Adipsin, MIP-1b, and IL-8 as CSF Biomarker Panels for ALS Diagnosis.

Amyotrophic lateral sclerosis (ALS) is an aggressive neurodegenerative disorder that selectively attacks motor neurons in the brain and spinal cord. Despite important advances in the knowledge of the etiology and progression of the disease, there are still no solid grounds in which a clinician could make an early objective and reliable diagnosis from which patients could benefit. Diagnosis is difficult and basically made by clinical rating scales (ALSRs and El Escorial). The possible finding of biomarkers to aid in the early diagnosis and rate of disease progression could serve for future innovative therapeutic approaches. Recently, it has been suggested that ALS has an important immune component that could represent either the cause or the consequence of the disease. In this report, we analyzed 19 different cytokines and growth factors in the cerebrospinal fluid of 77 ALS patients and 13 controls by decision tree and PanelomiX program. Results showed an increase of Adipsin, MIP-1b, and IL-6, associated with a decrease of IL-8 thresholds, related with ALS patients. This biomarker panel analysis could represent an important aid for diagnosis of ALS alongside the clinical and neurophysiological criteria.

Comparing stemness gene expression between stem cell subpopulations from peripheral blood and adipose tissue.

Cell therapy presents a promising alternative for the treatment of degenerative diseases. The main sources of adult stem cells are bone marrow, adipose tissue and peripheral blood. Within those tissues, there are cell subpopulations that share pluripotential characteristics. Nevertheless, there is insufficient data to determine which of these stem cell subtypes would have a better possibility to differentiate to a specific tissue. The objective of this research was to analyze and compare the stemness genes expression from peripheral blood and adipose tissue of plastic adherent cells, and those immune-selected by the CD133+ and CD271+ membrane markers. On all cell subpopulation groups, self-renew capacity, the membranes markers CD73, CD90 and CD105, as well as the stemness genes NANOG, OCT4, SOX2, REX1, NOTCH1 and, NESTIN expression were analyzed. Results showed that all samples presented the minimal criteria to define them as human stem cells. All cell subpopulation were capable of self-renewal. Nevertheless, the subpopulation cell types showed differences on the time needed to reach confluence. The slowest doubling times were for those cells bearing the CD133 marker from both sources. Surface markers determined by flow cytometry were positive for CD73, CD90 and, CD105, and negative for CD45. The stemness gene expression was positive in all subpopulation. However, there were significant differences in the amount and pattern of expression among them. Those differences could be advantageous in finding the best option for their application on cell therapy. Cells with high expression of OCT4 gene could be a better opportunity for neuron differentiation like CD133+ blood cells. On the other hand, lowest expression of NOTCH1 on CD271+ cells from the same source could be a better possibility for myoblast differentiation. The observed differences could be used as an advantage to find which cell type and from the different source; this represents the best option for its application on cell therapy. Experiments focused on the best response to specific differentiation, are conducted in order to confirm those possibilities.

Differentiation of CD133+ stem cells from amyotrophic lateral sclerosis patients into preneuron cells.

Improvements in quality of life and life expectancy have been observed in amyotrophic lateral sclerosis (ALS) patients transplanted with CD133(+) stem cells into their frontal motor cortices. However, questions have emerged about the capacity of cells from these patients to engraft and differentiate into neurons. The objective of this work was to evaluate the in vitro capacity of CD133(+) stem cells from 13 ALS patients to differentiate into neuron lineage. Stem cells were obtained through leukapheresis and cultured in a control medium or a neuroinduction medium for 2-48 hours. Expression of neuronal genes was analyzed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical techniques. Fluorescence microscopy demonstrated that CD133(+) stem cells from ALS patients incubated for 48 hours in a neuroinduction medium increased the detection of neuronal proteins such as nestin, ß-tubulin III, neuronal-specific enolase, and glial fibrillary acidic protein. RT-PCR assays demonstrated an increase in the expression of ß-tubulin III, nestin, Olig2, Islet-1, Hb9, and Nkx6.1. No correlation was found between age, sex, or ALS functional scale and the CD133(+) stem cell response to the neuroinduction medium. We conclude that CD133(+) stem cells from ALS patients, like the stem cells of healthy subjects, are capable of differentiating into preneuron cells.

In vitro amoebicidal activity of borage (Borago officinalis) extract on Entamoeba histolytica.

Borage (Borago officinalis) is a plant with nutritional value that is also used in traditional medicine to treat gastrointestinal disease. This study investigated the amoebicidal activity of a methanol extract of borage. The 50% inhibitory concentration (IC50) of the extract for Entamoeba histolytica was 33 µg/mL. The 50% lethal dose of the extract for brine shrimp was greater than 1,000 µg/mL. The IC50 of the extract for Vero cells was 203.9 µg/mL. These results support the use of borage to prevent diseases associated with E. histolytica infection.

Effect of Azadirachta indica leaf methanol extracts on stem cell reproduction.

Methanol extracts of Azadirachta indica leaves at concentration from 0.1 to 40 microg/ml showed in vitro an stimulatory activity in stem cell reproduction. These results suggest that the effect of methanol leaf extracts on stem cell reproduction could be of benefit to improve health.

DMPS reverts morphologic and mitochondrial damage in OK cells exposed to toxic concentrations of HgCl2.

Mercuric chloride (HgCl(2)) is a highly toxic compound, which can cause nephrotoxic damage. In the present study effects of HgCl(2) on mitochondria integrity and energy metabolism, as well as antidotal effects of 2,3-dimercaptopropane-1-sulfonate (DMPS) were investigated in the opossum kidney derived cell line (OK). OK cell monolayers were incubated during 0, 1, 3, 6, and 9 h in serum-free culture medium containing 15 microM HgCl(2), either in the absence or in the presence of 60 microM DMPS in a 1:4 ratio. Intracellular ATP content, MTT reduction, and HSP70/HSP90 induction were studied; confocal, transmission electron microscopy, and light microscopy studies were also performed. For confocal analysis, a mitochondrial selective probe (MitoTracker Red CMXH2Ros) was used. Antioxidant activity of DMPS was also studied by the scavenging of the free radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH) technique. A decrease of ATP content, an impaired ability to reduce tetrazolium, and dramatic changes on cellular and mitochondrial morphology, and energetic levels were found after either 6 or 9 h of HgCl(2) exposure. Increased expression of HSP90 and HSP70 were also seen. When OK cells were co-incubated with HgCl(2) and DMPS, cellular morphology, viability, intracellular ATP, and mitochondrial membrane potential were partially restored; a protective effect on mitochondrial morphology was also seen. DMPS also showed potent antioxidant activity in vitro. Mitochondrial protection could be the cellular mechanism mediated by DMPS in OK cells exposed to a toxic concentration of HgCl(2).

Morphologic and functional alterations induced by low doses of mercuric chloride in the kidney OK cell line: ultrastructural evidence for an apoptotic mechanism of damage.

Mercury produces acute renal failure in experimental animal models, but the mechanism of tubular injury has not completely been clarified. There is an increased interest in the role of apoptosis in the pathogenesis of renal diseases that result primarily from injury to renal tubular epithelial cells. However, detailed studies of morpho-functional alterations induced by mercuric chloride in kidney cell lines are scarce. This work characterizes these alterations in OK cell cultures. Morphological alterations were profiled using light microscopy, transmission electron microscopy, and confocal microscopy, as well as mitochondrial functional assays in the cells exposed to low concentrations of HgCl2. At concentrations of 1 and 10 microM of HgCl2 there were no morphological or ultrastructural alterations, but the mitochondrial function (MTT assay) and intracellular ATP content was increased, especially at longer incubation times (6 and 9 h). At 15 microM HgCl2, both the mitochondrial activity and the endogenous ATP decreased significantly. At this concentration the OK cells rounded up, had increased number of cytoplasmic vacuoles, and detached from the cell monolayer. At 15 microM HgCl2 ultrastructural changes were characterized by dispersion of the ribosomes, dilatation of the cisterns of the rough endoplasmic reticulum, increase of number of cytoplasmic vacuoles, chromatin condensation, invaginations of the nuclear envelope, presence of cytoplasmic inclusion bodies, and alterations in the size and morphology of mitochondria. At 15 microM HgCl2 apoptotic signs included membrane blebbing, chromatin condensation, mitochondrial alterations, apoptotic bodies, and nuclear envelope rupture. Using confocal microscopy and the mitochondrial specific dye MitoTracker Red, it was possible to establish qualitative changes induced by mercury on the mitochondrial membrane potential after incubation of the cells for 6 and 9h with 15 microM HgCl2. This effect was not observed at short times (1 and 3h) with this same concentration, neither with 1 and 10 microM HgCl2 in all the studied times. Taken together, these findings indicate that low concentrations of HgCl2 induce apoptosis by inhibiting mitochondrial function, and the OK cell line may be considered a useful tool for the study of programmed cell death involving mercurial species and other heavy metals.

Synthesis and in vitro antiprotozoal activity of 5-nitrothiophene-2-carboxaldehyde thiosemicarbazone derivatives.

Several thiosemicarbazone derivatives of 5-nitrothiophene-2-carboxaldehyde were prepared by the simple process in which N(4)-thiosemicarbazone moiety was replaced by aliphatic, arylic and cyclic amine. Among these thiosemicarbazones compound 11 showed significant antiamoebic activity whereas compound 3 was more active antitrichomonal than the reference drug.

Synthesis, characterization and antiamoebic activity of benzimidazole derivatives and their vanadium and molybdenum complexes.

Reaction of [MoO(2)(acac)(2)] (where, acac=acetyl acetone) and KVO(3) with 2-(salicylidieneimine) benzimidazole lead to form new complexes [MoO(2)(sal-BMZ)(2)] and K [VO(2)(sal-BMZ)(2)] [where, sal-BMZ=2-(salicylidieneimine) benzimidazole], which showed the monobasic bidentate nature of the ligand in which the phenolic oxygen and the imine nitrogen of the ligand are coordinated to the metal ion. These complexes were characterized along with nine other complexes of oxoperoxovanadium (V), molybdenum (Vl) and tungsten (Vl) with benzimidazole derivatives and screened in vitro by micro dilution technique for their amoebicidal activity with a view to search for a more effective agent against Entamoeba histolytica suggests that compound 2 and 3 might be endowed with important antiamoebic properties since they showed IC(50 )values in a microM range.

Leaf extracts of Carlowrightia cordifolia induce macrophage nitric oxide production.

Carlowrightia cordifolia (Acanthaceae) is a medicinal plant used in northeastern Mexico as a traditional remedy against inflammation. As tissue release of nitric oxide (NO) has been correlated with both inflammatory and anti-inflammatory processes, the aim of this study was to determine the effect of C. cordifolia leaf extracts on macrophage NO production. Lipopolysaccharide (LPS)-stimulated and non-LPS-stimulated mouse peritoneal macrophages were incubated with aqueous, ethanol, methanol and hexane extracts of C. cordifolia leaves. All extracts inhibited NO release from LPS-stimulated macrophages, with methanol and hexane extracts showing the greatest inhibition. On the other hand, macrophage cultures treated with extracts without LPS-stimulation produced high releases of NO. These unexpected results suggest two different ways by which leaf extracts may act, depending on cell status. On the other hand, data on NO activity in relation to inflammatory/anti-inflammatory auto-regulatory feedback and high concentrations of NO release by non-stimulated macrophages agreed with the hypothesis that NO may have an inhibitory effect in vascular inflammation.