Oxidized low‐density lipoprotein (oxLDL)‐induced cell death in dorsal root gangion cell cultures depends not on the lectin‐like oxLDL receptor‐1 but on the toll‐like receptor‐4

DRG cells have been found to undergo apoptosis and necrosis after oxidized low‐density lipoprotein (oxLDL) stimulation in vitro. However, the mechanism of oxLDL‐induced DRG cell death is unclear. For this reason, we studied the expression of two potential oxLDL receptors: lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1) and toll‐like receptor‐4 (TLR4) in dorsal root ganglion (DRG) cell cultures from postnatal rats. Cells were cultivated with and without oxLDL. In oxLDL‐treated DRG cell cultures, the increase of cleaved caspase‐3 protein was observed as a sign of enhanced apoptosis. Untreated and oxLDL‐treated DRG cell cultures expressed LOX‐1 and TLR4 at similar levels. The LOX‐1 expression remained unchanged after receptor blockade. However, the inhibition of LOX‐1 caused a significant increase of cleaved caspase‐3 and a decrease of TLR4 levels. The TLR4‐inhibited DRG cell cultures lacked changes in LOX‐1 expression for all experimental groups. The inhibition of TLR4 caused activation of jun N‐terminal kinase (JNK) and a significant decrease of cleaved caspase‐3 but did not change the TLR4 level. We conclude that LOX‐1 and TLR4 are expressed in cultivated rat DRG cells and that the oxLDL‐induced cell death in DRG cell cultures does not depend on the LOX‐1 but on the TLR4. © 2009 Wiley‐Liss, Inc.     

Baseline platelet size is increased in patients with acute coronary syndromes developing early stent thrombosis and predicts future residual platelet reactivity. A case-control study

Introduction

Pre-procedural predictors of early stent thrombosis (ST) and future response to platelet inhibitors are in demand. We sought to evaluate the impact of baseline platelet indices on the occurrence of early ST and future residual platelet reactivity.

Materials and methods

Hundred and eight patients with acute coronary syndromes (ACS) in whom stents were implanted were included: 36 consecutive ST cases and 72 matched controls. Platelet indices assessed with flow cytometry before stent implantation were retrieved from the department's data base. Residual platelet reactivity specific to aspirin (aspirin reaction units-ARU) and clopidogrel (P2Y12 reaction units-PRU) was assessed prospectively with VerifyNow^® under dual antiplatelet treatment.

Results

Platelet size reported as mean platelet volume (MPV) or proportion of large platelets (LPLT) was significantly higher in ST cases compared with controls (10.4, 95% confidence intervals [CI], 10.1-10.8 vs. 9.7, CI, 9.5-9.9, P = 0.0004 and 35.8, CI, 34.2-37.3 vs. 33.3, CI, 32.2-34.3, P = 0.007, respectively). Dual aspirin and clopidogrel poor-responsiveness was diagnosed significantly more often in ST cases than in controls (19.6% vs. 1.4%, P = 0.004), whereas no difference was observed for single aspirin or clopidogrel poor-responsiveness. A strong correlation was found between MPV and both, ARU (r = 0.66, P < 0.0001) and PRU (r = 0.55, P < 0.0001). Similarly, higher LPLT was associated with higher ARU (r = 0.47, P < 0.0001) and PRU (r = 0.38, P = 0.0001).

Conclusions

Baseline platelet size is increased in patients with ACS developing early ST and correlates with future residual platelet reactivity under aspirin and clopidogrel therapy. Dual but not isolated aspirin or clopidogrel poor-responsiveness appears to be associated with early ST.     

Surgical strategy for sporadic primary hyperparathyroidism an evidence-based approach to surgical strategy,patient selection,surgical access,and reoperations

Purpose  Progress in parathyroid imaging has brought substantial changes in the surgical strategy to approach patients with sporadic primary hyperparathyroidism (pHPT). The present review is focused on the safety and efficacy of limited parathyroid exploration. Materials and methods  Review of the literature focused on studies dealing with unilateral (two-gland exploration) or selective parathyroidectomy (one-gland exploration) in selected patients with pHPT and on the classification of published reports according to the degree of evidence. Results  Parathyroid exploration limited to a solitary parathyroid adenoma can be considered a minimally invasive procedure that can be performed by the minicervicotomy, video-assisted, or endoscopic approaches. In properly selected patients, it affords results comparable to those of four-gland bilateral exploration in terms of cure and recurrence. It causes less postoperative hypocalcemia. Conclusions  Selective parathyroidectomy is an option for patients with positive preoperative localization tests undergoing first-time surgery who have no family history of pHPT, no goiter for which surgical therapy is proposed, and are not on lithium therapy. This paper was presented at the “Primary HPT Symposium” organized by the European Society of Endocrine Surgeons (Lund, Sweden, March 19–21, 2009).     

Follow-up analysis of genome-wide association data identifies novel loci for type 1 diabetes

OBJECTIVE—Two recent genome-wide association (GWA) studies have revealed novel loci for type 1 diabetes, a common multifactorial disease with a strong genetic component. To fully utilize the GWA data that we had obtained by genotyping 563 type 1 diabetes probands and 1,146 control subjects, as well as 483 case subject–parent trios, using the Illumina HumanHap550 BeadChip, we designed a full stage 2 study to capture other possible association signals.RESEARCH DESIGN AND METHODS—From our existing datasets, we selected 982 markers with P < 0.05 in both GWA cohorts. Genotyping these in an independent set of 636 nuclear families with 974 affected offspring revealed 75 markers that also had P < 0.05 in this third cohort. Among these, six single nucleotide polymorphisms in five novel loci also had P < 0.05 in the Wellcome Trust Case-Control Consortium dataset and were further tested in 1,303 type 1 diabetes probands from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) plus 1,673 control subjects.RESULTS—Two markers (rs9976767 and rs3757247) remained significant after adjusting for the number of tests in this last cohort; they reside in UBASH3A (OR 1.16; combined P = 2.33 × 10⁻⁸) and BACH2 (1.13; combined P = 1.25 × 10⁻⁶).CONCLUSIONS—Evaluation of a large number of statistical GWA candidates in several independent cohorts has revealed additional loci that are associated with type 1 diabetes. The two genes at these respective loci, UBASH3A and BACH2, are both biologically relevant to autoimmunity.Type 1 diabetes is a multifactorial disease with a strong genetic component that results from autoimmune destruction of the pancreatic β-cells. The major type 1 diabetes susceptibility locus, mapping to the HLA class II genes at 6p21 (1) and encoding highly polymorphic antigen-presenting proteins, accounts for almost 50% of the genetic risk for type 1 diabetes (2). Several other loci with more modest effects are known, but they do not account for the remaining portion of the risk.The recent development of high-throughput single nucleotide polymorphism (SNP) genotyping array technologies has enabled us (3) and others (4) to perform high-density genome-wide association (GWA) studies in search of the remaining type 1 diabetes loci. We recently reported the outcome of our GWA for type 1 diabetes in a large pediatric type 1 diabetic cohort of European descent (3); in addition to confirming previously identified loci, we observed highly significant and replicated association with KIAA0350 (now renamed CLEC16A [C-type lectin domain family 16 member A]). Subsequent follow-up of our data also revealed a locus on 12q13 (5). In parallel and independently, the Wellcome Trust Case Control Consortium (WTCCC) (4) also demonstrated replicated (6) association to the same linkage disequlibrium blocks at 16p13 and 12q13, along with two additional loci on 12q24 and 18p11.The results that we have reported thus far were of loci that achieved statistical significance on the basis of the results of the GWA genotyping (stage 1) or replication in additional cohorts (stage 2) of only a small number of the most promising loci. Here, we describe the results of a full evaluation of all statistical candidates from the GWA phase.     

Clonotype analysis of cytomegalovirus-specific cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTL) control the replication of human cytomegalovirus (CMV). Previous studies assessed the clonotypic composition of CTL specific for individual immunodominant peptides within a certain HLA context. Such an approach has inherent limitations and may not assess the true clonal CTL response in vivo. Here, the clonotypic composition of CMV-specific CTL was determined in HLA-A2, CMV-seropositive kidney transplant recipients and healthy blood donors after stimulation of peripheral blood mononuclear cells with either a pp65 whole-peptide pool or a single immunodominant peptide. Even after stimulation with the whole peptide pool, CMV-specific CTL remained monoclonal or oligoclonal. Regarding intraindividual variation, the CDR3 motifs of the dominant clones were identical to those observed in CTL generated by the single immunodominant peptide. Sequencing of the CDR3 regions demonstrated significant interindividual variation; however, structural homology was observed for immunodominant clonotypes in three individuals. In conclusion, the highly focused T cell receptor repertoire found after stimulation with either a single immunodominant peptide or a peptide pool demonstrates a pivotal role for immunodominant epitopes in the generation of a clonal repertoire. These results provide new insights into the regulation of CMV clonal dominance and may contribute to the design and monitoring of adoptive immunotherapy.     

Endovascular treatment of arteriovenous malformation

Vascular anomalies are common congenital or neonatal abnormalities. According to the approved classification of vascular lesions by Glowacki and Mulliken, hemangiomas and vascular malformations are distinguishable. Hemangiomas usually appear during the first days or weeks after birth and grow faster than the whole body of the infant. They are proliferating benign tumors that often involute. The opposite of hemangiomas, vascular malformations are present at birth, grow commensurately with the patient, demonstrate normal endothelial turnover, and never involute. The case of a young woman with an arteriovenous malformation (AVM) located on the left side of her face beneath the lower lip is described. The patient did not have any specific complaints except the cosmetic effect, which was a reddish and bluish discoloration of the skin over the lesion. The AVM was embolized with polyvinyl alcohol, and no subsequent surgery was performed. Follow-up ultrasound examination after a 12-month period showed no flow within the lesion area.     

Chemical target validation studies of aminopeptidase in malaria parasites using alpha-aminoalkylphosphonate and phosphonopeptide inhibitors

During its intraerythrocytic phase, the most lethal human malarial parasite, Plasmodium falciparum, digests host cell hemoglobin as a source of some of the amino acids required for its own protein synthesis. A number of parasite endopeptidases (including plasmepsins and falcipains) process the globin into small peptides. These peptides appear to be further digested to free amino acids by aminopeptidases, enzymes that catalyze the sequential cleavage of N-terminal amino acids from peptides. Aminopeptidases are classified into different evolutionary families according to their sequence motifs and preferred substrates. The aminopeptidase inhibitor bestatin can disrupt parasite development, suggesting that this group of enzymes might be a chemotherapeutic target. Two bestatin-susceptible aminopeptidase activities, associated with gene products belonging to the M1 and M17 families, have been described in blood-stage P. falciparum parasites, but it is not known whether one or both are required for parasite development. To establish whether inhibition of the M17 aminopeptidase is sufficient to confer antimalarial activity, we evaluated 35 aminoalkylphosphonate and phosphonopeptide compounds designed to be specific inhibitors of M17 aminopeptidases. The compounds had a range of activities against cultured P. falciparum parasites with 50% inhibitory concentrations down to 14 muM. Some of the compounds were also potent inhibitors of parasite aminopeptidase activity, though it appeared that many were capable of inhibiting the M1 as well as the M17 enzyme. There was a strong correlation between the potencies of the compounds against whole parasites and against the enzyme, suggesting that M17 and/or M1 aminopeptidases may be valid antimalarial drug targets.     

Implication of active Erk in the classic type of human medulloblastoma

Molecular pathways underlying medulloblastoma (MB), the most common malignant brain tumour in children, are still under scrutiny. The mammalian target of the rapamycin (mTOR) pathway is one of the kinases that was recently found to be implicated in a number of human tumours. Also in the case of MB it is suspected that mTOR dysregulation may play an important role in pathogenesis. Active mTOR leads to translation of several proteins, some of which affect cellular proliferation. On the other hand, Akt/PKB (protein kinase B) and Erk (extracellular signal-regulated kinase, also called mitogen-activated protein kinase, MAPK) are two protein kinases whose hyperactivity leads to a number of downstream effects, including activation of mTOR. In our previous report we found that indeed Akt and Erk are variably activated in human MBs. However, because MBs are a highly heterogeneous group of tumours, we were unable to associate Akt or Erk activation with all the cases of MB. In this paper we evaluated six cases of MB, only of the classic subtype. We found that elements of the Erk pathway are hyperactive in all six tumours. Thus, we postulate that in classic type of MB, growth factor stimulation may lead to Erk upregulation and mTOR-dependent protein translation, causing malignant growth.