Components and Mechanisms of Import,Modification, Folding,and Assembly of Immunoglobulins in the Endoplasmic Reticulum

In mammalian cells, the endoplasmic reticulum (ER) plays a central role in biogenesis of secretory- and plasma membrane proteins as well as in cellular calcium (Ca²⁺) homeostasis. The protein biogenesis function involves an aqueous polypeptide conducting channel in the ER membrane, which is formed by the heterotrimeric Sec61 complex; the store- and receptor-controlled Ca²⁺- release function requires a steep ER to cytosol gradient, with more than 500 μM free Ca²⁺ in the ER and 50 nM Ca²⁺ in the cytosol. Recent work demonstrated that the Sec61 complex can transiently allow passive ER Ca²⁺ efflux. Therefore, gating of the Sec61 channel has to be tightly regulated by substrates as well as allosteric effectors. The ER lumenal Hsp70-type molecular chaperone, immunoglobulin heavy-chain binding protein (BiP), together with its membrane resident co-chaperone Sec63 facilitates channel opening in a precursor specific manner. In addition, BiP, together with its lumenal co-chaperones, ERj3 and ERj6, as well as cytosolic Ca²⁺-calmodulin (CaM) in collaboration with the membrane resident Sec62 protein represent allosteric effectors for channel closure. In the course of the last couple of years several human diseases were linked to the Sec61 complex and its effectors and were termed Sec61-channelopathies.     

Effect of Aryl Substituents and Fluorine Addition on the Optoelectronic Properties and Organic Solar Cell Performance of a High Efficiency Indacenodithienothiophene‐alt‐Quinoxaline π‐Conjugated Polymer

A series of donor‐acceptor (D‐A) π‐conjugated polymers, based on indacenodithienothiophene (IDTT) as an electron‐donating unit and quinoxaline as an electron‐deficient moiety, are synthesized via a Pd‐catalyzed Stille cross‐coupling polymerization. Molecular characteristics, photovoltaic parameters, and optoelectronic properties are examined through structural differences corresponding to thienyl versus phenyl side group substitutions on the IDTT and the non‐fluorinated versus the monofluoro quinoxaline derivatives. One of the most important outcome is that the power conversion efficiency (PCE) in the studied polymers is more device architecture dependent (conventional vs inverted) rather than chemical structure dependent. From single junction solar cells based on bulk heterojunction polymer:[6,6]‐phenyl‐C₇₁‐butyric acid methyl ester (PC₇₁BM) systems as the active layer, a maximum PCE of 5.33% has been achieved from the polymer containing the thienyl substituent on the IDTT and one fluorine atom on the quinoxaline. This demonstrates that finding the optimum molecular weight of ThIDTT‐QF or introducing the monofluoro‐quinoxaline in a regioregular motif in the polymer backbone significantly higher PCE can be expected versus the fully optimized high performance PhIDTT‐Q conjugated polymer.     

Non‐leukemic pediatric mixed phenotype acute leukemia/lymphoma: Genomic characterization and clinical outcome in a prospective trial for pediatric lymphoblastic lymphoma

Rare cases of hematological precursor neoplasms fulfill the diagnostic criteria of mixed phenotype acute leukemia (MPAL), characterized by expression patterns of at least two hematopoietic lineages, for which a highly aggressive behavior was reported. We present a series of 11 pediatric non‐leukemic MPAL identified among 146 precursor lymphoblastic lymphomas included in the prospective trial Euro‐LBL 02. Paraffin‐embedded biopsies of 10 cases were suitable for molecular analyses using OncoScan assay (n = 7), fluorescence in situ hybridization (FISH; n = 7) or both (n = 5). Except for one case with biallelic KMT2A (MLL) breaks, all cases analyzed by FISH lacked the most common translocations defining molecular subsets of lymphoblastic leukemia/lymphomas. Two non‐leukemic B‐myeloid MPALs showed the typical genomic profile of hyperdiploid precursor B‐cell lymphoblastic leukemia with gains of chromosomes 4, 6, 10, 14, 18, and 21. One B‐T MPAL showed typical aberrations of T‐cell lymphoblastic lymphoma, such as copy number neutral loss of heterozygosity (CNN‐LOH) at 9p targeting a 9p21.3 deletion of CDKN2A and 11q12.2‐qter affecting the ATM gene. ATM was also mutated in a T‐myeloid MPAL case with additional loss at 7q21.2‐q36.3 and mutation of NRAS, two alterations common in myeloid disorders. No recurrent regions of CNN‐LOH were observed. The outcome under treatment was good with all patients being alive in first complete remission after treatment according to a protocol for precursor lymphoblastic lymphoma (follow‐up 3‐10 years, median: 4.9 years). In summary, the present series of non‐leukemic MPALs widely lacked recurrently reported translocations in lymphoid/myeloid neoplasias and showed heterogeneous spectrum of chromosomal imbalances.     

Regulatory peptide receptors in human hepatocellular carcinomas

BACKGROUND: Overexpression of regulatory peptide receptors in selected human tumours is of diagnostic and therapeutic relevance. AIMS: To evaluate the expression of somatostatin, vasoactive intestinal peptide (VIP), substance P, cholecystokinin (CCK) A and B, and neurotensin receptors in hepatocellular carcinoma (HCC). METHODS: In vitro receptor autoradiography for the various peptide receptors using selective iodinated radioligands on tissue sections in 59 cases of HCC. RESULTS: 41% of HCC expressed somatostatin receptors; 47% expressed VIP receptors. VIP receptors were always identified in non-neoplastic liver tissue. Substance P receptors were only identified in 5% of HCC but in the majority of their peritumorous and intratumorous vessels. CCK-A and -B and neurotensin receptors were not detected in HCC. The somatostatin receptors showed high affinity for somatostatin and octreotide. The VIP receptors had high affinity for VIP, pituitary adenylate cyclase activating peptide (PACAP) 27, and a VIP1 selective analogue, suggesting the presence of VIP1/PACAP II type receptors. PACAP I receptors were identified in two cases. Substance P receptors were all of the NK1 subtype. The density of somatostatin receptors in HCC was low compared with the density found in liver metastases of neuroendocrine tumours. The VIP receptor density was always lower in HCC than in adjacent liver tissue. CONCLUSIONS: Somatostatin, VIP, and substance P may have a receptor mediated role in HCC. Substance P receptors may be involved in regulation of tumour associated blood flow; somatostatin receptors and VIP receptors may mediate tumour growth. Diagnostic and therapeutic evaluation of somatostatin and VIP analogues may be of interest in receptor positive HCC.     

The influence of automated plateletpheresis on systemic levels of hematopoietic growth factors

BACKGROUND: Megakaryocytopoiesis and platelet production are regulated by several hematopoietic growth factors. The present study focuses on the effects of automated plateletpheresis on systemic levels of different hematopoietic growth factors. STUDY DESIGN AND METHODS: Platelet count, mean platelet volume, and serum levels of thrombopoietin, erythropoietin, interleukin-1beta, interleukin-6, and stem cell factor in 21 healthy donors were measured before platelet collection, after the first half of the apheresis procedure, at the end of apheresis, and on Days 1, 2, and 7 thereafter. RESULTS: Thrombopoietin levels (initial level: 49.5 +/- 25.5 pg/mL) showed a significant increase between measurements taken at the end of apheresis and Day 1 (56.9 +/- 26.7 pg/mL; p = 0.01). There was a highly significant decrease in stem cell factor levels during apheresis (p<0.0005), reaching preapheresis values (1679 +/- 210 pg/mL) on Day 1. A highly significant increase in erythropoietin levels (initial level: 7.5 +/- 4.0 U/L) was seen after apheresis (p<0.0005 on Days 1 and 2). The level remained significantly elevated until Day 7 (p = 0.004). Interleukin-1beta and interleukin-6 levels (before donation: 1.4 +/- 1.8 pg/mL and 1.1 +/- 0.7 pg/mL, respectively) did not change during the observation period. Thrombopoietin levels correlated consistently and inversely with stem cell factor levels after apheresis (Day 1, r = -0.46, p = 0.035; Day 2, r = -0.50, p = 0.02; Day 7, r = -0.50, p = 0.02). CONCLUSION: The data show a coordinated response of the hematopoietic system to platelet loss. It is suggested that the decrease in serum stem cell factor levels during apheresis reflects the consumption of stem cell factor by early hematopoietic progenitors that expand to initiate early megakaryocytopoiesis. The temporary increase in thrombopoietin is the result of platelet loss and serves as a stimulus for subsequent thrombopoiesis. The pronounced elevation of erythropoietin after apheresis suggests a role for this primarily erythropoietic cytokine in thrombopoiesis, too.     

Endotoxin adsorbent based on immobilized human serum albumin.

Extracorporeal apheresis of endotoxins and pro-inflammatory cytokines is still a therapeutic option in the early hyper-inflammatory phase of gram-negative sepsis. There is therefore ongoing interest in adsorber materials suitable for that kind of clinical application. Here we describe lipopolysaccharide (LPS) and cytokine adsorption characteristics of a new adsorbent based on purified human serum albumin (HSA) covalently linked to macroporous polymer beads (iHSA). Multipoint attachment of HSA to acrylic beads via carboxyl groups of the protein resulted in an increased affinity to LPS. In adsorption experiments (adsorbent/ plasma ratio 1:3) a 70-80% reduction of limulus amoebocyte lysate (LAL) activity from 8.59+/-2.07 EU/ml (mean+/-SD) to 1.82+/-0.77 EU/ml (S. abortus equi; n=40) (p < 0.001) and from 115.13+/-53.76 EU/ml to 17.70+/-11.68 EU/ml (E. coli F583; n=6) (p < 0.01) was achieved. iHSA-purified plasma samples showed a decreased capability of inducing cytokine release from peripheral monocytes. Direct haemoperfusion of LPS pre-stimulated whole blood over iHSA resulted in decreased tumour necrosis factor alpha (TNFalpha) concentrations (30-40% reduction) whereas induced levels of interleukin (IL)-1beta and IL-6 were not affected. Depending on the means of immobilization, iHSA shows higher affinity for LPS than native albumin present in plasma. We demonstrated an efficient removal of LPS from plasma in vitro. Adsorption over immobilized HSA appears to be a simple and effective means of removing LPS and perhaps pro-inflammatory cytokines from the circulation.     

Reduction of viral load and immune complex load on CD4+ lymphocytes as a consequence of highly active antiretroviral treatment (HAART) in HIV-infected hemophilia patients.

BACKGROUND AND OBJECTIVES: Human immunodeficiency virus (HIV)-induced immune complex load on circulating CD4+ blood lymphocytes is associated with dysfunction and depletion of CD4+ lymphocytes and with increased monocyte/macrophage function. It was investigated whether HAART reduces both the viral load in plasma and the number of immune complex-coated CD4+ lymphocytes in the blood, and whether CD4+ counts are associated with viral load and/or immune complex load. MATERIALS AND METHODS: Twelve HIV+ hemophilia patients before and after conversion to HAART (group 1); eight HIV+ hemophilia patients without antiretroviral therapy (group 2). HIV-1 RNA copies in plasma using NASBA/Nuclisens kits; CD4+ lymphocytes coated in-vivo with immune complexes using flowcytometry on whole blood samples; in-vitro responses of immune complex-coated T lymphocytes in cell culture assays. RESULTS: After conversion to HAART there was a significant reduction of viral load, CD4+ gp120+, CD4+ IgM+, and CD4+ IgG+ circulating blood lymphocytes and plasma neopterin, paralleled by a significant increase of CD4+ and CD8+ counts. The percentage of immune complex-coated CD4+ lymphocytes of converted patients was significantly associated with CD4+ counts, in-vitro responses to concanavalin A (Con A), pokeweed mitogen (PWM), phytohaemagglutinin (PHA), anti-CD3 and pooled allogeneic stimulator cells, and with plasma neopterin levels. CONCLUSION: HAART reduces viral load and HIV-induced immune complex load on circulating CD4+ blood lymphocytes. The results of this study can be interpreted to suggest that HAART increases CD4+ lymphocyte counts in part by counteracting HIV-induced autoimmune phenomena.     

The R576 IL-4 receptor alpha allele correlates with asthma severity.

BACKGROUND: Atopic disorders, including asthma, are very prevalent, affecting up to 40% of populations, and their incidence is on the rise. Although environmental factors are important in the development of atopy, there is a strong genetic predisposition. Several genes and chromosomal regions have been linked to atopy and asthma, supporting the polygenic nature of these disorders. IL-4 and IL-13 are T(H)2 cytokines with numerous activities that contribute to allergic inflammation and asthma. Both IL-4 and IL-13 use the IL-4 receptor alpha chain (IL-4Ralpha) as a component of their respective receptor systems. Allelic variants of IL-4Ralpha have been reported, and the R576IL-4Ralpha allele was recently shown to be a risk factor for atopy. OBJECTIVE: We sought to determine whether the R576 allele was associated with the prevalence or clinical severity of asthma. METHODS: We developed a rapid, reliable, PCR-based assay to screen individuals for the R576IL-4Ralpha allele and used this assay to genotype prospectively recruited individuals with asthma (n = 149) and control subjects (n = 57). RESULTS: There was a strong association of R576IL-4Ralpha with the prevalence and clinical severity of asthma. In a prospective cohort, homozygosity for R576 was significantly increased in individuals with asthma (n = 149, P =.03; relative risk 8.2) compared with controls (n = 57). Furthermore, 1 or 2 copies R576IL-4Ralpha correlated with asthma severity establishing a genotype-phenotype relationship and suggesting a gene dosage effect. CONCLUSIONS: Thus R576IL-4Ralpha acts as an allergic asthma susceptibility and disease-modifying gene and may serve as a clinically useful marker of asthma severity.