The tyrosine-kinase receptor c-kit and its ligand, stem cell factor (SCF), are essential for the maintenance of primordial germ cells (PGCs) in both sexes. However, c-kit and a post-meiotic-specific ...alternative c-kit gene product play important roles also during post-natal stages of spermatogenesis. In the adult testis, the c-kit receptor is re-expressed in differentiating spermatogonia, but not in spermatogonial stem cells, whereas SCF is expressed by Sertoli cells under FSH stimulation. SCF stimulates DNA synthesis in type A spermatogonia cultured in vitro, and injection of anti-c-kit antibodies blocks their proliferation in vivo. A point mutation in the c-kit gene, which impairs SCF-mediated activation of phosphatidylinositol 3-kinase, does not cause any significant reduction in PGCs number during embryonic development, nor in spermatogonial stem cell populations. However males are completely sterile due to a block in the initial stages of spermatogenesis, associated to abolishment of DNA-synthesis in differentiating A1-A4 spermatogonia. With the onset of meiosis c-kit expression ceases, but a truncated c-kit product, tr-kit, is specifically expressed in post-meiotic stages of spermatogenesis, and is accumulated in mature spermatozoa. Microinjection of tr-kit into mouse eggs causes their parthenogenetic activation, suggesting that it might play a role in the final function of the gametes, fertilization.
The possibility of incorporating H
S slow-release donors inside biomimetic scaffolds can pave the way to new approaches in the field of tissue regeneration and anti-inflammatory treatment. In the ...present work, GYY4137, an easy-to-handle commercially available Lawesson's reagent derivative, has been successfully incorporated inside biomimetic silk fibroin-based electrospun scaffolds. Due to the instability of GYY4137 in the solvent needed to prepare silk fibroin solutions (formic acid), the electrospinning of the donor together with the silk fibroin turned out to be impossible. Therefore, a multilayer structure was realized, consisting of a PLGA mat containing GYY4137 sandwiched between two silk fibroin nanofibrous layers. Before their use in the multilayer scaffold, the silk fibroin mats were treated in ethanol to induce crystalline phase formation, which conferred water-resistance and biomimetic properties. The morphological, thermal, and chemical properties of the obtained scaffolds were thoroughly characterized by SEM, TGA, DSC, FTIR, and WAXD. Multilayer devices showing two different concentrations of the H
S donor, i.e., 2 and 5% w/w with respect to the weight of PLGA, were analyzed to study their H
S release and biological properties, and the results were compared with those of the sample not containing GYY4137. The H
S release analysis was carried out according to an "ad-hoc" designed procedure based on a validated high-performance liquid chromatography method. The proposed analytical approach demonstrated the slow-release kinetics of H
S from the multilayer scaffolds and its tunability by acting on the donor's concentration inside the PLGA nanofibers. Finally, the devices were tested in biological assays using bone marrow-derived mesenchymal stromal cells showing the capacity to support cell spreading throughout the scaffold and prevent cytotoxicity effects in serum starvation conditions. The resulting devices can be exploited for applications in the tissue engineering field since they combine the advantages of controlled H
S release kinetics and the biomimetic properties of silk fibroin nanofibers.
The c-kit gene plays a fundamental role during the establishment, the maintenance and the function of germ cells. In the embryonal gonad the c-kit tyrosine kinase receptor and its ligand Stem Cell ...Factor (SCF) are required for the survival and proliferation of primordial germ cells. In the postnatal animal, c-kit/SCF are required for the production of the mature gametes in response to gonadotropic hormones, i.e. for the survival and/or proliferation of the only proliferating germ cells of the testis, the spermatogonia, and for the growth and maturation of the oocytes. Finally, a truncated c-kit product, tr-kit, specifically expressed in post-meiotic stages of spermatogenesis and present in mature spermatozoa, causes parthenogenetic activation when microinjected into mouse eggs, suggesting that it might play a role in the final function of the gametes, fertilization.
To compare a full-dose epirubicin-cyclophosphamide (HEC) regimen with classical cyclophosphamide, methotrexate, and fluorouracil (CMF) therapy and with a moderate-dose epirubicin-cyclophosphamide ...regimen (EC) in the adjuvant therapy of node-positive breast cancer.
Node-positive breast cancer patients who were aged 70 years or younger were randomly allocated to one of the following treatments: CMF for six cycles (oral cyclophosphamide); EC for eight cycles (epirubicin 60 mg/m(2), cyclophosphamide 500 mg/m(2); day 1 every 3 weeks); and HEC for eight cycles (epirubicin 100 mg/m(2), cyclophosphamide 830 mg/m(2); day 1 every 3 weeks).
Two hundred fifty-five, 267, and 255 eligible patients were treated with CMF, EC, and HEC, respectively. Patient characteristics were well balanced among the three arms. One and three cases of congestive heart failure were reported in the EC and HEC arms, respectively. Three cases of acute myeloid leukemia were reported in the HEC arm. After 4 years of median follow-up, no statistically significant differences were observed between HEC and CMF (event-free survival EFS: hazards ratio HR = 0.96, 95% confidence interval CI, 0.70 to 1.31, P =.80; distant-EFS: HR = 0.97, 95% CI, 0.70 to 1.34, P =.87; overall survival OS: HR = 0.97, 95% CI, 0.65 to 1.44, P =.87). HEC is more effective than EC (EFS: HR = 0.73, 95% CI, 0.54 to 0.99, P =.04; distant-EFS: HR = 0.75, 95% CI, 0.55 to 1.02, P =.06; OS HR = 0.69, 95% CI, 0.47 to 1.00, P =.05).
This three-arm study does not show an advantage in favor of an adequately dosed epirubicin-based regimen over classical CMF in the adjuvant therapy of node-positive pre- and postmenopausal women with breast cancer. Moreover, this study confirms that there is a dose-response curve for epirubicin in breast cancer adjuvant therapy.
Exposure to hypoxia has shown beneficial adjustments in different species, including silver catfish (Rhamdia quelen), especially in situations of aquatic contamination with pollutants such as ...manganese (Mn). Considering that hypoxia is seasonal in the natural aquatic environment, we decided to assess whether these adaptive mechanisms could be maintained when reoxygenation is established. Silver catfish acclimated to moderate hypoxia (∼3 mg L−1, 41% O2 saturation) for 10 days and subsequently exposed to Mn (∼8.1 mg L−1) for additional 10 days displayed lower (47%) Mn accumulation in the gills, and it was maintained (62.6%) after reoxygenation, in comparison to normoxia. Oxidative status in the gills allowed us to observe increased reactive species (RS) generation and protein carbonyl (PC) level together with decreased mitochondrial viability induced by Mn under normoxia. Inversely, while hypoxia per se was beneficial on RS generation and PC level, this acclimation was able to minimize Mn toxicity, as observed by the minor increase of RS generation and the minor reduction of mitochondrial viability, together with decreased PC level. Interestingly, after reoxygenation, part of the protective influences observed during hypoxia against Mn toxicity were maintained, as observed through a lower level of PC and higher mitochondrial viability in relation to the group exposed to Mn under normoxia. Only groups exposed to Mn under hypoxia showed increased activity of both catalase (CAT) and Na+/K+-ATPase in the gills, but, while CAT activity remained increased after reoxygenation, Na+/K+-ATPase activity was decreased by Mn, regardless of the oxygen level. Based on these outcomes, it is possible to propose that environment events of moderate hypoxia are able to generate rearrangements in the gills of silver catfish exposed to Mn, whose influence persists after water reoxygenation. These responses may be related to the adaptive development, reducing Mn toxicity to silver catfish.
Moderate hypoxia generates rearrangements in the gills of Silver catfish, exerting beneficial and persistent protection against Mn toxicity.
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•Aquatic hypoxia acclimation and subsequent reoxygenation reduce Mn accumulation in silver catfish gill.•Hypoxia acclimation produces changes in gill morphology persistent after reoxygenation.•Hormesis by low oxygen can reduce Mn toxicity to silver catfish.
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•Rats developed preference for and relapse to amphetamine (AMPH).•The ultra-high diluted AMPH (successive dilutions of AMPH until 10-24 mg/mL), was used for treat addiction ...parameters.•Isotherapic reduced AMPH-induced relapse and anxiety behaviors.•Isotherapic and AMPH changed dopaminergic molecular targets.•Isotherapic per se reduced oxidative damage in prefrontal cortex of rats.
amphetamine (AMPH) is related to development of addiction, anxiety-like behaviors and impairments of memory after chronic use. In the current experiment, an ultra-high dilution (10−24mg/mL) of AMPH was used, consisting of the AMPH isotherapic (AMPH-ISO), which can be used as a replacement therapy to treat AMPH addiction.
To verify the influence of AMPH-ISO on toxicological aspects of AMPH addiction.
Rats received d,l-AMPH (4.0 mg/kg, i.p.) in the conditioned place preference (CPP) paradigm (8 days). Then, half of each experimental group (AMPH or saline) received AMPH-ISO/vehicle (0.2 mL per rat, once a day), for fourteen days. On the fifteenth day, animals were re-assessed in the CPP paradigm (to verify relapse behaviors) after a single dose of AMPH (2.0 mg/kg). Subsequently, anxiety-like behaviors were quantified, followed by ex vivo assays in the pre-frontal cortex.
AMPH-ISO prevented relapse-like behavior of AMPH and reduced anxiety-like behavior per se in animals co-treated with AMPH. Molecular analysis evidenced that AMPH-ISO modulated dopaminergic targets (dopamine transporter, tyrosine hydroxylase and D1-R), whose immunoreactivity was increased by AMPH. Also, AMPH-ISO increased catalase activity and NPSH levels and reduced lipid peroxidation and protein carbonyl levels in the prefrontal cortex.
This study shows that an ultra-high dilution of AMPH may be a useful alternative which can contribute with AMPH addiction treatment.
Abstract
Study question
Could in-utero exposure to cannabinoids alter the reproductive health of female offspring?
Summary answer
Prenatal exposure to cannabinoids causes a reduction of the ovarian ...reserve in F1 with compromised reproductive lifespan and low birth weight in F1/ F2 generations.
What is known already
Cannabis use during pregnancy is associated with adverse neonatal outcomes: preterm birth, intrauterine growth retardation, low birth weight, and neurological developmental changes. However, the impact on the germ line is still little known. Cannabis/cannabinoids exert their biological effect by activating two main cannabinoid receptors: CB1 and CB2. We previously demonstrated that fetal oocytes express CB2 receptors at a higher level than CB1. In vitro treatment of fetal oocytes with JWH-133, a selective CB2 agonist, induces an acceleration of meiosis and an increase in the percentage of γ-H2AX-positive cells and in TUNEL-positive cells (apoptotic cells).
Study design, size, duration
Five pregnant CD1 female mice were intraperitoneally injected with a single dose of JWH-133, at 1.5 mg/kg for 5 days between embryonic period (E) 12.5 to E16.5, while control pregnant females received a vehicle containing saline. At delivery, on post-natal day (PND) 2, 30 and 365, F1 pups were counted, weighted and tissues from females were collected. At adult age, F1 females were mated with control mice and F2 generation was morphologically analysed.
Participants/materials, setting, methods
Ovaries from control and in utero exposed mice were fixed in paraformaldehyde and stained with Haematoxylin and Eosin (H&E). Gonads were embedded in paraplast and sectioned at 5μm Leica-RM 2035 Microtome. Follicles in each ovary were counted serially in every third section through the entire ovary. Only healthy, non-atretic follicles with visible oocyte nuclei were scored.
Main results and the role of chance
We demonstrate that in vivo exposure to the cannabinoid JWH-133 during fetal life, through the administration of the drug to pregnant females, causes a significant reduction in the offspring body weight at birth (LBW). Histological analysis of the ovaries isolated from newborn females (PND2) shows a significant reduction of the primordial and primary follicles. This reduced ovarian reserve is observed also at PND30 and at PND365. At old adult age, these females, crossed with control males, show a drastic decrease in their litter size (F2) across the life course in comparison with control females. This suggests that the pool of primordial follicles becomes prematurely depleted in in-utero exposed F1 females, compromising their reproductive lifespan. Moreover, we find that F2 pups from prenatally exposed mothers show LBW but have an unaltered number of ovarian follicles in females. Since F2 pups have never been exposed to the drug, our results suggest that LBW is transmitted from the mother intergenerationally, while the reduced ovarian reserve is a consequence of direct exposure to the drug during fetal life of F1 females and it is not transmitted to the F2 generation.
Limitations, reasons for caution
The effects observed in mice with JWH-133 cannot be directly translated to cannabis effects since the active principle of cannabis (THC) activates both the cannabinoid receptors. Moreover, this study lacks molecular analysis of gene expression implicated in intergenerational transmission.
Wider implications of the findings
This is the first direct demonstration that prenatal exposure to cannabinoids could have long-term critical consequences for females’ reproductive health. In humans, diminished ovarian reserve is characterized by poor fertility outcomes and it represents a major challenge in reproductive medicine.
Trial registration number
not applicable
Background: Breast cancer is a heterogeneous disease. Predictive biological markers (BM) of responsiveness to therapy need to be identified. Evaluation of BM is mainly done at the primary site. ...However, in the adjuvant therapy of breast cancer, the main goal is control of micrometastases. It is still unknown whether heterogeneity in the expression of BM between the primary site and its micrometastases exists. Objective: To evaluate the expression of some BM with potential predictive value from the primary breast cancer site and metastatic ipsilateral axillary lymph nodes. Patients and methods: Focality (percentage of positive cells) and intensity staining scores were evaluated for each marker. Freshly cut sections (4 μm) from embedded blocks of breast cancer fixed in formalin or bouin were put onto superfrost slides (Menzel—Gläser). Protein expression was evaluated immunohistochemically (IHC) using monoclonal antibodies against: topo II-α (clone K1SI, 1 μg/ml, Roche) with a trypsine pre-treatment (P); HSP27 (clone G3.1, 1/60, Biogenex), HSP70 (clone BRM.22, 1/80, Biogenex) and HER2 (clone CBII, 1/40, Novocastra; without P); p53 (clone D07, 1/750, Dako) and bcl-2 (clone 124, 1/60, Dako) with citrate buffer as P. Results: Overall, the percentage of discordant marker status in the primary tumour and its metastatic lymph nodes was 2% for HER2, 6% for p53, 15% for bcl-2, 19% for topoisomerase II-α, 24% for HSP27 and 30% for HSP70. For the subgroup of patients with positive BM in the primary tumour, the percentage of discordance was 6% for HER2. 7% for p53, 14% for bcl-2, 19% for HSP70, 21% for topoisomerase II-α and 36% for HSP27. For the subgroup of patients with positive BM in the lymph nodes, the percentage of discordance was 9% for bcl-2, 15% for HER2 and p53, 21% for topoisomerase II-α, 22% for HSP27 and 25% for HSP70. Conclusions: 1) No biological marker had 100% concordant results. 2) Although some discordant cases might be explained by the limitations of the IHC technique, future studies aiming to evaluate the predictive value of BM in the adjuvant therapy of breast cancer should take into account a possible difference in BM expression between the primary and the metastatic sites.
Background: The predictive role of HER-2 in node-positive breast cancer patients receiving CMF or an anthracycline-based adjuvant therapy remains unclear. In addition, topo-isomerase II alpha (topo ...IIα), as the cellular target of anthracyclines. might have value as a predictive marker. Patients and methods: Four hundred eighty-one archival primary tumor samples were collected among 777 patients entered into a multicenter phase III trial comparing classical CMF with epirubicin-cyclophosphamide (HEC) as adjuvant therapy of node-positive breast cancer. HER-2 was evaluated by immunohistochemistry (IHC) using different antibodies (Abs). Topo IIα was evaluated by (IHC) using the Ab KiS 1. In each subgroup of patients identified by HER-2 and topo IIα, adjusted hazard ratios for event-free survival (EFS) and the corresponding 95% confidence intervals have been calculated for the different study comparisons. An interaction test has been performed to investigate the role of HER-2 and topo IIα, as predictive markers. Results: When HER-2 was evaluated by CB-11 and 4D5 mAbs, the EFS adjusted hazard ratios (HR) for the main study comparison HEC vs. CMF were: HER-2 positive: 0.33 (95% confidence interval (95% Cl): 0.09–1.27, P=0.08), HER-2 negative: 1.16 (95% Cl: 0.71–1.90, P=0.56); the P-value for the interaction test was 0.10. When HER-2 was evaluated by TAB-250+pAb1 Abs, the adjusted HR for the same comparison were: HER-2 positive: 1.06 (95% Cl: 0.45–2.52, P=0.90), HER-2 negative: 0.99(95% Cl: 0.58–1.68. P=0.97); the P-value for the interaction test was 0.84. With regard to topo 11α, the adjusted HR for the EFS comparison HEC vs. CMF were: topo 11α positive: 0.66 (95% Cl: 0.32–1.36, P=0.25), topo 11α negative: 1.26(95% Cl: 0.63–2.50, P=0.51): the P-value for the interaction test was 0.13. Conclusions: This study suggests that in node-positive breast cancer patients randomly treated with CMF or an epirubicin-based regimen, the predictive value of HER-2 may vary according to the Abs used in the immunohistochemistry assay. In addition, the study supports the concept that topo 11α might be involved in the determination of tumor responsiveness to an anthracycline-based adjuvant therapy.
This work deals with the preparation and physical-chemical characterization of new ultrasmall iron oxide superparamagnetic nanoparticles (USPIONs) functionalized with titanium-N,N-dialkylcarbamato. ...The preparation was performed starting with monodispersed USPIONs covered with oleic acid, synthesized by thermal-decomposition, and subsequently functionalized with metal-carbamato by a ligand-exchange reaction. The surface and coating structure was characterized by infrared (FT-IR) spectroscopy on the solid powders and thermogravimetry (TG) coupled with an FT-IR detector in order to better investigate the self-assembling properties of the coating. A detailed dimensional and morphological study was carried out by transmission electron microscopy (TEM) and atomic force microscopy (AFM) analysis. Zero-field-cooled (ZFC) and field-cooled (FC) magnetic susceptibility curves as well as the magnetization behavior as a function of temperature were investigated on both the starting oleic-USPIONs and those covered by titanium-N,N-dialkylcarbamato. These results confirmed the superparamagnetic properties of the new nanoparticles (NPs), highlighting the quite high saturation value of the magnetization. Based on the results obtained by combining different experimental techniques, a model of the coating structure and ligand organization around the magnetic core is proposed for both NPs, i.e. the starting USPIONs covered by oleic acid and the new USPIONs functionalized by titanium-N,N-dialkylcarbamato.
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