95% Confidence Interval (CI) 1.17-1.38; P < 0.0001; I 2  = 97.28%), cardiovascular death (RR = 1.29; 95% CI 1.03-1.60; P < 0.0001; I 2  = 55.16%), myocardial infarction (RR = 1.32; 95% CI 1.09-1.59; P = 0.0037; I 2  = 97.14%), stroke (RR = 1.21; 95% CI 1.19-1.24; P < 0.0001; I 2  = 0.06%), and compound CVD outcomes (RR = 1.36; 95% CI 1.08-1.73; P = 0.01; I 2  = 92.41%). Similar RRs were observed regarding BMI variability and per unit standard deviation (SD) increase in body weight variability. Comparable effects were seen in people with and without diabetes, in White Europeans and Asians. In conclusion, body weight variability is associated with increased risk of CV diseases regardless of ethnicity or diabetes status. Future research is needed to prove a causative link between weight variability and CVD risk, as appropriate interventions to maintain stable weight could positively influence CVD., (© 2023. The Author(s).)

Purpose: Exercise intolerance is a common complication in survivors of allogeneic hematopoietic stem-cell transplantation (allo-HSCT). The aim of this study was to determine if cardiac function measured with echocardiography is associated with exercise capacity measured with cardio-pulmonary exercise tests in long-term survivors treated in their youth with allo-HSCT., Methods: The study included 96 patients, of which 54.2% were female, aged 34.9 ± 11.6 years and 17.7 ± 9.3 years after allo-HSCT. Reduced exercise capacity was defined as <85% of predicted-peak oxygen uptake (VO 2peak ). Linear regression was used in the prediction of VO 2peak (ml/kg/min). Receiver operating characteristic evaluated the accuracy of predicting reduced exercise capacity., Results: VO 2peak was 36.2 ± 7.7 ml/kg/min and 43 (44.8%) had reduced exercise capacity. Left ventricular ejection fraction was 55.4 ± 5.9% and global longitudinal strain (GLS) was -17.6% ± 2.0%. Left and right ventricular functions were significantly lower in survivors with reduced exercise capacity. Increased body mass index, lower physical activity score, reduced pulmonary function (by forced expiratory volume in 1-s) and reduced left ventricular systolic function (by GLS) were significant independent predictors for reduced VO 2peak . GLS was superior to other echocardiographical indices for identifying reduced exercise capacity (area under curve = 0.64, p = 0.014)., Conclusions: Left ventricular systolic dysfunction measured by GLS is associated with reduced exercise capacity in long-term allo-HSCT survivors., (© 2022 The Authors. Journal of Clinical Ultrasound published by Wiley Periodicals LLC.)

Objective: Dysregulation of the complement system has been described in patients with heart failure (HF). However, data on the alternative pathway are scarce and it is unknown if levels of factor B (FB) and the C3 convertase C3bBbP are elevated in these patients. We hypothesized that plasma levels of FB and C3bBbP would be associated with disease severity and survival in patients with HF., Methods: We analyzed plasma levels of FB, C3bBbP, and terminal C5b-9 complement complex (TCC) in 343 HF patients and 27 healthy controls., Results: Compared with controls, patients with HF had elevated levels of circulating FB (1.6-fold, p < 0.001) and C3bBbP (1.3-fold, p < 0.001). In contrast, TCC, reflecting the terminal pathway, was not significantly increased (p = 0.15 vs controls). FB was associated with NT-proBNP, troponin, eGFR, and i.e., C-reactive protein. FB, C3bBbP and TCC were not associated with mortality in HF during a mean follow up of 4.3 years., Conclusion: Our findings suggest that in patients with HF, the alternative pathway is activated. However, this is not accompanied by activation of the terminal pathway., Competing Interests: NS is currently an employee of Johnson and Johnson; the current work was conducted when she was employed by Oslo University Hospital. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Holt, Michelsen, Shahini, Bjørkelund, Bendz, Massey, Schjalm, Halvorsen, Broch, Ueland, Gullestad, Nilsson, Aukrust, Mollnes and Louwe.)

Aims: Survivors of allogeneic haematopoietic stem-cell transplantation (allo-HSCT) are at higher risk of cardiovascular disease. We aimed to describe right ventricular (RV) systolic function and risk factors for RV dysfunction in long-term survivors of allo-HSCT performed in their youth., Methods and Results: This cohort included 103 survivors (53% female), aged (mean±SD) 17.6±9.5 years at allo-HSCT, with a follow-up time of 17.2±5.5 years. Anthracyclines were used as first-line therapy for 44.7% of the survivors. The RV was evaluated with echocardiography, and found survivors to have reduced RV function in comparison to a group of healthy control subjects: Tricuspid annular plane systolic excursion, (TAPSE, 20.8±3.7 mm vs 24.6±3.8 mm, p<0.001), RV peak systolic velocity (RV-s', 11.2±2.3 cm/s vs 12.3±2.3 cm/s, p=0.001), fractional area change (FAC, 41.0±5.2% vs 42.2±5.1%, p=0.047) and RV free-wall strain (RVFWS, -27.1±4.2% vs -28.5±3.3%, p=0.043). RV systolic dysfunction (RVSD) was diagnosed in 14 (13.6%), and was strongly associated with progressive left ventricular systolic dysfunction (LVSD). High dosages of anthracyclines were associated with greater reductions in RV and LV function. Multivariable linear regressions confirmed global longitudinal strain to be a significant independent predictor for reduced RV function., Conclusion: Impaired RV function was found in long-term survivors of allo-HSCT who were treated in their youth. This was associated with progressive left ventricle dysfunction, and pretransplant therapies with anthracyclines. The occurrence of RVSD was less frequent and was milder than coexisting LVSD in this cohort., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Rationale: Sleep apnea (SA) is highly prevalent in patients with atrial fibrillation (AF), and both conditions are associated with adverse cardiovascular outcomes. Objectives: To determine the effect of continuous positive airway pressure (CPAP) on AF burden. Methods: This open-label, parallel-group, randomized controlled trial included patients with paroxysmal AF and moderate to severe SA (apnea-hypopnea index ⩾15). A computerized system randomized eligible patients (1:1) to 5 months' treatment with CPAP plus usual care (CPAP, n  = 55) or usual care alone (control, n  = 54). The outcome assessment was blinded. The planned primary outcome was the difference between CPAP treatment and control groups in change of AF burden (percentage of time in AF) as measured by implantable loop recorder. Measurements and Main Results: A total of 579 patients with paroxysmal AF had respiratory polygraphy, of whom 244 (42%) had moderate to severe SA. Of these, 158 (65%) participated in the CPAP run-in period, of whom 39 (25%) patients did not tolerate the treatment. A total of 108 patients were available for the primary analysis. The mean time in AF decreased from 5.6% at baseline to 4.1% during the last 3 months of CPAP intervention and from 5.0% to 4.3% in the control group. The adjusted between-group difference at follow-up was -0.63 (95% confidence interval, -2.55 to 1.30) percentage points ( P  = 0.52). Seven serious adverse events (13%) occurred in the CPAP group, and two (4%) occurred in the control group. Conclusions: In patients with paroxysmal AF and SA, treatment with CPAP did not result in a statistically significant reduction in the burden of AF.Clinical trial registered with www.clinicaltrials.gov (NCT02727192).

Purpose: Survivors of allogeneic hematopoietic stem cell transplantation (allo-HSCT) are at risk for cardiopulmonary adverse events. Data on long-term effects on cardiorespiratory fitness are limited. To address the gap in knowledge, we aimed to determine peak oxygen uptake (V̇O 2 peak) and identify associations between cardiorespiratory fitness and clinical characteristics, self-reported physical activity, cardiac, and pulmonary function., Methods: In a nationwide, single-center cross-sectional study, 90 survivors [aged median (range) 35 (17-54) years, 56% females] were examined, 17 (6-26) years after allo-HSCT. Myeloablative conditioning comprised busulfan/cyclophosphamide or cyclophosphamide only. Methods included pulmonary function tests, echocardiography, and cardiopulmonary exercise test., Results: Chronic graft-versus-host disease (cGVHD) was found in 31% of the subjects, of whom 40% had bronchiolitis obliterans syndrome (BOS). Seventy-one percent of the survivors did not meet WHO recommendations for physical activity and 42% were overweight. Reduced gas diffusion (DL CO ) and systolic ventricular dysfunction (LVEF) were found in 44% and 31%, respectively. For the group, mean (95% CI), V̇O 2 peak was 36.4 (34.7-38.0) mL/min/kg [89 (85-93)% of predicted]. V̇O 2 peak was low at 43%. Cardiopulmonary factors and deconditioning were equally common limitations for exercise. In a multiple linear regression model, low V̇O 2 peak was associated with low DL CO , low LVEF, BOS, overweight, and inactivity., Conclusion: Half of the survivors had reduced cardiorespiratory fitness median 17 years after allo-HSCT. Cardiopulmonary factors and deconditioning were equally common limitations to exercise. We encourage long-term cardiopulmonary monitoring of allo-HSCT survivors and targeted advice on modifiable lifestyle factors.

White sponge nevus (WSN) is a benign autosomal dominant disorder characterized by the formation of white spongy plaques in the oral mucosa. Keratin (KRT) 13 is highly expressed in the mucosa, and mutations in this gene have been commonly associated with WSN patients. However, it remains unknown whether there is a causal relationship between KRT13 mutations and WSN and what the underlying mechanisms might be. Here, we use mouse genetic models to demonstrate that Krt13 is crucial for the maintenance of epithelial integrity. Krt13 knockout mice show a WSN-like phenotype in several tissues, including the tongue, buccal mucosa, and esophagus. Transcriptome analyses uncover that Krt13 regulates a cohort of gene networks in tongue epithelial cells, including epithelial differentiation, immune responses, stress-activated kinase signaling, and metabolic processes. We also provide evidence that epithelial cells without Krt13 are susceptible to mechanical stresses experienced during postnatal life, resulting in unbalanced cell proliferation and differentiation. These data demonstrate that Krt13 is essential for maintaining epithelial homeostasis and loss of Krt13 causes the WSN-like phenotype in mice., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT), a potentially curative therapy for malignant and nonmalignant diseases, is being increasingly used in younger patients. Although allo-HSCT survivors have an established increased risk of cardiovascular disease, there is limited knowledge of the long-term effects on cardiac function in survivors., Objectives: The purpose of this study was to describe left ventricular (LV) systolic function in long-term allo-HSCT survivors treated in childhood, adolescence, or early adulthood., Methods: Our cross-sectional cohort study included 104 patients (56% women), age 18 ± 10 years at time allo-HSCT with 17 ± 6 years of follow-up. Echocardiography included 2-dimensional (2D) and 3-dimensional (3D) analyses and speckle tracking imaging. In total, 55 healthy control subjects with a similar age, sex, and body mass index were used for comparison. Left ventricular systolic dysfunction (LVSD) was defined as reduced 2D left ventricular ejection fraction (LVEF) of <52% in men and <54% in women, and/or a reduced global longitudinal strain (GLS) of ≥-17%. Multivariable linear regression was used to determine independent predictors of 2D-LVEF and GLS., Results: Allo-HSCT survivors had significantly reduced LV systolic function compared with control subjects: 2D-LVEF (55.2 ± 5.8% vs. 59.0 ± 2.9%; p < 0.001), 3D LVEF (54.0 ± 5.1% vs. 57.6 ± 2.7%; p < 0.001), and GLS (-17.5 ± 2.2% vs. -19.8 ± 1.4%; p < 0.001). LVSD was found in 44.2%, of whom 28.3% were symptomatic. Clinical factors independently associated with 2D-LVEF and/or GLS included age, anthracyclines, graft versus host disease (GVHD), heart rate, and hypertension. In the 45% of survivors pre-treated with anthracyclines, the effect of anthracyclines on 2D-LVEF and GLS was dose-dependent., Conclusions: LVSD is common in long-term survivors of allo-HSCT treated in their youth. Pre-HSCT therapies with anthracyclines, age, heart rate, hypertension, and graft versus host disease are associated with measures of LV function., (© 2020 The Authors.)

At a fraction of the total cost of an equivalent orbital mission, scientific balloon-borne platforms, operating above 99.7% of the Earth's atmosphere, offer attractive, competitive, and effective observational capabilities-namely, space-like seeing, transmission, and backgrounds-which are well suited for modern astronomy and cosmology. The Super-pressure Balloon-borne Imaging Telescope (SUPERBIT) is a diffraction-limited, wide-field, 0.5 m telescope capable of exploiting these observing conditions in order to provide exquisite imaging throughout the near-infrared to near-ultraviolet. It utilizes a robust active stabilization system that has consistently demonstrated a 48 mas 1σ sky-fixed pointing stability over multiple 1 h observations at float. This is achieved by actively tracking compound pendulations via a three-axis gimballed platform, which provides sky-fixed telescope stability at < 500 mas and corrects for field rotation, while employing high-bandwidth tip/tilt optics to remove residual disturbances across the science imaging focal plane. SUPERBIT's performance during the 2019 commissioning flight benefited from a customized high-fidelity science-capable telescope designed with an exceptional thermo- and opto-mechanical stability as well as a tightly constrained static and dynamic coupling between high-rate sensors and telescope optics. At the currently demonstrated level of flight performance, SUPERBIT capabilities now surpass the science requirements for a wide variety of experiments in cosmology, astrophysics, and stellar dynamics.

Quantum mechanics-based design of useful catalytic antibodies (catabodies) failed because of the uncertain structure of the dynamic catalyst-substrate complex. The Catabody Platform emerged from discovery of beneficial germline gene catabodies that hydrolyzed self-proteins by transient covalent pairing of the strong catabody nucleophile with a weak target protein electrophile. Catabodies have evolved by Darwinian natural selection for protection against misfolded self-proteins that threatened survival by causing amyloid disease. Ancient antibody scaffolds upregulate the catalytic activity of the antibody variable (V) domains. Healthy humans universally produce beneficial catabodies specific for at least 3 misfolded self-proteins, transthyretin, amyloid β peptide and tau protein. Catabody are superior to ordinary antibodies because of catalyst reuse for thousands of target destruction cycles with little or no risk of causing inflammation, a must for non-toxic removal of abundant targets such as amyloids. Library mining with electrophilic target analogs (ETAs) isolates therapy-grade catabodies (fast, specific). Ex vivo- and in vivo-verified catabodies specific for the misfolded protein are available to dissolve brain, cardiac and vertebral amyloids. Immunization with ETAs overcomes important ordinary vaccine limitations (no catabody induction, poor immunogenicity of key target epitopes). We conceive electrophilic longevity vaccines that can induce catabody synthesis for long-lasting protection against amyloid disease., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: In recipients with type 1 diabetes, we aimed to determine whether long-term normoglycemia achieved by successful simultaneous pancreas and kidney (SPK) transplantation could beneficially affect progression of coronary artery disease (CAD) when compared with transplantation of a kidney-alone from a living donor (LDK)., Methods: In 42 kidney transplant recipients with functioning grafts who had received either SPK (n = 25) or LDK (n = 17), we studied angiographic progression of CAD between baseline (pretransplant) and follow-up at 7 years or older. In addition, computed tomography scans for measures of coronary artery calcification and echocardiographic assessment of left ventricular systolic function were addressed at follow-up., Results: During a median follow-up time of 10.1 years (interquartile range [IQR], 9.1-11.5) progression of CAD occurred at similar rates (10 of 21 cases in the SPK and 5 of 14 cases in the LDK group; P = 0.49). Median coronary artery calcification scores were high in both groups (1767 [IQR, 321-4035] for SPK and 1045 [IQR, 807-2643] for LDK patients; P = 0.59). Left ventricular systolic function did not differ between the 2 groups. The SPK and LDK recipients were similar in age (41.2 ± 6.9 years vs 40.5 ± 10.3 years; P = 0.80) and diabetes duration at engraftment but with significant different mean HbA1c levels of 5.5 ± 0.4% for SPK and 8.3 ± 1.5% for LDK patients (P < 0.001) during follow-up., Conclusions: In patients with both type 1 diabetes and end-stage renal disease, SPK recipients had similar progression of CAD long-term compared with LDK recipients. Calcification of coronary arteries is a prominent feature in both groups long-term posttransplant.

With the advancement of three-dimensional (3-D) real-time echocardiography in recent years, automatic creation of patient specific geometric models is becoming feasible and important in clinical decision making. However, the vast majority of echocardiographic segmentation methods presented in the literature focus on the left ventricle (LV) endocardial border, leaving segmentation of the right ventricle (RV) a largely unexplored problem, despite the increasing recognition of the RV's role in cardiovascular disease. We present a method for coupled segmentation of the endo- and epicardial borders of both the LV and RV in 3-D ultrasound images. To solve the segmentation problem, we propose an extension of a successful state-estimation segmentation framework with a geometrical representation of coupled surfaces, as well as the introduction of myocardial incompressibility to regularize the segmentation. The method was validated against manual measurements and segmentations in images of 16 patients. Mean absolute distances of [Formula: see text], [Formula: see text], and [Formula: see text] between the proposed and reference segmentations were observed for the LV endocardium, RV endocardium, and LV epicardium surfaces, respectively. The method was computationally efficient, with a computation time of [Formula: see text].

Bacterial biofilms play key roles in environmental and biomedical processes, and understanding their activities requires comprehension of their nanoarchitectural characteristics. Electron microscopy (EM) is an essential tool for nanostructural analysis, but conventional EM methods are limited in that they either provide topographical information alone, or are suitable for imaging only relatively thin (<300 nm) sample volumes. For biofilm investigations, these are significant restrictions. Understanding structural relations between cells requires imaging of a sample volume sufficiently large to encompass multiple cells and the capture of both external and internal details of cell structure. An emerging EM technique with such capabilities is bright-field scanning transmission electron microscopy (BF-STEM) and in the present report BF-STEM was coupled with tomography to elucidate nanostructure in biofilms formed by the polycyclic aromatic hydrocarbon-degrading soil bacterium, Delftia acidovorans Cs1-4. Dual-axis BF-STEM enabled high-resolution 3-D tomographic recontructions (6-10 nm) visualization of thick (1250 and 1500 nm) sections. The 3-D data revealed that novel extracellular structures, termed nanopods, were polymorphic and formed complex networks within cell clusters. BF-STEM tomography enabled visualization of conduits formed by nanopods that could enable intercellular movement of outer membrane vesicles, and thereby enable direct communication between cells. This report is the first to document application of dual-axis BF-STEM tomography to obtain high-resolution 3-D images of novel nanostructures in bacterial biofilms. Future work with dual-axis BF-STEM tomography combined with correlative light electron microscopy may provide deeper insights into physiological functions associated with nanopods as well as other nanostructures., (© 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.)

As the right ventricle's (RV) role in cardiovascular diseases is being more widely recognized, interest in RV imaging, function and quantification is growing. However, there are currently few RV quantification methods for 3D echocardiography presented in the literature or commercially available. In this paper we propose an automated RV segmentation method for 3D echocardiographic images. We represent the RV geometry by a Doo-Sabin subdivision surface with deformation modes derived from a training set of manual segmentations. The segmentation is then represented as a state estimation problem and solved with an extended Kalman filter by combining the RV geometry with a motion model and edge detection. Validation was performed by comparing surface-surface distances, volumes and ejection fractions in 17 patients with aortic insufficiency between the proposed method, magnetic resonance imaging (MRI), and a manual echocardiographic reference. The algorithm was efficient with a mean computation time of 2.0 s. The mean absolute distances between the proposed and manual segmentations were 3.6 ± 0.7 mm. Good agreements of end diastolic volume, end systolic volume and ejection fraction with respect to MRI ( -26±24 mL , -16±26 mL and 0 ± 10%, respectively) and a manual echocardiographic reference (7 ± 30 mL, 13 ± 17 mL and -5±7% , respectively) were observed.

Classical immunization methods do not generate catalytic antibodies (catabodies), but recent findings suggest that the innate antibody repertoire is a rich catabody source. We describe the specificity and amyloid β (Aβ)-clearing effect of a catabody construct engineered from innate immunity principles. The catabody recognized the Aβ C terminus noncovalently and hydrolyzed Aβ rapidly, with no reactivity to the Aβ precursor protein, transthyretin amyloid aggregates, or irrelevant proteins containing the catabody-sensitive Aβ dipeptide unit. The catabody dissolved preformed Aβ aggregates and inhibited Aβ aggregation more potently than an Aβ-binding IgG. Intravenous catabody treatment reduced brain Aβ deposits in a mouse Alzheimer disease model without inducing microgliosis or microhemorrhages. Specific Aβ hydrolysis appears to be an innate immune function that could be applied for therapeutic Aβ removal., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objective: HIV is vulnerable to antibodies that recognize a linear CD4 binding site epitope of gp120 (C), but inducing C-directed antibody synthesis by traditional vaccine principles is difficult. We wished to understand the basis for deficient C-directed antibody synthesis and validate correction of the deficiency by an electrophilic gp120 analog (E-gp120) immunogen that binds B-cell receptors covalently., Methods: Serum antibody responses to a C peptide and full-length gp120 epitopes induced by HIV infection in humans and immunization of mice with gp120 or E-gp120 were monitored. HIV neutralization by monoclonal and variable domain-swapped antibodies was determined from tissue culture and humanized mouse infection assays., Results: We describe deficient C-directed IgG but not IgM antibodies in HIV-infected patients and mice immunized with gp120 accompanied by robust synthesis of IgGs to the immunodominant gp120 epitopes. Immunization with the E-gp120 corrected the deficient C-directed IgG synthesis without overall increased immunogenicity of the C or other gp120 epitopes. E-gp120-induced monoclonal IgGs neutralized diverse HIV strains heterologous to the immunogen. A C-directed IgG neutralized HIV more potently compared to its larger IgM counterpart containing the same variable domains, suggesting obstructed access to HIV surface-expressed C. An E-gp120-induced IgG suppressed HIV infection in humanized mice, validating the tissue culture neutralizing activity., Conclusion: A C-selective physiological defect of IgM→IgG class-switch recombination (CSR) or restricted post-CSR B-cell development limits the functional utility of the humoral immune response to gp120. The E-gp120 immunogen is useful to bypass the restriction and induce broadly neutralizing C-directed IgGs (see Supplemental Video Abstract, http://links.lww.com/QAD/A551).

Peptide bond-hydrolyzing catalytic antibodies (catabodies) could degrade toxic proteins, but acquired immunity principles have not provided evidence for beneficial catabodies. Transthyretin (TTR) forms misfolded β-sheet aggregates responsible for age-associated amyloidosis. We describe nucleophilic catabodies from healthy humans without amyloidosis that degraded misfolded TTR (misTTR) without reactivity to the physiological tetrameric TTR (phyTTR). IgM class B cell receptors specifically recognized the electrophilic analog of misTTR but not phyTTR. IgM but not IgG class antibodies hydrolyzed the particulate and soluble misTTR species. No misTTR-IgM binding was detected. The IgMs accounted for essentially all of the misTTR hydrolytic activity of unfractionated human serum. The IgMs did not degrade non-amyloidogenic, non-superantigenic proteins. Individual monoclonal IgMs (mIgMs) expressed variable misTTR hydrolytic rates and differing oligoreactivity directed to amyloid β peptide and microbial superantigen proteins. A subset of the mIgMs was monoreactive for misTTR. Excess misTTR was dissolved by a hydrolytic mIgM. The studies reveal a novel antibody property, the innate ability of IgMs to selectively degrade and dissolve toxic misTTR species as a first line immune function.

The immunodominant epitopes expressed by the HIV-1 envelope protein gp120 are hypermutable, defeating attempts to develop an effective HIV vaccine. Targeting the structurally conserved gp120 determinant that binds host CD4 receptors (CD4BD) and initiates infection is a more promising route to vaccination, but this has proved difficult because of the conformational flexibility of gp120 and immune evasion mechanisms used by the virus. Mimicking the outer CD4BD conformational epitopes is difficult because of their discontinuous nature. The CD4BD region composed of residues 421-433 (CD4BD(core)) is a linear epitope, but this region possesses B cell superantigenic character. While superantigen epitopes are vulnerable to a small subset of spontaneously produced neutralizing antibodies present in humans without infection (innate antibodies), their non-covalent binding to B cell receptors (BCRs) does not stimulate an effective adaptive response from B cells. Covalent binding at naturally occurring nucleophilic sites of the BCRs by an electrophilic gp120 (E-gp120) analog is a promising solution. E-gp120 induces the synthesis of neutralizing antibodies the CD4BD(core). The highly energetic covalent reaction is hypothesized to convert the abortive superantigens-BCR interaction into a stimulatory signal, and the binding of a spatially distinct epitope at the traditional combining site of the BCRs may furnish a second stimulatory signal. Flexible synthetic peptides can detect pre-existing CD4BD(core)-specific neutralizing antibodies. However, induced-fit conformational transitions of the peptides dictated by the antibody combining site structure may induce the synthesis of non-neutralizing antibodies. Successful vaccine targeting of the CD4BD will require a sufficiently rigid immunogen that mimics the native epitope conformation and bypasses B cell checkpoints restricting synthesis of the neutralizing antibodies.

Failure to induce synthesis of neutralizing Abs to the CD4 binding determinant (CD4BD) of gp120, a central objective in HIV vaccine research, has been alternately ascribed to insufficient immunogen binding to Abs in their germline V region configuration expressed as BCRs, insufficient adaptive mutations in Ab V regions, and conformational instability of gp120. We employed peptide analogs of gp120 residues 421-433 within the CD4BD (CD4BD(core)) to identify Abs produced without prior exposure to HIV (constitutive Abs). The CD4BD(core) peptide was recognized by single-chain Fv fragments from noninfected humans with lupus that neutralized genetically diverse strains belonging to various HIV subtypes. Replacing the framework region (FR) of a V(H)4-family single-chain Fv with the corresponding V(H)3-family FRs from single-chain Fv JL427 improved the CD4BD(core) peptide-binding activity, suggesting a CD4BD(core) binding site outside the pocket formed by the CDRs. Replacement mutations in the FR site vicinity suggested the potential for adaptive improvement. A very small subset of serum CD4BD(core)-specific serum IgAs from noninfected humans without autoimmune disease isolated by epitope-specific chromatography neutralized the virus potently. A CD4BD(core)-specific, HIV neutralizing murine IgM with H and L chain V regions (V(H) and V(L) regions) free of immunogen-driven somatic mutations was induced by immunization with a CD4BD(core) peptide analog containing an electrophilic group that binds B cells covalently. The studies indicate broad and potent HIV neutralization by constitutive Abs as an innate, germline-encoded activity directed to the superantigenic CD4BD(core) epitope that is available for amplification for vaccination against HIV.

Immunoglobulins (antibodies) frequently express constitutive functions. Two such functions are nucleophilic catalysis and the reversible binding to B-cell superantigens. Constitutive or "naturally-occurring" antibodies are produced spontaneously from germline genetic information. The antibody structural elements mediating the constitutive functions have originated over millions of years of phylogenic evolution, contrasting with antigen-driven, somatic sequence diversification of the complementarity determining regions (CDR) that underlies the better-known high affinity antigen binding function of antibodies. Often, the framework regions (FRs) play a dominant role in antibody constitutive functions. Catalytic antibody subsets with promiscuous, autoantigen-directed and microbe-directed specificities have been identified. Mucosal antibodies may be specialized to express high-level catalytic activity against microbes transmitted by the mucosal route, exemplified by constitutive production of IgA class antibodies in mucosal secretions that catalyze the cleavage of HIV gp120. Catalytic specificity can be gained by constitutive noncovalent superantigen binding at the FRs and by adaptive development of noncovalent classical antigen or superantigen binding, respectively, at the CDRs and FRs. Growing evidence suggests important functional roles for catalytic antibodies in homeostasis, autoimmune disease and protection against infection. Adaptive antibody responses to microbial superantigens are proscribed underphysiological circumstances. Covalent electrophilic immunogen binding to constitutively expressed nucleophilic sites in B-cell receptors bypasses the restriction on adaptive antibody production, and simultaneous occupancy of the CDR binding site by a stimulatory antigenic epitope can also overcome the downregulatory effect of superantigen binding at the FRs. These concepts may be useful for developing novel vaccines that capitalize and improve on constitutive antibody functions for protection against microbes.

Subcellular distribution of Aedes aegypti sterol carrier protein-2 (AeSCP-2) and AeSCP-x was studied using electron microscopy. In both cultured A. aegypti cells and in the larval midgut, AeSCP-2 was detected mostly in the cytosol, with some labeling mitochondria and nucleus, but not in membranous vesicles. The widespread distribution of AeSCP-2 in the midgut epithelium is consistent with its potential lipid transfer function in all phases of cholesterol absorption. In contrast, AeSCP-x was found mostly in the peroxisome. Differences in the subcellular distribution of AeSCP-2 and AeSCP-x suggest that these two members of the SCP-2 gene family are functionally distinct. Overexpression of AeSCP-2 in A. aegypti cells showed increased localization of AeSCP-2 to cytosol, mitochondria, and nucleus. This is the first report on the nuclear distribution of an SCP. Overexpression of AeSCP-2 resulted in increased cholesterol incorporation in cells, suggesting that AeSCP-2 enhances cholesterol uptake., (Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.)

Fibroblasts produce extracellular matrix and play an important role in wound healing and scarring. Hepatocyte growth factor (HGF) has strong antifibrotic activity, and has been suggested to have therapeutic potential for treatment of fibrotic diseases. In the present in vitro study, morphological and functional changes of human vocal fold fibroblasts with HGF were examined by transmission electron microscopy and enzyme-linked immunosorbent assay to help clarify the potential use of HGF in the prevention or treatment of vocal fold scarring. The HGF stimulated the production of hyaluronic acid (HA) and decreased the production of collagen type I from the fibroblasts in Reinke's space (FbRS), whereas fibronectin production was not affected. The HGF also changed the shape of the FbRS from an oval shape toward a spindle and stellate shape, and developed Golgi apparatus (GA) and rough endoplasmic reticulum (rER) in the FbRS. The fibroblasts in the macula flava (FbMF) presented with much more production of HA and collagen type I than did FbRS, and were more frequently formed in a stellate shape with well-developed GA and rER. The HGF decreased the production of collagen type I from the FbMF, but barely affected the FbMF in terms of the shape of the cells, the development of GA and rER, or the production of HA. These results were interpreted to suggest that the FbMF are not as susceptible to HGF as are FbRS. On the contrary, HGF appeared to activate the FbRS and modify the function. The increased HA and decreased collagen type I production from the FbRS suggest that HGF may be useful in the prevention or treatment of fibrotic vocal fold scarring.

Enzymes within the biosynthetic pathway of mycolic acid (C(60)-C(90) a-alkyl,b-hydroxyl fatty acid) in Mycobacterium tuberculosis are attractive targets for developing new anti-tuberculosis drugs. We have turned to the simple model system of Corynebacterium matruchotii to study the terminal steps in the anabolic pathway of a C32 mycolic acid called corynomycolic acid. By transposon-5 mutagenesis, we transformed C. matruchotii into a mutant that is unable to synthesize corynomycolic acid. Instead, it synthesized two related series of novel fatty acids that were released by saponification from the cell wall fraction and from two chloroform/methanol-extractable glycolipids presumed to be analogues of trehalose mono- and di-corynomycolate. By chemical analyses and MS, we determined the general structure of the two series to be 2,4,6,8,10-penta-alkyl decanoic acid for the larger series (C(70)-C(77)) and 2,4,6,8-tetra-alkyl octanoic acid for the smaller series (C(52)-C(64)), both containing multiple keto groups, hydroxy groups and double bonds. The mutant was temperature-sensitive, aggregated extensively, grew very slowly relative to the wild type, and was resistant to the presence of lysozyme. We suggest that a regulatory protein that normally prevents the transfer of the condensation product back to b-ketoacyl synthase in the corynomycolate synthase system of the wild type was inactivated in the mutant. This will result in multiple Claisen-type condensation and the formation of two similar series of these complex hybrid fatty acids. A similar protein in M. tuberculosis would be an attractive target for new drug discovery.

The cDNA for an antibody light chain raised by immunization against vasoactive intestinal peptide (VIP) was cloned in a bacterial expression vector, and the recombinant light chain was purified to electrophoretic homogeneity. The light chain catalyzed the hydrolysis of VIP efficiently owing to its comparatively high affinity for the substrate. In control experiments, the catalytic activity was preserved at a constant level after further chromatography of the light chain on anion-exchange and gel-filtration fast protein liquid chromatography columns, and it was removed by immunoadsorption with immobilized anti-mouse light chain antibody. The amide bond linking methylcoumarinamide (MCA) and arginine in a tripeptide unrelated in sequence to VIP was cleaved by the light chain with lower affinity and kinetic efficiency (kcat/Km). Hydrolysis of the peptidyl-MCA conjugate was inhibited competitively by the alternate substrate, VIP. The Ki and Km values for VIP were in the same range, indicating that peptide-MCA and VIP hydrolysis occurs at a common catalytic site in the light chain. Molecular modeling suggested the presence of a serine protease-like site in the light chain. This was supported by inhibition of the hydrolytic activity by serine protease inhibitors, but not by inhibitors of other classes of proteases. These observations suggest a poorly discriminatory catalytic site, with specificity for VIP arising chiefly by means of the antigen recognition function of the light chain combining site.

Described are rapid assays for the analysis of PCR products in a one step, non-separation assay based on the use of electrochemiluminescence generated from a tris-bipyridine ruthenium (II) label. The assay uses PCR incorporation of a biotinylated oligonucleotide as a primer, with the inclusion of a labelled oligonucleotide. Oligonucleotides were labelled with an N-hydroxy succinimide ester of tris-bipyridine ruthenium (II) dihexafluorophosphate (Origen-label) by modifying the 3' and 3' 5' ends of the oligonucleotide probes. The assay makes use of the inherent thermal stability and absence of polymerase activity on such probes to allow the PCR and probe hybridization to be completed automatically on the thermocycler. The assay is concluded by the addition of PCR samples to streptavidin beads on an electrochemiluminescence analyser for binding and analysis. Target genes evaluated were the HIV-1 gag gene, and cystic fibrosis delta F-508 deletion mutation. The results obtained from these assays demonstrated the detection of 10 copies of the HIV-1 gag gene, and cystic fibrosis delta F-508 mutation in 1 ng of human DNA within 15 min. This assay format allows a rapid and simple determination of specific amplified DNA sequences, reducing the contamination risks due to washes and multiple pipetting.

We report evidence that a monoclonal antibody raised by immunization with a vasoactive intestinal peptide (VIP)-carrier protein conjugate selectively hydrolyzes VIP and a fluorescence quenched decapeptide (FQ14-22D), representing the region of VIP most susceptible to autoantibody-mediated cleavage (residues 14-22). A high affinity of the antibody for VIP and a lower affinity for FQ14-22D were revealed by kinetic studies and further substantiated by potent inhibition of FQ14-22D cleaving activity by full-length VIP. Sequencing of FQ14-22D hydrolysis products indicated selective cleavage at one peptide bond. These observations suggest that antibodies induced against naturally occurring polypeptide antigens can express peptidolytic activity targeted for specific sequences in the recognition epitope.

We describe the characterization and utility of a new electrochemiluminescent (ECL) label for oligonucleotides, utilizing phosphoramidite chemistry. This phosphoramidite of the tris(2,2-bipyridine)ruthenium(II) complex, bis(2,2-bipyridine)(4-[4-(2-cyanoethoxy-N,N-diisopropyl-amino) phosphinoxybutyl]4'-methyl)2,2-bipyridine ruthenium(II) dihexafluorophosphate or Origen phosphoramidite, enables the direct incorporation of the label during automated DNA synthesis. Efficiency of this automated synthesis allows the direct utilization of probes without further purification. Introduction of this labeling group is reproducible, and the ECL signal recovered is not influenced by hybridization. Furthermore, neither hybridization kinetics nor hybrid stability was affected by our label. We also demonstrate the utility of these labels for the development of rapid assays with oligonucleotides direct from automated synthesis. The clinical utility of these labeled oligonucleotides is shown with assays of total nucleic acid, extracted from peripheral blood lymphocytes of patients with acquired immunodeficiency syndrome (AIDS), to detect the human immunodeficiency virus (HIV-1). The results demonstrate the ability of the assay to quantify 30-2000 copies of HIV1 gag genes and to rapidly detect (less than 45 min) HIV-1 gag genes in a nonseparation assay. The application of this assay to clinical samples demonstrates the utility of these assays for rapid and quantitative analysis.

Vasoactive intestinal peptide (VIP) fragments generated by autoantibodies purified from the blood of two human beings were separated and sequenced. Based on the identity of these fragments, seven peptide bonds cleaved by the antibodies were identified. Six of the seven scissile bonds are clustered in the region of VIP spanning residues 14-22 and were cleaved by antibodies from both human subjects. The seventh scissile bond is located at residues 7-8 and was cleaved by antibodies from one of the subjects. The scissile bonds link amino acid residues with different size, charge, and hydrophobicity. The hydrolytic activity of the antibodies was selective in that they failed to hydrolyze polypeptides unrelated in sequence to VIP (insulin and atrial natriuretic peptide). These observations demonstrate substrate specific hydrolysis by naturally occurring antibodies and expand the range of peptide bonds hydrolyzed by these antibodies.

Ten fragments of vasoactive intestinal peptide (VIP) were tested for reactivity with a human catalytic autoantibody that cleaves full-length VIP(1-28) at the Gln16-Met17 peptide bond. A large COOH-terminal subsequence, VIP(15-28), was bound by the autoantibody with high affinity (Ki 1.25 nM), suggesting that it is the antibody binding epitope. VIP(22-28), a short subsequence distant from the scissile bond, inhibited the binding (Ki 242 microM) and hydrolysis (Ki 260 microM) of full-length VIP by the catalytic autoantibody in a competitive fashion. The autoantibody did not show detectable binding of short VIP subsequences that encompass the scissile bond (VIP(15-21), VIP(11-17), and VIP(13-20]. These data show that residues 22-28, located four amino acids distant from the scissile bond, contribute in recognition of VIP by the catalytic autoantibody.

Hybrid cell lines producing monoclonal antibodies against the C3H strain of mouse mammary tumor virus (C3H MMTV) were prepared by the fusion of mouse myeloma cells with the lymphocytes of BALB/c mice that were immunized with C3H MMTV. Approximately 10% of the hybrid cells initially plated after cell fusion produced immunoglobulins that reacted in antibody-binding assays with C3H MMTV; 40 of these cells were cloned, and 6 eventually yielded stable cell lines. High concentrations of monoclonal antibodies (5 to 20 mg/ml) were obtained from serum and ascites fluid of syngeneic mice inoculated with the hybrid cells. All of the monoclonal antibodies were directed against the envelope glycoprotein gp52. Three of the hybrid cell lines produced immunoglobulins of the immunoglobulin M subclass and three produced immunoglobulin G2a. The monoclonal antibodies showed limited charge heterogeneity in light and heavy chains when analyzed by high-resolution, two-dimensional gel electrophoresis. Three serologically distinct specificities were observed when these ascites fluids were tested against different strains of MMTV. The antigenic determinants detected were the following: (i) a type-specific determinant unique to the C3H strain of MMTV; (ii) class-specific determinants shared between C3H and GR MMTVs; and (iii) a group-specific determinant found on C3H, GR, RIII, and the endogenous C3H (C3Hf) MMTVs. Because monoclonal antibodies recognize single antigenic determinants, these results demonstrate for the first time that the three patterns of antigenic reactivity for MMTV are related to individual determinants on the gp52 molecule and also clearly show that one strain of MMTV can be distinguished from other strains.

Twenty-three healthy human adults with naturally occurring (spontaneous) mononuclear cell cytotoxicity (MCC) to cultured human tumor cells were evaluated in a serial study over 23 months to determine whether their initial MCC levels remained stable. A 3-hour 51Cr-release microcytotoxicity assay, in which the HBT-3 cancer-derived cell line with HeLa markers and the ALAB breast cancer-derived cell line were used, was standardized and its suitability for such serial studies was evaluated. The data showed that the 51Cr-release assay is reproducible and suitable for use in serial studies of MCC. The intrinsic technical error for a single assay averaged 12.5% (2-26%) coefficient of variation. The greatest sources of technical variation with time were the passage level of the target cells and the temperature at which the effector cells were stored and handled. When these technical factors were controlled, naturally occurring MCC was fairly reproducible with time. Of the healthy individuals with initial high or low MCC levels, 61% tended to retain these levels in a stable fashion, but the remaining 39% studied showed fluctuations of MCC greater than 33% coefficient of variation.

Hemocyanin (Hcy) from whelks, Busycon canniculatum has been developed as a visual marker for the identification of virus and cell surface antigens by the correlative techniques of fluorescence microscopy, TEM and SEM. A series of hybridomas producing monoclonal antibodies that allow the identification of type-, group-, and class-specific antigenic determinants on the major envelope glycoprotein of mouse mammary tumor virus (MMTV) gp 52 and two group-specific monoclonal antibodies to MMTV gp 36 have been developed. We used these antibodies in the unlabeled antibody Hcy bridge for immunoelectron microscopy (IEM) and found that each gp 52 antigenic determinant was expressed on virus during all stages of morphogenesis and on the infected cell surface while the gp 36 determinants were not detected. The positive labeling of gp 52 by IEM correlated well with immuno-experiments using the 125I Staph protein A plate binding assay (125IPA). The anti-gp 36 monoclonals in contrast, however, gave positive results only in the 125IPA assay. Hybridomas to murine and primate type C virion envelope proteins [gp 70 and p15(E)] have also been developed. None of the monoclonal antibodies to murine type C virus gp 70 or p15(E) gave positive labeling by IEM when tested with Rauscher murine leukemia virus but two were positive by the 125IPA assay. Monoclonal antibodies to the gp 70 of a baboon type C virus, M-7 were positive in IEM, labeling both the cell surface and viral envelope and reacted with virus in the 125IPA assay. Since monoclonal antibodies provide a much better resolution of the molecular structure and antigenic differences of viruses, interpretations of labeling results are thoroughly discussed. Methods for testing the specificity and titer of monoclonals are presented. The unlabeled antibody Hcy bridge technique and recent advances in methodology are reviewed.

The tumors induced in white-lipped marmosets (Saguinus fuscicollis, S. nigricollis) by Rous sarcoma virus (RSV) of chicken origin (RSV-SR) were not transplantable to allogeneic hosts. In contrast, RSV rescued from these tumors (RSV-M) induced sarcomas that were transplantable to young but not to adult marmosets. The tumors induced by RSV-M and the transplants rapidly enlarged, metastasized to various organs, and killed the recipients 29-59 days post inoculation. Cell lines were readily established from all transplantable sarcomas. No virus expression was detected in transplantable tumor cell lines by electron microscopy or by biochemical and biological assays. However, RSV of the same subgroup as RSV-SR was rescued from both short-term and long-term tumor cell cultures by cocultivation with chicken embryo fibroblasts (CEF). The rescued viruses transformed marmoset cells 100-fold more efficiently than CEF cells, although CEF cells remained permissive for virus replication. Cytogenetic studies revealed extensive chromosome abnormalities in tumor transplants but not in RSV-M-induced sarcomas. All cell lines were hyperploid and contained structurally abnormal, large metacentric and telocentric chromosomes. Immunologic studies failed to detect group-specific (gs) antigen of the avian sarcoma-leukemia complex in either RSV-M-induced, transformed cells or tumor transplants. By complement-dependent cytotoxicity assays, with the use of marmoset anti-gs serum, RSV-associated antigen could be detected on the surfaces of tumor cells. No differences in the expression of this antigen existed between transplantable and nontransplantable marmoset sarcomas. All transplantable cell lines contained abnormal amounts of lipids and glycogen in comparison to RSV-SR-induced tumors and normal marmoset cell lines. The glycogen was associated with unique cytoplasmic membrane complexes and was surrounded by either single- or double-membraned vesicles.

The interference among viruses is a well-documented biological phenomenon, both in animals and tissue culture systems. In two of our previous in vivo experiments and in four independent animal experiments, which are described in this presentation, interferences were successfully used to influence the outcomes of viral diseases by using non-pathogenic viruses. In this study, four pathogenic viruses were studied in their natural hosts, and against these viruses, in different combinations, 15 non-pathogenic viruses were tested. There was great variation in mutual effects among pathogenic and non-pathogenic viruses. In our four experiments, the viruses were either simultaneously inoculated or the non-pathogenic viruses were preinoculated. Newcastle disease vaccine (Strain H) had remarkable effects in the development of mouse ascites-associated lymphoma virus. The 50% mortality rate in mice caused by a vaccine strain of rabies virus was reduced to 15% using avian encephalitis virus. The clinical manifestations of rabbit myxoma virus effects were significantly delayed by Newcastle disease vaccine (Strain H). The 72% mortality rate due to Rous sarcoma virus in chickens was decreased to 33.3% when the animals were preinoculated with avian bursa virus vaccine.

The inability of pathogenic animal viruses to be completely neutralized by antibodies can lead to chronic viral infections in which infectious virus persists even in the presence of excess neutralizing antibody. A mechanism that results in this nonneutralized fraction of virus was defined by the topographical relationships of viral epitopes identified with monoclonal antibodies wherein monoclonal antibodies bind to virus and sterically block the binding of neutralizing antibodies.