Parathyroid hormone-related protein is a hypertrophy factor for human mesangial cells: Implications for diabetic nephropathy

Hypertrophy of human mesangial cells (HMC) is among the earliest characteristics in patients with diabetic nephropathy (DN). Recently, we observed the upregulation of parathyroid hormone (PTH)-related protein (PTHrP) in experimental DN, associated with renal hypertrophy. Herein, we first examined whether PTHrP was overexpressed in human DN, and next assessed the putative role of this protein on high glucose (HG)-induced HMC hypertrophy. As previously found in mice, kidneys from diabetic patients showed an increased tubular and glomerular immunostaining for PTHrP. In HMC, HG medium increased PTHrP protein expression associated with the development of hypertrophy as assessed by cell protein content. This effect was also induced by PTHrP(1-36). HG and PTHrP(1-36)-induced hypertrophy were associated with an increase in cyclin D1 and p27Kip1 protein expression, a decreased cyclin E expression, and the prevention of cyclin E/cdk2 complex activation. Both PTHrP neutralizing antiserum (α-PTHrP) and the PTH/PTHrP receptor antagonist (JB4250) were able to abolish HG induction of hypertrophy, the aforementioned changes in cell cycle proteins, and also TGF-β1 up-regulation. Moreover, the capability of both HG and PTHrP(1-36) to induce HMC hypertrophy was abolished by α-TGFβ1. These data show for the first time that PTHrP is upregulated in the kidney of patients with DN. Our findings also demonstrate that PTHrP acts as an important mediator of HG-induced HMC hypertrophy by modulating cell cycle regulatory proteins and TGF-β1.     

Hyaluronan receptors in the human ocular surface: a descriptive and comparative study of RHAMM and CD44 in tissues, cell lines and freshly collected samples

The purpose of this study was to demonstrate the presence of the receptor for hyaluronan-mediated motility (RHAMM) in human conjunctival epithelium and in two widely used cell lines from human corneal (HCE) and conjunctival (IOBA-NHC) epithelia. We compared the distribution of RHAMM proteins and mRNAs in human ocular surface tissues (corneal, limbal and conjunctival), HCE and IOBA-NHC cell lines, and corneal and conjunctival epithelia primary samples from healthy donors with the previously identified hyaluronan receptor CD44. We also aimed to determine if soluble CD44 (sCD44) was present in human tears, as it could have a role in the interaction of the tear fluid with hyaluronan. Protein expression was evaluated by Western blots and immunofluorescence microscopy. mRNA expression was evaluated by RT-PCR and Q-PCR. sCD44 was analyzed by ELISA in culture supernatants and in human tears. We describe the expression of RHAMM in human healthy conjunctiva and in HCE and IOBA-NHC cells at both protein and mRNA levels, and the presence of sCD44 in human tears. Furthermore, we detected CD44 and sCD44 expression variations in in vitro inflammatory conditions. This study also focused on the necessary caution with which the conclusions extracted from cell lines should be made, and in the great value of using primary samples as often as possible.     

On the mechanism of Candida spp. photoinactivation by hypericin

The photoprocesses involved in hypericin photoinactivation of three different Candida species (C. albicans, C. parapsilosis and C. krusei) have been examined. Production of singlet oxygen from the triplet state and of superoxide from both the triplet state and the semiquinone radical anion are demonstrated. Hydrogen peroxide is formed downstream of these early events. The outcome of the photodynamic treatments is dictated by the intracellular distribution of hypericin, which is different in the three species and affects the ability of hypericin to produce the different reactive oxygen species and trigger cell-death pathways. The results are in line with the previously-observed different susceptibilities of the three Candida species to hypericin photodynamic treatments.     

Ih current is necessary to maintain normal dopamine fluctuations and sleep consolidation in Drosophila

HCN channels are becoming pharmacological targets mainly in cardiac diseases. But apart from their well-known role in heart pacemaking, these channels are widely expressed in the nervous system where they contribute to the neuron firing pattern. Consequently, abolishing Ih current might have detrimental consequences in a big repertoire of behavioral traits. Several studies in mammals have identified the Ih current as an important determinant of the firing activity of dopaminergic neurons, and recent evidences link alterations in this current to various dopamine-related disorders. We used the model organism Drosophila melanogaster to investigate how lack of Ih current affects dopamine levels and the behavioral consequences in the sleep:activity pattern. Unlike mammals, in Drosophila there is only one gene encoding HCN channels. We generated a deficiency of the DmIh core gene region and measured, by HPLC, levels of dopamine. Our data demonstrate daily variations of dopamine in wild-type fly heads. Lack of Ih current dramatically alters dopamine pattern, but different mechanisms seem to operate during light and dark conditions. Behaviorally, DmIh mutant flies display alterations in the rest:activity pattern, and altered circadian rhythms. Our data strongly suggest that Ih current is necessary to prevent dopamine overproduction at dark, while light input allows cycling of dopamine in an Ih current dependent manner. Moreover, lack of Ih current results in behavioral defects that are consistent with altered dopamine levels.     

Appraisal of a Leishmania major Strain Stably Expressing mCherry Fluorescent Protein for Both In Vitro and In Vivo Studies of Potential Drugs and Vaccine against Cutaneous Leishmaniasis

Background

Leishmania major cutaneous leishmaniasis is an infectious zoonotic disease. It is produced by a digenetic parasite, which resides in the phagolysosomal compartment of different mammalian macrophage populations. There is an urgent need to develop new therapies (drugs) against this neglected disease that hits developing countries. The main goal of this work is to establish an easier and cheaper tool of choice for real-time monitoring of the establishment and progression of this pathology either in BALB/c mice or in vitro assays. To validate this new technique we vaccinated mice with an attenuated Δhsp70-II strain of Leishmania to assess protection against this disease.

Methodology

We engineered a transgenic L. major strain expressing the mCherry red-fluorescent protein for real-time monitoring of the parasitic load. This is achieved via measurement of fluorescence emission, allowing a weekly record of the footpads over eight weeks after the inoculation of BALB/c mice.

Results

In vitro results show a linear correlation between the number of parasites and fluorescence emission over a range of four logs. The minimum number of parasites (amastigote isolated from lesion) detected by their fluorescent phenotype was 10,000. The effect of antileishmanial drugs against mCherry+L. major infecting peritoneal macrophages were evaluated by direct assay of fluorescence emission, with IC₅₀ values of 0.12, 0.56 and 9.20 µM for amphotericin B, miltefosine and paromomycin, respectively. An experimental vaccination trial based on the protection conferred by an attenuated Δhsp70-II mutant of Leishmania was used to validate the suitability of this technique in vivo.

Conclusions

A Leishmania major strain expressing mCherry red-fluorescent protein enables the monitoring of parasitic load via measurement of fluorescence emission. This approach allows a simpler, faster, non-invasive and cost-effective technique to assess the clinical progression of the infection after drug or vaccine therapy.     

Polysaccharides composition from tropical brown seaweeds

Polysaccharides composition of the tropical brown seaweeds Turbinaria turbinata, Sargassum filipendula, Dictyota caribaea and Padina perindusiata collected at Yucatan Peninsula (Mexico) was determined in this study. Crude fucoidan extracted with HCl and alginate extracted with a hot alkali solution were characterized in terms of their molecular weight, sulfate content, uronic acid, total carbohydrate and neutral sugar components. Low molecular weight sulfated‐fucoidan was the major component in all species studied. Fucoidan from T. turbinata and from D. caribaea were characterized as a homofucan, with fucose as the neutral sugar. Fucoidan from S. filipendula was composed of a galactofucan, and fucoidan from P. perindusiata was characterized as a heterofucan consisting of fucose, glucose and galactose. The Fourier transform infrared (FT‐IR) spectra of fucoidan extracted from species studied indicated that the majority of sulfate groups are located at C‐4 and to a lesser extent at C‐2 and/or C‐3 of the fucopyranose residues. This could be advantageous since several therapeutic effects have been reported for fucoidans with similar characteristics. FT‐IR spectra from D. caribaea and P. perindusiata revealed the presence of O‐acetyl groups in crude fucoidan, which could be potentially utilized as an immune stimulant. Molecular weight of alginate varied between 595 and 1301 kDa with similar uronic acid content in all species. Alginate M : G ratio inferred from FT‐IR spectra suggests a high content of G‐block in all species. Potential applications of these polysaccharides are discussed.     

β-catenin confers resistance to PI3K and AKT inhibitors and subverts FOXO3a to promote metastasis in colon cancer

The Wnt–β-catenin and PI3K-AKT-FOXO3a pathways have a central role in cancer. AKT phosporylates FOXO3a, relocating it from the cell nucleus to the cytoplasm, an effect that is reversed by PI3K and AKT inhibitors. Simultaneous hyperactivation of the Wnt–β-catenin pathway and inhibition of PI3K-AKT signaling promote nuclear accumulation of β-catenin and FOXO3a, respectively, promoting cell scattering and metastasis by regulating a defined set of target genes. Indeed, the anti-tumoral AKT inhibitor API-2 promotes nuclear FOXO3a accumulation and metastasis of cells with high nuclear β-catenin content. Nuclear β-catenin confers resistance to the FOXO3a-mediated apoptosis induced by PI3K and AKT inhibitors in patient-derived primary cultures and in corresponding xenograft tumors in mice. This resistance is reversed by XAV-939, an inhibitor of Wnt–β-catenin signaling. In the presence of high nuclear β-catenin content, activation of FOXO3a by PI3K or AKT inhibitors makes it behave as a metastasis inductor rather than a proapoptotic tumor suppressor. We show that it is possible to evaluate the β-catenin status of patients' carcinomas and the response of patient-derived cells to target-directed drugs that accumulate FOXO3a in the nucleus before deciding on a course of treatment. We propose that this evaluation could be essential to the provision of a safer and more effective personalized treatment.     

IL-15 expression on RA synovial fibroblasts promotes B cell survival

Introduction

The purpose of this study was to examine the role of RA Synovial Fibroblast (RASFib) IL-15 expression on B cell survival.

Methods

Magnetically sorted peripheral blood memory B cells from 15 healthy subjects were cocultured with RASFib.

Results

RASFib constitutively expressed membrane IL-15. Survival of isolated B cells cultured for 6 days, below 5%, was extended in coculture with RASFib to 52+/−8% (p<0.001). IL-15 neutralizing agents but not isotype controls, reduced this rate to 31+/−6% (p<0.05). Interestingly, rhIL-15 had no effect on isolated B cells but significantly increased their survival in coculture with RASFib. In parallel, B cell IL-15R chains were upregulated in cocultures. BAFF and VCAM-1, that are expressed on RASFib, were tested as potential candidates involved in upregulating B cell IL-15R. Culture of B cells in the presence of rhBAFF or rhVCAM-1 resulted in significantly increased survival, together with upregulation of all three IL-15R chains; in parallel, rhIL-15 potentiated the anti-apoptotic effect of BAFF and VCAM-1. Both BAFF and VCAM-1 neutralizing agents downmodulated the effect of RASFib on B cell survival and IL-15R expression. In parallel, rhIL-15 had a lower effect on the survival of B cells cocultured with RASFib in the presence of BAFF or VCAM-1 neutralizing agents. Peripheral blood B cells from 15 early RA patients demonstrated an upregulated IL-15R and increased survival in cocultures.

Conclusion

IL-15 expression on RASFib significantly contributes to the anti-apoptotic effect of RASFib on B cells. IL-15 action is facilitated by BAFF and VCAM-1 expressed on RASFib, through an upregulation of IL-15R chains.     

Risk assessment studies on succinate dehydrogenase inhibitors, the new weapons in the battle to control Septoria leaf blotch in wheat

Chemical control of Septoria leaf blotch, caused by Mycosphaerella graminicola, is essential to ensure wheat yield and food security in most European countries. Mycosphaerella graminicola has developed resistance to several classes of fungicide and, with the efficacy of azoles gradually declining over time, new modes of action and/or improvements in host varietal resistance are urgently needed to ensure future sustainable disease control. Several new‐generation carboxamide fungicides with broad‐spectrum activity have recently been introduced into the cereal market. Carboxamides inhibit succinate dehydrogenase (Sdh) of the mitochondrial respiratory chain (complex II) but, because of their single‐site specificity, these fungicides may be prone to resistance development. The objective of this study was to assess the risk of resistance development to different Sdh inhibitor (SDHI) fungicides in M. graminicola. UV mutagenesis was conducted to obtain a library of carboxin‐resistant mutants. A range of SDHI resistance‐conferring mutations was found in Sdh subunits B, C and D. Pathogenicity studies with a range of Sdh variants did not detect any fitness costs associated with these mutations. Most of the amino acid residues identified (e.g. B‐S221P/T, B‐H267F/L/N/Y, B‐I269V and D‐D129E/G/T) are directly involved in forming the cavity in which SDHI fungicides bind. Docking studies of SDHI fungicides in structural models of wild‐type and mutated Sdh complexes also indicated which residues were important for the binding of different SDHI fungicides and showed a different binding for fluopyram. The predictive power of the model was also shown. Further diagnostic development, enabling the detection of resistant alleles at low frequencies, and cross‐resistance studies will aid the implementation of anti‐resistance strategies to prolong the cost‐effectiveness and lifetime of SDHI fungicides.