Increased Expression of Apolipoprotein D Following Experimental Traumatic Brain Injury

Increasing evidence suggests that apolipoprotein D (apoD) could play a major role in mediating neuronal degeneration and regeneration in the CNS and the PNS. To investigate further the temporal pattern of apoD expression after experimental traumatic brain injury in the rat, male Sprague-Dawley rats were subjected to unilateral cortical impact injury. The animals were killed and examined for apoD mRNA and protein expression and for immunohistological analysis at intervals from 15 min to 14 days after injury. Increased apoD mRNA and protein levels were seen in the cortex and hippocampus ipsilateral to the injury site from 48 h to 14 days after the trauma. Immunohistological investigation demonstrated a differential pattern of apoD expression in the cortex and hippocampus, respectively: Increased apoD immunoreactivity in glial cells was detected from 2 to 3 days after the injury in cortex and hippocampus. In contrast, increased expression of apoD was seen in cortical and hippocampal neurons at later time points following impact injury. Concurrent histopathological examination using hematoxylin and eosin demonstrated dark, shrunken neurons in the cortex ipsilateral to the injury site. In contrast, no evidence of cell death was observed in the hippocampus ipsilateral to the injury site up to 14 days after the trauma. No evidence of increased apoD mRNA or protein expression or neuronal pathology by hematoxylin and eosin staining was detected in the contralateral cortex and hippocampus. Our results reveal induction of apoD expression in the cortex and hippocampus following traumatic brain injury in the rat. Our data also suggest that increased apoD expression may play an important role in cortical neuronal degeneration after brain injury in vivo. However, increased expression of apoD in the hippocampus may not necessarily be indicative of neuronal death.     

Immunoreactive luteinizing hormone, estradiol, progesterone, testosterone, and androstenedione levels during the breeding season and anestrus in Siberian tigers

Seasonal analysis of 1239 captive births of Siberian tigers (Panthera tigris altaica) indicated a peak in April to June (P less than 0.001). Studies on seven animals in Minnesota indicated that behavioral heat cycles and ovarian follicular phase cycles began in late January and ceased in early June. Behavioral observation of 12 heat cycles in four tigers yielded an estrous length of 5.3 +/- 0.2 days and an interestrous interval of 25.0 +/- 1.3 days. Hormone assays on weekly blood samples (N = 180) from three female tigers indicated 16 cycles in two breeding seasons. Peak estradiol-17 beta levels were 46.7 +/- 6.0 pg/ml (N = 17) and interestrous concentrations were 8.7 +/- 0.66 pg/ml (N = 28) during the breeding season. Anestrous estradiol levels were 4.2 +/- 0.5 pg/ml (N = 70). The interestrous interval between estradiol peaks was 24.9 +/- 1.3 days (N = 9) with two outliers of 42 days. Serum progesterone concentrations from February to June were 1.2 +/- 0.15 ng/ml (N = 32), providing no evidence for ovulation or corpus luteum formation. Luteinizing hormone (LH) levels were 0.56 +/- 0.04 ng/ml (N = 180). Serum testosterone (r=0.71, P less than 0.001) and androstenedione levels (r=0.75, P less than 0.001) were correlated with estradiol during the breeding season. The duration of anestrus was 8 mo in two of these tigers. The interval was shortened in one tiger by exposure to a 16L:8D photoperiod. The Siberian tiger appears to be a polyestrous seasonal breeder and an induced ovulator whose breeding season may be synchronized by photoperiod.     

Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin,a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10^?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.     

An extensive review on antifungal approaches in the treatment of mucormycosis

During the period of COVID-19, the occurrences of mucormycosis in immunocompromised patients have increased significantly. Mucormycosis (black fungus) is a rare and rapidly progressing fungal infection associated with high mortality and morbidity in India as well as globally. The causative agents for this infection are collectively called mucoromycetes which are the members of the order Mucorales. The diagnosis of the infection needs to be performed as soon as the occurrence of clinical symptoms which differs with types of Mucorales infection. Imaging techniques magnetic resonance imaging or computed tomography scan, culture testing, and microscopy are the approaches for the diagnosis. After the diagnosis of the infection is confirmed, rapid action is needed for the treatment in the form of antifungal therapy or surgery depending upon the severity of the infection. Delaying in treatment declines the chances of survival. In antifungal therapy, there are two approaches first-line therapy (monotherapy) and combination therapy. Amphotericin B ( 1 ) and isavuconazole ( 2 ) are the drugs of choice for first-line therapy in the treatment of mucormycosis. Salvage therapy with posaconazole ( 3 ) and deferasirox ( 4 ) is another approach for patients who are not responsible for any other therapy. Adjunctive therapy is also used in the treatment of mucormycosis along with first-line therapy, which involves hyperbaric oxygen and cytokine therapy. There are some drugs like VT-1161 ( 5 ) and APX001A ( 6 ), Colistin, SCH 42427, and PC1244 that are under clinical trials. Despite all these approaches, none can be 100% successful in giving results. Therefore, new medications with favorable or little side effects are required for the treatment of mucormycosis.     

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Effects of oral casokefamide on plasma levels, tolerance, and intestinal transit in man

Food-derived opioid peptides such as beta-casomorphins are of interest for treatment of chronic diarrhea. The beta-casomorphin analog casokefamide was administered orally at doses of 5.5, 8.0, and 16.0 mg to 10 healthy male volunteers, respectively. Dose-dependent increases of plasma levels with a maximum of 350 fmol/l were determined. No side-effects due to casokefamide has been observed. In comparison to placebo, casokefamide showed a trend toward prolongation of oro-caecal transit time. Orally applied casokefamide is well tolerated and may represent a useful tool for treatment of diarrhea in the future.     

Mesenchymal stem cells in a transgenic mouse model of multiple system atrophy: immunomodulation and neuroprotection

Background

Mesenchymal stem cells (MSC) are currently strong candidates for cell-based therapies. They are well known for their differentiation potential and immunoregulatory properties and have been proven to be potentially effective in the treatment of a large variety of diseases, including neurodegenerative disorders. Currently there is no treatment that provides consistent long-term benefits for patients with multiple system atrophy (MSA), a fatal late onset α-synucleinopathy. Principally neuroprotective or regenerative strategies, including cell-based therapies, represent a powerful approach for treating MSA. In this study we investigated the efficacy of intravenously applied MSCs in terms of behavioural improvement, neuroprotection and modulation of neuroinflammation in the (PLP)-αsynuclein (αSYN) MSA model.

Methodology/Principal Findings

MSCs were intravenously applied in aged (PLP)-αSYN transgenic mice. Behavioural analyses, defining fine motor coordination and balance capabilities as well as stride length analysis, were performed to measure behavioural outcome. Neuroprotection was assessed by quantifying TH neurons in the substantia nigra pars compacta (SNc). MSC treatment on neuroinflammation was analysed by cytokine measurements (IL-1α, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, GM-CSF, INFγ, MCP-1, TGF-β1, TNF-α) in brain lysates together with immunohistochemistry for T-cells and microglia.Four weeks post MSC treatment we observed neuroprotection in the SNc, as well as downregulation of cytokines involved in neuroinflammation. However, there was no behavioural improvement after MSC application.

Conclusions/Significance

To our knowledge this is the first experimental approach of MSC treatment in a transgenic MSA mouse model. Our data suggest that intravenously infused MSCs have a potent effect on immunomodulation and neuroprotection. Our data warrant further studies to elucidate the efficacy of systemically administered MSCs in transgenic MSA models.     

Nogo-receptors NgR1 and NgR2 do not mediate regulation of CD4 T helper responses and CNS repair in experimental autoimmune encephalomyelitis

Myelin-associated inhibition of axonal regrowth after injury is considered one important factor that contributes to regeneration failure in the adult central nervous system (CNS). Blocking strategies targeting this pathway have been successfully applied in several nerve injury models, including experimental autoimmune encephalomyelitis (EAE), suggesting myelin-associated inhibitors (MAIs) and functionally related molecules as targets to enhance regeneration in multiple sclerosis. NgR1 and NgR2 were identified as interaction partners for the myelin proteins Nogo-A, MAG and OMgp and are probably mediating their growth-inhibitory effects on axons, although the in vivo relevance of this pathway is currently under debate. Recently, alternative functions of MAIs and NgRs in the regulation of immune cell migration and T cell differentiation have been described. Whether and to what extent NgR1 and NgR2 are contributing to Nogo and MAG-related inhibition of neuroregeneration or immunomodulation during EAE is currently unknown. Here we show that genetic deletion of both receptors does not promote functional recovery during EAE and that NgR1 and NgR2-mediated signals play a minor role in the development of CNS inflammation. Induction of EAE in Ngr1/2-double mutant mice resulted in indifferent disease course and tissue damage when compared to WT controls. Further, the development of encephalitogenic CD4(+) Th1 and Th17 responses was unchanged. However, we observed a slightly increased leukocyte infiltration into the CNS in the absence of NgR1 and NgR2, indicating that NgRs might be involved in the regulation of immune cell migration in the CNS. Our study demonstrates the urgent need for a more detailed knowledge on the multifunctional roles of ligands and receptors involved in CNS regeneration failure.     

Quantitative characterization of binding of small molecules to extracellular matrix

Extracellular matrix (ECM) is a major tissue component that, besides its cell support function, is implicated in cell-cell signaling, wound repair, cell adhesion, and other cell and tissue functions. For small molecules acting in tissues, including chemicals, signaling peptides, effectors, inhibitors, and other man-made and physiological compounds, non-specific binding to ECM is a critical phenomenon affecting their disposition. We describe here a method for a quantitative characterization of the ECM binding, using a solidified ECM layer incubated with medium containing studied small molecules. Working conditions of Matrigel, a commercial basement membrane preparation, were optimized in terms of the protein concentration, surface area, gel layer thickness, solidification time, and mixing speed. The release of proteins from the solidified layer into the buffer was monitored and taken into account. Two major proteins, laminin and collagen IV, dissolve at different rates. The Matrigel stability data, obtained under varying incubation conditions and gentle mixing, can also be useful in other ECM-related research. The experimental binding data, averaged over all binding sites, were analyzed assuming a fast linear binding. The binding constants were determined for 10 small organic molecules for both dissolved proteins and the solidified layer. The binding constants tend to increase with lipophilicity of the compounds, as characterized by the 1-octanol/water partition coefficients.