Non-T cell activation linker (NTAL): a transmembrane adaptor protein involved in immunoreceptor signaling

A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non-T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcgamma- and Fc epsilon -receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non-T cells.     

Prognosis of children with mixed phenotype acute leukemia treated on the basis of consistent immunophenotypic criteria

Background

Mixed phenotype acute leukemia (MPAL) represents a diagnostic and therapeutic dilemma. The European Group for the Immunological Classification of Leukemias (EGIL) scoring system unambiguously defines MPAL expressing aberrant lineage markers. Discussions surrounding it have focused on scoring details, and information is limited regarding its biological, clinical and prognostic significance. The recent World Health Organization classification is simpler and could replace the EGIL scoring system after transformation into unambiguous guidelines.

Design and Methods

Simple immunophenotypic criteria were used to classify all cases of childhood acute leukemia in order to provide therapy directed against acute lymphoblastic leukemia or acute myeloid leukemia. Prognosis, genotype and immunoglobulin/T-cell receptor gene rearrangement status were analyzed.

Results

The incidences of MPAL were 28/582 and 4/107 for children treated with acute lymphoblastic leukemia and acute myeloid leukemia regimens, respectively. In immunophenotypic principal component analysis, MPAL treated as T-cell acute lymphoblastic leukemia clustered between cases of non-mixed T-cell acute lymphoblastic leukemia and acute myeloid leukemia, while other MPAL cases were included in the respective non-mixed B-cell progenitor acute lymphoblastic leukemia or acute myeloid leukemia clusters. Analogously, immunoglobulin/T-cell receptor gene rearrangements followed the expected pattern in patients treated as having acute myeloid leukemia (non-rearranged, 4/4) or as having B-cell progenitor acute lymphoblastic leukemia (rearranged, 20/20), but were missing in 3/5 analyzed cases of MPAL treated as having T-cell acute lymphobastic leukemia. In patients who received acute lymphoblastic leukemia treatment, the 5-year event-free survival of the MPAL cases was worse than that of the non-mixed cases (53±10% and 76±2% at 5 years, respectively, P=0.0075), with a more pronounced difference among B lineage cases. The small numbers of MPAL cases treated as T-cell acute lymphoblastic leukemia or as acute myeloid leukemia hampered separate statistics. We compared prognosis of all subsets with the prognosis of previously published cohorts.

Conclusions

Simple immunophenotypic criteria are useful for therapy decisions in MPAL. In B lineage leukemia, MPAL confers poorer prognosis. However, our data do not justify a preferential use of current acute myeloid leukemia-based therapy in MPAL.     

A prospective longitudinal study of BK virus infection in 120 Czech renal transplant recipients

Polyomavirus BK (BKV) is a common human polyomavirus that rarely causes clinical symptoms in immunocompetent individuals. However, BK virus reactivation occurs in 20-40% of kidney transplant patients and 1-10% of cases present with BK virus-associated nephropathy (BKVN) and reduced kidney allograft survival. In this study, 120 consecutive renal allograft recipients were monitored for BK virus replication by real-time PCR (qPCR) in the blood and urine during the first year post-transplantation and risk factors for BK viremia, viruria, and polyoma BKV-associated nephropathy were evaluated. Receiver operating characteristic curve analysis was used to determine the cutoff points for assessing the risk of developing BKVN. In total, 1,243 samples were tested. BK-DNAuria >10(7) copies/ml and BK-DNAemia >10(4) copies/ml were found in 25.8% and 5% of the samples screened, respectively, during the 12 month follow-up period. BKVN was confirmed histologically in 3/120 patients and viremic patients were treated with dialysis for longer time periods and had higher levels of panel [corrected] reactive antibodies. Patients with viruria were also treated longer with dialysis and had impaired graft function 12 months post-transplantation. Patients with sustained viruria exhibited more acute rejection episodes than patients with transient viruria. Using receiver operating characteristic curve analysis, the cutoff point for viremia and viruria was redefined to 10(3) copies/ml serum for BK viremia and a cutoff point of 6.7 × 10(7) copies/ml in urine. In conclusion, polyoma BK viremia and viruria are frequent findings in kidney transplant recipients that warrant intensive monitoring as a means of preventing graft failure [corrected].     

Conversion to sirolimus of patients with chronic allograft nephropathy—a retrospective analysis of outcome and influencing factors

Purpose

The purpose of this study was to analyse the outcome and its influencing factors in patients whose therapy was converted from calcineurin inhibitors (CNI) to sirolimus (SRL) due to chronic allograft nephropathy (CAN).

Materials and methods

Therapies of 78 patients (44 men) with CAN from three European transplant centres were converted from CNI therapy to SRL and followed 24 months. Slopes for creatinine clearance before and after conversion were calculated. Influencing factors were analysed by a multivariance analysis.

Results

The slope of the creatinine clearance improved significantly (?0.90 vs. ?0.34 ml min^?1 month^?1; p?<?0.01). In patients whose therapy was converted from cyclosporine A (CyA) to SRL, the slope improved significantly, whereas conversion from Tacrolimus (Tac) to SRL did not affect the slope. The benefit was more pronounced in (1) patients with low or moderate baseline creatinine clearance, (2) patients receiving SRL after conversion without additional mycophenolate mofetil and (3) patients with low or moderate proteinuria.

Conclusion

Conversion from CyA to SRL but not from Tac to CRL is associated with a reduced loss of renal allograft function in patients with CAN.     

Evaluation of clinical relevance of examining K-ras, p16 and p53 mutations along with allelic losses at 9p and 18q in EUS-guided fine needle aspiration samples of patients with chronic pancreatitis and pancreatic cancer

AIM： To establish an optimum combination of molecular markers resulting in best overall diagnostic sensitivity and specificity for evaluation of suspicious pancreatic mass.
METHODS： Endoscopic ultrasound （EUS）-guided fine needle aspiration cytology （FNA） was performed on 101 consecutive patients （63 males, 38 females, 60 ± 12 years; 81 with subsequently diagnosed pancreatic cancer, 20 with chronic pancreatitis） with focal pancreatic mass. Samples were evaluated on-site by an experienced cytopathologist. DNA was extracted from Giemsa stained cells selected by laser microdissection and the presence of K-ras, p53 and p16 somatic mutations was tested by cycling-gradient capillary electrophoresis （CGCE） and single-strand conformation polymorphism （SSCP） techniques. In addition, allelic losses of tumor suppressor genes p16 （INK4, CDKN2A） and DPC4 （MADH4, SMAD4） were detected by monitoring the loss of heterozygosity （LOH） at 9p and 18q, respectively.
RESULTS： Sensitivity and specificity of EUS-guided FNA were 75% and 85%, positive and negative predictive value reached 100%. The remaining 26% samples were assigned as inconclusive. Testing of molecular markers revealed sensitivity and specificity of 70% and 100% for K-ras mutations （P 〈 0.001）, 24% and 90% for p53 mutations （NS）, 13% and 100% for p16 mutations （NS）, 85% and 64% for aUelic losses at 9p （P 〈 0.001） and 78% and 57% for allelic losses at 18q （P 〈 0.05）. When tests for different molecular markers were combined, the best results were obtained with K-ras ＋ LOH at 9p （92% and 64%, P 〈 0.001）, K-ras ＋ LOH at 18q （92% and 57%, P 〈 0.001）, and K-ras ＋ LOH 9q ＋ LOH 18q （96% and 43%, P 〈 0.001）. When the molecular markers were used as complements to FNA cytology to evaluate inconclusive samples only, the overall sensitivity of cancer detection was 100% in all patients enrolled in the study.
CONCLUSION： EUS-guided FNA cytology combined with screening of K-ras mutations an     

Cerebral blood supply with aging: normal, stenotic and recanalized

The prosperity of brain parenchyma during aging depends on the preservation of cerebral blood flow (CBF) parameters. We have analysed ultrasonographic measurements of peak systolic (PSV) and end diastolic velocities (EDV) along with pulsatility (PI) and resistance indexes (RI) in common (CCA), internal (ICA) and external carotid artery (ECA) (N=199) and in vertebral arteries (VA) (N=200) in patients without any signs of stenosis. In two other cohorts patients with internal carotid artery stenosis (N=231) and patients prior to and after therapeutic recanalization (N=81) were evaluated in the same parameters. Results: in the range of 21-92 years PSV in CCA decreases by 7 mm/s/year, while in ICA only by 2.31 mm/s/year. The decrease of EDV in carotid arteries occurs between 1.72 and 2.28 mm/s/year. PSV in VA drops down by 0.91 mm/s/year, EDV by 0.86 mm/s/year. PI and RI increase with age in all vessels, but not significantly. Stenotic ICAs are associated with increased PSV in the range of 0.7-2.9 m/s, but also with an increasing PSV variability along the growing stenosis in individual patients. In all degrees of stenoses some patients preserve normal velocities. In average the increment for each 10% of the stenosis below 50% makes 8 cm/s, while above 50% it makes already 50 cm/s. In persons with bilateral stenoses the increment with growing stenosis is steeper. The restoration of normal ICA lumen by means of carotid endarterectomy or by angioplasty with stenting results in an average drop by 1.23 m/s in PSV and by 0.4 m/s in EDV. We have investigated the ophthalmic artery and other substitution supplies and deduce, that the remarkable differences in blood flow velocity reactions to a compromised carotid lumen depend on the formation of collaterals in mutual interplay with peripheral resistance.