Genotype-phenotype correlation and frequency of the 3199del6 cystic fibrosis mutation among I148T carriers: results from a collaborative study.

PURPOSE: We expect that the mutation panel currently recommended for preconception/prenatal CF carrier screening will be modified as new information is learned regarding the phenotype associated with specific mutations and allele frequencies in various populations. One such example is the I148T mutation, originally described as a severe CF mutation. After implementation of CF population-based carrier screening, we learned that I148T exists as a complex allele with 3199del6 in patients with clinical CF, whereas asymptomatic compound heterozygotes for I148T and a second severe CF mutation were negative for 3199del6. METHODS: We performed reflex testing for 3199del6 on 663 unrelated specimens, including I148T heterozygotes, compound heterozygotes, and a homozygous individual. RESULTS: Less than 1% of I148T carriers were also positive for 3199del6. Excluding subjects tested because of a suspected or known CF diagnosis or positive family history, 0.6% of I148T-positive individuals were also positive for 3199del6. We identified 1 I148T homozygote and 6 unrelated compound heterozygous individuals with I148T and a second CF variant (2 of whom also carried 3199del6). In addition, one fetus with echogenic bowel and one infertile male were heterozygous for I148T (3199del6 negative). CONCLUSIONS: Reflex testing for 3199del6 should be considered whenever I148T is identified. Reflex testing is of particular importance for any symptomatic patient or whenever one member of a couple carries a deleterious CF mutation and the other member is an I148T heterozygote. Further population data are required to determine if I148T, in the absence of 3199del6, is associated with mild or atypical CF or male infertility.     

Inhibition of the production of a soluble helper mediator by cyclosporin A results in the failure to generate alloreactive cytolytic cells in mixed-lymphocyte culture

The mechanism of action of cyclosporin A (CsA) in inhibiting the induction of alloreactive cytolytic T lymphocytes (CTL) in mixed-lymphocyte culture (MLC) was investigated. CsA at concentrations of 10(-3) to 10(-1) micrograms/ml completely prevented the generation of CTL. However, the addition of culture supernatants from mitogen-activated lymphocytes to MLC not only significantly reversed the suppressive effect of CsA but also fully restored the reactivity of lymphocytes already treated with CsA. By measuring the presence of a soluble helper mediator (SHF) in MLC supernatants, we found that CsA-treated lymphocytes produced no SHF, possibly interleukin 2 (IL-2). The effect of CsA on receptors for IL-2 was subsequently studied and it was found that the binding capacity of 125I-labeled IL-2 to lymphocytes was not altered by the presence of CsA. These findings suggest that the prevention of helper cells from producing SHF, rather than the inhibition of the response of effector cells to SHF, is a possible explanation for the immunosuppression mediated by CsA.     

Immunohistochemical detection of c-erbB-2 protein in human benign and neoplastic prostate.

Both the polyclonal anti-c-erbB-2 peptide antiserum pAB 60 and the monoclonal anti-c-erbB-2 protein antibody mAB-1 detect the c-erbB-2 protein in human breast adenocarcinomas. We investigated c-erbB-2 expression in adult human benign hyperplastic and neoplastic prostates, using the avidin-biotin complex immunoperoxidase method. Formalin-fixed, paraffin-embedded specimens of benign hyperplastic prostate (13), prostatic adenocarcinoma (22), and prostatic adenocarcinoma lymph node metastases (two) were tested with pAB 60. Ten formalin-fixed, paraffin-embedded specimens of prostate adenocarcinoma, 11 frozen sections of benign hyperplastic specimens, and eight frozen sections of prostate adenocarcinoma were tested with mAB-1. Our results demonstrated consistent detection of c-erbB-2 immunohistochemically in frozen sections of both benign and malignant prostate. Preincubation of pAB 60 with the immunizing peptide blocked subsequent reactivity with prostatic tumor tissue, indicating specificity. However, fixation and processing protocols significantly affected the reactivity of the antigenic determinants detected by these antibodies, as mAB-1 was nonreactive with formalin-fixed, paraffin-embedded prostatic tissues. Differential reactivity of pAB 60 with malignant rather than benign glands was maximized by exposure of the specimen to the antibody at 4 degrees C rather than 22 degrees C. The most frequently observed staining pattern with both antibodies was cytoplasmic. However, mAB-1 produced distinctly membranous staining in two frozen specimens of benign hyperplasia and one specimen of prostate cancer.     

Alterations in epithelial glycocalyx of rabbit uteri during early pseudopregnancy and pregnancy,and following ovariectomy

Pseudopregnant, pregnant, and ovariectomized rabbits were utilized to study hormonal mediation of uterine epithelial surface negativity and glycocalyx morphology, and to seek local effects of blastocysts at sites of implantation. A loss of surface negativity [polycationic ferritin (PCF) binding] by day 6 of pregnancy or pseudopregnancy was noted, accompanied by alterations in epithelial glycocalyx. Uteri from estrous animals, or ovariectomized animals receiving oil or estradiol injections, bound PCF and exhibited a “globular” glycocalyx. Uteri from day 6 pseudopregnant or pregnant animals, or ovariectomized animals receiving progesterone injections, did not bind PCF or exhibit a globular glycocalyx. Both PCF binding and the globular character of the epithelial glycocalyx were sensitive to neuraminidase and trypsin treatment, suggesting sialoglycoprotein contribution to surface negativity. Implanting blastocysts had no detectable local effect on surface negativity, but did induce local reduction of epithelial glycocalyx at sites of implantation. Results of this study suggest that uterine epithelial glycocalyx alterations during the preimplantation period reflect a general response to prosgesterone stimulation, primarily qualtitative in nature, related to the acquisition of receptivity to ovo-implantation.     

In vivo and in vitro comparison of fire ant venom and fire ant whole body extract

Thirty-four patients with a history of immediate hypersensitivity to the sting of the imported fire ant were evaluated in a study designed to compare the diagnostic usefulness of fire ant whole body extract (WBE) preparations with that of fire ant venom (IFAV). Ninety-one percent ($\text{31}\text{34}$) of the hypersensitive patients skin tested with IFAV at a maximal concentration of 1:5 × 10³, $\text{v}\text{v}$, demonstrated a wheat equal to or greater than the histamine control. Fifty-three percent ($\text{18}\text{34}$) of the group were skin test positive to a WBE preparation. When the criteria for a positive skin test were relaxed, 82% of the hypersensitive group could be identified with the IFAWBE. A comparison of skin test results in sensitive patients revealed variability in the sensitivity of the WBE preparations utilized in the study. Leukocyte histamine release demonstrated a dose-response release of histamine with both IFAV and SIWBEa preparations. Specific venom antisera produced in rabbits identified a precipitin line of common identity in a gel-diffusion system containing IFAWBE and IFAV. This finding was verified by the competitive inhibition of IFAWBE with IFAV in a solid-phase radioimmunoassay system. Fire ant WBEs contain venom constituents and are effective diagnostic agents in up to 82% of patients with hypersensitivity to the sting of the imported fire ant. Marked variability in the responsiveness of sensitive patients to different WBE preparations mandates standardization of these diagnostic preparations.     

Protein synthesis is required for the enhancement of long-term potentiation and long-term memory by spaced training

Spaced training is generally more effective than massed training for learning and memory, but the molecular mechanisms underlying this trial spacing effect remain poorly characterized. One potential molecular basis for the trial spacing effect is the differential modulation, by distinct temporal patterns of neuronal activity, of protein synthesis-dependent processes that contribute to the expression of specific forms of synaptic plasticity in the mammalian brain. Long-term potentiation (LTP) is a type of synaptic modification that may be important for certain forms of memory storage in the mammalian brain. To explore the role of protein synthesis in the trial spacing effect, we assessed the protein synthesis dependence of hippocampal LTP induced by 100-Hz tetraburst stimulation delivered to mouse hippocampal slices in either a temporally massed (20-s interburst interval) or spaced (5-min interburst interval) fashion. To extend our studies to the behavioral level, we trained mice in fear conditioning using either a massed or spaced training protocol and examined the sensitivity of long-term memory to protein synthesis inhibition. Larger LTP was induced by spaced stimulation in hippocampal slices. This improvement of synaptic potentiation following temporally spaced synaptic stimulation in slices was attenuated by bath application of an inhibitor of protein synthesis. Further, the maintenance of LTP induced by spaced synaptic stimulation was more sensitive to disruption by anisomycin than the maintenance of LTP elicited following massed stimulation. Temporally spaced behavioral training improved long-term memory for contextual but not for cued fear conditioning, and this enhancement of memory for contextual fear was also protein synthesis dependent. Our data reveal that altering the temporal spacing of synaptic stimulation and behavioral training improved hippocampal LTP and enhanced contextual long-term memory. From a broad perspective, these results suggest that the recruitment of protein synthesis-dependent processes important for long-term memory and for long-lasting forms of LTP can be modulated by the temporal profiles of behavioral training and synaptic stimulation.     

Preimplantation diagnosis for Fanconi anemia combined with HLA matching.

CONTEXT: The advent of single-cell polymerase chain reaction (PCR) has presented the opportunity for combined preimplantation genetic diagnosis (PGD) and HLA antigen testing. This is a novel and useful way to preselect a potential donor for an affected sibling requiring stem cell transplantation. OBJECTIVE: To perform in vitro fertilization (IVF) and preimplantation HLA matching combined with PGD for Fanconi anemia (FA). DESIGN: DNA analysis for the IVS 4 + 4 A-->T (adenine to thymine) mutation in the FA complement C (FANCC) gene in single blastomeres, obtained by biopsy of embryos, to identify genetic status and HLA markers of each embryo before intrauterine transfer. SETTING: In vitro fertilization programs at large medical centers in Chicago, Ill, and Denver, Colo. PARTICIPANTS: A couple, both carriers of the IVS 4 + 4 A-->T mutation in the FANCC gene with an affected child requiring an HLA-compatible donor for cord blood transplantation. MAIN OUTCOME MEASURES: DNA analysis of single blastomeres to preselect unaffected embryos representing an HLA match for the affected sibling. RESULTS: Of 30 embryos tested in 4 IVF attempts, 6 were homozygous affected and 24 were unaffected. Five of these embryos were also found to be HLA-compatible, of which 2 were transferred in the first and 1 in each of the other 3 cycles, resulting in a pregnancy and birth of an unaffected child in the last cycle. CONCLUSION: To our knowledge, this is the first PGD with HLA matching, demonstrating feasibility of preselecting unaffected embryos that can also be an HLA-compatible source for stem cell transplantation for a sibling.     

ASO Author Reflections: National Analysis of Breast Surgery Malpractice Cases: A Teachable Moment?

Annals of Surgical Oncology -     

“It’s a tool,not a cure”: the preoperative teen perspective on bariatric surgery

BackgroundBariatric surgery is well established in adults as the most effective tool for sustained weight loss and reduction of obesity-related co-morbidities, and is an emerging option for adolescents in whom conservative approaches have failed. Narratives are vital in understanding the motivating reasons and psychosocial profiles of adolescents considering bariatric surgery during a developmental period of evolving self-concept, body image sensitivity, peer pressure, and increased opportunity for risky behaviors.ObjectivesTo explore preoperative adolescent patient perspectives on their decision to pursue bariatric surgery, anticipated physical and psychosocial effects, and preparation process.SettingsThe Hospital for Sick Children (SickKids) in Toronto, Ontario, Canada.MethodsWe conducted 14 semi-structured interviews with adolescents (16–18 yr old) 2–4 months before bariatric surgery. A theoretical thematic analysis was conducted with 3 independent reviewers (interrater reliability, Cronbach’s α= .81) and conflicts were resolved through discussion.ResultsPatients’ perspectives are captured in 4 themes: motivation for surgery, effects on health and habits, psychosocial changes, and support systems during preparation. Participants perceived surgery as a tool but not a solution for weight loss. Most were motivated for health and daily functional improvement rather than aesthetic reasons, and some anticipated improved social interactions while maintaining their self-identity. Participants were selective in sharing news of their surgery, and received varying levels of support from families, friends, and healthcare teams.ConclusionAdolescents seem generally prepared for and informed about surgery, with expectations for weight loss and psychosocial improvements, although uncertainty and contradictory thoughts were prevalent. Insights provided by these adolescent patients will help optimize bariatric surgery assessments and support from clinical teams and inform preoperative education for future patients and families.