Low-Level Laser Irradiation (InGaAlP-660 nm) Increases Fibroblast Cell Proliferation and Reduces Cell Death in a Dose-Dependent Manner

Background and Objective: Impaired cell metabolism and increased cell death in fibroblast cells are physiological features of chronic tendinopathy. Although several studies have shown that low-level laser therapy (LLLT) at certain parameters has a biostimulatory effect on fibroblast cells, it remains uncertain if LLLT effects depend on the physiological state. Study Design/Material and Methods: High-metabolic immortal cell culture and primary human keloid fibroblast cell culture were used in this study. Trypan blue exclusion and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test were used to determine cell viability and proliferation. Propidium iodide stain was used for cell-cycle analysis by flow cytometry. Laser irradiation was performed daily on three consecutive days with a GaAlAs 660-nm laser (mean output: 50 mW, spot size 2 mm(2), power density = 2.5 W/cm(2)) and a typical LLLT dose and a high LLLT dose (irradiation times: 60 or 420 s; fluences: 150 or 1050 J/cm(2); energy delivered: 3 or 21 J). Results: Primary fibroblast cell culture from human keloids irradiated with 3 J showed significant proliferation by the trypan blue exclusion test (p < 0.05), whereas the 3T3 cell culture showed no difference using this method. Propidium iodide staining flow cytometry data showed a significant decrease in the percentage of cells being in proliferative phases of the cell cycle (S/g(2)/M) when irradiated with 21 J in both cell types (hypodiploid cells increased). Conclusions: Our data support the hypothesis that the physiological state of the cells affects the LLLT results, and that high-metabolic rate and short-cell-cycle 3T3 cells are not responsive to LLLT. In conclusion, LLLT with a dose of 3 J reduced cell death significantly, but did not stimulate cell cycle. A LLLT dose of 21 J had negative effects on the cells, as it increased cell death and inhibited cell proliferation.

Chopper, a new death domain of the p75 neurotrophin receptor that mediates rapid neuronal cell death

The cytoplasmic juxtamembrane region of the p75 neurotrophin receptor (p75(NTR)) has been found to be necessary and sufficient to initiate neural cell death. The region was named Chopper to distinguish it from CD95-like death domains. A 29-amino acid peptide corresponding to the Chopper region induced caspase- and calpain-mediated death in a variety of neural and nonneural cell types and was not inhibited by signaling through Trk (unlike killing by full-length p75(NTR)). Chopper triggered cell death only when bound to the plasma membrane by a lipid anchor, whereas non-anchored Chopper acted in a dominant-negative manner, blocking p75(NTR)-mediated death both in vitro and in vivo. Removal of the ectodomain of p75(NTR) increased the potency of Chopper activity, suggesting that it regulates the association of Chopper with downstream signaling proteins.

Gene expression profile of residual breast cancer after doxorubicin and cyclophosphamide neoadjuvant chemotherapy

In breast cancer patients, primary chemotherapy is associated with the same survival benefits as adjuvant chemotherapy. Residual tumors represent a clinical challenge, Lis they may be resistant to additional cycles of the same drugs. Our aim was to identify differential transcripts expressed in residual tumors, after neoadjuvant chemotherapy, that might be related with tumor resistance. Hence, 16 patients with paired tumor samples, collected before and after treatment (4 cycles doxorubicin/cyclophosphamide, AC) had their gene expression evaluated on cDNA microarray slides containing 4,608 genes. Three hundred and eighty-nine genes were differentially expressed (paired Student`s t-test, pFDR<0.01) between pre- and post-chemotherapy samples and among the regulated functions were the JNK cascade and cell death. Unsupervised hierarchical clustering identified one branch comprising exclusively, eight pre-chemotherapy samples and another branch, including the former correspondent eight post-chemotherapy samples and other 16 paired pre/post-chemotherapy samples. No differences in clinical and tumor parameters could explain this clustering. Another group of I I patients with paired samples had expression of selected genes determined by real-time RT-PCR and CTGF and DUSP1 were confirmed more expressed in post- as compared to pre-chemotherapy samples. After neoadjuvant chemotherapy some residual samples may retain their molecular signature while others present significant changes in their gene expression, probably induced by the treatment. CTGF and DUSP1 overexpression in residual samples may be a reflection of resistance to further administration of AC regimen.

Effect of Femtosecond Laser Energy Level on Corneal Stromal Cell Death and Inflammation

PURPOSE: To analyze the effects of variations in femtosecond laser energy level on corneal stromal cell death. and inflammatory cell influx following flap creation in a rabbit model. METHODS: Eighteen rabbits were stratified in three different groups according to level of energy applied for flap creation (six animals per group). Three different energy levels were chosen for both the lamellar and side cut; 2.7 mu J (high energy), 1.6 mu J (intermediate energy), and 0.5 mu J (low energy) with a 60 kHz, model II, femtosecond laser (IntraLase). The opposite eye of each rabbit served as a control. At the 24-hour time point after surgery, all rabbits were euthanized and the comeoscleral rims were analyzed for the levels of cell death and inflammatory cell influx with the terminal uridine deoxynucleotidyl transferase dUTP-nick end labeling (TUNEL) assay and immunocytochemistry for monocyte marker CD11b, respectively. RESULTS: The high energy group (31.9 +/- 7.1 [standard error of mean (SEM) 2.9]) had significantly more TUNEL positive cells in the central flap compared to the intermediate (22.2 +/- 1.9 [SEM 0.8], P=.004), low (17.9 +/- 4.0 [SEM 1.6], P <= .001), and control eye (0.06 +/- 0.02 [SEM 0.009], P <= .001) groups. The intermediate and low energy groups also had significantly more TUNEL positive cells than the control groups (P <= .001). The difference between the intermediate and low energy levels was not significant (P=.56). The mean for CD11b-positive cells/400x field at the flap edge was 26.1 +/- 29.3 (SEM 11.9), 5.8 +/- 4.1 (SEM 1.6), 1.6 +/- 4.1 (SEM 1.6), and 0.005 +/- 0.01 (SEM 0.005) for high energy, intermediate energy, low energy, and control groups, respectively. Only the intermediate energy group showed statistically more inflammatory cells than control eyes (P = .015), most likely due to variability between eyes. CONCLUSIONS: Higher energy levels trigger greater cell death when the femtosecond laser is used to create corneal flaps: Greater corneal inflammatory cell infiltration is observed with higher femtosecond laser energy levels. [J Refract Surg. 2009;25:869-874.] doi:10.3928/1081597X-20090917-08

Genetic disruption of the growth hormone receptor does not influence motoneuron survival in the developing mouse

In the rodent central nervous system (CNS) during the five days prior to birth, both growth hormone (GH) and its receptor (GHR) undergo transient increases in expression to levels considerably higher than those found postnatally. This increase in expression coincides with the period of neuronal programmed cell death (PCD) in the developing CNS. To evaluate the involvement of growth hormone in the process of PCD, we have quantified the number of motoneurons in the spinal cord and brain stem of wild type and littermate GHR-deficient mice at the beginning and end of the neuronal PCD period. We found no change in motoneuron survival in either the brachial or lumbar lateral motor columns of the spinal cord or in the trochlear, trigeminal, facial or hypoglossal nuclei in the brain stem. We also found no significant differences in spinal cord volume, muscle fiber diameter, or body weight of GHR-deficient fetal mice when compared to their littermate controls. Therefore, despite considerable in vitro evidence for GH action on neurons and glia, genetic disruption of GHR signalling has no effect on prenatal motoneuron number in the mouse, under normal physiological conditions. This may be a result of compensation by the signalling of other neurotrophic cytokines.

NEUROPATHOPHYSIOLOGICAL MECHANISMS IN GLUTARIC ACIDURIA TYPE I: 3-HYDROXYGLUTARIC ACID LEADS TO AMMONIA INCREASE AND NON-APOPTOTIC CELL DEATH

A 3D in vitro model of rat organotypic brain cell cultures in aggregates was used to investigate neurotoxicity mechanisms in glutaric aciduria type I (GA-I). 1 mM glutarate (GA) or 3-hydroxyglutarate (3OHGA) were repeatedly added to the culture media at two different time points. In cultures treated with 3OHGA, we observed an increase in lactate in the medium, pointing to a possible inhibition of Krebs cycle and respiratory chain. We further observed that 3OHGA and to a lesser extend GA induced an increase in ammonia production with concomitant decrease of glutamine concentrations, which may suggest an inhibition of the astrocytic enzyme glutamine synthetase. These previously unreported findings may uncover a pathogenic mechanism in this disease which has deleterious effects on early stages of brain development. By immunohistochemistry we showed that 3OHGA increased non-apoptotic cell death. On the cellular level, 3OHGA and to a lesser extend GA led to cell swelling and loss of astrocytic fibers whereas a loss of oligodendrocytes was only observed for 3OHGA. We conclude that 3OHGAwas the most toxic metabolite in our model for GA-I. 3OHGA induced deleterious effects on glial cells, an increase of ammonia production, and resulted in accentuated cell death of non-apoptotic origin.

Ammonium accumulation and cell death in a rat 3D brain cell model of glutaric aciduria type I.

Glutaric aciduria type I (glutaryl-CoA dehydrogenase deficiency) is an inborn error of metabolism that usually manifests in infancy by an acute encephalopathic crisis and often results in permanent motor handicap. Biochemical hallmarks of this disease are elevated levels of glutarate and 3-hydroxyglutarate in blood and urine. The neuropathology of this disease is still poorly understood, as low lysine diet and carnitine supplementation do not always prevent brain damage, even in early-treated patients. We used a 3D in vitro model of rat organotypic brain cell cultures in aggregates to mimic glutaric aciduria type I by repeated administration of 1 mM glutarate or 3-hydroxyglutarate at two time points representing different developmental stages. Both metabolites were deleterious for the developing brain cells, with 3-hydroxyglutarate being the most toxic metabolite in our model. Astrocytes were the cells most strongly affected by metabolite exposure. In culture medium, we observed an up to 11-fold increase of ammonium in the culture medium with a concomitant decrease of glutamine. We further observed an increase in lactate and a concomitant decrease in glucose. Exposure to 3-hydroxyglutarate led to a significantly increased cell death rate. Thus, we propose a three step model for brain damage in glutaric aciduria type I: (i) 3-OHGA causes the death of astrocytes, (ii) deficiency of the astrocytic enzyme glutamine synthetase leads to intracerebral ammonium accumulation, and (iii) high ammonium triggers secondary death of other brain cells. These unexpected findings need to be further investigated and verified in vivo. They suggest that intracerebral ammonium accumulation might be an important target for the development of more effective treatment strategies to prevent brain damage in patients with glutaric aciduria type I.

Leishmania major metacaspase can replace yeast metacaspase in programmed cell death and has arginine-specific cysteine peptidase activity.

The human protozoan parasite Leishmania major has been shown to exhibit several morphological and biochemical features characteristic of a cell death program when differentiating into infectious stages and under a variety of stress conditions. Although some caspase-like peptidase activity has been reported in dying parasites, no caspase gene is present in the genome. However, a single metacaspase gene is present in L. major whose encoded protein harbors the predicted secondary structure and the catalytic dyad histidine/cysteine described for caspases and other metacaspases identified in plants and yeast. The Saccharomyces cerevisiae metacaspase YCA1 has been implicated in the death of aging cells, cells defective in some biological functions, and cells exposed to different environmental stresses. In this study, we describe the functional heterologous complementation of a S. cerevisiae yca1 null mutant with the L. major metacaspase (LmjMCA) in cell death induced by oxidative stress. We show that LmjMCA is involved in yeast cell death, similar to YCA1, and that this function depends on its catalytic activity. LmjMCA was found to be auto-processed as occurs for caspases, however LmjMCA did not exhibit any activity with caspase substrates. In contrast and similarly to Arabidopsis thaliana metacaspases, LmjMCA was active towards substrates with arginine in the P1 position, with the activity being abolished following H147A and C202A catalytic site mutations. These results suggest that metacaspases are members of a family of peptidases with a role in cell death conserved in evolution notwithstanding possible differences in their catalytic activity.

Plasmodium falciparum metacaspase PfMCA-1 triggers a z-VAD-fmk inhibitable protease to promote cell death.

Activation of proteolytic cell death pathways may circumvent drug resistance in deadly protozoan parasites such as Plasmodium falciparum and Leishmania. To this end, it is important to define the cell death pathway(s) in parasites and thus characterize proteases such as metacaspases (MCA), which have been reported to induce cell death in plants and Leishmania parasites. We, therefore, investigated whether the cell death function of MCA is conserved in different protozoan parasite species such as Plasmodium falciparum and Leishmania major, focusing on the substrate specificity and functional role in cell survival as compared to Saccharomyces cerevisae. Our results show that, similarly to Leishmania, Plasmodium MCA exhibits a calcium-dependent, arginine-specific protease activity and its expression in yeast induced growth inhibition as well as an 82% increase in cell death under oxidative stress, a situation encountered by parasites during the host or when exposed to drugs such as artemisins. Furthermore, we show that MCA cell death pathways in both Plasmodium and Leishmania, involve a z-VAD-fmk inhibitable protease. Our data provide evidence that MCA from both Leishmania and Plasmodium falciparum is able to induce cell death in stress conditions, where it specifically activates a downstream enzyme as part of a cell death pathway. This enzymatic activity is also induced by the antimalarial drug chloroquine in erythrocytic stages of Plasmodium falciparum. Interestingly, we found that blocking parasite cell death influences their drug sensitivity, a result which could be used to create therapeutic strategies that by-pass drug resistance mechanisms by acting directly on the innate pathways of protozoan cell death.

Discordant increases in CD4+ T cells in human immunodeficiency virus-infected patients experiencing virologic treatment failure: role of changes in thymic output and T cell death.

Some patients infected with human immunodeficiency virus (HIV) who are experiencing antiretroviral treatment failure have persistent improvement in CD4+ T cell counts despite high plasma viremia. To explore the mechanisms responsible for this phenomenon, 2 parameters influencing the dynamics of CD4+ T cells were evaluated: death of mature CD4+ T cells and replenishment of the CD4+ T cell pool by the thymus. The improvement in CD4+ T cells observed in patients with treatment failure was not correlated with spontaneous, Fas ligand-induced, or activation-induced T cell death. In contrast, a significant correlation between the improvement in CD4+ T cell counts and thymic output, as assessed by measurement of T cell receptor excision circles, was observed. These observations suggest that increased thymic output contributes to the dissociation between CD4+ T cell counts and viremia in patients failing antiretroviral therapy and support a model in which drug-resistant HIV strains may have reduced replication rates and pathogenicity in the thymus.