Multiplexed high-throughput immune cell imaging in patients with high-risk triple negative early breast cancer: Analysis from the International Breast Cancer Study Group (IBCSG) Trial 22-00.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer (BC) subtype, with dismal prognosis and limited option in advanced settings, yet stromal tumor infiltrating lymphocytes (sTILs) in this subtype has a predictive role. PATIENTS AND METHODS: The International Breast Cancer Study Group (IBCSG) Trial 22-00 is a randomized phase III clinical trial testing the efficacy of low-dose metronomic oral Cyclophosphamide-Methotrexate (CM) maintenance following standard adjuvant chemotherapy treatment for early-stage hormone receptor-negative breast cancer patients. A case-cohort sampling was used. We characterized immune cells infiltrates in patients with TNBC by 6 plex immunofluorescence (IF) staining for CD4, FOXP3, CD3, cytokeratine and CD8 RESULTS: We confirmed that high immune CD3+ T cells as well as stromal and intra-epithelial Tregs (CD4+Foxp3+ T cells) infiltrates were associated with a better Distant Recurrence-Free Interval (DRFI), especially in LN+ patient, regardless of the treatment. More importantly, we showed that the spatial distribution of immune cells at baseline is crucial, as CM maintenance was detrimental for T cells excluded LN+ TNBC patients. CONCLUSIONS: immune spatial classification on immune cells infiltrates seems crucial and could help patients' selection in clinical trial and greatly improve responses to specific therapies.

Propentofylline-induced astrocyte modulation leads to alterations in glial glutamate promoter activation following spinal nerve transection.

We have previously shown that the atypical methylxanthine, propentofylline, reduces mechanical allodynia after peripheral nerve transection in a rodent model of neuropathy. In the present study, we sought to determine whether propentofylline-induced glial modulation alters spinal glutamate transporters, glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST) in vivo, which may contribute to reduced behavioral hypersensitivity after nerve injury. In order to specifically examine the expression of the spinal glutamate transporters, a novel line of double transgenic GLT-1-enhanced green fluorescent protein (eGFP)/GLAST-Discosoma Red (DsRed) promoter mice was used. Adult mice received propentofylline (10 mg/kg) or saline via i.p. injection starting 1 h prior to L5-spinal nerve transection and then daily for 12 days. Mice receiving saline exhibited punctate expression of both eGFP (GLT-1 promoter activation) and DsRed (GLAST promoter activation) in the dorsal horn of the spinal cord, which was decreased ipsilateral to nerve injury on day 12. Propentofylline administration reinstated promoter activation on the injured side as evidenced by an equal number of eGFP (GLT-1) and DsRed (GLAST) puncta in both dorsal horns. As demonstrated in previous studies, propentofylline induced a concomitant reversal of L5 spinal nerve transection-induced expression of glial fibrillary acidic protein (GFAP). The ability of propentofylline to alter glial glutamate transporters highlights the importance of controlling aberrant glial activation in neuropathic pain and suggests one possible mechanism for the anti-allodynic action of this drug.

Full-length single-gene cDNA libraries: applications in splice variant analysis.

Alternative splicing of pre-mRNA may generate many distinct proteins from a single gene: regulation of alternative exon selection constitutes control of molecular structure downstream of transcription. Identifying natural splice variants among hundreds or thousands of theoretical alternatives, and examining the regulation of exon selection at multiple sites, may require screening many full-length cDNAs. We describe methods for preparing full-length cDNA libraries comprising the splice variants from single genes. The methods employ robust long distance reverse transcription, gene-specific second strand synthesis, long PCR, and cloning: with these methods cDNAs coding full-length open reading frames were prepared for 21 ion channels (1.2-15 kb). Exon combinations in isolated clones are determined by multiplex PCR. Approximately 85% of the clones contain full-length inserts. Screening can detect even rare variants (0.1%) in linear proportion to their abundance in initial mRNA pools. Tissue-specific expression patterns are reproducible. We describe methods for quantifying and minimizing artifactual exon recombination by template switching. These methods can be used to generate thousands of full-length clones of even large transcripts (>8 kb) for the systematic identification of splice variants and the analysis of regulation of alternative exon selection.

The type 1 inositol 1,4,5-trisphosphate receptor gene is altered in the opisthotonos mouse.

The opisthotonos (opt) mutation arose spontaneously in a C57BL/Ks-db2J colony and is the only known, naturally occurring allele of opt. This mutant mouse was first identified based on its ataxic and convulsive phenotype. Genetic and molecular data presented here demonstrate that the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) protein, which serves as an IP3-gated channel to release calcium from intracellular stores, is altered in the opt mutant. A genomic deletion in the IP3R1 gene removes two exons from the IP3R1 mRNA but does not interrupt the translational reading frame. The altered protein is predicted to have lost several modulatory sites and is present at markedly reduced levels in opt homozygotes. Nonetheless, a strong calcium release from intracellular stores can be elicited in cerebellar Purkinje neurons treated with the metabotropic glutamate receptor (mGluR) agonist quisqualate (QA). QA activates Group 1 mGluRs linked to GTP-binding proteins that stimulate phospholipase C and subsequent production of the intracellular messenger IP3, leading to calcium mobilization via the IP3R1 protein. The calcium response in opt homozygotes shows less attenuation to repeated QA application than in control littermates. These data suggest that the convulsions and ataxia observed in opt mice may be caused by the physiological dysregulation of a functional IP3R1 protein.

A glial-specific voltage-sensitive Na channel gene maps close to clustered genes for neuronal isoforms on mouse chromosome 2.

A variety of glial cell types express saxitoxin (STX)-binding voltage-sensitive Na channels (1,2), although the possible role of impulse conduction in these cells is not understood. Gautron et al. (1992) recently identified a 7.5 kb species of mRNA in type 1 astrocytes cultured from rat brain cerebrum that hybridized with a "common" Na channel probe but not with brain isoform-specific cDNA probes. Sequence data from cloned cDNAs demonstrate that it encodes a structurally atypical Na channel isoform. We have prepared a cDNA probe specific for a portion of subunit domain IV of the glial channel and mapped the location of the corresponding gene (Scn7a) to mouse chromosome 2. The Scn7a gene mapped 0.9 (+/- 0.9) cM distal to the Gcg locus; the location of the corresponding human gene (SCN7A) is predicted to be in the q36-q37 region of chromosome 2. This site lies just outside a cluster of genes for the brain-specific Na channel isoforms RI, RII and RIII which map proximal to Gcg (17). The presence of at least four genes from two distinct Na channel subfamilies suggests that multiple genetic defects for central and peripheral nervous system disorders ultimately may be linked to this area.