Use of class II tetramers for identification of CD4+ T cells

Multivalent MHC class II molecules containing peptide antigens are useful tools for the detection of antigen specific human CD4+ T cells. Tetramers produced by exogenous peptide loading onto empty class II molecules are comparable to tetramers with peptide tethered to the class II chain covalently, but have many practical advantages. Conditions for optimal peptide loading to generate tetramers are discussed and optimal conditions of using tetramers for staining T cells are examined. As the frequency of antigen specific CD4+ T cells in peripheral blood is low, we demonstrate that an in vitro expansion step is effective in detecting low frequency T cells. Two new applications with tetramers, their uses for mapping T cell epitopes and for the detection of low affinity T cells are described. In a clinical setting, potential applications include using these reagents for monitoring disease progression during clinical intervention.     

The history of contraction of the wrist flexors can change cortical excitability

Many freshwater turtles in temperate climates may experience winter periods trapped under ice unable to breathe, in anoxic mud, or in water depleted of O₂. To survive, these animals must not only retain function while anoxic, but they must do so for extended periods of time. Two general physiological adaptive responses appear to underlie this capacity for long-term survival. The first is a coordinated depression of metabolic processes within the cells, both the glycolytic pathway that produces ATP and the cellular processes, such as ion pumping, that consume ATP. As a result, both the rate of substrate depletion and the rate of lactic acid production are slowed greatly. The second is an exploitation of the extensive buffering capacity of the turtle's shell and skeleton to neutralize the large amount of lactic acid that eventually accumulates. Two separate shell mechanisms are involved: release of carbonate buffers from the shell and uptake of lactic acid into the shell where it is buffered and sequestered. Together, the metabolic and buffering mechanisms permit animals to survive for 3–4 months at 3 °C with no O₂ and with circulating lactate levels of 150 mmol l⁻¹ or more.     

BRCA1 and pancreatic cancer: pedigree findings and their causal relationships

Anecdotal reports and series studies indicate that 5-10% of pancreatic cancer (PC) cases are familial. In addition, PC is associated with a variety of hereditary cancer syndromes. PC appears to be an integral cancer in the hereditary breast-ovarian cancer (HBOC) syndrome, with most notice given to the role of BRCA2. Our purpose is to call attention to BRCA1, which also predisposes to PC. Using data from our familial breast cancer registry, we identified 19 BRCA1/2 families that contain PC affecteds in the pedigrees, 15 with BRCA1 mutations and 4 with BRCA2 mutations. The association between BRCA2 and pancreatic cancer is well established; however, a definite link with pancreatic cancer in families carrying a BRCA1 mutation has been far less studied. Thus, the focus of this report is on 9 of the 15 BRCA1 families, in which PC affecteds were either confirmed carriers of the BRCA1 mutation or were inferred as probable obligate BRCA1 mutation carriers. The numbers are small, but nevertheless illustrate the finding of others of an apparent association between PC and BRCA1-mutation-bearing families. Given the dismal prognosis of PC, with the only current hope for survival being through surgical extirpation of the pancreas prior to metastasis, it is prudent that we realize the potential predisposition toward PC via BRCA1, in the hope of early diagnosis and prevention.     

An investigation of the disclosure process and support needs of BRCA1 and BRCA2 carriers

Disclosure of the results of a positive genetic mutation to offspring can be challenging. The purpose of this study was to investigate the content and process of disclosure from BRCA1/2 carriers to their offspring. A semi-structured questionnaire focused on the disclosure processes between parent and offspring. Thirty-one/40 mothers with BRCA1/2 mutations completed the cross-sectional survey. Sixteen carriers (51.6%) chose to disclose their results to all of their children, thirteen carriers (41.9%) chose not to disclose their results, and two carriers (6.5%) chose to disclose to some of their children. The age of a child appeared to be the most significant contributing factor in the decision to disclose. The mean age of the offspring who learned of the positive test result was 24.3 years with most carriers advocating the ideal age range for disclosure from 19 to 25 years. There was a discrepancy between actual and potential disclosure topics between those who had disclosed and those who had not disclosed at the time of the survey. Women who disclosed their result tended to do so alone, within a week of learning their own results, equally to male and female offspring and expressed that the relationships between themselves and their children had strengthened since revealing the presence of a genetic mutation in the family. Women who had not disclosed the results of their genetic test to offspring were significantly more interested in receiving additional individual counseling, educational videos, and email newsletters that focus on disclosure of this complex and life altering information compared to those who had already disclosed. Disclosure of BRCA1/2 results is determined primarily by age of offspring, is usually done by women alone, relatively soon after receiving results and appears to enhance the relationships between mothers and offspring. Both disclosed and non-disclosed carriers demonstrated significant interest in a variety of interventions to support the disclosure process.     

Expression and localization of mutant p16 proteins in melanocytic lesions from familial melanoma patients

Little is known about the correlation between the loss of p16 expression and tumor progression in familial melanoma; no systematic study has been conducted on p16 expression in melanocytic tumors from patients carrying germline CDKN2A mutations. We analyzed 98 early primary lesions from familial patients, previously tested for germline CDKN2A status, by quantitative immunohistochemistry using 3 p16 antibodies. We found that p16 expression was inversely correlated with tumor progression and was significantly lower in melanomas, including in situ lesions, than in nevi. Of other features analyzed, tumor thickness showed the most significant correlation with p16 levels. Lesions from mutation-negative patients displayed combined nuclear and cytoplasmic staining. However, some mutation-positive lesions (ie, G101W, 113insR, M53I, R24P, and 33ins24), including benign nevi, showed nuclear mislocalization, confirming previous studies suggesting that subcellular distribution indicates functional impairment of p16.     

Selection of strains for quality assessment of the disk induction method for detection of inducible clindamycin resistance in Staphylococci: a CLSI collaborative study

A nine-laboratory collaborative study was conducted to select positive and negative quality assessment control strains for the detection of inducible clindamycin resistance in staphylococci. Four strains of Staphylococcus aureus were tested as unknowns on 10 different days in each laboratory using the recently recommended CLSI (formerly NCCLS) disk diffusion method and the inoculum purity control method. Strains contained either macrolide-lincosamide-streptogramin B (MLSB) resistance genes encoded by erm(A) or erm(C) or a macrolide resistance efflux pump encoded by msr(A). Based upon the results of this study, strain UT 32 (now designated ATCC strain BAA-977) containing erm(A) is recommended as the positive control organism for inducible clindamycin resistance. Strain UT 25 (now designated ATCC BAA-976), which harbors the efflux pump encoded by msr(A), is recommended as the negative control organism.     

Structure and genomic sequence of the myotonic dystrophy (DM kinase) gene

The mutation causing myotonic dystrophy (DM) has recently beenidentified as an unstable CTG trinucleotide repeat located inthe 3' untranslated region of a gene encoding for a proteinwith putative serine-threonine protein kinase activity. In thisreport we present the genomic sequences of the human and murineDM kinase gene. A comparison of these sequences with each otherand with known cDNA sequences from both species, led us to predicta translation initiation codon, as well as determine the organizationof the DM kinase gene. Several polymorphisms within the humanDM kinase gene have been identified, and PCR assays to detecttwo of these are described. The complete sequence and characterizationof the structure of the DM kinase gene, as well as the identificationof novel polymorphisms within the gene, represent an importantstep in a further understanding of the genetics of myotonicdystrophy and the molecular biology of the gene.