Dysregulation of interleukin 5 expression in familial eosinophilia.

BACKGROUND: Familial eosinophilia (FE) is a rare autosomal dominant inherited disorder characterized by the presence of lifelong peripheral eosinophilia (>1500/µL). Mapped to chromosome 5q31-q33, the genetic cause of FE is unknown, and prior studies have failed to demonstrate a primary abnormality in the eosinophil lineage. OBJECTIVE: The aim of this study was to identify the cells driving the eosinophilia in FE. METHODS: Microarray analysis and real-time PCR were used to examine transcriptional differences in peripheral blood mononuclear cells (PBMC), and in purified cell subsets from affected and unaffected family members belonging to a single large kindred. Cytokine levels in serum and PBMC culture supernatants were assessed by suspension array multiplexed immunoassays. RESULTS: Whereas IL-5 mRNA expression was significantly increased in freshly isolated PBMC from affected family members, this was not accompanied by increased mRNA expression of other Th2 cytokines (IL-4 or IL-13). Serum levels of IL-5 and IL-5 receptor α, but not IgE, were similarly increased in affected family members. Of note, IL-5 mRNA expression was significantly increased in purified CD3+ CD4+, CD14+, CD19+, and ILC2 cells from affected family members, as were IL-5 protein levels in supernatants from both stimulated PBMC and ILC2 cultures. CONCLUSIONS: These data are consistent with the hypothesis that the eosinophilia in FE is secondary to dysregulation of IL-5 production in PBMC (and their component subsets).

Clinical features predict responsiveness to imatinib in platelet-derived growth factor receptor-alpha-negative hypereosinophilic syndrome.

BACKGROUND: With the exception of the presence of the FIP1L1-PDGFRA fusion gene, little is known about predictors of imatinib response in clinically-defined hypereosinophilic syndrome (HES). METHODS: Subjects with FIP1L1-PDGFRA-myeloid neoplasm (FP; n =12), PDGFRA-negative HES with ≥4 criteria suggestive of a myeloid neoplasm (MHES; n =10), or steroid-refractory PDGFRA-negative HES with <4 myeloid criteria (SR; n = 5) were enrolled in a prospective study of imatinib therapy (NCT00044304: registered at clinicaltrials.gov). The primary outcome was an eosinophil count <1.5 × 109/L at one month and improvement of clinical symptoms. Clinical, molecular, and bone marrow responses to imatinib were assessed. A retrospective cohort of 18 subjects with clinically-defined HES who received imatinib (300-400 mg daily ≥ 1 month) were classified according to the criteria used in the prospective study. RESULTS: Overall, imatinib response rates were 100% in the FP group (n = 16), 54% in the MHES group (n = 13) and 0% in the SR group (n = 16). The presence of ≥ 4 myeloid features was the sole predictor of response. After ≥ 18 months in complete remission, imatinib was tapered and discontinued in 8 FP and 1 MHES subjects. Seven subjects (6 FP, 1 MHES) remain in remission off therapy for a median of 29 months (range 14-36). CONCLUSIONS: Clinical features of MHES predict imatinib response in PDGFRA-negative HES.

Mutation frequencies of GNAQ, GNA11, BAP1, SF3B1, EIF1AX and TERT in uveal melanoma: detection of an activating mutation in the TERT gene promoter in a single case of uveal melanoma.

BACKGROUND: Uveal melanoma is the most frequent primary tumour of the eye. It is molecularly clearly distinct from cutaneous melanoma and shows a different pattern of driver mutations. The influence of sunlight ultraviolet (UV) exposure on the aetiology of uveal melanoma is a matter of debate. The recent identification of driver mutations in the promoter of the telomerase reverse transcriptase (TERT) gene with UV-induced cytidine-to-thymidine transitions in cutaneous melanoma prompted us to investigate whether these mutations also occur in uveal melanoma. METHODS: We analysed 50 cases of uveal melanoma obtained from enucleation surgery for mutations in the genes GNAQ, GNA11, BAP1, SF3B1, EIFAX1 and TERT, measured gene expression using microarrays and analysed gene copy numbers by SNP arrays. RESULTS: We detected a TERT mutation in only one case of a 57-year-old white male patient with clinical and histopathological features typical for uveal melanoma. The tumour showed mutations in GNA11 and EIF1AX that are typical for uveal melanoma and absent from cutaneous melanoma. No mutations were detected in GNAQ, BAP1 and SF3B1 that are frequently mutated in uveal melanoma. Both copies of chromosome 3 were retained. Several tumours among which the one carrying the TERT promoter mutation showed elevated TERT expression. Consistent with previous reports, GNAQ is inversely associated with chromosome 3 monosomy and metastasis. BAP1 mutations are significantly associated with chromosome 3 monosomy but not with relapse. CONCLUSION: These data indicate that TERT mutations are rare in uveal melanoma. No conclusion can be drawn on their potential influence on tumour progression.

Microstructural alterations of femoral head articular cartilage and subchondral bone in osteoarthritis and osteoporosis.

OBJECTIVE: Explore whether osteoporosis (OP) in humans influences the morphological status of the articular cartilage and the subchondral bone. Explore the relationship between the macroscopic aspect of the articular surface and the rate of microscopic changes of both the cartilage and the subchondral bone in OP and osteoarthritis (OA). METHODS: Femoral heads after total hip replacement were obtained from patients with OP or hip OA (OP, n = 56; OA, n = 12). Cartilage degeneration was assessed using the Mankin grading system whereas subchondral bone was evaluated using histomorphometry and Micro-computed Tomography (µCT) scanning system. Thickness of the cartilage layers and subchondral cortical bone (SCB) was measured. RESULTS: Samples with higher total Mankin score have significantly reduced cartilage thickness. Mankin score differed between all OP specimens. In OP samples with lower Mankin scores the thickness of SCB shows a trend of an increase caused by increased levels of bone remodeling. In OP samples with higher Mankin scores we observed thinning of SCB. Structural indices of subchondral trabecular bone (STB) were significantly lower in OP than in OA samples. CONCLUSION: Thinning of SCB, found in OP samples with higher Mankin scores could be related with the progression of the cartilage degeneration indicating an early-stage OA. Increased levels of bone remodeling and evidently changed morphology of subchondral bone found in OP samples with lower Mankin score indicated that bony bed level must have a role in the progression of the cartilage degeneration.

Detection of multi-class pesticide residues with surface-enhanced Raman spectroscopy.

The excessive use of pesticides disturbs the natural balance in the environment, creates resistance to pesticides and leads to water and food contamination. Therefore, the implementation of fast, robust and cost effective techniques for the monitoring of pesticides is required. In this work surface-enhanced Raman spectroscopy (SERS) was used for the detection of four common pesticides: atrazine, simazin, irgarol, and diuron. SERS is nowadays considered an effective technique for detection of various analytes in low concentration. Sensitivity of the SERS method depends on the type of substrate that can be either a colloidal solution of metal nanoparticles (NPs) or a metal surface with a suitable nanostructured topology. Here, we have investigated the application of silver nanospheres and silver nanoprisms as SERS substrates in pesticides detection. Colloids with spherical NPs were produced by chemical reduction while Ag nanoprisms were prepared by reducing silver nitrate with borohydride (with citrate as a stabilizing agent) and stirring under a UV lamp for 4 and 10 h. The SERS results have shown that, in the presence of synthesized NPs, it was possible to detect millimolar concentrations of aforementioned pesticides with the exception of diuron.

Today Balkan endemic nephropathy is a disease of the elderly with a good prognosis.

BACKGROUND: The outcome for Balkan endemic nephropathy (BEN) patients diagnosed in 1992 was analyzed in 2006 with the aim of detecting factors associated with disease progression and patient outcome. METHODS: In 1992 BEN was detected in 119 patients (53 males, 56.9 +/- 13.8 years) from the village of Sopic. Changes in creatinine clearance as well as outcome (death or onset of regular hemodialysis) were analyzed retrospectively in 2006. RESULTS: During the 14-year period 47 patients deceased (5 on hemodialysis) at the mean age of 72.2 +/- 8.2 years, while no data were available for 13 cases. Out of 59 remaining patients 3 were on hemodialysis in 2006 and 56 participated in the control examination. Of these 12 had creatinine clearance at least 50% lower than in 1992 and 44 had unchanged creatinine clearance. Logistic regression revealed age and proteinuria, but linear regression only age as significant prognostic factors for changes in creatinine clearance. The all-cause mortality rate varied between 1.1 and 5.3% per year and was similar to the mortality rate of the general population. The main cause of death was cardiovascular disease (40.5%) followed by malignant diseases (17%), most frequently (11%) due to upper urothelial tumors. Urine protein and age were found to be a significant independent predictor of all-cause mortality. CONCLUSION: In the village of Sopic BEN was commonly detected in patients in their fifties, progressed slowly, but most patients died from other causes at old age before end-stage renal disease occurred. Kidney function remained stable over the decade in three quarters of the surviving patients. Age and proteinuria were found to be prognostic factors for both disease progression and patient mortality.

Efficacy of hyperphosphatemia control in the progression of chronic renal failure and the prevalence of cardiovascular calcification.

BACKGROUND AND AIMS: Chronic kidney disease mineral- and bone disorder (CKD-MBD) has been studied more often in dialysis than in predialysis CKD patients. The association between efficacy of hyperphosphatemia control and chronic renal failure (CRF) progression, prevalence of bone disease and cardiovascular calcification was the objective of the present investigation. MATERIAL AND METHODS: 42 patients with CKD in Stage 5, regularly monitored for 5 years, were divided into Group 1 of 20 patients with normal serum phosphate (sPO4) levels and Group 2 of 22 patients with hyperphosphatemia registered at the majority of checks. Serum urea, creatinine, calcium (sCa) and sPO4 levels were regularly determined in the retrospective 5-year period. At the end of this period iPTH, bone alkaline phosphatase-BAP and inflammation markers (CRP, fetuin-A) were measured, valvular and arterial calcifications were detected by B mode echocardiogram and soft-tissue native radiograms of the pelvis and the wrist. RESULTS: Progression of CRF (1/sCr over time) was faster in Group 2 than in Group 1 (b = -0.0577 vs. -0.0288, p = 0.003) during the study period. Average BAP and iPTH values were similar in both groups and 23/42 patients had PTH > 300 pg/ml. Arterial and valvular calcifications were found in 5/23 patients from Group 1 and 14/22 patients from Group 2 (p = 0.011). Linear regression analysis revealed sPO4 as a predictor for total calcification number, inflammatory diseases as a predictor for valvular calcifications, while sPO4 and iPTH were predictors for arterial calcifications. CONCLUSIONS: More than half the patients with Stage 5 CKD not yet on dialysis exhibited elevated PTH. Faster CRF progression and frequent arterial and valvular calcifications were seen in patients with poor phosphate control and sPO4 was selected as an independent predictor of total calcification score.

Hemodialysis headache.

AIM: The aim of this study was to evaluate and analyze the incidence and features of headaches in patients undergoing hemodialysis. MATERIAL AND METHODS: In this prospective study 318 patients, 119 women and 199 men, undergoing chronic HD in four hemodialysis centers in Serbia, were questioned about their problems with headaches using a questionary designed according to the diagnostic criteria of the International Headache Classification of Headache Disorders (ICHD) from 2004. Patients were distributed in two groups according to the presence of hemodialysis headaches (HDH). The groups were compared regarding sex, age, duration of HD, primary diseases that lead to ESRD, arterial systolic and diastolic blood pressure (BP) and serum levels of hemoglobin, urea nitrogen, creatinine, sodium, potassium, calcium, phosphates, albumin, glucose and calcium-phosphate product. We also analyzed features of HDH. The results were statistically compared. RESULTS: Patients with HDH had significantly lower serum glucose, but higher serum phosphates and albumin than patients without headaches. Furthermore, HDH patients had higher calcium phosphate product and systolic blood pressure than non-HDH patients. Out of 318 patients included in the study, 21 (6.6%) patients had HDH. According to our results, HDH appeared more frequently in men, during the 3rd hour of HD in more than a half of the patients and lasted less then 4 h in the majority of HDH patients. In the majority of patients HDH was bilateral, non-pulsating, without associated symptoms and it appeared mostly during HD. Personal history was negative for primary headaches in all patients with HDH. CONCLUSION: We believe that the results of our investigation of more than 300 HD patients pointed to some biochemical changes, possibly implicated by pathophysiology of HDH and disclose some specific HDH features that might contribute to a better understanding of this secondary headache disorder.

Analysis of mural paintings in Istria.

Fragments of wall paintings from Istria, coming from the Basilica of Guran near Vodnjan, from the cemeterial Church of Saint Simeon in Guran and from the Benedictine monastery of Santa Maria Alta near Bale were studied. The analytical instrumental techniques used were Optical Microscopy, Scanning Electron Microscopy equipped with an EDS microanalysis detector, X Ray diffraction, FTIR infrared Spectroscopy and Raman Spectroscopy. Red and yellow pigments used in Guran and Bale have bean derived from red and yellow istrian bauxites, as already demonstrated for works from 11th to 15th century. The blue pigment found in the paintings of the Bale Chapel is a lapislazzuli blue; this fact confirms the literature data referring to the period from 11th to the 16th century. The materials and pigments used at Bale and Guran fit with the Istrian tradition and history of painting going back to the first Carolingian period.

GABAA receptor subunit composition and functional properties of Cl- channels with differential sensitivity to zolpidem in embryonic rat hippocampal cells.

Using flow cytometry in conjunction with a voltage-sensitive fluorescent indicator dye (oxonol), we have identified and separated embryonic hippocampal cells according to the sensitivity of their functionally expressed GABAA receptors to zolpidem. Immunocytochemical and RT-PCR analysis of sorted zolpidem-sensitive (ZS) and zolpidem-insensitive (ZI) subpopulations identified ZS cells as postmitotic, differentiating neurons expressing alpha2, alpha4, alpha5, beta1, beta2, beta3, gamma1, gamma2, and gamma3 GABAA receptor subunits, whereas the ZI cells were neuroepithelial cells or newly postmitotic neurons, expressing predominantly alpha4, alpha5, beta1, and gamma2 subunits. Fluctuation analyses of macroscopic Cl- currents evoked by GABA revealed three kinetic components of GABAA receptor/Cl- channel activity in both subpopulations. We focused our study on ZI cells, which exhibited a limited number of subunits and functional channels, to directly correlate subunit composition with channel properties. Biophysical analyses of GABA-activated Cl- currents in ZI cells revealed two types of receptor-coupled channel properties: one comprising short-lasting openings, high affinity for GABA, and low sensitivity to diazepam, and the other with long-lasting openings, low affinity for GABA, and high sensitivity to diazepam. Both types of channel activity were found in the same cell. Channel kinetics were well modeled by fitting dwell time distributions to biliganded activation and included two open and five closed states. We propose that short- and long-lasting openings correspond to GABAA receptor/Cl- channels containing alpha4beta1gamma2 and alpha5beta1gamma2 subunits, respectively.

GABA expression dominates neuronal lineage progression in the embryonic rat neocortex and facilitates neurite outgrowth via GABA(A) autoreceptor/Cl- channels.

GABA emerges as a trophic signal during rat neocortical development in which it modulates proliferation of neuronal progenitors in the ventricular/subventricular zone (VZ/SVZ) and mediates radial migration of neurons from the VZ/SVZ to the cortical plate/subplate (CP/SP) region. In this study we investigated the role of GABA in the earliest phases of neuronal differentiation in the CP/SP. GABAergic-signaling components emerging during neuronal lineage progression were comprehensively characterized using flow cytometry and immunophenotyping together with physiological indicator dyes. During migration from the VZ/SVZ to the CP/SP, differentiating cortical neurons became predominantly GABAergic, and their dominant GABA(A) receptor subunit expression pattern changed from alpha4beta1gamma1 to alpha3beta3gamma2gamma3 coincident with an increasing potency of GABA on GABA(A) receptor-mediated depolarization. GABA(A) autoreceptor/Cl(-) channel activity in cultured CP/SP neurons dominated their baseline potential and indirectly their cytosolic Ca(2+) (Ca(2+)c) levels via Ca(2+) entry through L-type Ca(2+) channels. Block of this autocrine circuit at the level of GABA synthesis, GABA(A) receptor activation, intracellular Cl(-) ion homeostasis, or L-type Ca(2+) channels attenuated neurite outgrowth in most GABAergic CP/SP neurons. In the absence of autocrine GABAergic signaling, neuritogenesis could be preserved by depolarizing cells and elevating Ca(2+)c. These results reveal a morphogenic role for GABA during embryonic neocortical neuron development that involves GABA(A) autoreceptors and L-type Ca(2+) channels.

The cellular immune system in myelomagenesis: NK cells and T cells in the development of myeloma [corrected] and their uses in immunotherapies.

As vast strides are being made in the management and treatment of multiple myeloma (MM), recent interests are increasingly focusing on understanding the development of the disease. The knowledge that MM develops exclusively from a protracted phase of monoclonal gammopathy of undetermined significance provides an opportunity to study tumor evolution in this process. Although the immune system has been implicated in the development of MM, the scientific literature on the role and status of various immune components in this process is broad and sometimes contradictory. Accordingly, we present a review of cellular immune subsets in myelomagenesis. We summarize the current literature on the quantitative and functional profiles of natural killer cells and T-cells, including conventional T-cells, natural killer T-cells, γδ T-cells and regulatory T-cells, in myelomagenesis. Our goal is to provide an overview of the status and function of these immune cells in both the peripheral blood and the bone marrow during myelomagenesis. This provides a better understanding of the nature of the immune system in tumor evolution, the knowledge of which is especially significant considering that immunotherapies are increasingly being explored in the treatment of both MM and its precursor conditions.

Neuroepithelial cells in the rat spinal cord express glutamate decarboxylase immunoreactivity in vivo and in vitro.

It is unknown whether neuroepithelial cells in the mammalian central nervous system express neurotransmitter-synthesizing enzymes. In this study, expression of glutamate decarboxylase (GAD), the gamma-aminobutyric acid (GABA)-synthesizing enzyme, was examined in proliferative cells and postmitotic neuroblasts in embryonic rat spinal cord. Immunostaining coronal sections of the embryonic spinal cord with K2 antiserum, which recognizes GAD proteins encoded by the GAD67 gene, revealed intensely stained neuroepithelial cells in the basal plate at embryonic day (E) 13, in the intermediate plate between E 13-16, and last seen in the alar plate at E 16. Nissl counterstaining demonstrated that a small number of these GAD-immunoreactive cells adjacent to the neural tube lumen were mitotic. The ventral-to-dorsal gradient of GAD expression in precursor cells and postmitotic neuroblasts correlates anatomically and temporally with the sequential generation of motoneurons, commissural neurons, and interneurons in the dorsal horn. Some of these GAD-immunoreactive neuroepithelial cells may re-enter the mitotic cycle, while others are postmitotic neuroblasts presumably migrating to the intermediate zone to differentiate into young neurons. Double-immunostaining cells acutely dissociated from E 11-18 spinal cords with K2 and anti-bromodeoxyuridine antisera, following a bromodeoxyuridine pulse in vivo, revealed considerable numbers of DNA-synthesizing cells immunoreactive for GAD. The absolute number of double-stained cells peaked during E 12-15, coinciding with terminal cell division in most spinal neurons. These observations suggest that spinal neuronal precursors can synthesize GAD-related proteins prior to, or during, the terminal cell cycle. Although GAD immunoreactivity revealed by K2 antiserum was detected in proliferative cells and in migrating postmitotic neuroblasts, GABA immunoreactivity was never detectable in these cells. These early embryonic GAD-immunoreactive neuroepithelial cells may either synthesize levels of GABA that cannot be detected immunocytochemically, and/or express enzymatically inactive GAD-related proteins.

Increased TUNEL staining in brains of autoimmune Fas-deficient mice.

Profound changes in brain morphology and behavior coincide with the spontaneous development of systemic autoimmune/inflammatory disease in Fas-deficient MRL-lpr mice. The dendrites atrophy, the density of hippocampal and cortical neurons decreases, and an anxious/depressive-like behavior emerges while lymphoid cells infiltrate into the choroid plexus of MRL-lpr mice. We hypothesized that the inherited lack of the Fas-dependent anti-inflammatory mechanism would lead to unsuppressed immune activity, characterized by reduced apoptosis in the MRL-lpr brain. Using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeled (TUNEL) method as an indicator of apoptosis, a surprisingly high incidence of TUNEL-positive cells was observed in the hippocampus, choroid plexus and periventricular regions of MRL-lpr mice, 5-10-fold higher than that found in the MRL +/+ control brain. Immunostaining with anti-CD3, CD4 and CD8 monoclonal antibodies showed limited overlap between CD-positive and TUNEL-positive cells, suggesting that the dying cells are for the most part (approximately 70%) not T-lymphocytes. Although further characterization of the phenotype of the dying cells and the mechanism of cell death are required, the present results suggest the involvement of a Fas-independent apoptotic process in neurodegeneration induced by systemic autoimmune disease.

Striatal matrix neurons of the rat differentiate in culture from dissociated fetal progenitor cells isolated by buoyant density centrifugation.

The adult striatum is composed of interlacing compartments known as patches (striosomes) and matrix, which differ with respect to a host of architectonic, biochemical and developmental parameters. We have exploited the 2-phase development of the striatum, employing buoyant-density fractionation to separate proliferating/undifferentiated neural precursors from the differentiated neurons of the E19 striatum. Primary cell cultures were established for the collected fractions, and immunohistochemistry for maturational and compartment-specific markers performed. The results indicate that the least buoyant, striatal precursors concentrate principally in the low buoyancy fraction of the gradient, and in culture express known matrix phenotype markers in an appropriate time frame.

Creatinine clearance and kidney size in Balkan endemic nephropathy patients.

BACKGROUND/AIMS: Symmetrically shrunken kidneys have been considered as one of the characteristics of Balkan endemic nephropathy (BEN), but there is no agreement when the shrinking does occur in the course of the disease. In the present study, the relation between creatinine clearance (Ccr) and kidney length was compared between the patients in the early phase of BEN and other renal diseases. METHODS: The study included 84 patients with BEN (39 males, aged 54 +/- 12 years), 31 patients with other renal diseases (15 males, aged 54 +/- 14 years) and 15 healthy subjects as controls. Only the patients with Ccr above 90 ml/min were included into the study. The kidney length was measured by sonography using sector sound of 3.5 MHz. RESULTS: In healthy controls and patients with renal disease other than BEN, Ccr varied between 90 and 177 ml/min, and only 3 patients had Ccr above 150 ml/min. On the contrary, 15 (18%) BEN patients had Ccr between 90 and 120 ml/min, and 37 (44%) BEN patients had Ccr above 150 ml/min. Sonographically measured kidney length of all healthy subjects and patients with renal diseases other than BEN was above 10.5 cm. Out of 84 BEN patients, 21 (25%) patients had kidney length less than 10.5 cm. CONCLUSION: BEN patients in the early phase of the disease had significantly higher Ccr and smaller kidney in comparison to the patients with other renal diseases.

Variations of the great arteries in the carotid triangle.

The variations of the common carotid artery, as well as of the external and internal carotid arteries, are described. During anatomic dissection on adult cadavers, we investigated the variability of appearance of 40 carotid arterial systems. Special consideration was given to the topographic relations such as the level of the bifurcation of the common carotid artery, the relationship between the external and internal carotid arteries, and the origin of the great collateral branches. Special attention was paid to the origin of the superior thyroid artery. In this article the practical importance of these variations is stressed.

Ibrutinib-induced lymphocytosis in patients with chronic lymphocytic leukemia: correlative analyses from a phase II study.

Ibrutinib and other targeted inhibitors of B-cell receptor signaling achieve impressive clinical results for patients with chronic lymphocytic leukemia (CLL). A treatment-induced rise in absolute lymphocyte count (ALC) has emerged as a class effect of kinase inhibitors in CLL and warrants further investigation. Here we report correlative studies in 64 patients with CLL treated with ibrutinib. We quantified tumor burden in blood, lymph nodes (LNs), spleen and bone marrow, assessed phenotypic changes of circulating cells and measured whole-blood viscosity. With just one dose of ibrutinib, the average increase in ALC was 66%, and in>40% of patients the ALC peaked within 24 h of initiating treatment. Circulating CLL cells on day 2 showed increased Ki67 and CD38 expression, indicating an efflux of tumor cells from the tissue compartments into the blood. The kinetics and degree of the treatment-induced lymphocytosis was highly variable; interestingly, in patients with a high baseline ALC the relative increase was mild and resolution rapid. After two cycles of treatment the disease burden in the LN, bone marrow and spleen decreased irrespective of the relative change in ALC. Whole-blood viscosity was dependent on both ALC and hemoglobin. No adverse events were attributed to the lymphocytosis.

Expression of receptor activator of nuclear factor-kappa B ligand in leukocytes during acute kidney rejection after transplantation in rats.

BACKGROUND: Acute cellular rejection of the transplanted kidney is an important cause of impaired graft function. One of the basic characteristics of acute cellular rejection according to the latest Banff classification of renal allograft pathology is the presence of a large number of T lymphocytes in the allografted tissue. Osteoprotegerin, receptor activator of nuclear factor-kappa B (RANK) and RANK ligand (RANKL), three relatively novel members of the tumor necrosis factor superfamily, have crucial roles not only in physiologic and pathologic bone metabolism but also in immunologic processes. The aim of our study was to determine the expression of RANKL and RANK by T lymphocytes and macrophages in acute cellular kidney allograft rejection in rats. METHODS: The study included 15 male Wistar rats of 3 months old and 250-300 g as recipients and 15 male DA rats donors of 3 months old; and weight 250-300 g. When animals were sacrificed at 3 weeks to extract the transplanted kidney for pathohistologic analysis and immunoflorescence. all samples showed acute cellular rejection. Kidney sections were examined by dual-labeled immunofluorescence to detect CD4, CD8, or CD68 (red) and RANK or RANKL (green) with coexpressing cells as orange. RESULTS: RANKL-positive expression colocalized with CD4(+) and CD8(+) T lymphocytes in acutely rejected kidney tissue. There was no association between CD4(+) and CD8(+) T cells with RANK expression, which was evident by infiltrating CD68-positive macrophages in the kidney tissue interstitium. CONCLUSION: RANK and RANKL were expressed by T lymphocytes and macrophages in acute cellular kidney rejection after transplantation in rats.

Targeting the KIT activating switch control pocket: a novel mechanism to inhibit neoplastic mast cell proliferation and mast cell activation.

Activating mutations in the receptor tyrosine kinase KIT, most notably KIT D816V, are commonly observed in patients with systemic mastocytosis. Thus, inhibition of KIT has been a major focus for treatment of this disorder. Here we investigated a novel approach to such inhibition. Utilizing rational drug design, we targeted the switch pocket (SP) of KIT, which regulates its catalytic conformation. Two SP inhibitors thus identified, DP-2976 and DP-4851, were examined for effects on neoplastic mast cell proliferation and mast cell activation. Autophosphorylation of both wild-type and, where also examined, KIT D816V activation was blocked by these compounds in transfected 293T cells, HMC 1.1 and 1.2 human mast cell lines, and in CD34(+)-derived human mast cells activated by stem cell factor (SCF). Both inhibitors induced apoptosis in the neoplastic mast cell lines and reduced survival of primary bone marrow mast cells from patients with mastocytosis. Moreover, the SP inhibitors more selectively blocked SCF potentiation of FcɛRI-mediated degranulation. Overall, SP inhibitors represent an innovative mechanism of KIT inhibition whose dual suppression of KIT D816V neoplastic mast cell proliferation and SCF-enhanced mast cell activation may provide significant therapeutic benefits.