An Historical Overview of the Amyloidoses

The amyloidoses are a heterogenous group of clinical disorders that share the common finding of the abnormal deposition of insoluble proteins into various organs, with the result that these proteinaceous deposits disrupt cellular function and impair the integrity of the organs involved. Most typically, the abnormal protein deposition is the consequence of abnormal three dimensional folding of the culprit protein. The abnormal folding of the protein, in turn, may be due to a germ line mutation, may be due to an acquired mutation, or may be due to a polymorphism or characteristic of a normal protein that leads to abnormal folding, precipitation, and deposition of the protein, particularly when that protein is expressed at unusually high levels for a prolonged period of time. The clinical manifestations of an amyloid disorder are the consequences of the array of organs involved, the extent of amyloid deposition, and co-morbid conditions present in the individual patient. The array of organs involved, and the extent of organ involvement, in turn, depend in large part on the specific protein that is responsible for the amyloid deposition, and the process driving that protein’s production. In this chapter, a chronological overview is intended to summarize the critical insights into the patho-biology of amyloid accumulation of various types. These insights have allowed an improved understanding over time of the of the major subgroups and disease entities of the amyloidoses, leading to some degree of improvement in diagnosis and treatment outcomes. Unfortunately, as of this writing, treatment outcomes still remain poor for a large fraction of patients, and there is need for improvement in all aspects of the evaluation and management of these diseases

Two potential biomarkers identified in mesenchymal stem cells and leukocytes of patients with sporadic amyotrophic lateral sclerosis.Dis.Markers

Abstract. Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disorder caused by degeneration of motor neurons. The cause for most cases of ALS is multi-factorial,this enhances the need to characterize and isolate specific biomarkers found in biological samples from ALS patients. To this end we use human mesenchymal stem cells (hMSC) derived from the bone marrow of six ALS patients (ALS hMSC) and identified two genes, Cytoplasmic FMR Interacting Protein 2 (CyFIP2) and Retinoblastoma (Rb) Binding Protein 9 (RbBP9) with a significant decrease in post transcriptional A to I RNA editing compared to hMSC of healthy individuals. At the transcriptional level we show abnormal expression of these two genes in ALS hMSC by quantitative real time-PCR (qRT-PCR) and Western blot suggesting a problem in the regulation of these genes in ALS. To strengthen this view we tested by qRT-PCR the expression of these genes in peripheral blood leukocytes (PBL) isolated from blood samples of 17 ALS patients and found that CyFIP2 and RbBP9 levels of expression were significantly different compared to the levels of expression of these two genes in 19 normal PBL samples. Altogether we found two novel ALS potential biomarkers in non-neural tissues from ALS patients that may have direct diagnostic and therapeutic implications to the disease. Keywords: ALS biomarkers, CyFIP2, RbBP9, human mesenchymal stem cells, leukocytes Background ALS is a fatal and incurable neurodegenerative disorder characterized by progressive loss of motor neurons in the spinal cord, brainstem and motor cortex, resulting in generalized weakness and muscle atrophy. ALS is the most common motor neuron disorder, with a prevalence of 6 per 100,000 at any given time RNA editing is a post-transcriptional mechanism for expanding the proteomic repertoire. Adenosineto-inosine (A to I) RNA editing by enzymes named ADARs (adenosine deaminases acting on RNAs) is essential for normal life and development of both invertebrates and vertebrates Here we show that ALS hMSC have abnormal pattern of RNA editing in two novel genes, CyFIP2 and RbBP9, which also expressed significantly different in hMSC and PBL of ALS patients compared with samples from normal individuals. Overall, we show that non-neural samples of ALS patients could serve as model tools to reveal potential biomarkers of the disease that could be used for future direct diagnosis of ALS in blood samples. Results hMSC were isolated from bone marrow samples of 6 ALS patients (ALS hMSC) and from 6 non-ALS donors, based on their known property to adhere onto a plastic surface + , CD45 − , CD19 − and CD34 − measured by FACS (Fluorescence-activated cell sorting) and by their ability to differentiate into adipocytes, osteocytes and chondrocytes, as established by The International Society for Cellular Therapy From these common biological features between the hMSC donors we tested the hypothesis that ALS hMSC may serve as a model tools for ALS biomarker identification. We investigated in ALS hMSC the possibility that A to I conversion by RNA editing may be altered in specific target substrate genes of the ADAR-mediated RNA editing enzymes To verify these results we performed RT-PCR analysis on the same RNA samples from 6 ALS and 3 non-ALS hMSC with the addition of 3 new non-ALS hMSC samples as we show in These results lead us to investigate the mRNA expression levels of CyFIP2 and RbBP9 genes in ALS and non-ALS hMSC cultures using qRT-PCR analysis. In these experiments we found significant expression differences for these genes between ALS and non-ALS Comparative analysis of cell survival of ALS hMSC (n = 4) and non-ALS (n = 2) donors at two low densities cultured for up to 3 days in the presence or absence of serum. For comparative analysis between different samples and experiments the mean values are expressed as Log of the live/dead ratio as indicated in y axis. The conditions were scored in triplicates of two independent experiments. C. ALS and non-ALS hMSC differentiate into adipocytes and osteoblasts. Cells were grown either in adipogenesis, or osteogenesis differentiation medium or in control medium (DMEM + 10%FCS) for 21 days. The cells were fixed and stained specifically to detect differentiated cells with oil red for adipocytes and Alizarin red for osteoblasts. Note that control cells show typical hMSC morphologies and did not stain with the differentiation specific dyes. hMSC as shown in Overall, the ALS specific RNA editing pattern, found consistently in all ALS individuals for CyFIP2 and RbBP9, and the differences found at the transcriptional and translational levels of these genes, strongly support our view that ALS hMSC are useful model tool to identify possible ALS biomarkers. To strengthen the possibility that these genes could become future diagnostic ALS biomarkers, we performed several experiments on PBL samples from ALS and non-ALS individuals. After verification of the expression of these genes in ALS and non-ALS PBL samples we performed RNA editing analysis on RNA samples and found that both genes,CyFIP2 and RbBP9 are not RNA edited in PBL while the ADAR1 editing control gene BLCAP (bladder cancer associated protein) H. Nachmany et al. / Detection of ALS biomarkers in ALS cell samples editing in PBL, we wanted to investigate whether these genes are differentially expressed in ALS PBL as shown above in hMSC (see Discussion Here we have identified two genes CyFIP2 and RbBP9 that are differentially expressed in bone marrow derived hMSC and in PBL of ALS patients. Interestingly, these genes were isolated from 10 RNA editing target genes by their different A to I RNA editing pattern found in all six samples of ALS hMSC after being kept for several passages in culture. It is still unknown what functional effect does the lack of A to I conversion of RNA editing may have on CyFIP2 and RbBP9 proteins and how this may affect ALS hMSC biology. Moreover, we also don't know the meaning of the difference in the transcriptional levels of gene expression of these two genes in the hMSC and PBL derivates from several ALS individuals. It is possible that this transcriptional difference between PBL and hMSC in ALS samples may indicate abnormal transcriptional regulation of CyFIP2 and RbBP9, which expresses itself in a different manner according to the cell type. We believe that this abnormal gene expression is product of an intrinsic molecular mechanism that is deregulated in cells of ALS patients. Transcriptional regulators such as TDP43 and FUS/TLS are good candidates to test this hypothesis since both are similar DNA/RNA binding proteins that could alter RNA processing and have been directly implicated in ALS pathogenesis in familial and in sporadic cases for TDP4

Two Potential Biomarkers Identified in Mesenchymal Stem Cells and Leukocytes of Patients with Sporadic Amyotrophic lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disorder caused by degeneration of motor neurons. The cause for most cases of ALS is multi-factorial,this enhances the need to characterize and isolate specific biomarkers found in biological samples from ALS patients. To this end we use human mesenchymal stem cells (hMSC) derived from the bone marrow of six ALS patients (ALS hMSC) and identified two genes, Cytoplasmic FMR Interacting Protein 2 (CyFIP2) and Retinoblastoma (Rb) Binding Protein 9 (RbBP9) with a significant decrease in post transcriptional A to I RNA editing compared to hMSC of healthy individuals. At the transcriptional level we show abnormal expression of these two genes in ALS hMSC by quantitative real time-PCR (qRT-PCR) and Western blot suggesting a problem in the regulation of these genes in ALS. To strengthen this view we tested by qRT-PCR the expression of these genes in peripheral blood leukocytes (PBL) isolated from blood samples of 17 ALS patients and found that CyFIP2 and RbBP9 levels of expression were significantly different compared to the levels of expression of these two genes in 19 normal PBL samples. Altogether we found two novel ALS potential biomarkers in non-neural tissues from ALS patients that may have direct diagnostic and therapeutic implications to the disease

Serum free cultured bone marrow mesenchymal stem cells as a platform to characterize the effects of specific molecules.

Human mesenchymal stem cells (hMSC) are easily isolated from the bone marrow by adherence to plastic surfaces. These cells show self-renewal capacity and multipotency. A unique feature of hMSC is their capacity to survive without serum. Under this condition hMSC neither proliferate nor differentiate but maintain their biological properties unaffected. Therefore, this should be a perfect platform to study the biological effects of defined molecules on these human stem cells. We show that hMSC treated for five days with retinoic acid (RA) in the absence of serum undergo several transcriptional changes causing an inhibition of ERK related pathways. We found that RA induces the loss of hMSC properties such as differentiation potential to either osteoblasts or adipocytes. We also found that RA inhibits cell cycle progression in the presence of proliferating signals such as epidermal growth factor (EGF) combined with basic fibroblast growth factor (bFGF). In the same manner, RA showed to cause a reduction in cell adhesion and cell migration. In contrast to these results, the addition of EGF+bFGF to serum free cultures was enough to upregulate ERK activity and induce hMSC proliferation and cell migration. Furthermore, the addition of these factors to differentiation specific media instead of serum was enough to induce either osteogenesis or adipogenesis. Altogether, our results show that hMSC's ability to survive without serum enables the identification of signaling factors and pathways that are involved in their stem cell biological characteristics without possible serum interferences

The clinical response of West Nile virus neuroinvasive disease to intravenous immunoglobulin therapy

The aim of the study was to determine whether intravenous gamma globulin (IVIG) treatment is effective in patients with West Nile Virus (WNV) neuroinvasive disease. We contacted hospital based infectious disease experts in Israeli hospitals to identify patients with WNV neuroinvasive disease who were treated with IVIG. The main outcome measure was neurological response after treatment. There were 12 patients who received IVIG and four improved within 48 h. Three patients died, 6 had partial recovery, and 3 recovered completely. Eleven of the 12 patients were infected with Israeli genotypes that are highly homologous to Europe/Africa viruses. The rapid response in some patients suggests that IVIG is effective, and might be used to treat patients with WNV neuroinvasive disease with IVIG

RA and EGF+bFGF effects on Erk phosphorylation and on cell cycle progression.

<p>A: Western blot analysis of hMSC that were cultured for 5 days in either DMEM alone (DMEM) or in the presence of 0.5 µM RA (RA) or in the presence of 20 ng/ml EGF+ 5 ng/ml bFGF (EGF+bFGF), with or without the addition of 0.5 µM RA (EGF+bFGF+RA). As control, cells were cultured with 10%FBS (FBS), or with 0.5 µM RA in 10%FBS (FBS+RA). Whole-cell protein extracts from these differently treated cells were fractionated on a denaturating 12% polyacrylamide gel, transferred to nitrocellulose and detected with anti phosphorylated Erk antibody (pErk1/2). The membrane was stripped twice, one for detection with anti total Erk antibody (Erk1) and the second for β-actin antibody detection used as loading control. B: Densitometry analysis of A. The bars represent relative expression normalized to β-actin expression and referred to this ratio in DMEM. C–J: Cell cycle progression by FACS of hMSC that were cultured for 5 days with DMEM (C), 0.5 µM RA in DMEM (D), 20 ng/ml EGF (E), 5 ng/ml bFGF (F), 5 ng/ml bFGF +20 ng/ml EGF (G), or 5 ng/ml bFGF +20 ng/ml EGF +0.5 µM RA (H). In addition, hMSC were cultured for 2 days in DMEM (I) or in the presence of 0.5 µM RA (J) before replacement of the medium with 20 ng/ml EGF +5 ng/ml bFGF in DMEM for further 2 days. At the end of the experiment the cells were harvested by trypsinization, permeabilized and stained with propidium iodide to measure the DNA content by FACS.</p

Microarray bioinformatical analysis of the RA effects on hMSC cultured for 5 days without serum.

<p>A: Venn diagram showing the number of differentially expressed transcripts in DMEM and RA treated hMSC when compared to 10%FBS treated cells. B: The genes regulated specifically by RA (i.e. blue or yellow in the diagram depicted in A) were clustered by Ingenuity software. This software contains a database with different transcripts arranged in networks according to their known biological interactions. According to the number of transcripts regulated in each of these networks, the program scores them. The best scored network is depicted here. Direct interactions are represented by continuous arrows and direct by dotted ones. Increase in expression is represented by red color and decrease by green. The number under each transcript name is the logarithmic change in comparison to 10%FBS control cells RNA. The type of interaction is indicated by a label and the type of molecule is indicated by the shape of the box, being both detailed at the right of the figure.</p