Coordinate expression of β1 and β2 integrin “activation” epitopes during T cell responses in secondary lymphoid tissue

The monoclonal antibodies (mAb) 15/7 and 24 recognize unique activation-dependent, conformational epitopes on β1 and β 2-integrins, respectively. The expression of both of these epitopes closely correlates with the ligand binding ability of their respective integrins, and thus serves as indicators of functional integrin “activation”. Here, we have used six-parameter flow cytometry to examine the expression of these epitopes and conventional β1- and β2-integrin epitopes during human T cell activation in secondary lymphoid tissues in vivo, focusing particularly on the virgin to memory/effector cell transition. Fresh tonsil lymphocytes were stained with mAb against conventional or activation-dependent integrin epitopes, followed by staining with mAb against CD3, CD45RA, and CD45RO, thus allowing the determination of integrin epitope expression on virgin (CD3⁺) T cells (CD45RA⁺/RO^?to±), memory/effector (CD45RA^?/RO⁺⁺) T cells, and T cells undergoing the virgin to memory/effector transition: transition region-1 (T1; CD45RA^+to++/RO⁺); -2 (T2; CD45RA⁺⁺/RO⁺⁺); and -3 (T3; CD45RA⁺/RO⁺⁺). Conventional β1- and β2-integrin epitopes progressively increase during the virgin to T3 stages of the transition in tonsil, in keeping with the generally higher levels of these adhesion molecules on memory/effector vs. virgin T cells. Expression of both the β1 (15/7)-and β2 (24)-integrin activation epitopes first appears on transitional T cells, and is maintained on a relatively constant number of cells (averaging 25-30%) throughout the T1-T3 stages. These epitopes are also noted on a subset of activated memory/effector T cells. Importantly, on both transitional and activated memory/effector T cell subsets, the expression patterns of the 15/7 and 24 epitopes vs. a variety of T cell activation antigens are identical, and the expression of these epitopes relative to each other is linearly correlated, findings strongly supporting the coordinate activation of β1 and β2 integrins duringT cell activation in vivo. These results provide the first evidence of integrin activation during an in vivo immunologic response, and demonstrate the usefulness of mAb recognizing conformational epitopes and multiparameter flow cytometry in delineating the dynamic interplay of adhesion molecules during complex physiologic processes.     

Peripheral blood tumor cell load reflects the clinical activity of the disease in patients with carcinoma of the breast

Peripheral blood was examined for the presence of tumor cells at multiple time points over a one-year period in 45 patients with a history of surgical resection of breast carcinoma. The number of circulating epithelial cells in 8 of 8 patients with clinically active disease concurred or preceded changes in the disease activity. In 12 of 37 patients with no evidence of disease epithelial cells were found at least at one time point at a frequency larger than the control group. One patient had a recurrence of the breast cancer 4 weeks after circulating epithelial cells were detected in the blood.     

Functional properties of alpha7 nicotinic acetylcholine receptors co-expressed with RIC-3 in a stable recombinant CHO-K1 cell line

Heterologous functional expression of alpha7 nicotinic acetylcholine receptors (nAChRs) is difficult to achieve in mammalian cell lines, and the reasons have been associated with a lack of expression of the putative chaperone factor RIC-3. Here, we describe the generation and functional and pharmacological characterization of a Chinese hamster ovary (CHO)-K1 cell line co-expressing the human alpha7 nAChR and RIC-3. Stable recombinant cells expressing alpha7 nAChR on the plasma membrane were selected by binding of fluorochrome-conjugated alpha-bungarotoxin and fluorescence-activated cell sorting. The presence of functional alpha7 channels was demonstrated by whole cell patch clamp recordings. Nicotine and acetylcholine induced rapid desensitizing currents with 50% effective concentration values of 14 and 37 microM, respectively, with agonist-evoked currents detected in approximately 75% of the cell population. Surprisingly, when tested in a FLIPR (Molecular Devices, Sunnyvale, CA) Ca(2+) assay, activation of alpha7 nAChRs was measured only when nicotinic agonists were applied either in the presence of the positive allosteric modulator (PAM) PNU-120596 or after pretreatment of cells with the tyrosine kinase inhibitor genistein. No Ca(2+) influx was measured upon addition of agonists alone or together with allosteric potentiators such as 5-hydroxyindole that predominantly increase the apparent peak amplitude without robustly affecting the current desensitization rate, as exemplified by PNU-120596. These results show that functional alpha7 nAChRs can stably be expressed in the non-neuronal CHO-K1 cell line. This recombinant cell system is useful for characterization of alpha7 nAChRs and to study the mechanism of action of chemical modulators, in particular the detection of PAMs capable of slowing receptor desensitization kinetics.     

Control of head lice with a coconut-derived emulsion shampoo

Objective   To evaluate a novel coconut-derived emulsion (CDE) shampoo against head lice infestation in children.
Design   A school trial in which pupils were treated on days 0 and 7 and checked on days 8 and 15 and a family trial where product was applied by parents three times in 2 weeks or used as a cosmetic shampoo and checked on days 14 and days 70.
Setting   UK schools in Bristol and Western-super-Mare and families in Northern Somerset.
Main outcome measure   Numbers of children free from infestation after treatment.
Results   In the school trial, percentage cures at day 8 were 14% (permethrin, n  = 7) and 61% (CDE, n  = 37). In the family trial where all family members were treated, cure rate was 96% ( n  = 28), and if the shampoo was subsequently used as a cosmetic shampoo, only 1 of 12 children became re-infested after 10 weeks.
Conclusion   CDE shampoo is a novel effective method of controlling head lice and used after treatment as a cosmetic shampoo can aid in the reduction of re-infestation.     

Pigmented basal cell carcinoma--comparing the diagnostic methods of SIAscopy and dermoscopy

Pigmented basal cell carcinomas can be difficult to distinguish clinically from melanoma. Dermoscopy has proven to be useful in the differential diagnosis of the two tumour types. SIAscopy (Spectrophotometric intracutaneous analysis) is a fairly new technique of imaging pigmented skin lesions that has been presented previously as a useful tool in diagnosing melanoma. The aim of this study was to evaluate whether SIAscopy can be useful in diagnosing pigmented basal cell carcinomas. Twenty-one pigmented basal cell carcinomas were analysed, comparing dermoscopic and SIAscopic findings. The results, in this limited setting, show that SIAscopy has no advantages over dermoscopy when diagnosing pigmented basal cell carcinomas. On the contrary, pigmented basal cell carcinomas show, in SIAscopy, similar features to those previously reported for melanoma.     

Single tube liquid biopsy for advanced non-small cell lung cancer

The need for a liquid biopsy in non-small cell lung cancer (NSCLC) patients is rapidly increasing. We studied the relation between overall survival (OS) and the presence of four cancer biomarkers from a single blood draw in advanced NSCLC patients: EpCAM^high circulating tumor cells (CTC), EpCAM^low CTC, tumor-derived extracellular vesicles (tdEV) and cell-free circulating tumor DNA (ctDNA). EpCAM^high CTC were detected with CellSearch, tdEV in the CellSearch images and EpCAM^low CTC with filtration after CellSearch. ctDNA was isolated from plasma and mutations present in the primary tumor were tracked with deep sequencing methods. In 97 patients, 21% had ≥2 EpCAM^high CTC, 15% had ≥2 EpCAM^low CTC, 27% had ≥18 tdEV and 19% had ctDNA with ≥10% mutant allele frequency. Either one of these four biomarkers could be detected in 45% of the patients and all biomarkers were present in 2%. In 11 out of 16 patients (69%) mutations were detected in the ctDNA. Two or more unfavorable biomarkers were associated with poor OS. The presence of EpCAM^high CTC and elevated levels of tdEV and ctDNA was associated with a poor OS; however, the presence of EpCAM^low CTC was not. This single tube approach enables simultaneous analysis of multiple biomarkers to explore their potential as a liquid biopsy.     

RRM1 variants cause a mitochondrial DNA maintenance disorder via impaired de novo nucleotide synthesis

Mitochondrial DNA (mtDNA) depletion/deletions syndromes (MDDS) encompass a clinically and etiologically heterogenous group of mitochondrial disorders caused by impaired mtDNA maintenance. Among the most frequent causes of MDDS are defects in nucleoside/nucleotide metabolism, which is critical for synthesis and homeostasis of the deoxynucleoside triphosphate (dNTP) substrates of mtDNA replication. A central enzyme for generating dNTPs is ribonucleotide reductase, a critical mediator of de novo nucleotide synthesis composed of catalytic RRM1 subunits in complex with RRM2 or p53R2. Here, we report 5 probands from 4 families who presented with ptosis and ophthalmoplegia as well as other clinical manifestations and multiple mtDNA deletions in muscle. We identified 3 RRM1 loss-of-function variants, including a dominant catalytic site variant ({"type":"entrez-protein","attrs":{"text":"NP_001024.1","term_id":"4506749"}}NP_001024.1: p.N427K) and 2 homozygous recessive variants at p.R381, which has evolutionarily conserved interactions with the specificity site. Atomistic molecular dynamics simulations indicate mechanisms by which RRM1 variants affect protein structure. Cultured primary skin fibroblasts of probands manifested mtDNA depletion under cycling conditions, indicating impaired de novo nucleotide synthesis. Fibroblasts also exhibited aberrant nucleoside diphosphate and dNTP pools and mtDNA ribonucleotide incorporation. Our data reveal that primary RRM1 deficiency and, by extension, impaired de novo nucleotide synthesis are causes of MDDS.     

Classification of chronic daily headache by International Headache Society criteria: limits and new proposals

We conducted a retrospective study of 150 patients with chronic daily headache (CDH) to determine how to categorize their headache according to the classification of the International Headache Society (IHS). All patients were first evaluated at Parma and Pavia Headache Centres (from January 1992 to March 1993) and had had headache for at least 15 days a month during the previous 6 months. Four patients were thereafter excluded due to poor reliability. The 146 patients who met our CDH criteria (92 with and 54 without clear-cut migraine attacks) could be classified into four groups: (i) chronic tension-type headache (CTTH)-27 patients; (ii) coexisting migraine plus CTTH-65 patients; (iii) unclassifiable daily headache-27 patients; and (iv) migraine and an unclassifiable interval headache-27 patients. Seventy-two percent of patients with CDH had migraine as the initial form of their headache. We therefore propose to revise the IHS classification for migraine, taking into account its evolution, and add two subcategories, migraine with interparoxysmal headache and chronic migraine.