Transplantation of embryonic noradrenergic neurons in two models of adult rat spinal cord injury: ultrastructural immunocytochemical study

The synaptic connections established by grafted noradrenergic (NA) neurons into the lesioned adult rat spinal cord were analysed using immunocytochemistry at the electron microscopic level. An embryonic cell suspension of the locus coeruleus region from E-13 rat embryos was transplanted into the spinal cord following either: (1) spinal cord transection or (2), partial selective denervation by 6-hydroxy dopamine (6-OH DA). One month after grafting, the NA-neurons established, in the two models, an innervation pattern similar to that found in the intact spinal cord. In both models, the transplanted NA-immunoreactive neurons formed extensive synaptic contacts with dendrites, spines and perikarya. The proportion of axodendritic and axospinous contacts was inverse in the two models. The first model thus reproduced more closely the normal synaptic pattern prefering dendritic targets, which could correspond to a better integration of the graft. In the second model, a partially NA-denervated spinal cord, there existed a competition between residual intrinsic and grafted neuron-derived fibres, which presumably affects synaptogenesis. In conclusion, the present study illustrate the complexity of cell interations conducting to the formation of a specific circuitry. Recognition phenomenon are likely modulated by space constraints, which ultimately shape-up the geometry of synaptic contacts.     

Strain-Dependent Migration of CD4 and CD8 Lymphocyte Subsets to Lymph Nodes in NOD (Nonobese Diabetic) and Control Mice

Subpopulations of lymphoid cells were compared with respect to their ability to migrate into peripheral lymphoid organs of nonobese diabetic (NOD) mice and various strains of control mice. In short-term, in vivo homing studies, no major differences in the pattern of homing of B and T cells were observed among all mouse strains studied. On the other hand, CD4 cells localized consistently more efficiently than CD8 cells in both PP and LN of adult NOD and BALB/c mice, whereas both populations migrated roughly equivalently in LN of adult DBA/2, CBA, and C57BL/6 mice. No age-dependent differences in the homing of CD4 and CD8 cells were observed in BALB/c mice. On the contrary, in 2-week-old NOD mice, CD4 and CD8 cells migrated equally well. The preferential entry of CD4 cells in adult NOD and BALB/c did not result from increased blood transit time of CD8 cells. On the other hand, the preferential migration of CD8 cells was observed in the liver, whereas the two T-cell subsets migrated equally well in the lungs. The differences in the homing characteristics of CD4 and CD8 cells among NOD, BALB/c, and C57BL/6 mice were not related to modifications in the level of expression of adhesion molecules such as MEL-14, LFA-1, and Pgp-1.     

Circulating tumor DNA predicts efficacy of a dual AKT/p70S6K inhibitor (LY2780301) plus paclitaxel in metastatic breast cancer: plasma analysis of the TAKTIC phase IB/II study

The phosphatidylinositol‐3‐kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is frequently activated in HER2‐negative breast cancer and may play a role in taxane resistance. The phase IB/II TAKTIC trial ({"type":"clinical-trial","attrs":{"text":"NCT01980277","term_id":"NCT01980277"}}NCT01980277) has shown that combining a dual AKT and p70 ribosomal protein S6 kinase (p70S6K) inhibitor (LY2780301) taken orally with weekly paclitaxel in HER2‐negative advanced breast cancer is feasible, with preliminary evidence of efficacy. We wanted to explore whether circulating tumor DNA (ctDNA) may be a surrogate marker of treatment efficacy in this setting. Serial plasma samples were collected and cell‐free DNA was sequenced using low‐coverage whole‐genome sequencing, and analysis was completed with droplet digital polymerase chain reaction (PCR) for some patients with driver mutations. Baseline tumor fraction (TF) and TF after 7 weeks on treatment were compared to progression‐free survival (PFS) and the overall response rate. We also explored circulating copy number alterations associated with treatment failure. Of the 51 patients enrolled in the TAKTIC trial, at least one plasma sample was available for 44 cases (96 timepoints). All patients with tumor TP53, PI3KCA, or AKT1 mutations harbored at least one of these alterations in plasma. TF at inclusion was correlated with PFS (6m‐PFS was 92% for ctDNAneg patients vs 68% for ctDNApos cases; hazard ratio [HR] = 3.45, 95% confidence interval [CI] [1.34–8.90], P = 0.007). ctDNA status at week 7 was not correlated with prognosis. Even though most circulating copy number alterations were conserved at disease progression, some genomic regions of interest were altered in post‐progression samples. In conclusion, ctDNA detection at baseline was associated with shorter PFS in patients included in the TAKTIC trial. Plasma‐based copy number analysis may help to identify alterations involved in resistance to treatment.     

Intracellular Tracking of Single-plasmid DNA Particles After Delivery by Electroporation

Electroporation is a physical method of transferring molecules into cells and tissues. It takes advantage of the transient permeabilization of the cell membrane induced by electric field pulses, which gives hydrophilic molecules access to the cytoplasm. This method offers high transfer efficiency for small molecules that freely diffuse through electrically permeabilized membranes. Larger molecules, such as plasmid DNA, face several barriers (plasma membrane, cytoplasmic crowding, and nuclear envelope), which reduce transfection efficiency and engender a complex mechanism of transfer. Our work provides insight into the way electrotransferred DNA crosses the cytoplasm to reach the nucleus. For this purpose, single-particle tracking experiments of fluorescently labeled DNA were performed. Investigations were focused on the involvement of the cytoskeleton using drugs disrupting or stabilizing actin and tubulin filaments as the two relevant cellular networks for particle transport. The analysis of 315 movies (~4,000 trajectories) reveals that DNA is actively transported through the cytoskeleton. The large number of events allows a statistical quantification of the DNA motion kinetics inside the cell. Disruption of both filament types reduces occurrence and velocities of active transport and displacements of DNA particles. Interestingly, stabilization of both networks does not enhance DNA transport.     

Direct Control of Brown Adipose Tissue Thermogenesis by Central Nervous System Glucagon-Like Peptide-1 Receptor Signaling

We studied interscapular brown adipose tissue (iBAT) activity in wild-type (WT) and glucagon-like peptide 1 receptor (GLP-1R)–deficient mice after the administration of the proglucagon-derived peptides (PGDPs) glucagon-like peptide (GLP-1), glucagon (GCG), and oxyntomodulin (OXM) directly into the brain. Intracerebroventricular injection of PGDPs reduces body weight and increases iBAT thermogenesis. This was independent of changes in feeding and insulin responsiveness but correlated with increased activity of sympathetic fibers innervating brown adipose tissue (BAT). Despite being a GCG receptor agonist, OXM requires GLP-1R activation to induce iBAT thermogenesis. The increase in thermogenesis in WT mice correlates with increased expression of genes upregulated by adrenergic signaling and required for iBAT thermogenesis, including PGC1a and UCP-1. In spite of the increase in iBAT thermogenesis induced by GLP-1R activation in WT mice, Glp1r^−/− mice exhibit a normal response to cold exposure, demonstrating that endogenous GLP-1R signaling is not essential for appropriate thermogenic response after cold exposure. Our data suggest that the increase in BAT thermogenesis may be an additional mechanism whereby pharmacological GLP-1R activation controls energy balance.The increasing incidence of type 2 diabetes (T2D) and obesity worldwide has prompted the need for new therapies. Agonism of the receptor for glucagon-like peptide-1 (GLP-1) is currently one of the most successfully and widely used therapies for T2D. GLP-1 is a product of proglucagon that also gives rise to glucagon (GCG) and oxyntomodulin (OXM) (1). Both GLP-1 and its receptor (GLP-1R) are expressed in peripheral tissues and in areas of the central nervous system (CNS) involved in the control of energy balance. Treatment with GLP-1R agonists improves glycemic control and reduces body weight in diabetic humans (2). Studies in animals have demonstrated that CNS–GLP-1R signaling contributes to the body weight–reducing effect of these agonists (3).GCG is produced in the α cells of the pancreatic islets and is involved in the maintenance of euglycemia. Although its exogenous administration induces body weight loss associated with anorexia and increased energy expenditure (4), GCG has been traditionally dismissed as a potential drug target because of its diabetogenic effects. However, recent preclinical data have shown that simultaneous activation of both GLP-1R and GCG receptor (GCGR) leads to greater efficacy in both glycemic control and weight loss than the activation of GLP-1R alone (5,6).OXM can bind to and activate both GLP-1R and GCGR (7), and studies with rodents (8,9) and humans (10) suggest that it may be efficacious in treating obesity and diabetes. OXM regulates feeding, at least in part, through GLP-1R (7,11). There is evidence that OXM action in the CNS reduces body weight by increasing energy expenditure (12). This may involve activation of brown adipose tissue (BAT) metabolism, since intracerebroventricular (ICV) administration of OXM reduces the weight of interscapular BAT (iBAT) pads and increases body temperature in rats (12). The relative contribution of GLP-1R and GCGR to this process has never been investigated; however, it is known that GCG regulates iBAT activity, and this may be, at least in part, centrally mediated (13). The contribution of GLP-1R to the control of energy expenditure, and more specifically to BAT metabolism, remains largely unknown.The sympathetic nervous system (SNS) is essential for control of BAT metabolism by the CNS (14) and is involved in the CNS–GLP-1R control of lipid metabolism in white adipose tissue (WAT) (15). This, in addition to the evidence that GCG may increase BAT thermogenesis through actions in the CNS (13), led us to hypothesize that the action(s) of GCGR and GLP-1R in the brain controls BAT thermogenesis through the SNS. Here, we show that central administration of both GCGR and GLP-1R agonists increased SNS activity to iBAT and induced thermogenesis. Thus, we propose that CNS–GLP-1R may contribute to the control of energy balance by regulating BAT thermogenesis. The existence of functional BAT in adult humans has now been determined (16–18), and effort needs to be directed toward a better understanding of the regulation of this tissue as a target for antiobesity therapeutics. The increase in BAT metabolism described here may contribute to the weight loss induced by GCGR and GLP-1R agonists in both animal models and humans.     

PET/CT assessment in follicular lymphoma using standardized criteria: central review in the PRIMA study

Purpose

We aimed to compare the standardized central review of ¹⁸F-fluorodeoxyglucose (FDG) positron emission tomography (PET)/CT scans performed after induction therapy for follicular lymphoma (FL) in the PRIMA study (Salles et al., Lancet 377:42–51, 2011; Trotman et al., J Clin Oncol 29:3194–3200, 2011) to scan review at local centres.

Methods

PET/CT scans were independently evaluated by two nuclear medicine physicians using the 2007 International Harmonization Project (IHP) criteria (Cheson et al., J Clin Oncol 25:579–586, 2007; Juweid et al., J Clin Oncol 25:571–578, 2007; Shankar et al., J Nucl Med 47:1059–1066, 2006) and Deauville 5-point scale (5PS) criteria (Meignan et al., Leuk Lymphoma 50:1257–1260, 2009; Meignan et al., Leuk Lymphoma 51:2171–2180, 2010; Barrington et al., Eur J Nucl Med Mol Imaging 37:1824–1833, 2010). PET/CT status was compared with prospectively recorded patient outcomes.

Results

Central evaluation was performed on 119 scans. At diagnosis, 58 of 59 were recorded as positive, with a mean maximum standardized uptake value (SUV_max) of 11.7 (range 4.6–35.6). There was no significant association between baseline SUV_max and progression-free survival (PFS). Sixty post-induction scans were interpreted using both the IHP criteria and 5PS. Post-induction PET-positive status failed to predict progression when applying the IHP criteria [p?=?0.14; hazard ratio (HR) 1.9; 95 % confidence interval (CI) 0.8–4.6] or 5PS with a cut-off ≥3 (p?=?0.12; HR 2.0; 95 % CI 0.8–4.7). However, when applying the 5PS with a cut-off ≥4, there was a significantly inferior 42-month PFS in PET-positive patients of 25.0 % (95 % CI 3.7–55.8 %) versus 61.4 % (95 % CI 45.4–74.1 %) in PET-negative patients (p?=?0.01; HR 3.1; 95 % CI 1.2–7.8). The positive predictive value (PPV) of post-induction PET with this liver cut-off was 75 %. The 42-month PFS for patients remaining PET-positive by local assessment was 31.1 % (95 % CI 10.2–55.0 %) vs 64.6 % (95 % CI 47.0–77.6 %) for PET-negative patients (p?=?0.002; HR 3.3; 95 % CI 1.5–7.4), with a PPV of 66.7 %.

Conclusion

We confirm that FDG PET/CT status when applying the 5PS with a cut-off ≥4 is strongly predictive of outcome after first-line immunochemotherapy for FL. Further efforts to refine the criteria for assessing minimal residual FDG uptake in FL should provide a reproducible platform for response assessment in future prospective studies of a PET-adapted approach.     

Alport syndrome associated with diffuse leiomyomatosis: COL4A5-COL4A6 deletion associated with a mild form of Alport nephropathy.

BACKGROUND: The X-linked Alport syndrome (AS) is an inherited nephropathy due to mutations in the COL4A5 gene, encoding the alpha5 chain of type IV collagen, a major component of the glomerular basement membrane (GBM). Here, we report a new kindred with the rare association of X-linked AS and diffuse leiomyomatosis (DL), which is a tumourous process involving smooth muscle cells of the oesophagus, the tracheobronchial tree and, in females, the genital tract. For this syndrome, an almost constant association of large COL4A5 rearrangements with a severe juvenile form of nephropathy has been described for male patients. METHODS: DNA rearrangement at the COL4A5-COL4A6 locus was studied in several members of this family using polymerase chain reaction and pulsed field gel electrophoresis. Furthermore, immunohistochemical staining of tumour and skin samples was performed. RESULTS: The affected patients in this family carry a 120 kb deletion by which the COL4A5 exon 1 and COL4A6 exons 1, 1', and 2 are removed. Immunohistochemical investigation of a skin biopsy of an affected male patient confirmed the absence of both the alpha5 and the alpha6 chains of type IV collagen in the basement membrane of the skin. Surprisingly, both affected male patients had a rather mild renal phenotype. CONCLUSIONS: This report shows that, contrary to what has been reported to date, patients suffering from AS associated with DL can be associated with a late onset renal failure (adult) form of nephropathy.     

Autosomal dominant polycystic kidney disease: risk factor for nonmelanoma skin cancer following kidney transplantation

Nonmelanoma skin cancers (NMSC) are the most common malignant tumors following solid organ transplantation. Risk factors for NMSC mainly include immunosuppression, age, sun exposure and patient phototype. Recent findings have suggested that autosomal dominant polycystic kidney disease (ADPKD) may increase the risk of developing NMSC. We performed a monocenter retrospective study including all kidney recipients between 1985 and 2006 (n = 1019). We studied the incidence of NMSC, solid cancers and post‐transplantation lymphoproliferative disease (PTLD), and analyzed the following parameters: age, gender, phototype, time on dialysis, graft rank, immunosuppressive regimen, history of cancer and kidney disease (ADPKD versus others). Median follow‐up was 5.5 years (range: 0.02–20.6; 79 838 patient‐years). The cumulated incidence of NMSC 10 years after transplantation was 12.7% (9.3% for solid cancers and 3.5% for PTLD). Autosomal dominant polycystic kidney disease and age were risk factors for NMSC (HR 2.63; P < 0.0001 and HR 2.21; P < 0.001, respectively) using univariate analysis. The association between ADPKD and NMSC remained significant after adjustments for age, gender and phototype using multivariate analysis (HR 1.71; P = 0.0145) and for immunosuppressive regimens (P < 0.0001). Autosomal dominant polycystic kidney disease was not a risk factor for the occurrence of solid cancers after transplantation (HR 0.96; P = 0.89). Our findings suggest that ADPKD is an independent risk factor for developing NMSC after kidney transplantation.