Susceptibility to multiple sclerosis (MS) is associated with the human histocompatibility leukocyte antigen (HLA)-DR2 haplotype, suggesting that major histocompatibility complex class II-restricted ...presentation of central nervous system-derived antigens is important in the disease process. Antibodies specific for defined HLA-DR2-peptide complexes may therefore be valuable tools for studying antigen presentation in MS. We have used phage display technology to select HLA-DR2-peptide-specific antibodies from HLA-DR2-transgenic mice immunized with HLA-DR2 molecules complexed with an immunodominant myelin basic protein (MBP) peptide (residues 85-99). Detailed characterization of one clone (MK16) demonstrated that both DR2 and the MBP peptide were required for recognition. Furthermore, MK16 labeled intra- and extracellular HLA-DR2-MBP peptide complexes when antigen-presenting cells (APCs) were pulsed with recombinant MBP. In addition, MK16 inhibited interleukin 2 secretion by two transfectants that expressed human MBP-specific T cell receptors. Analysis of the structural requirement for MK16 binding demonstrated that the two major HLA-DR2 anchor residues of MBP 85-99 and the COOH-terminal part of the peptide, in particular residues Val-96, Pro-98, and Arg-99, were important for binding. Based on these results, the antibody was used to determine if the HLA-DR2-MBP peptide complex is presented in MS lesions. The antibody stained APCs in MS lesions, in particular microglia/macrophages but also in some cases hypertrophic astrocytes. Staining of APCs was only observed in MS cases with the HLA-DR2 haplotype but not in cases that carried other haplotypes. These results demonstrate that HLA-DR2 molecules in MS lesions present a myelin-derived self-peptide and suggest that microglia/macrophages rather than astrocytes are the predominant APCs in these lesions.
Introduction: Multiple myeloma (MM) is a hematological malignancy of clonal plasma cells (PCs) in the bone marrow (BM). MM cells are dependent on the BM microenvironment (e.g. BM stromal cells, ...macrophages etc) and create a network with surrounding cells. These cells play a pivotal role in the regulation of MM cell survival and drug resistance bydirect interactions through adhesion molecules causing cell adhesion mediated drug resistance (CAM-DR) or soluble factors including supportive cytokines (e.g. IL-6, IL-8, and VEGF) or exosomes (or extracellular vesicles). Exosomes (EV) are endosome-derived membrane-covered cell fragments, which contain specific protein and RNA cargo. The protein content of EVs in MM has not been readily explored. Recently our group established the use a global label-free quantification method to determine the relative amount of proteins identified from EVs obtained from MM cell lines. Here we are reporting a systematic proteomic analysis of EVs derived from MM cell lines, blood from MM patients, and BM from MM patients. We also report preliminary data showing the biological importance of specific EV enriched proteins in MM cell lines and MM patients.
Methods: Liquid Chromatography Mass Spectrometry (MS) and Label-free relative quantitation were used to assess the protein content of MM EV and cellular lysates. Cryo-Transmission Electron Microscopy (cryo-TEM) and Nanoparticle Tracking Analysis were used to assess size distribution and specific EV surface markers in EV isolated from the supernatant of MM cell lines and from the serum of non-cancer, MGUS, smoldering MM and active MM donors. Western blot and enzyme-linked immunosorbent assays were used to validate MS data in a bigger cohort of primary MM patients and to assess the biological effect of MM derived EV in the BM stromal cells.
Results: Our data show that the Major Histocompatibility Complex Class I (MHCI) and its associated binding protein β2-microglobulin (β2-MG) are the most abundant communally enriched proteins in the EV derived from MM cell lines and from the serum of MM patients. Although it is well known that the serum level of β2-MG is an important prognostic factor in MM, our data indicate that β2-MG represents only a small percentage of the total serum β2-MG, suggesting that two separate β2-microglobulin populations coexist in the serum of patients. Additionally, we show that the main receptor of hyaluronic acid, CD44, is highly expressed in the EVs isolated from the corticosteroid resistant MM cell line, MM.1R, and is a protein that is differentially expressed in EVs isolated from newly diagnosed MM patients and which seems to play a key role in communicating with the BM microenvironment. By using a large cohort of serum obtained from MM patients prospectively treated on a randomized phase 3 trial (233), we establish the potential of serum CD44 as a predictive biomarker of overall survival. These results support the analysis of EVs as easily accessible MM biomarker.
Conclusions: Our results generate a foundation for the potential use of circulating EVs as novel serum markers of MM and provide the rationale to further explore previously unconsidered molecular players associated with MM disease.
Palumbo:Array BioPharma: Consultancy; Onyx Pharmaceuticals: Consultancy; Millennium Pharmaceuticals Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Genmab A/S: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy; Sanofi Aventis: Consultancy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Introduction: Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of plasma cells (PCs) in the bone marrow (BM). Over 50% of patients die within 5 years of diagnosis. ...The transition from normal PCs to active MM, via premalignant condition (monoclonal gammopathy of undetermined significance; MGUS), consists of many oncogenic events, including upregulation of cyclin D1 and IRF4 genes, as well as activating mutations in NRAS, and KRAS. Despite recent advances in oncogenomics, further studies are needed to identify critical players in MM pathogenesis that could be targeted for pharmacological intervention to improve outcome. Recently, aberrations in epigenetic modifications, including DNA modifications and miRNA expression have been shown to play a crucial role in development and progression of MM. miRNAs are short non-coding RNAs that bind to complementary sequences on target messenger RNA (mRNAs) and downregulate their expression by inhibiting mRNA translation, or inducing its degradation. miRNA analysis, may lead to improved understanding of MM biology and classification, by establishing associations between gene expression changes and MM molecular and clinical features. To assess whether miRNA deregulation plays a critical role in the development of MM we performed small RNA next generation sequencing in the PCs isolated from 3 patients at the MGUS stage and after that they developed active disease, but still untreated. miRNA deregulation was also validated in an independent set of newly diagnosed MM patients (n.34) compared to non-cancer age matched donors (n.6). Mechanisms of transcriptional regulation and biological roles of the differentially expressed miRNAs were also analyzed.
Methods: 1x106 CD138+ frozen PCs (purity>90%) from different 3 donors before and after the disease development were used for the analysis. RNA was extracted with RNA-DNA-protein kit (Norgen Biotek) and 0.8µg of total RNA was used to generate the cDNA libraries using TruSeq Small RNA Library Preparation Kit. The obtained cDNA libraries were sequenced on an ILLUMINA system through the OSU Genomic Shared Resource (GSR). Myeloma cells (MM.1S) were treated with pan-HDACi for 24 hours and total miRNA expression was analyzed by nCounter microRNA array (NanoString Technologies, Inc.). miRNA deregulation upon use of several pan-HDAC'i in clinical use (LBH589, SAHA and AR-42) were validated in 4 different cell lines and in the MM cells of newly diagnosed MM patients. Chromatin immunoprecipitation, silencing RNA for specific histone deacetylase enzymes (HDACI, HDAC2, HDAC3, and HDAC6), western blot analysis, luciferase assays, stem loop RT-PCR, q-RT-PCR and cell proliferation assays were also performed.
Results: We found that several miRNAs are commonly deregulated during disease transition. Some of these miRNAs, including miR-223, miR-221, miR-222, miR-92a and miR-93 (p<0.05) were commonly upregulated, where instead the majority of c-Myc transcriptionally repressed miRNAs, including miR-29s family, were found to be downregulated in the MM-PCs compared to the MGUS-PCs obtained from the same patients. Our data also show that IRF4 expression in MM cells is directly under the control of c-Myc regulated miRNAs, which we found downregulated during the multi-step transformation process of the PCs. We also observed that the use of pan-HDACi's in clinical use, including the recently FDA approved panobinostat (LBH589), in MM cells is able to upregulate the expression of those miRNAs and subsequently downregulate IRF4 at the posttranscriptional level. We also dissected the role of different histone deacetylases to specifically modulate miRNA expression and consequently IRF4-MYC pathway in MM cells.
Conclusions: Collectively, we believe, that these findings support the key role of miRNA aberrant expression in PC transformation. Their role in regulating the expression of IRF4 during myeloma development and lead us to speculate that this explains why IRF4 and c-Myc mRNA levels are higher in newly diagnosed MM patients, without obvious chromosomal abnormalities, compared to MGUS patients. Understanding of the molecular bases of how c-Myc expression can be regulated by a specific histone deacetylase via modulation of miRNA levels will have important impact not only on myeloma therapy, but also other hematopoietic malignancies involving c-Myc deregulation.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The adverse consequences of herbicide drift towards sensitive crops have been extensively reported in the literature. However, no information is available on the consequences of herbicide drift onto ...weed species inhabiting boundaries of agricultural fields. Exposure to herbicide drift could be detrimental to long-term weed management as several weed species have evolved herbicide resistance after recurrent selection with low herbicide rates. Despite the herbicide drift exposure and its potential implications on resistance evolution and weed management, resistance prone weed species such as Palmer amaranth (Amaranthus palmeri) and waterhemp (Amaranthus tuberculatus) are often neglected and not properly managed in agricultural field margins.The first study of this research investigated the frequency and distribution of glyphosate-resistant Amaranthus spp. in Nebraska. The study also investigated how agronomic practices influenced the occurrence of glyphosate resistance in Amaranthus spp. in Nebraska. While glyphosate resistance was widespread in waterhemp, few glyphosate-resistant Palmer amaranth populations were reported in Nebraska. Weed species, geographic region within the state, and current crop were the most important factors predicting the occurrence of glyphosate resistance in fields infested with Amaranthus spp. in Nebraska. Moreover, glyphosate resistance was widespread in waterhemp populations collected on field borders and ditches.The second study investigated the near-field deposition of glyphosate, 2,4-D, and dicamba spray drift from applications with two different nozzles in a low-speed wind tunnel, and their impact on Palmer amaranth and waterhemp growth and development. Herbicide drift was influenced by nozzle design and resulted in Amaranthus spp. biomass reduction or complete plant mortality. Herbicide drift can expose weeds inhabiting field margins to herbicide rates previously reported to select for herbicide-resistant biotypes.The third study investigated if recurrent selection with glyphosate, 2,4-D, and dicamba spray drift could select for Amaranthus spp. biotypes with reduced herbicide-susceptibility over two generations. The study results confirmed that herbicide drift towards field margins can rapidly select for weed biotypes with reduced herbicide sensitivity. Preventing the establishment of resistance prone weeds on field margins is an important management strategy to delay herbicide resistance. Weed management programs should consider strategies to mitigate near-field spray drift, and suppress weed populations on field borders.
This is a small pilot study to compare hemodynamic stability during carotid endarterectomy performed with either regional anesthesia or combined regional/general anesthesia.
Twenty-eight patients ...undergoing elective carotid endarterectomy were randomly assigned to: (1) superficial cervical plexus block (n = 14); or (2) superficial cervical plexus block plus general anesthesia (n = 14). The primary outcome measure was hemodynamic stability as indicated by blood pressure changes. Mean arterial pressure was recorded at different times (T(0), T(1), T(2), T(3)). Surgery duration, carotid cross-clamping time, time to recovery of consciousness, perioperative complications, requirement for antihypertensive or antihypotensive drugs, and lidocaine supplementation were recorded. Patients were asked to grade their intraoperative comfort.
In group 1, mean arterial pressure was higher at T(1) and T(2) compared with group 2 (P < .001). Hedges' g effect size was 2.47 (95% confidence interval CI, 1.48-3.45) at T(1) (very large effect) and 1.36 (95% CI, 0.54-2.18) at T(2) (large effect). Surgery duration was 106 +/- 28 minutes in group 1 versus 91 +/- 16 in group 2 (P = .1026). Carotid cross-clamping time was 45 +/- 14 minutes in group 1 versus 36 +/- 9 in group 2 (P = .0503). Comfort and analgesia were better in group 2 (1.43 +/- 0.65) than in group 1 (2.21 +/- 0.7) (P = .0053).
This pilot study suggests that combined regional/general anesthesia provides greater hemodynamic stability and patient comfort compared with regional anesthesia during carotid endarterectomy. Further studies are warranted before conclusions can be drawn as to the efficacy and safety of the technique.
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