Enriched Plasma Research Articles

PDL1 test to assess the dynamic range of mRNA expression from platelet enriched plasma in patients with NSCLC.

e23063 Background: Therapeutic options for patients with non-small cell lung cancer (NSCLC) continue to expand with the advent of immunotherapies. Lack of tissue and drawbacks with IHC have increased the need for blood-based diagnostics that measure continuous variables. Thus these tests are of increasing relevance for clinical testing. Methods: We focused on mechanisms of blood-based testing for the sensitive measurement of circulating RNA using ddPCR. Specifically, we optimized methods for the detection of PD-L1 and cytokeratin 19 (CK19) transcripts recovered from plasma. Specimens included tumor derived cell-lines, activated and resting immune cells, normal donor plasma and NSCLC (stages 1-4) donor plasma. Results: Assessing PD-L1 in circulation is complicated by its expression in both immune and cancer cells. Analytic performance was initially evaluated with cancer cell lines and activated lymphocytes and monocytes expressing variable levels of cytokeratins and PD-L1. A preliminary threshold was defined based on CK19 expression levels in normal donor plasma (n = 35). Of the 79 NSCLC donors tested 49% expressed CK19 above the threshold, indicating they contained sufficient circulating RNA derived from tumor. These donor samples tested either positive for EGFR del19 E746-A750 or L858R (n = 13), negative for EGFR (n = 10) or positive for KRAS G12C/V (n = 16). We found no differentiation in PD-L1 levels on the basis of EGFR sensitizing and EGFR wild-type status. We observed only three donors that expressed PD-L1 at high levels, and this was regardless of EGFR mutation status. In contrast, 30% of KRAS mutation-positive donor samples were above the pre-defined PDL1 threshold (n = 6 of 16 samples tested). These data are consistent with tissue-based studies that report trends of higher PD-L1 expression in KRAS positive NSCLC patients. Conclusions: We have developed sensitive and specific methods to measure the dynamic range of PD-L1 in circulation and have shown utility of these methods by evaluating key immune and cancer-specific RNAs.

Superior Identification of Prognostic Relevant Copy Number Abnormalities By SNP-Based Genomic Arrays As Compared to Interphase FISH in Multiple Myeloma

Multiple myeloma (MM) is a neoplasm that exhibits a broad heterogeneity in both biological behavior and clinical presentation. Specific copy number abnormalities (CNAs) such as hyperdiploidy, 1p loss, 1q gain, 13q loss and 17p loss (including the TP53 gene), and IGH translocations, such as t(4;14)(p16;q32) and t(14;16)(q32;q23), provide important information regarding prognosis and treatment response.Interphase fluorescence in situ hybridization (FISH) on enriched plasma cells, currently used in clinical diagnostics of MM, is a targeted test aimed at specific genomic loci. However, it is laborious and provides only genetic information of the probe targets. Microarray-based genomic profiling is a high-resolution tool that enables genome-wide analyses for copy number alterations (CNA), including focal CNA (<5 Mb) and regions of copy neutral loss of heterozogosity (CNLOH) that cannot be identified by FISH. A limitation of SNP-based array is its inability to identify balanced translocations.The aim of this study was to compare FISH with SNP-based genomic arrays with respect to the detection yield for prognostic relevant genetic copy number abnormalities in enriched plasma cell samples from MM patients. In addition we have set up a diagnostic work flow in which on one sample of enriched plasma cells interphase FISH for (balanced) IGH translocations as well as SNP-based array for identification of CNA can be performed.SNP-based genomic array profiling and FISH were performed in 37 MM patients. After enrichment of CD138 plasma cells half of each sample was treated with 0.075M KCl and, subsequently, fixed with 3:1 methanol/acetic acid and transferred to a microscopic slide for subsequent FISH. From the remaining part of the CD138-enriched plasma cell fraction DNA was extracted to perform SNP-based genomic array. Interphase FISH was performed according to standard methods using the following probes D5S23/D5S721/CEP9/CEP15, LSI13 (13q14), LSI TP53 (17p13.1) (all from Abbott Molecular, USA), and CDKN2C/CKS1B (from Cytocell, UK). 200 nuclei were analyzed by two different investigators and the detection limit was set at 20% as proposed by the EMN (Ross et al 2012; Haematologica 97:1272-1277). SNP-based array was performed using the CytoScan HD array platform (Affymetrix, USA), using the interpretation criteria as proposed by Schoumans (Schoumans et al 2016; Genes Chromosomes Cancer 55:480-491). Data regarding FISH and SNP-based array were obtained in a fully blinded fashion.All prognostic relevant CNA as observed by FISH were also identified when only SNP-based genomic arrays would have been performed, including 4 cases with loss of 1p, 19 cases with gain of 1q, 14 cases with loss of 13q, 4 cases with loss of 17p, and 20 cases with a hyperdiploid karyotype. However, SNP-based arrays identified 20 additional prognostic relevant abnormalities which were not observed by interphase FISH for several reasons. Due to a higher detection limit of the applied SNP-based array platform, 2 cases with loss of 17p (abnormality present in 15-20% of the cells) and 1 case with loss of 13q and a hyperdiploid karyotype (present in 15% of the cells) were observed by SNP-array only. Four cases showed a 1p21 or 1p16 loss, which were not observed by FISH since these deleted regions were outside the 1p32 probe target region. In 3 cases tumor-associated regions of CNLOH were observed involving the regions 13q and 17p. Finally, in 4 cases in which FISH was suggestive for a hyperdiploid karyotype, the SNP-based array information regarding whole genome analysis and allele frequencies demonstrated that these 4 cases appeared to have a doubled up DNA content in their plasma cells, and therefore the losses of 1p, 13q and 17p were not observed by interphase FISH.In conclusion, we demonstrate that SNP-based arrays are superior in the identification of prognostic relevant CNA in MM. SNP-based array do identify all CNA as observed by FISH, and in addition, identifies additional prognostic relevant abnormalities, such as loss of 1p, 13q, and 17p, that escaped the detection by FISH. The prognostic relevance of the CNLOH and the loss of 1p21 and 1p16 regions requires further evaluation in prospective clinical trials. DisclosuresZweegman:Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding.

Glutathione species and metabolomic prints in subjects with liver disease as biological markers for the detection of hepatocellular carcinoma

BackgroundThe incidence of liver disease is increasing in USA. Animal models had shown glutathione species in plasma reflects liver glutathione state and it could be a surrogate for the detection of hepatocellular carcinoma (HCC). MethodsThe present study aimed to translate methods to the human and to explore the role of glutathione/metabolic prints in the progression of liver dysfunction and in the detection of HCC. Treated plasma from healthy subjects (n = 20), patients with liver disease (ESLD, n = 99) and patients after transplantation (LTx, n = 7) were analyzed by GC- or LC/MS. Glutathione labeling profile was measured by isotopomer analyzes of 2H2O enriched plasma. Principal Component Analyzes (PCA) were used to determined metabolic prints. ResultsThere was a significant difference in glutathione/metabolic profiles from patients with ESLD vs healthy subjects and patients after LTx. Similar significant differences were noted on patients with ESLD when stratified by the MELD score. PCA analyses showed myristic acid, citric acid, succinic acid, l-methionine, d-threitol, fumaric acid, pipecolic acid, isoleucine, hydroxy-butyrate and glycolic, steraric and hexanoic acids were discriminative metabolites for ESLD-HCC+ vs ESLD-HCC− subject status. ConclusionsGlutathione species and metabolic prints defined liver disease severity and may serve as surrogate for the detection of HCC in patients with established cirrhosis.

Identification of leukemia enriched plasma membrane proteins allows prospective isolation and analysis of distinct subclones providing new opportunities for diagnosis and targeting of acute myeloid leukemia

Identification of leukemia enriched plasma membrane proteins allows prospective isolation and analysis of distinct subclones providing new opportunities for diagnosis and targeting of acute myeloid leukemia

PRGF exerts more potent proliferative and anti-inflammatory effects than autologous serum on a cell culture inflammatory model

Ocular graft versus host disease (oGVHD) is part of a systemic inflammatory disease that usually affects ocular surface tissues manifesting as a dry eye syndrome. Current treatments provide unsatisfactory results. Blood-derived products, like plasma rich in growth factors (PRGF) emerge as a potential therapy for this disease. The purpose of this study was to evaluate the tissue regeneration and anti-inflammatory capability of PRGF, an autologous platelet enriched plasma eye-drop, compared to autologous serum (AS) obtained from oGVHD patients on ocular surface cells cultured in a pro-inflammatory environment. PRGF and AS were obtained from four GVHD patients. Cell proliferation and inflammation markers, intercellular adhesion molecule-1 (ICAM-1) and cyclooxygenase-2 (COX-2), were measured in corneal and conjunctival fibroblastic cells cultured under pro-inflammatory conditions and after treatment with PRGF or AS eye drops. Moreover, cell proliferation increased after treatment with PRGF and AS, though this enhancement in the case of keratocytes was significantly higher with PRGF. PRGF eye drops showed a significant reduction of both inflammatory markers with respect to the initial inflammatory situation and to the AS treatment. Our results concluded that PRGF exerts more potent regenerative and anti-inflammatory effects than autologous serum on ocular surface fibroblasts treated with pro-inflammatory IL-1β and TNFα.

A detailed proteomic profiling of plasma membrane from zebrafish brain.

Zebrafish (Danio rerio) is a well-established model organism in developmental biology and disease modeling. In recent years, an increasing amount of studies used zebrafish to analyze the genetic changes underlying various neurological disorders. The brain plasma membrane proteome represents the major subsets of signaling proteins and promising drug targets, but is often understudied due to traditional experimental difficulties including problems with solubility, detergent removal, or low abundance. Here, we report a comprehensive dataset of the proteins identified in the enriched plasma membrane of the zebrafish brain by applying sequential trypsin/chymotrypsin digestion with multidimensional LC-MS/MS. A total number of 97017 peptide groups corresponding to 9201 proteins were identified. These were annotated in various molecular functions or neurological disorders. The dataset of the current study provides a useful data source for further utilizing zebrafish in basic and clinical neuroscience.

Quantitative proteomics revealed novel proteins associated with molecular subtypes of breast cancer

The early diagnosis and successful treatment of breast cancer (BC) is still a challenging task due to the diverse origin and functional heterogeneity of cancer cells. The heterogeneity of BC may likely to explained by molecular BC subtypes, comprises Luminal-A (LA), Luminal-B (LB), Triple-negative (TN) and HER2-positive (HP), which are governed by a variety of cancer associated pathways. To identify protein signatures in different BC subtypes, we performed isobaric tag for absolute and relative quantitation (iTRAQ) of enriched blood plasma samples of BC subtypes (N=32) and healthy subjects (N=8). After analyses of data, 58 proteins were found to be modulated in BC subtypes from healthy subjects (p<0.05) and among these; Fibronectin (FN1), Alpha-2-macroglobulin (A2M), and Complement component-4-binding protein-alpha (C4BPA) and Complement factor-B (CFB) were selected for validation in BC subtypes and healthy subjects in the independent set of blood plasma (N=100) and tissue samples (N=25). Statistical analysis showed the significant modulation of FN1 and C4BPA in LB, and A2M in TN patients in both plasma as well as tissues comparatively control (p<0.05). Further, FN1 and C4BPA in LB subtype revealed a good diagnostic accuracy in plasma level validation. The receiver operating characteristic (ROC) curve and regression analysis demonstrated that these proteins with associated criterion of expression could act as discriminating signatures among BC subtypes with diagnostic and prognostic relevance. SignificanceThe heterogeneity of breast cancer (BC) has gained many challenges for successful management of BC, thus, the delineating proteomic alterations BC subtypes may provide great clinical values in diagnostic, prognostic and therapeutics of BC. The findings from the present quantitative proteomic study have deciphered the altered proteomic patterns and their possible molecular interactions in each BC subtype. The study showed a strong association of FN1, A2M, C4BPA and CFB in molecular subtypes of BC, in which, C4BPA and A2M demonstrated a potent signature in blood plasma and tissue samples of LB and TN subtypes in BC patients, respectively. The findings also revealed the altered level expressions of these selected proteins could classify BC subtypes through plasma and tissue based expression analysis in patients and control samples. Hence, these proteins could have clinical importance for the diagnosis and prognosis purposes among molecular BC subtypes.

Optic nerve involvement in immunoglobulin G4-related disease: A case report.

The present study reports the case of a 79-year-old woman manifesting skin changes, a pancreatic neoplasm, enlarged lymph nodes, an eyelid mass and interstitial pneumonia over a 30-year period. At 2 months before admission to our hospital, the patient presented rapid vision loss in the left eye. Left optic nerve atrophy with a focal hyperintense lesion was documented on a T2-weighted magnetic resonance imaging (MRI) scan, and visual evoked potential implicit times were prolonged. Elevated serum immunoglobulin G4 (IgG4) concentrations and the presence of enriched IgG4-positive plasma cells in the lymph nodes established the diagnosis of IgG4-related optic neuropathy. Following oral treatment with methylprednisolone, the serum IgG4 levels fell to normal levels, and the left eye visual acuity improved to a level that remained stable over a 1-year follow-up period. After 4 months of methylprednisolone administration, the optic nerve appeared to be normal on an MRI scan. Prior reports on IgG4-related optic neuropathy involved an infiltrating mass, which was not observed in the present case. Similarly to the current study, previous cases have responded to treatment with glucocorticoids, indicating that the underlying mechanism of the disease may be common.

IgG4 Associated Cholangitis

IgG4-related disease (IgG4-RD) is a systemic inflammatory disease characterized by an infiltration of lymphoplasmacytic enriched IgG4-positive plasma cells, variable degrees of fibrosis and mass forming lesions. Previously, this disorder was known as IgG4 multi-organ lymphoproliferative syndrome (IgG4 MOSLP), IgG4 sclerosing disease, or IgG4-related systemic plasmocytic syndrome (SIPS). The most common clinical manifestations are parotid inflammation, lymphadenopathy and autoimmune pancreatitis.

Experimental substantiation of platelet enriched plasma application for stimulation of cartilage autografts regeneration

The objective of this work was to study the influence of platelet enriched plasma on regeneration of cartilage tissue after its mechanical destruction using morphological methods. In the process of study, withdrawal and fragmentation of cartilage tissue sections of both ear conches of rabbits was performed with subsequent reimplantation of the crushed cartilage tissue to the same place in the left ear and crushed cartilage tissue mixed with platelet­enriched plasma obtained from the blood of the same laboratory animal - to the right auricle. After that a cut was made and the peculiarities of regeneration of the cartilage on the 10th, 20th days and after 1, 2 and 3 months after surgery were studied. After that the platelet enriched plasma was injected in the defect site on the right ear conch once a week. The study revealed that in the experimental group in terms of platelet enriched plasma application, regeneration of cartilage tissue occurred more adequately in comparison with the control group, and was accompanied by the acceleration of healing processes, which was expressed in activation of fibroblastic and chondroblastic of programed differentiation.

Optimal strategy for obtaining routine chromosome analysis by using negative fractions of CD138 enriched plasma cells

Fluorescence in situ hybridization (FISH) is superior to routine chromosome analysis (RCA) in detecting important prognostic genetic abnormalities in plasma cell dyscrasia (PCD); however, its sensitivity is hampered due to paucity of plasma cells (PC) in whole bone marrow (BM). Studies showed that the abnormality detection rate in enriched plasma cells (EPC) is greater than unselected plasma cells (UPC), but purification techniques are limiting to only FISH when sample volumes are inadequate. Not performing RCA may compromise patient care since RCA is equally important for detecting non-PC related abnormalities when the diagnosis is undefined. To resolve this critical issue, we designed a study where an immuno-magnetic CD138 enriched positive selection was used for FISH while the negative fraction (NF) was used to retrieve other myeloid elements for RCA. Parallel FISH studies were performed using UPC and CD138 EPC, while karyotyping was achieved using whole BM and discarded myeloid elements from the NF. Results showed that the abnormality rate of EPC was doubled compared to UPC for FISH, and CA displayed 100% success rate using the NF. PCD related chromosome abnormalities were confined to whole BM while non-PCD related abnormalities were found in both whole BM and NF. Our results demonstrate the feasibility of using the NF for RCA.

Immunolocalization of hyperpolarization-activated cationic HCN1 and HCN3 channels in the rat nephron: regulation of HCN3 by potassium diets.

Hyperpolarization-activated cationic and cyclic nucleotide-gated channels (HCN) comprise four homologous subunits (HCN1-HCN4). HCN channels are found in excitable and non-excitable tissues in mammals. We have previously shown that HCN2 may transport ammonium (NH4 (+)), besides sodium (Na(+)), in the rat distal nephron. In the present work, we identified HCN1 and HCN3 in the proximal tubule (PT) and HCN3 in the thick ascending limb of Henle (TALH) of the rat kidney. Immunoblot assays detected HCN1 (130kDa) and HCN3 (90KDa) and their truncated proteins C-terminal HCN1 (93KDa) and N-terminal HCN3 (65KDa) in enriched plasma membranes from cortex (CX) and outer medulla (OM), as well as in brush-border membrane vesicles. Immunofluorescence assays confirmed apical localization of HCN1 and HCN3 in the PT. HCN3 was also found at the basolateral membrane of TALH. We evaluated chronic changes in mineral dietary on HCN3 protein abundance. Animals were fed with three different diets: sodium-deficient (SD) diet, potassium-deficient (KD) diet, and high-potassium (HK) diet. Up-regulation of HCN3 was observed in OM by KD and in CX and OM by HK; the opposite effect occurred with the N-terminal truncated HCN3 in CX (KD) and OM (HK). SD diet did not produce any change. Since HCN channels activate with membrane hyperpolarization, our results suggest that HCN channels may play a role in the Na(+)-K(+)-ATPase activity, contributing to Na(+), K(+), and acid-base homeostasis in the rat kidney.

Simvastatin nanolipid carriers decreased hypercholesterolemia induced cholesterol inclusion and phosphatidylserine exposure on human erythrocytes

This study aimed to develop simvastatin (SV) loaded lipid nanocarriers as solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs) and lipid nanoemulsion (LNE) and to estimate their in vitro erythrocyte uptake to reduce the cholesterol level. Therefore, numerous SLNs were prepared using Imwitor 900K, Stearic acid, Softisan 154 and Dynasan 118. For NLC, 30% w/v of Miglyol 812 was added to the previous lipids and LNE consisting 100% w/v of Miglyol 812 was also prepared for comparison. These lipid nanocarriers exhibited spherical-shapes with negative charges and particle size lower than 400nm (PDI<0.3). Moreover, the optimized formulations were LNE (Miglyol 812), NLC (Stearic acid and Miglyol 812) and SLN (Stearic acid), which showed the highest entrapment efficiency and drug release. In addition, the percent of erythrocyte uptake of SV from free SV solution, optimized SLN, NLC and LNE was 28%, 78%, 85% and 72%, respectively. Furthermore, incubation of erythrocytes with cholesterol enriched plasma resulted in a significant increase in the percent of cholesterol inclusion, hemolysis and eryptosis compared to control. In contrast, the incubation of erythrocytes with cholesterol rich plasma in the presence of the optimized nanocarriers resulted in a significant decrease percent of cholesterol inclusion, hemolysis and eryptosis. However, effect of the lipid nanocarriers on the measured parameters was more pronounced than that of the free drug. Thus, these results suggested that the lipid nanocarriers might be a promising nanocarrier for SV to maintain the native erythrocyte function in hypercholesterolemia.

The effect of platelet enriched plasma on clinical outcomes in patients with femoroacetabular impingement following arthroscopic labral repair and femoral neck osteoplasty.

To compare the clinical outcome of patients treated with and without platelet-rich plasma (PRP) injection while undergoing arthroscopic labral repair and femoral neck osteoplasty for femoral acetabular impingement. Patients were randomized at the time of surgery to receive either an intra-articular injection of 5 cc of PRP, or an equal volume of 0.9% normal saline. All patients underwent arthroscopic labral repair and osteoplasty of the femoral neck and, at the conclusion of the case, received the injection. One week following surgery, thigh circumference (measured 10 cm distal to the tip of the greater trochanter) and the presence of ecchymosis of the thigh were recorded. Clinical outcome scores, including Non-Arthritic Hip Score, Modified Harris Hip Score and Hip Outcome Score were collected prior to surgery at 1, 3, 6 and a minimum of 12 months post-operatively. Thirty-five patients were enrolled into this study. Twenty patients received a PRP injection and 15 received a saline injection. Thigh circumference was compared pre-operatively and 1 week post-operatively. There was no significant difference between the two groups. Ecchymosis was compared between the two groups at 1 week post-operatively. Four of the 20 patients in the PRP group and 10 of the 15 in the placebo group demonstrated bruising on the lateral thigh. This was compared with a Chi-Square test and found to be statistically significant (P = 0.005). There was no significant difference in any of the outcome scores between the two groups. An intra-articular injection of PRP after labral repair did not improve the clinical outcome up to 1 year post-operatively in patients undergoing arthroscopic labral repair and osteoplasty of the femoral neck. Level of evidence is level I study.

Influence of L-lysine amino acid on the HIV-1 RNA replication invitro.

Virus replication strongly depends on host metabolic machinery and essential cellular factors, in particular, on amino acid profiles. Amino acids play an important role in the pathogenesis of all virus-related infections both as basic substrates for protein synthesis and as regulators in many metabolic pathways, including gene expression. The inhibitory effects of deficiency or excess of these essential elements on virus replication are widely appreciated. Although the same interrelationship between host cellular factors and HIV have been recognized for a long time, the effects of amino acids on HIV-1 RNA replication dynamic is not yet well documented. Our aim was to determine in this pilot study the direct effect of L-lysine amino acid on HIV-1 RNA replication in vitro in HIV-infected patients. A total of 100 HIV-1-infected males without highly active antiretroviral therapy (HAART) were monitored in our center. The patients were in stage A of the disease according to the 1993 Centers for Disease Control (CDC) classification system for HIV-infection. Patients with HIV were enrolled in one stage (A) of the disease with the average amount CD4 lymphocytes in the range of 200-300 cells/µL at the time of sample acquisition. For evaluation of the effects of essential L-lysine amino acid on HIV-1 RNA replication level, we used a model of amino acid-excess system in vitro following incubation of plasma samples for 24 h at 25 °C. Quantitative HIV-1 RNA assay was performed using (RT-PCR) reverse-transcriptase polymerase chain reaction (Rotor-Gene Q, QIAGEN, Germany). The mean HIV-1 RNA levels were significantly higher in the enriched peripheral blood mononuclear cells plasma samples HIV-infected subjects after 24 h incubation at 25 °C temperature than in the plasma samples the same patients studied on the date of blood tests (p < 0.0001). The number of HIV-1 RNA copies increased in 1.5 times. We observed that in plasma of the same HIV-infected patients after adding L-lysine and following incubation in vitro, viral load increased significantly in comparison with standard samples (p < 0.0001). The increased viral load was found in 100/92 (92%) of HIV-infected subjects. The average number of HIV-1 RNA copies in samples had increased by 4.0 times. However, we found no difference in HIV-1 RNA levels after replacement of L-lysine for L-arginine in comparison samples in the same HIV-infected patients. It is obvious that the addition of L-arginine does not increase viral replication in vitro as L-lysine amino acid supplement does. Additionally, no increase in viral load was determined after adding L-lysine and non toxic doses of its inhibitor (L-lysine alpha-oxidase) in plasma samples. The results show that L-lysine amino acid excess is characterized by significant increased of HIV-1 RNA copies in enriched peripheral blood mononuclear cells plasma samples of HIV-infected patients. There was evidence for an association between L-lysine supplementation and HIV-1 RNA replication and the level changes of this host essential nutritional element play a key role in the synthesis of the virus proteins and in transcription initiation of the retrovirus life cycle. High intake of L-lysine amino acid may increase the risk of high viral load, subsequent acceleration of immunosuppression and HIV progression. Overall results demonstrate that the simple L-lysine-related model in vitro can be widely used for practical purposes to evaluate HIV-1 RNA replication dynamic, disease prognosis and new approaches in treatment of the patients with human immunodeficiency virus. Although the impact mechanism of L-lysine amino acid on the viral load in the pathogenesis of HIV-infection is at present conjectural and requires further development, the results highlight an interesting target in antiviral therapy, and this statement remains to be proved in further research and clinical trials.

Fluorescence in situ hybridization on enriched CD138-positive cells in plasma cell myeloma

To validate plasma cell enrichment technique for improving the detection of cytogenetic abnormalities in the Plasma cell myeloma (PCM)/multiple myeloma (MM). We compared the abnormality detection rate for overnight unstimulated bone marrow cultures to that for the plasma cell enriched fractions obtained with the use of CD138-coated immunomagnetic beads. Average enrichment factor (EF) was 11. One or more abnormalities were detected in 90% of enriched samples vs. 65% of non-enriched samples, thus resulting in a significantly higher detection rate of total cytogenetic abnormalities in enriched plasma cells (p=0.0038). Additional findings of RB1 deletion, TP53-, 1p-, 1q+ and IGH@ rearrangement seen in the 25% of enriched samples could contribute to the altered risk in the patient. One of the three cases with plasma cells as low as 1% by morphology was positive for a residual disease marker in the enriched sample and negative in the non-enriched sample. The plasma cell enrichment technique increased the detection rate of diagnostic and prognostic markers and is a very sensitive method for detecting minimal residual disease.

In vitro investigations of ABC transporters of the human liver – advantages and surprises

The hepatocyte contains a battery of membrane transporters in the sinusoidal and canalicular membrane that guarantee the regulated import and export of nutrients, messengers and toxic compounds. In the canalicular membrane, a number of important ABC (ATP binding cassette) transporters are located. Three of these primary active transporters, BSEP (bile salt export pump, ABC B11), MDR3 (multidrug resistance protein 3, ABC B4) and ABC G5/G8 catalyze the secretion of bile acids (BSEP), phosphatidylcholine (MDR3) and cholesterol (ABC G5/G8). These three molecules form mixed micelles in bile and thereby reduce the harmful action of unbound bile salts on membranes. BSEP, which was initially named s-Pgp (sister of P-gp) and MDR3 share a high sequence identity with MDR1 (P-glycoprotein, ABC B1), which is the hallmark of the family of multidrug resistance ABC transporter present in every eukaryotic cell. For BSEP the identity is approximately 70% and for MDR3 even 85%, respectively. Nevertheless, MDR3 is specific for phosphatidylcholine lipids and BSEP is specific for bile acids, while MDR1 is capable of translocating a myriad of structurally unrelated compound across biological membranes. This of course raises the question, which residues impose specificity in the case of BSEP and MDR3 and which residues impose promiscuity in the case of MDR1. Homology models of BSEP and MDR3 were generated based on the crystal structures of Sav1866 and mouse P-gp to obtain initial information about the putative three-dimensional location of certain amino acid residues of BSEP and MDR3. However, one has to stress that these “structures” represent only models that have to be used with proper caution. The homology model of MDR3 was helpful in providing a structural explanation for the non-functionality of the MDR3 mutant H1231Y. The mutant protein was properly located to the plasma membrane, but displayed a severe substrate transport phenotype. Histidine 1231 corresponds to the histidine of the H-loop, a residue that is critical for ATP hydrolysis in many other ABC transporter systems. Thus, exchange of His against Tyr in position 1231 of MDR3 will impair ATP hydrolysis in one of the two ATP binding site drastically reducing transport activity of MDR3 H1231Y [1]. To study the transport cycle and the importance of mutations within these transporters on their function, overexpression systems are required. We decided to use the methylotrophic yeast Pichia pastoris for the overexpression of two of the three transporters, BSEP and MDR3, respectively. Initially we encountered severe problems in cloning the transporters genes in E. coli employing standard approaches. Only after establishing a cloning strategy that is entirely based on Saccharomyces cerevisiae, both genes could be successfully cloned [2]. This allowed us to combine the speed and ease of cloning of this yeast system for the wild type genes and mutations that were identified in patients. Having this tool in hand, we overexpressed BSEP in yeast to study its transport function in plasma membrane vesicles. This allowed us to establish structure-function relations for a new clinically relevant mutation (G374S) that resulted in a phenotype between PFIC-2 and BRIC-2. Although membrane targeting was normal, e.g. targeting to the canalicular membrane was evident, transport activity of the G374S was strongly impaired [3]. The Sav1866-based homology model of BSEP suggested that Gly 374 is lining the translocation pore of BSEP. Thus, an exchange to serine would impose steric restriction within the pore, thereby influencing the substrate spectrum as evident from the bile acid transport assays. Based on the expression levels of BSEP and MDR3, purification protocols for both membrane proteins were successfully established [4]. Thus, next to the transport assay that is based only highly enriched yeast plasma membranes, purified protein is now also available in the detergent-solubilized state. While the transport assay can be used for BSEP to analyze a possible influence of a mutation on the transporter activity of the protein, purified MDR3 can be used to determine the influence of a mutation on ATPase activity and / or stimulation or inhibition of this activity. In summary, the established yeast overexpression system combines the ease and speed of yeast cloning approaches with the possibility to overexpress and purify both proteins in a quantity and homogeneity sufficient for biochemical and biophysical investigations of the function of these two ABC transporters. Furthermore, the speed with which mutants can be obtained will allow an in depth characterization of clinically relevant mutations of both transporters with respect to function and spectrum of transported substrates.

A novel autologous plasma based viscosupplementation

Purpose: Combined long acting viscosupplement and autologous cartilage promoting factors for the treatment of osteoarthritis. Methods: RegenoGel-SP is a novel elastomer made of fibrinogen enriched plasma chemically linked to high molecular weight Hyaluronic acid (HA). Plasma fibrinogen-HA conjugate solution polymerized by CaCl2 was seeded with primary human articular chondrocytes. Cell viability and potency in the 3D constructs were evaluated using Alamar blue, DMMB for sulphated glycosaminoglycans (sGAGs) and quantitative rtPCR for chondro-specific markers. Results: In the last OARSI meeting we presented a novel Fibrin-HA conjugate as a long acting viscosupplement and as a carrier for the disease modifying agent FGF18 or its FGF receptor selective variants. To further enhance the biological properties of the hydrogel, a similar chemical approach was implemented using whole human plasma conjugated to high molecular weight Hyaluronic acid. This self plasma based hydrogel designated Regenogel-SP demonstrated remarkable physical endurance and biocompatibility to diverse primary cell types. Specifically, Regenogel-SP demonstrated superior capacity to support cell proliferation, viability and differentiation of human articular chondrocytes from both normal and OA cartilage. Conclusions: We have previously reported on the regenerative and cell supportive activity of Fibrin-HA compositions combined with FGF18 variants. The newly developed Regenogel-SP provides a novel and safe approach for delivering autologous plasma rich constituents in a stable, long acting, viscous support for cartilage preservation and repair in OA.

New Insights on the Versatile Role of the Cholesterol Binding Motif of the HIV-1 Glycoprotein Gp41

Recent experimental results indicate that host cell invasion as well as assembly and budding of the Human Immunodeficiency Virus (HIV) are highly cholesterol dependent. Supposably, cholesterol enriched plasma membrane microdomains, so called rafts, play an important role in different steps of the virus lifecycle. However, the exact function and molecular background of this sensitivity to bilayer compositions remains unknown.We produced different variants of the HIV transmembrane protein gp41 labeled with a yellow fluorescent protein. Fluorescence lifetime imaging microscopy was used to report Forster Resonance Energy Transfer (FRET) between a raft marker labelled with a cyan fluorescent protein and gp41 chimeras in living cells. Since it is highly distance dependent, occurring FRET reports a co-clustering of both fluorescent protein species in microdomains. By comparison of FRET efficiencies from different truncation and mutation variants of gp41, the Cholesterol Recognition Amino Acid Consensus (CRAC) was identified as main determinant of the protein's raft partitioning in the plasma membrane and interestingly in the Golgi apparatus as well. Moreover, using fluorescence polarization anisotropy microscopy we found indications, that wildtype gp41 oligomers are stabilized in plasma membrane microdomains. However, oligomerization of CRAC mutants was found to be significantly impaired, suggesting a pooling function of the lipid rafts for the assembly and sustainment of functional homo-oligomers. Moreover, flow cytometer experiments revealed a remarkable influence of CRAC mutations on the well-known plasma membrane perturbation properties of gp41. Finally, different biophysical methods were applied to further reveal the role of the cholesterol-CRAC interaction in more details.Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral protein sorting for the virus assembly processes at cellular membranes.

Utility of CD-138 negative fraction for chromosome analysis in Plasma Cell Dyscrasias (PCD): a novel approach

Background FISH analysis is superior to chromosome analysis in detecting important prognostic genetic abnormalities in PCD. However, its sensitivity is hampered due to paucity of plasma cells in whole bone marrow and often shows false-negative results when frequency of abnormal cells is below the cut-off values. Studies have shown that the abnormality detection rate in enriched plasma cells (EPC) is greater than unselected plasma cells, but purification techniques are limiting to only FISH when bone marrow volumes are inadequate. The inability to perform chromosome analysis may compromise patient care since chromosome analysis is equally important for detecting non-plasma cell related abnormalities, such as secondary myelodysplastic syndrome. To resolve this critical issue and optimize limited quantity received, we designed a study where an immuno-magnetic CD138 enriched positive selection of plasma cells was used for FISH while the negative selection was used to retrieve the remaining cellular components (RCC) for chromosome analysis. After validating this approach in a pilot study, we implemented this strategy in 2012 for routine clinical diagnosis in patients with PCD. When there was adequate sample volume available, both whole bone marrow and RCC were used for chromosome analysis.