Efficient generation of respiratory syncytial virus (RSV)-neutralizing human MoAbs via human peripheral blood lymphocyte (hu-PBL)-SCID mice and scFv phage display libraries

RSV is one of the major causes of pneumonia and bronchiolitis in infants and young children and is associated with high mortality. RSV neutralizing human antibody (hu-Ab) is known to mediate resistance to viral infection as well as to be an effective treatment for severe lower respiratory tract RSV infection. We have previously demonstrated that human primary and secondary immune responses can be established in severe combined immunodeficient mice engrafted with human peripheral blood lymphocytes (hu-PBL-SCID). By combining this animal model with the single-chain Fv antibody (scFv) phage display library technique, we were able to investigate further its clinical potential by generating a panel of human scFvs that exhibit both high F glycoprotein (RSV-F) binding affinities ( approximately 108 M(-1)) and strong neutralizing activities against RSV infection in vitro. Sequencing analysis of the randomly isolated anti-RSV-F scFv clones revealed that they were derived from different VH families with mutations in the complementarity-determining region 1 (CDR1). The results suggest that: (i) RSV-F-specific human immune responses and affinity maturation can be induced in hu-PBL-SCID mice; and (ii) this approach can be applied to generate large numbers of human scFvs with therapeutic potential. Despite the fact that hu-PBL-SCID mouse and human scFv phage display library have individually been established, our approach contributes a simple and significant step toward the generalization of antigen-specific human monoclonal antibody (hu-MoAb) production and their clinical applications.     

Isolation of human single chain antibodies (scFv) against human TNF-alpha from human peripheral blood lymphocyte-SCID mice

By developing an appropriate immunization protocol for SCID (hu-PBL-SCID) mice engrafted with human peripheral blood lymphocytes in combination with scFv phage display library, we were able to establish an efficient strategy to obtain human scFv clones against a human self-antigen, TNF-alpha. The mice pretreated with gamma-radiation (3Gy) and anti-asialo GM1 antibody were immunized with a mixture of human TNF-alpha-keyhole limpet hemocyanin and Freund's adjuvant. Human antibody maturation was suggested to be induced in the mice with the immunization protocol. The scFv clones obtained from the mice were shown to exhibit binding affinities in the range of 10(7)-10(8) M(-1). Together with our previously published work on the isolation of respiratory syncytial virus neutralizing scFvs, the results of this study have implicated that this combined approach is one of the effective alternatives for the cloning of human monoclonal antibodies specific for a wide range of antigens of interest.     

Identification and characterization of aquaporin-2 water channel mutations causing nephrogenic diabetes insipidus with partial vasopressin response

Congenital nephrogenic diabetes insipidus (NDI) is a rare disease caused most often by mutations in the vasopressin V2 receptor (AVPR2). We studied a family which included a female patient with NDI with symptoms dating from infancy. The patient responded to large doses of desmopressin (dDAVP) which decreased urine volume from 10 to 4 I/day. Neither the parents nor the three sisters were polyuric. The patient was found to be a compound heterozygote for two novel recessive point mutations in the aquaporin-2 (AQP2) gene: L22V in exon 1 and C181W in exon 3. Residue Cys181 in AQP2 is the site for inhibition of water permeation by mercurial compounds and is located near to the NPA motif conserved in all aquaporins. Osmotic water permeability (Pf) in Xenopus oocytes injected with cRNA encoding C181W-AQP2 was not increased over water control, while expression of L22V cRNA increased the Pf to approximately 60% of that for wild-type AQP2. Co-injection of the mutant cRNAs with the wild-type cRNA did not affect the function of the wild-type AQP2. Immunolocalization of AQP2-transfected CHO cells showed that the C181W mutant had an endoplasmic reticulum-like intracellular distribution, whereas L22V and wild-type AQP2 showed endosome and plasma membrane staining. Water permeability assays showed a high Pf in cells expressing wild-type and L22V AQP2. This study indicates that AQP2 mutations can confer partially responsive NDI.      

High vs. low entrustment behaviors in the operating room

BackgroundOperative experience with an appropriate degree of supervised autonomy is critical to resident training. Progressively greater intraoperative entrustment has been associated with gradually higher levels of resident autonomy. This study attempts to identify consistently observed intraoperative behaviors that are linked with higher resident entrustment.MethodsThis qualitative study analyzed observational notes recorded by trained raters who provided entrustment scores for 204 surgical cases at Michigan Medicine from 2015 to 2017. Notes were coded in NVivo12. Thematic analysis was used to identify themes and patterns within the data.ResultsThe analysis generated 144 codes. Codes were clustered into 10 themes. These themes manifested differently in intraoperative behaviors strongly associated with high entrustment versus low entrustment.ConclusionThis study demonstrates key differences in intraoperative behaviors exhibited by residents and faculty in high and low entrustment interactions. Awareness of behaviors that enhance entrustment can help faculty augment resident learning and enable higher resident operative autonomy.     

Balloon aortic valvuloplasty for aortic stenosis in neonates, children, and young adults

Balloon aortic valvuloplasty is a safe and effective treatment for aortic stenosis in neonates, children, and young adults. The indication for balloon valvuloplasty in children is a peak systolic gradient > 70 mmHg or > 50 mmHg in any patient with symptoms. Published results indicate that the procedure results in an acute reduction in gradient ranging from 49%-70%, and this reduction appears to persist through at least an intermediate follow-up. The percentage reduction in gradient is similar for neonates as well as older children. In neonates, it is imperative to evaluate the size of the left ventricle prior to balloon valvuloplasty since mortality is highest in those with variants of hypoplastic left heart syndrome. Though morbidity and mortality is higher in the neonatal age group, the results are similar to those following surgical intervention. In children older than 1 month of age, the major complication is the development of aortic regurgitation, although it usually appears to be well-tolerated. Previous surgical valvotomy is not a contraindication to balloon aortic valvuloplasty. In the current era, we believe that balloon aortic valvuloplasty should be considered as the first option in neonates, children and young adults with significant aortic valve obstruction.     

Hepatitis C prevalence and risk factors in the northern Alberta dialysis population.

Hepatitis C virus (HCV) is an emerging global public health issue with particular relevance in multiply transfused renal dialysis patients. This cross-sectional study evaluated the prevalence and risk factors for HCV infection among renal dialysis patients in northern Alberta, Canada. Ninety-two percent of eligible patients (n = 336) provided informed consent to participate. Participants were interviewed to gather risk factor information and, using multiple logistic regression analysis with exact inference, a predictive model for HCV infection in this population was developed. The prevalence of HCV infection in the population was 6.5%, and all positive patients had at least one identifiable risk factor. The multivariate analysis showed that the risk of HCV infection was greater for those in the 18-55 years age category (odds ratio (OR) = 4.9, 95% confidence interval (CI) 1.2-27.9), patients who had been on dialysis > 5 years (OR = 3.7, 95% CI 1.2-12.0), and patients who had > or = 2 high risk life-style behaviors (OR = 5.0, 95% CI 1.5-16.7). Transfusion prior to 1990 was marginally associated with HCV status (OR = 4.0, 95% CI 0.96-16.3). This study documented previously unreported life-style risk factors for HCV infection in patients with renal failure, confirmed the expected decline in transfusion-acquired HCV infection in this population, and provided evidence against nosocomial transmission of HCV.     

Ventricular function

Multiple modalities contribute to the evaluation of ventricular function. The role of cineangiography, echocardiography, MR imaging, ultrafast CT, and nuclear medicine continue to evolve and improve our understanding of the physiology and pathophysiology of ventricular function. This article discusses the use and limitation of each modality.     

Cardiac reoperations in octogenarians: analysis of outcomes

BACKGROUND: With the rapid growth of the elderly segment of the population, more octogenarians are referred for complex cardiac interventions, including reoperations. Data regarding the outcomes, quality of life, and long-term results after reoperative open-heart surgical procedures in octogenarians are scarce. METHODS: We retrospectively studied 113 consecutive octogenarians (mean age, 83+/-2.6 years) who underwent reoperative cardiac procedures within a 13-year period. Coronary artery bypass grafting (CABG) was performed in 49 patients (CABG group), valvular procedures (aortic, mitral, or tricuspid valve, alone or in combination) in 35 (valve group), and combined CABG and valve intervention in 29 (combined CABG and valve group). RESULTS: The 30-day mortality rate was 8% (4 of 49) for the CABG group, 9% (3 of 35) for the valve group, and 17% (5 of 29) for the combined CABG and valve group. One- and 5-year actuarial survival rates were, respectively, 85%+/-5% and 58%+/-10% for the CABG group, 78%+/-7% and 53%+/-12% for the valve group, and 69%+/-9% and 63%+/-10% for the combined CABG and valve group. Sixty-one percent of patients in the CABG group, 40% in the valve group, and 38% in the combined CABG and valve group were in New York Heart Association class I or II postoperatively at a mean follow-up time of 2.1+/-2.4 years. Similarly, 91%, 85%, and 80%, respectively, thought that they had an improved quality of life and were satisfied with their functional status. CONCLUSIONS: Cardiac reoperations can be performed successfully in most octogenarians, although with an increased risk, particularly in the combined CABG and valve group. Long-term survival is acceptable with improved quality of life and functional status. However, it is possible that these results could be improved in this high-risk group of patients with earlier referral and surgical intervention, for the effective use of health care resources.