Fas,FasL, and cleaved caspases 8 and 3 in glioblastomas: A tissue microarray-based study

This investigation analyzed the immunoexpression of FasL, Fas, cleaved caspase-8, and cleaved caspase-3 in glioblastomas. Formalin-fixed and paraffin-embedded glioblastoma tissues and control brain tissues from 97 patients were analyzed by tissue microarrays and immunohistochemistry. Patients with glioblastomas that were negative or weakly stained (<50% of cells positive) for cleaved caspase-8 had worse cancer-specific overall survival (median = 8.5 months) than did patients with tumors that highly expressed cleaved caspase-8 (median = 11.7 months; P = 0.0325), independent of clinical variables. There was no association of other markers with survival, treatment, sex, age, tumor size, and primary site. Among the tumors, there were reasonable to good positive correlations between the expression of FasL and Fas (r = 0.47) and between Fas and cleaved caspase-8 (r = 0.41), and there were poor positive correlations between Fas and cleaved caspase-3 (r = 0.26), FasL and cleaved caspase-8 (r = 0.22), and cleaved caspase-8 and -3 (r = 0.31). Our results suggest that Fas-Fas-ligand signal transduction could be inhibited, especially at the stage of caspase-8 activation, thereby establishing a major mechanism for evasion of apoptosis by these tumors. The absence or low expression of cleaved caspase-8 in the tumors was a negative prognostic indicator for patient survival.     

Phosphorylation and cytoplasmic localization of MAPK p38 during apoptosis signaling in bone marrow granulocytes of mice irradiated in vivo and the role of amifostine in reducing these effects

We studied p38 phosphorylation and its intracellular localization during p53 and Puma (a p53 upregulated modulator of apoptosis) apoptotic signaling pathway in bone marrow granulocytes in mice irradiated in vivo and the role of the radioprotector amifostine in ameliorating these responses. Sixty-four C57BL mice were randomly assigned in two non-irradiated (Ami−/rad− and Ami+/rad−) and two irradiated (Ami−/rad+ and Ami+/rad+) groups. Animals received 400 mg/kg of amifostine i.p. 30 min prior to a single whole body radiation dose of 7 Gy. The experiments were performed using immunohistochemistry for caspase-3, cleaved caspase-3, p53, p-p53 (Ser 15), Puma, p38 and p-p38 (Thr 180/Tyr 182) protein expression. In addition transmission electron microscopy was used for ultrastructural characterization of apoptosis. Data showed that: (i) amifostine significantly reduced the number of apoptotic cells, (ii) p-p53 and Puma proteins were strongly immunostained in granulocytes after irradiation (Ami−/rad+), (iii) amifostine decreased the immunostaining of the proteins (Ami+/rad+), (iv) p38 was immunolocalized in physiological conditions in the nucleus and cytoplasm of granulocytes and neither radiation nor amifostine changed the protein immunostaining or its subcellular distribution, but influenced its activation, (v) radiation-induced p38 phosphorylation and its cytoplasmic accumulation during apoptosis signaling in granulocytes after whole body high radiation dose and amifostine markedly reduced these effects.     

Atorvastatin prevents early apoptosis after thoracic spinal cord contusion injury and promotes locomotion recovery

The systemic administration of atorvastatin has been shown to be neuroprotective after spinal cord injury (SCI), by decreasing the inflammatory response at the lesion site and by reducing neuronal and oligodendrocyte apoptosis. The latter effect spares white matter at the injury site and improves locomotion. The aim of this study was to confirm the neuroprotective efficacy of atorvastatin as well as its early action in limiting apoptosis with its administration post-SCI. Female Sprague-Dawley rats received an intra peritoneal injection of: (1) statin/saline (5 mg/kg) at 2 h after the contusion injury; (2) physiological saline at 2 h post-SCI; or (3) physiological saline without injury. Statin-treated rats showed significant (p < 0.05) improvement in locomotion at week 4 post-SCI compared to vehicle-treated animals. Explaining this outcome, caspase-3 activity decreased by 50% (p < 0.05), and the histological TUNEL method revealed a decrease of approximately 20% in apoptotic cells at the injury site (p < 0.01) at 4 h post-SCI in atorvastatin-treated rats in comparison to vehicle-treated controls. These data demonstrate that atorvastatin is effective after experimental spinal cord contusion injury in preventing early apoptosis at the injury site within 2 h post-administration.     

Thapsigargin-induced apoptosis was prevented by glycogen synthase kinase-3 inhibitors in PC12 cells

Uncontrolled calcium stress has been linked causally to a variety of neurodegenerative diseases, including ischemia, excitotoxicity and Alzheimer's disease. Thapsigargin, which increases [Ca²⁺]_i, induces apoptotic cell death (chromatin condensation and DNA fragmentation) accompanied by caspase-3 activation in PC12 cells. We examined whether GSK-3 is involved in thapsigargin-induced cell death by using GSK-3 inhibitors in PC12 cells. Cells treated with 0.1 μM thapsigargin for 24 h shrank. The injured cells underwent chromatin condensation and nuclear fragmentation, indicating apoptotic cell death. We assayed the effects of selective GSK-3 inhibitors, SB216763, azakenpaullone and alsteropaullone on thapsigargin-induced apoptosis. These inhibitors completely protected cells from thapsigargin-induced apoptosis. Alsterpaullone did not reduce the GRP78 protein expression induced by thapsigargin, suggesting that GSK-3 activation is not involved in induction of GRP78. In addition, GSK-3 inhibitors inhibited caspase-3 activation accompanied by thapsigargin-induced apoptosis. We showed in this report that thapsigargin-induced apoptosis is prevented by GSK-3 inhibitors, suggesting that thapsigargin induces caspase-dependent apoptosis mediated through GSK-3 activation in PC12 cells.     

Abnormal innervation patterns in the anorectum of ETU-induced fetal rats with anorectal malformations

Valproate (VPA) is commonly used in the treatment of bipolar disorder and epilepsy. The mechanism underlying its clinical efficacy is complicated, including its ability to inhibit histone deacetylase (HDAC). Here, we show that VPA promoted endoplasmic reticulum (ER) chaperone expression and attenuated ER-induced apoptosis after ischemia/reperfusion (I/R) injury in retina. Male Wistar rats were randomly divided into four groups: sham (group A), sham + VPA (group B), I/R + vehicle (group C), and I/R + VPA (group D). VPA was administered subcutaneously at 300 mg/kg twice daily before insult. Morphological changes were analyzed on stained histological sections and flat-mounted retinas labeled by Fluoro-gold. Western blot analysis was used to determine protein levels of GRP78, CHOP, caspase-12 and acetylation of histone H3 in each group. In group C, the severe retinal damage was shown in histological sections, however, the damage was reduced by VPA in group D. Significant loss of retinal ganglion cells (RGCs) was observed in group C, whereas, the density of RGCs was significantly higher in group D at 7 days post-insult. VPA increased GRP78 expression and acetylation of histone H3, attenuated upregulation of CHOP and activation of caspase-12 in group D. Our results suggest that VPA can protect ischemic retinas from ER stress-induced apoptosis by mechanisms that may involve HDAC inhibition.     

Elevated cleaved caspase-3 is associated with shortened overall survival in several cancer types

Emerging evidence has indicated that apoptotic cells have a compensatory effect on the proliferation of neighboring cells. Recent studies have shown that apoptotic tumor cells stimulate the repopulation of tumors from a small number of surviving cells by cleaved caspase-3 regulation and elevated tumor cleaved (and thus activated) caspase-3 expression levels predict worse treatment outcomes in cancer patients. The prognostic significance of cleaved caspase-3 should be demonstrated in more human cancer types and larger subjects. Here, we examined the cleaved caspase-3 expression in 367 human tumor samples (gastric cancer: 97 cases, ovarian cancer: 65 cases, cervical cancer: 104 cases; colorectal cancer: 101 cases) with immunohistochemistry (IHC) and the relationship between the expression of cleaved caspase-3 and various clinicopathological factors were also detected. We found that, cleaved caspase-3 was significantly associated with pathological risk factors (P < 0.005) for the studied cancers, such as tumor stage, lymph-node metastasis, differentiation and so on. In univariate and multivariate analysis, patients with high expression of cleaved caspase-3 had a significant shorter overall survival time compared with those with low cleaved caspase-3 expression in gastric cancer (P < 0.001), ovarian cancer (P < 0.001), cervical cancer (P = 0.002), colorectal cancer (P < 0.001) individually and in the patients combined (P < 0.001). Cox regression results suggested cleaved caspase-3 as an independent prognosis predictor for the studied four cancer types. Our study showed cleaved caspase-3 was well correlated to progression, aggressive behaviors in the studied cancer, and implicated it as a potential predictive factor for the prognosis of the four cancer types. It also indicated cleaved caspase-3 as a potential therapeutic target for cancer patients.     

Mechanism of tyrosine phosphorylation of procaspase-9 and Apaf-1 in cytosolic fractions of the cerebral cortex of newborn piglets during hypoxia

Previous studies have shown that cerebral hypoxia results in increased activity of caspase-9 in the cytosolic fraction of the cerebral cortex of newborn piglets. The present study tests the hypothesis that hypoxia results in increased tyrosine phosphorylation of procaspase-9 and apoptotic protease activating factor-1 (Apaf-1) and the hypoxia-induced increased tyrosine phosphorylation of procaspase-9 and Apaf-1 is mediated by nitric oxide. To test this hypothesis, 15 newborn piglets were divided into three groups: normoxic (Nx, n = 5), hypoxic (Hx, n = 5) and hypoxic treated with nNOS inhibitor I (Hx + nNOS I 0.4 mg/kg, i.v., 30 min prior to hypoxia) [16]. The hypoxic piglets were exposed to an FiO₂ of 0.06 for 1 h. Tissue hypoxia was documented by ATP and phosphocreatine (PCr) levels. Cytosolic fractions were isolated and tyrosine phosphorylated procaspase-9 and Apaf-1 were determined by immunoblotting using specific anti-procaspase-9, anti-Apaf-1 and anti-phosphotyrosine antibodies. ATP levels (μmoles/g brain) were 4.3 ± 0.2 in the Nx and 1.4 ± 0.3 in the Hx and 1.7 ± 0.3 in Hx + nNOS I group (p < 0.05 vs. Nx) groups. PCr levels (μmoles/g brain) were 3.8 ± 0.3 in the Nx and 0.9 ± 0.2 in the Hx and 1.0 ± 0.4 in the Hx + nNOS I (p < 0.05 vs. Nx) group. Density (OD × mm²) of tyrosine phosphorylatd procaspase-9 was 412 ± 8 in the Nx, 1286 ± 12 in the Hx (p < 0.05 vs. Nx) and 421 ± 10 in the Hx + nNOS I (p < 0.05 vs. Hx) group. Density of tyrosine phosphorylated Apaf-1 was 11.72 ± 1.11 in Nx, 24.50 ± 2.33 in Hx (p < 0.05 vs. Nx) and 16.63 ± 1.57 in Hx + nNOS I (p < 0.05 vs. Hx) group. We conclude that hypoxia results in increased tyrosine phosphorylation of procaspase-9 and Apaf-1 proteins in the cytosolic compartment and the hypoxia-induced increased tyrosine phosphorylation of procaspase-9 and Apaf-1 is mediated by nNOS derived nitric oxide. We propose that increased interaction between the tyrosine phosphorylated procaspase-9 and Apaf-1 molecules lead to increased activation of procaspase-9 to caspase-9 in the hypoxic brain that initiates programmed neuronal death.     

Mechanism of caspase-3 activation during hypoxia in the cerebral cortex of newborn piglets

We have previously shown that the activity and the expression of caspase-9 and caspase-3 were increased during hypoxia in the cerebral cortex of newborn piglets. The present study was conducted to test the hypothesis that the hypoxia-induced activation of caspase-3 in the cerebral cortex of newborn piglets is mediated by caspase-9. Twenty-two newborn piglets were randomly assigned to four groups: normoxic (Nx), normoxic pretreated with a selective caspase-9 inhibitor, Z-Leu-Glu(OMe)-His-Asp(OMe)-Fluoromethyl ketone (Z-LEHD-FMK) (Nx + LEHD), hypoxic (Hx), and hypoxic pretreated with Z-LEHD-FMK (Hx + LEHD). Cerebral tissue hypoxia was confirmed biochemically by measuring ATP and phosphocreatine. Caspase-9 and -3 activities were determined spectrofluorometrically. The expression of caspase-9 and -3 proteins was measured by Western blot analysis using active enzyme specific antibodies. Cytosolic caspase-9 activity (nmol/mg protein/h) was 3.70 ± 0.40 in Nx, 3.56 ± 0.31 in Nx + LEHD (p = NS versus Nx), 4.99 ± 0.64 in Hx (p < 0.05 versus Nx), and 3.73 ± 0.80 in Hx + LEHD (p < 0.05 versus Hx, p = NS versus Nx). Cytosolic caspase-3 activity (nmol/mg protein/h) was 7.80 ± 1.17 in Nx, 8.15 ± 0.87 in Nx + LEHD (p = NS versus Nx), 13.07 ± 0.78 in Hx (p < 0.05 versus Nx), and 10.05 ± 2.09 in Hx + LEHD (p < 0.05 versus Hx) The density (OD × mm²) of active caspase-9 protein was 18.52 ± 1.89 in Nx, 20.53 ± 1.12 in Nx + LEHD (p = NS versus Nx), 32.36 ± 5.03 in Hx (p < 0.05 versus Nx), and 19.94 ± 3.59 in Hx + LEHD (p < 0.05 versus Hx, p = NS versus Nx). The density (OD × mm²) of active caspase-3 protein was 55.87 ± 8.73 in Nx, 55.69 ± 8.18 in Nx + LEHD (p = NS versus Nx), 94.10 ± 12.05 in Hx (p < 0.05 versus Nx), and 56.12 ± 14.56 in Hx + LEHD (p < 0.05 versus Hx, p = NS versus Nx). These data show that administration of a selective caspase-9 inhibitor, Z-LEHD-FMK, prior to hypoxia prevents the hypoxia-induced increase in caspase-3 activity and the expression of active caspase-3 protein. We conclude that the hypoxia-induced activation of caspase-3 during hypoxia in the cerebral cortex of newborn piglets is mediated by caspase-9.     

Morphological changes and molecular expressions of hepatocellular carcinoma cells in three-dimensional culture model

Metastatic processes of hepatocellular carcinoma (HCC) are highly associated with the breakdown of extracellular matrix (ECM). However, the regular two-dimensional (2D) culture system, in which only little ECM is involved, fails to provide a well-defined microenvironment for HCC functional research. HAb18G/CD147, a HCC-associated antigen, plays important roles in HCC progression, migration and invasion. In this study, we investigated whether HAb18G/CD147 enhanced the HCC migration and invasion in three-dimensional (3D) culture model through affecting the key molecules and enzymes involved in the metastatic processes, such as focal adhesion kinase (FAK), matrix metalloproteinases (MMPs) and cytoskeleton proteins. We found that, compared with those in 2D cell culture model, the expression of HAb18G/CD147 was significantly increased in 3D cell culture model, together with a high production of MMPs (P < 0.01), an enhanced expression and activation of FAK (P < 0.01) and a changed distribution of F-actin. In addition, the expressions of paxillin and E-cadherin, which enhance the adhesion and migration potentials, were also significantly increased in 3D cell culture model (P < 0.01). All the results suggest that the enhanced expressions of HAb18G/CD147, MMPs, paxillin and FAK changed the distributions of cytoskeleton in the 3D reconstituted basement membrane (BM) and increased the adhesion and invasion potentials of HCC cells.     

Yersinia enterocolitica inactivates NK cells

Natural Killer (NK) cells serve as an important source of proinflammatory cytokines early during infection. Hypothesizing that Yersinia enterocolitica might interact with and inactivate NK cells, we examined NK cell–Y. enterocolitica interactions in vitro and in vivo. Y. enterocolitica adheres to NK cells in an Invasin dependent manner and inhibits NK cell cytotoxicity and IFN-γ production induced by IL-12 + IL-18 or IL-12 alone. YopP, an acetyltransferase known to inhibit MAPK and NFκB signaling, suppresses IL-12 and IL-12 + IL-18 mediated IFN-γ production in NK cells by inhibiting phosphorylation of Tyk2 and STAT4 in addition to MAPK. YopP inhibits induction of all genes whose expression is induced by IL-12 + IL-18 in NK cells. Y. enterocolitica-mediated adherence to and inactivation of NK cells also occurs after infection in vivo. Thus, we present the first report of a bacterial pathogen inactivating NK cells, and report interaction with Tyk2–STAT4 signaling as a novel function of YopP.     

A kinetic study of SDF-1, VEGF and MCP-1 blood and tissue levels after aortic transplantation in mice

Vascular rejection is characterized by intimal proliferation and perivascular inflammation. We hypothesize that recipient stem cell therapy could prevent or ameliorate the development of the obliterative lesion. We studied the kinetic expression of three cytokines (SDF-1, MCP-1, VEGF) implicated in mobilization, homing and differentiation of progenitor cells during vascular aggression. An aortic allograft mouse model was used (BALBc donor-C57BL6/j recipient). Ten mice were sacrificed at Day 0, D1, D3, D6, D9, D12, and D20. Cytokine rates were measured in blood and in graft tissue by an ELISA technique. Results showed that in the allograft, SDF-1 and VEGF tissue levels were significantly increased at D12 as compared to the isograft (SDF-1: 22.16 ng/mg vs. 5.69 ng/mg, t = 3.38; VEGF: 28.3 pg/mg vs. 9.3 pg/mg, t = 3.06). In allografted and isografted groups, MCP-1 tissue levels were higher at D0 as compared to the other time points, without any difference between the two groups. These results prompt us to consider cell therapy at D0 and D12 in this mouse model of aortic graft.     

Mild as well as severe insults produce necrotic,not apoptotic,cells: Evidence from 60-min seizures

We tested the hypothesis that mild insults produce apoptotic, and severe insults necrotic, cells by subjecting adult Wistar rats to 60-min instead of 3-h generalized seizures. Rats’ brains were evaluated 6 and 24 h later for evidence of neuronal necrosis by light and electron microscopy, the presence of TUNEL staining and active caspase-3 immunoreactivity, and for evidence of DNA laddering 24 h after seizures. Apoptotic neurons from the retrosplenial cortex of postnatal day 8 rat pups served as positive controls. Six and 24 h after seizures, 16 and 15 brain regions respectively out of 24 showed significant numbers of acidophilic neurons by hematoxylin and eosin stain. Three brain regions had significant numbers of TUNEL-positive neurons 24 h after seizures. No neurons showed active caspase-3 immunoreactivity. Acidophilic neurons were necrotic by electron-microscopic examination. Ultrastructurally, they were shrunken and electron-dense, with shrunken, pyknotic nuclei and swollen mitochondria with disrupted cristae. Nuclei did not contain the irregular chromatin clumps found after 3-h seizures. None of the six brain regions studied ultrastructurally that show DNA laddering 24 h after 3-h seizures showed DNA laddering 24 h after 60-min seizures, probably because there were too few damaged neurons, although the lack of chromatin clumping might have been a contributing factor. Following seizures, a mild as well as a severe insult produces caspase-3-negative necrotic neurons. These results do not support the hypothesis that mild insults produce apoptotic, and severe insults, necrotic, cells.     

Olfactory bulb implantation and methylprednisolone administration in the treatment of spinal cord injury in rats

Estimate the effects of methylprednisolone (MP) administration and olfactory bulb (OB) implantation independently and in combination after a spinal trauma model in Wistar rats, evaluated with BBB scale and CBS with remark of inclined plane test and Tarlov scale. Thirty adult rats were divided into six different groups, evaluated before trauma, one day post-surgery and weekly up to six weeks post-lesion. Group A (control); group B (sham) laminectomy without lesion; group C (SCI) lesion only; group D (MP) SCI and MP; group E (OB) SCI and OB implantation; group F (MP/OB) SCI and both therapeutics. Intragroup data at three weeks showed evident significance in groups D, E and F for Tarlov (p = 0.001) and BBB (p < 0.01); groups C, D, E and F for CBS (p < 0.05); and only group D with inclined plane (p < 0.05). On the sixth week differences were present in groups C, D, E, and F for Tarlov, BBB and CBS (p < 0.001); and C and F for inclined plane (p < 0.05). For intergroup analysis any treatment showed differences with Tarlov scale; for BBB and inclined plane, statistical differences were evident in groups E and F; and for CBS only in group F (p < 0.05). Real effects of MP are obtained at immediate follow-up, without notorious augmentation after time. OB improvement is achieved only after weeks. None of these therapies used independently achieve a constant and sustained improvement. Combined treatments were more effective and reached higher functional levels for longer periods of time.     

Suppressive effect of perinatal testes on the differentiation of fetal ovaries transplanted into adult males in the rat

A 14 d ovarian primordium was transplanted with a fetal testis (13–18 d and 21 d of gestation) or a neonatal testis (15, 20, 30 and 45 d after birth) into the renal subcapsular position of an adult male rat. Two weeks after transplantation, transplants were examined as to the degree of ovarian and testicular differentiation. In the combination of a 14 d ovary and a 13 d testis, there were 3 types of result: either the ovary or the testis alone developed or both gonads developed well. Ovaries transplanted in union with 15–18 d testes did not develop, although the testes developed normally. Some ovaries in union with 21 d testes developed normally. In combination with infantile testes, the incidence of developed ovaries increased as the age of testes advanced. These results suggest that the 13 d fetal testes begin to suppress the development of cotransplanted 14 d ovaries, that 14–18 d fetal testes maintain such suppressive effects and that this effect gradually diminishes in infantile testes as they progress toward 45 d after birth.     

Virulence plasmid of Aeromonas hydrophila induces macrophage apoptosis and helps in developing systemic infection in mice

Pathogenic Aeromonas hydrophila Strain AO1 bears a 21 kb plasmid encoding several virulence determinants. Infection studies revealed that this isolate induced cytotoxicity in BALB/c mice splenic macrophages involving reactive oxygen species generation. DNA gel, Hoechst 33342, annexin-V and TUNEL assay documented macrophage death induced by 21 kb plasmid bearing isolates to be apoptotic in nature. Apoptosis induced by the plasmid bearing isolates involved initiator caspase-8 and caspase-9 and executed by effector caspase-3. ELISA revealed the wild-type isolate as weak inducer of pro-inflammatory cytokine IL-1β. Oral infection with wild-type isolates caused systemic infection in BALB/c mice. With plasmid curing the isolate looses several virulence attributes including cytotoxic potential. The cured isolate induced significant amounts of IL-1β from infected macrophages, disseminated into Peyer's patches, spleen and liver but never attained the bacterial loads recorded with wild-type isolates and were rapidly cleared. Transformation of 21 kb plasmid helped the cured bacteria regain wild-type virulence attributes, apoptotic potential and ability to cause systemic infection in mice. Thus the 21 kb plasmid is a virulence factor in mice. It helps in suppressing the production of pro-inflammatory cytokine IL-1β and induced apoptosis of host macrophages enabling A. hydrophila to evade host immune responses and establish systemic infection in mice.     

Oral ultra-low dose continuous combined hormone replacement therapy with 0.5 mg 17β-oestradiol and 2.5 mg dydrogesterone for the treatment of vasomotor symptoms: Results from a double-blind,controlled study

Objectives

Guidelines recommend using the lowest effective dose of oestrogen for the management of vasomotor symptoms in postmenopausal women. The primary aim of this double-blind, multi-centre, randomised study was to assess the efficacy of oral ultra-low dose continuous combined hormone replacement therapy with 17β-oestradiol and dydrogesterone.

Study design

313 women with ≥50 moderate to severe hot flushes during the previous week were randomised to 0.5 mg 17β-oestradiol/2.5 mg dydrogesterone (E 0.5 mg/D 2.5 mg), 1 mg 17β-oestradiol/5 mg dydrogesterone (E 1 mg/D 5 mg) or placebo for 13 weeks. The placebo group then switched to E 0.5 mg/D 2.5 mg for a further 39 weeks, whilst the other groups continued on the same treatment.

Results

After 13 weeks, the reduction in the number of moderate to severe hot flushes/day in the E 0.5 mg/D 2.5 mg group was greater than in the placebo group (−6.4 vs. −4.9, p < 0.001) and comparable to that in the 1/5 mg group (−6.3). E 0.5 mg/D 2.5 mg and E 1 mg/D 5 mg significantly improved the total Menopause Rating Scale score. The number of bleeding/spotting days was lower with E 0.5 mg/D 2.5 mg than with E 1 mg/D 5 mg. The overall amenorrhoea rate with E 0.5 mg/D 2.5 mg was 81%; this increased to 91% in months 10–12.

Conclusions

Continuous combined 0.5 mg 17β-oestradiol and 2.5 mg dydrogesterone was effective in alleviating vasomotor symptoms and improving quality of life, and was associated with a high amenorrhoea rate and a good tolerability profile.     

Human papillomaviruses 53 and 66: clinical aspects and genetic analysis

Variants of HPV types may have different oncogenic potential. While HPV 16 and 18 variants have been extensively studied, little is known on the less frequent high-risk types such as HPV 53 and 66. Here, we analyzed the genetic variability of HPV 53 and 66 by sequencing the E6, E7, L1 genes and the Long Control Region (LCR) sequences of HPV 53 and HPV 66 from infected women. Fisher's exact-test was performed to correlate viral variants with cervical lesions. Higher-order interactions among identified mutations were analyzed by co-variation and cluster analyses. Antigenic-index alterations following L1 mutations were predicted by Jameson-Wolf algorithm.In HPV53, novel variants were identified in L1 (N = 9) and E6 (N = 1) genes. The novel L1 mutation P432L was statistically associated with L-SIL lesions (P = 0.04) and its development reduced the L1 predicted antigenicity (up to −2.3 for Glu433). HPV 53 E6 and L1 sequences clustered phylogenetically into two main clades.In HPV 66, novel polymorphisms were identified in L1 (N = 4) and E6 (N = 4) genes. The L1 protein mutations S405P and D458N were exclusively found in patients with L-SILs. Seven E7 variants and 10 LCR variants were for the first time analyzed.Novel HPV 53 and 66 variants were identified in this study. Some of these mutations were significantly associated with L-SIL lesions and affected the antigenic index of the L1 protein with possible interesting implications in vaccine design.     

Clinical and medial temporal features in a family with mood disorders

It is debated whether non-affected relatives of patients with affective disorders share a specific brain structure endophenotype. Aim of this work is to explore the medial temporal morphology in affected and non-affected members of a family with mood disorders. Hippocampi and amygdalae were manually traced from the 3D magnetic resonance imaging of five affected family members, 10 non-affected relatives, and 15 unrelated matched controls. Affected and non-affected relatives were characterized by larger left amygdalae (18%, p = 0.030), smaller right hippocampus (up to 18%, p < 0.0005), and reduced hippocampal asymmetry (p < 0.001) than controls. Abnormal, albeit non significant, positive correlations of MTL volumes with age were observed, with the exception of smaller volume of the left hippocampus with advancing age (r = −0.76) in the affected relatives. These data add to the evidence that abnormal medial temporal structures may constitute an endophenotype for affective disorders.     

Regulation of oxidative stress and somatostatin,cholecystokinin, apelin gene expressions by ghrelin in stomach of newborn diabetic rats

The aim of the study was to determine whether ghrelin treatment has a protective effect on gene expression and biochemical changes in the stomach of newborn streptozotocin (STZ) induced diabetic rats. In this study, four groups of Wistar rats were used: control, ghrelin control, diabetic and diabetic + ghrelin. The rats were sacrificed after four weeks of treatment for diabetes. The gene expressions of: somatostatin, cholecystokinin, apelin and the altered active caspase-3, active caspase-8, proliferating cell nuclear antigen, were investigated in the pyloric region of the stomach and antioxidant parameters were measured in all the stomach. Although ghrelin treatment to diabetic rats lowered the stomach lipid peroxidation levels, the stomach glutathione levels were increased. Exogenous ghrelin caused an increased activities of stomach catalase, superoxide dismutase, glutathione reductase and glutathione peroxidase in diabetic rats. Numbers of somatostatin, cholecystokinin and proliferating cell nuclear antigen immunoreactive cells decreased in the diabetic + ghrelin group compared to the diabetic group. Apelin mRNA expressions were remarkably less in the diabetic + ghrelin rats than in diabetic rats. The results may indicate that ghrelin treatment has a protective effect to some extent on the diabetic rats. This protection is possibly accomplished through the antioxidant activity of ghrelin observed in type 2 diabetes. Consequently exogenous ghrelin may be a candidate for therapeutic treatment of diabetes.     

Osteopontin expression correlates with nuclear factor-κB activation and apoptosis downregulation in clear cell renal cell carcinoma

Osteopontin (OPN) is a phosphoglycoprotein implicated in tumorigenesis and tumor cell metastasis. Apoptosis inhibition is one of the mechanisms that contribute to development and progression of cancer, and might be initiated by OPN interaction with tumor cells. The aim of this study was to analyze the relation between OPN and nuclear factor-kappa B (NF-κB) expression in clear cell renal cell carcinoma (CCRCC), as well as their relation to apoptotic activity of tumor cells.Expression of OPN protein and p65 NF-κB subunit was analyzed immunohistochemically in 87 CCRCC samples, and compared mutually and with apoptotic index. Expression of OPN mRNA was analyzed using quantitative real-time PCR and compared with OPN and NF-κB protein expression in 22 CCRCC samples.Statistical analysis showed an association of p65 NF-κB with OPN mRNA (p = 0.015) and protein (p < 0.001). Also, we found an inverse relationship of OPN with NF-κB protein expression and apoptotic activity of tumor cells (p = 0.006 and p = 0.022, respectively). Our results indicate that p65 NF-κB signaling pathway may be involved in OPN-mediated CCRCC progression, partly by protecting tumor cells from apoptosis. Therefore, both molecules can constitute potential targets for therapeutic intervention in CCRCC.