Proliferating Cell Nuclear Antigen Immunoexpression Research Articles

Mitigating effect of gallic acid on zinc oxide nanoparticles and arsenic trioxide-induced spermatogenesis suppression, testicular injury, hormonal imbalance, and immunohistochemical changes in rats.

The current study compared the effects of incorporated exposure to arsenic trioxide (As) and zinc oxide nanoparticles (ZnONPs) on male reproductive hormones, oxidative stress, and inflammatory biomarkers in adult rats to each metal alone. A defensive trial with gallic acid (GA) has also been studied. A total of 60 adult male Sprague Dawley rats were categorized into six groups: control, GA (20 mg/kg), ZnONPs (100 mg/kg), As (8 mg/kg), ZnONPs with As, and GA concurrently with ZnONPs and As at the same previous doses. The regimens were applied for 60 days in sequence. Current findings showed significant weight loss in all study groups, with testicular weights significantly decreased in the As and combined groups. Testosterone, follicular stimulating hormone, and luteinizing hormone serum levels were also considerably reduced, while serum levels of estradiol increased. Inducible nitric oxide synthase (iNOS) immunoexpression was significantly upregulated while proliferating cell nuclear antigen (PCNA) was downregulated. Moreover, there was a significant elevation of testicular malondialdehyde, reduction of testicular superoxide dismutase, and glutathione peroxidase with disruptive testes, prostate glands, and seminal vesicle alterations in all experimental groups with marked changes in the combined group. Additionally, the present results revealed the protective effects of GA on ZnONPs and As adverse alterations in rats. GA enhanced sperm picture, oxidant status, and hormonal profile. Also, it modulates iNOS and PCNA immunoexpression and recovers the histoarchitecture of the testes, prostate glands, and seminal vesicles. Ultimately, GA may be a promising safeguarding agent against ZnONPs and As-induced disturbances to reproductive parameters.

Reparative potential of mesenchymal stem cells and platelet-rich plasma on irradiated submandibular glands of male albino rats

ObjectiveTo appraise and compare the reparative role of bone marrow-mesenchymal stem cells (BM-MSCs) and platelet-rich plasma (PRP) against irradiation damage on albino rats’ submandibular gland. DesignSeventy four male albino rats were used, one for BM-MSCs harvesting, 10 for PRP preparation, seven as control group (Group 1). The remaining 56 rats were subjected to single dose (6 Gy) gamma irradiation and were divided into equal four groups; (Group 2): received no treatment, (Group 3): each rat was injected with 1 × 105 BM-MSCs, (Group 4): each rat was injected with 0.5 ml/kg PRP, and (Group 5): each rat was injected with 1 × 105 BM-MSCs and 0.5 ml/kg PRP. Each group was further subdivided into two subgroups in which rats sacrificed after one and two weeks from irradiation. Any structural changes were examined histopathologically, immunohistochemically using proliferating cell nuclear antigen (PCNA) and CD31 primary antibodies and histochemically using picrosirius red (PSR) stain, then analyzed statistically. ResultsHistopathological examination of Group 2 showed atrophied acini, with nuclear changes and signs of degeneration in duct systems. Treated groups revealed signs of regeneration in form of uniform acini and regenerated duct systems especially in Group 5 and in a time depended manner. Immunohistochemical examination revealed increased immunoexpression of PCNA and CD31, while histochemical examination showed decreased PSR in all treated groups in relation to the irradiated group and this was proved statistically. ConclusionsBM-MSCs and PRP are effective as treatment for irradiation-induced submandibular gland damage. However, the combined therapy is recommended over each one separately.

Prognostic relevance of clinicopathological factors in sporadic and syndromic odontogenic keratocysts: A comparative study.

Current evidence suggests that nevoid basal cell carcinoma syndrome (NBCCS)-associated odontogenic keratocysts (OKCs) exhibit more aggressive clinical behavior and a higher tendency to relapse. The prognostic efficacy of various markers in sporadic and syndromic OKCs is unclear, and so are the results of studies on the usefulness of immunohistochemistry in distinguishing syndromic from sporadic OKCs. This retrospective study aimed to compare the prognostic relevance of various clinicoradiological and histopathological features, as well as the immunoexpression of COX-2, Bcl-2, proliferating cell nuclear antigen (PCNA), p53, Ki-67, osteoprotegerin (OPG), receptor activator of nuclear factor κ B (RANK) and receptor activator of nuclear factor κ B ligand (RANKL), as well as RANKL/OPG balance between sporadic and syndromic OKCs, and to test their utility in distinguishing the 2 types of OKC. We compared the immunoexpression of the aforementioned markers between 31 sporadic and 12 syndromic OKCs, and tested clinicopathological findings and levels of immunostaining against recurrence. We found a significant association between NBCCS and OKC recurrence. There were significant differences in PCNA, p53 and OPG immunoexpression between sporadic and syndromic OKCs. We also found that recurrent sporadic OKCs were significantly larger and markedly more often associated with cortical perforation. Recurrent sporadic OKCs exhibited COX-2 upregulation, but we failed to demonstrate its prognostic relevance. Recurrent syndromic OKCs showed a markedly higher RANKL > OPG ratio. The NBCCS-associated OKCs are significantly more prone to recur than their sporadic counterparts. Larger size and radiological signs of cortical perforation in sporadic OKCs may indicate a higher risk of recurrence. The COX-2 is upregulated in recurrent sporadic OKCs, whereas recurrent syndromic OKCs exhibit higher RANKL and lower OPG expression; however, these findings have no prognostic relevance. The immunoexpression of p53, PCNA and OPG may help to distinguish syndromic from sporadic OKCs.

Therapeutic Effects of Bee Bread on Obesity-Induced Testicular-Derived Oxidative Stress, Inflammation, and Apoptosis in High-Fat Diet Obese Rat Model.

Obesity is a debilitating disorder with a variety of problems including oxidative stress, inflammation, and apoptosis. The aim of our study was to investigate the therapeutic role of bee bread on oxidative stress, apoptosis, and inflammation in the testis of obese rats. Thirty-two adult male Sprague Dawley rats, with weights between 230–300 g, were distributed into four groups (n = 8/group), namely normal control (C), obese (Ob), obese + BB or obese + OR [high-fat diet (HFD) for 6 weeks then HFD plus bee bread or orlistat for another 6 weeks] groups. Bee bread (0.5 g/kg) or orlistat (10 mg/kg/day) was diluted with distilled water and administered daily for 6 weeks by oral gavage. There were significant decreases in the activities of antioxidant enzymes [glutathione-S-transferase (GST), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione reductase (GR)], glutathione (GSH)] and total antioxidant capacity (TAC) levels and mRNA expressions of nuclear factor erythroid 2–related factor 2 (Nrf2), superoxide dismutase (Sod), catalase (Cat) and glutathione peroxidase (Gpx) in the obese group relative to the control group. Meanwhile, the mRNA levels of pro-inflammatory markers, namely: inducible nitric oxide synthase (Inos), nuclear factor kappa B (Nf-κβ), tumour necrotic factor α (Tnf-α) and interleukin 1β (Il-1β) were significantly increased while interleukin (Il-10) was decreased in the obese group relative to the control group. Further, proliferating cell nuclear antigen (PCNA) immunoexpressions decreased while cleaved caspase-3 immunohistochemical staining increased significantly in the obese group, in addition to increases in the mRNA levels of p53, Bax, Caspases-8, 9 and 3, relative to the control group. Treatment with bee bread showed increases in antioxidant enzymes and PCNA immunoexpression, as well as decreases in inflammation and apoptosis markers in the testes. This study has shown that bee bread has therapeutic effects against oxidative stress, inflammation, apoptosis in the testis of HFD-induced obese male rats, thereby suggesting its role as a natural supplement capable of treating obesity-induced male reproductive impairment.

Suppression of the inflammation and fibrosis in Asherman syndrome rat model by mesenchymal stem cells: histological and immunohistochemical studies.

Asherman syndrome (AS) is a symptomatic intrauterine adhesion caused by endometrial basal layer fibrosis as a result of either uterine cavity surgery or infection leading to many complications. There is a concern to repair the injured tissues by using bone marrow mesenchymal stem cells (BM-MSCs). We aimed in this study to develop an animal model of AS and evaluate the anti-inflammatory and anti-fibrotic effects of BM-MSCs in this model through histological, immunohistochemical, and morphometric studies. Forty-two adult female adult albino rats were divided into (i) donor group composed of 2 rats used for isolation and propagation of BM-MSCs, and (ii) experimental groups: 40 rats equally divided into 4 groups: GpI (control), GpII (AS model), GpIII (BM-MSCs-treated AS rats), GpIV (untreated AS rats). Histological staining and immunohistochemical (IHC) detection of proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), and nuclear factor-kappa beta (NF-kB) were performed. The results were evaluated by morphometric and statistical analysis. Significant endometrial thinning, fibrosis, and degeneration of the endometrial epithelium with a significant decrease in PCNA and VEGF immunoexpression and a significant increase in NF-kB immunoexpression were detected in GpII and GpIV groups. These changes were substantially reversed in BM-MSCs-treated animals. BM-MSCs treatment resulted in substantial improvement of intrauterine adhesion in the rat model of Asherman syndrome.

Protective effects of bee bread on testicular oxidative stress, NF-κB-mediated inflammation, apoptosis and lactate transport decline in obese male rats

Oxidative stress, chronic inflammation and apoptosis are associated with obesity. Herein, we investigated the potential protective effect of bee bread, a natural bee product, on testicular oxidative stress, inflammation and apoptosis, as well as lactate transport in the testis of high-fat diet (HFD)-induced obese rats. Adult male Sprague-Dawley rats were either fed with normal chow (NC), HFD, HFD + bee bread (0.5 g/kg b.w./day) or HFD + orlistat (10 mg/kg b.w./day) for 12 weeks. Our results show significant decreases in the activities and mRNA expression of antioxidant genes (Nrf2, Sod, Cat and Gpx), with significant increase in pro-inflammatory (Nf-κb, Tnf-α, iNos, Il-1β) and pro-apoptotic (p53, Bax, Bax/Bcl2, Caspase-8, Caspase-9 and Caspase-3) genes in the testis of HFD group relative to the NC group. Furthermore, proliferating cell nuclear antigen (PCNA) was poorly expressed in the testis of the HFD group relative to the NC group. Similarly, the mRNA levels of glucose transporters (Glut1 and Glut3), monocarboxylate transporters (Mct2 and Mct4) and lactate dehydrogenase type C (Ldhc) decreased significantly, with decrease in lactate utilisation. Treatment with bee bread upregulated testicular antioxidant enzymes, downregulated inflammation and apoptosis, and increased PCNA immunoexpression, in addition to improving lactate transport. Taken together, our results suggest that bee bread is a promising natural product with the potential to improve male fertility.

The protective effect of fenofibrate, triptorelin, and their combination against premature ovarian failure in rats.

Cyclophosphamide (CP) is a chemotherapy alkylating agent that causes a lot of side effects including premature ovarian failure (POF). This study aimed to evaluate the possible protective effect of fenofibrate (FEN) in CP-induced POF. Rats were randomly divided into five groups as follows: negative control, CP, triptorelin (TRI)-treated, FEN (FEN)-treated, and FEN + TRI-treated. Histological study, collagen area fraction, and immunoexpression of proliferating cell nuclear antigen (PCNA) were evaluated. Also, estrogen, anti-mullerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and ovarian malondialdehyde (MDA), nitric oxide (NOx), reduced glutathione (GSH), superoxide dismutase (SOD), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF) were measured. CP significantly reduced ovarian follicle count, as compared with the control group (1.00 ± 0.76 versus 7.75 ± 1.83, respectively). Meanwhile, FEN, either solely or in combination with TRI, significantly increased ovarian follicle count, as compared with the CP group (3.88 ± 0.83 and 5.75 ± 1.39, respectively). As compared with the control group, CP increased the levels of MDA, NOx, IL-10, TNF-α, FSH, LH, and collagen area fraction; however, levels of GSH, SOD, VEGF, AMH, estrogen, and PCNA immunoexpression were reduced with CP. Administration of FEN either solely or in combination with TRI showed significant improvement in all the parameters previously mentioned. FEN can protect the ovary from CP-induced side effects possibly through antioxidant and anti-inflammatory actions.

Protective effect of Spirulina platensis against physiological, ultrastructural and cell proliferation damage induced by furan in kidney and liver of rat.

The modulatory role of the Spirulina platensis (SP) against furan-induced (FU) hepatic and renal damage was assessed in this study. For achieving this, sixty rats were distributed into six groups: control group, SP-administered group (300mg/kg b.wt orally for 28 days), a FU-intoxicated group (16mg/kg b.wt, orally, daily for 28 days), protective co-treated group SP/F (administered SP 300mg/kg b.wt, one week before, and concurrently with FU intoxication), therapeutic co-treated group FU/SP (administered SP 300mg/kg b.wt, one week after FU intoxication for 28 days) and protective/therapeutic co-treated group SP/FU/SP (administered SP one week before and after, concurrently with FU intoxication). Subsequently, the biochemical responses and the histology of hepatic and renal tissues in treated rats were assessed. The results indicated that FU intoxication induced a significant hepato- and nephropathy represented by the elevation in the values of tissue injury biomarkers and reduction in protein levels. Histologically, a wide range of morphological, cytotoxic, inflammatory, and vascular alterations as well as downregulation in the immunoexpression of the proliferating cell nuclear antigen (PCNA) and the proliferation-associated nuclear antigen (Ki-67) were induced by FU intoxication. Oral SP administration, particularly in the protective/therapeutic co-treated group, markedly supressed the serum levels of the tissue injury biomarkers, diminished the inflammatory response, restored the cytotoxic alterations, upregulated the immunoexpression of PCNA and Ki-67, and restored the perturbed morphology of the hepatic and renal tissues. In conclusion, the obtained data demonstrated that SP co-administration elicits both protective and therapeutic potential against the FU-induced hepato- and nephropathy.

Malaysian Bee Bread Attenuates Apoptosis and Improves Cell Proliferation in Testis of High-Fat Diet-Induced Obese Rats

Introduction: Obesity has been reported to impair male reproductive function and testicular apoptosis while bee bread has been traditionally consumed to enhance male fertility.Objective: The objective of this study was to determine the effects of Malaysian bee bread on apoptosis and proliferation of testicular germ cells in high-fat diet-induced obese rats.Methods: Twenty-four adult male Sprague Dawley rats weighing between 250-300 g were randomised into four groups (n=6/group), namely normal control (NC), high-fat diet (HFD), HFD plus bee bread (HFD+B) and HFD plus an anti-obesity drug orlistat (HFD+O) groups. Bee bread (0.5g/kg/day) and orlistat (10 mg/kg/day) were suspended in distilled water and given by oral gavage for 12 weeks. Markers for apoptosis and proliferation of testicular germ cells were assessed.Results: mRNA transcript levels of caspase-3, caspase-8, caspase-9, p53 and Bax/Bcl2 ratio in the testis of HFD group were significantly increased while immunohistochemical staining of cleaved caspase-3 increased and proliferating cell nuclear antigen (PCNA) immunoexpress ions decreased relative to NC group. Treatment with bee bread significantly decreased the apoptotic markers, significantly decreased mRNA transcript levels of anti-apoptotic markers (Bcl2) and increased PCNA immunoexpression in HFD+B, relative to NC group and HFD+O.Conclusion: Bee bread improved proliferation of testicular germ cells by attenuating apoptosis in high- fat diet-induced obese male rats.International Journal of Human and Health Sciences Supplementary Issue: 2019 Page: 44

Diabetes-induced testicular oxidative stress, inflammation, and caspase-dependent apoptosis: the protective role of metformin

Context: Metformin’s effect on glycaemic control is well documented, but its effect on diabetes-induced testicular impairment has been scarcely reported.Objective: To investigate the effects of metformin on testicular oxidative stress, inflammation, and apoptosis, which largely contribute to fertility decline in diabetic state.Methods: Male Sprague–Dawley rats were divided into 3 groups (n = 6/group) namely: normal control (NC), diabetic control (DC), and metformin (300 mg/kg b.w./d)-treated diabetic groups. Metformin was administrated for 4 weeks.Results: Decreased mRNA expressions and activities of antioxidant enzymes were seen in the testes of DC group. mRNA and protein expressions of pro-inflammatory and pro-apoptotic markers increased, while interleukin-10 and proliferating cell nuclear antigen (PCNA) decreased in the testes of DC group. Treatment with metformin up-regulated antioxidant enzymes, down-regulated inflammation, and apoptosis and increased PCNA immunoexpression in the testes.Conclusions: Metformin protects the testes from diabetes-induced impairment and may improve male reproductive health in diabetic state.

Histological Study on the Protective Effect of Endogenous Stem Cell Mobilization in Busulfan-Induced Testicular Injury in Albino Rats.

Background:Testicular damage is one of the most hazardous effects of chemotherapy as it is frequently associated with oligozoospermia and azoospermia.Aim of the Work:This study aimed at evaluating the protective effect of hematopoietic stem cell mobilization by granulocyte colony-stimulating factor (G-CSF) in a rat model of busulfan-induced testicular injury.Materials and Methods:Twenty-four adult albino rats were divided into four groups: group I, the control, Group II: rats received two doses of busulfan (each 15 mg/kg) intraperitoneally (IP) with 14 days interval, Group III: rats received busulfan and left untreated, and Group IV received busulfan IP then G-CSF (70 μg/kg/day) subcutaneously for 5 consecutive days. Testicular sections were stained with H and E and immunohistochemically for CD34, proliferating cell nuclear antigen (PCNA) and caspase-3, and semithin sections were stained with toluidine blue.Results:Groups II and III showed loss of the normal histological architecture of the testis and spermatogenic cells, with increased apoptosis confirmed by significantly increased caspase-3 and significantly decreased PCNA immunoexpression. While Group IV revealed improved testicular histology, decreased apoptosis, and increased proliferative capacity of spermatogenic cells. This was confirmed by significantly decreased caspase-3 immunoexpression and increased PCNA immunoreaction.Conclusion:Mobilization of stem cells with G-CSF was found to improve the testicular histology following busulfan chemotherapy in albino rats.

EXPRESSION OF METASTASIS ASSOCIATED PROTEIN 1 AS A POSSIBLE PROGNOSTIC INDICATOR IN ORAL SQUAMOUS CELL CARCINOMA

INTRODUCTION: Lymph node (LN) metastasis is one of the most important prognostic factors related to survival of oral squamous cell carcinoma (OSCC). Although, metastasis associated protein 1 (MTA1) overexpression is closely correlated with the metastasis of several human cancers, its role in determining lymph node metastases in OSCC patients remains undefined. Proliferating Cell Nuclear Antigen (PCNA) is the most representative proliferating marker; yet there is a controversy as to whether it can be used as a prognostic marker in OSCC. OBJECTIVES: To evaluate the immunoexpression of MTA1 and PCNA in metastatic versus non-metastatic OSCC; and correlate it with LN metastasis. MATERIALS AND METHODS: MTA1 and PCNA immunoexpression was detected immunohistochemically in 20 OSCC specimens; 10 of which had metastatic LNs and 10 with non-metastatic LNs (used as a control group). Their correlation with LN metastasis was evaluated. RESULTS: MTA1 immunoexpression showed significant correlation with LN metastasis but not with the histologic grade of the tumor. Whereas, PCNA immunoexpression revealed a significant correlation with the histologic grade of the tumor, but not with nodal metastasis. CONCLUSIONS: The present study suggests that MTA1 protein may have a clinical potential to be used as a prognostic factor to identify pathologically node-negative OSCC patients who may be at high risk to develop regional metastasis. On the other hand, PCNA is unlikely to play an important role in predicting LN metastasis in OSCC patients. KEYWORDS: OSCC, MTA1, PCNA, LN metastasis.

Role of topical dehydroepiandrosterone in ameliorating isotretinoin-induced Meibomian gland dysfunction in adult male albino rat

Isotretinoin broad effectiveness is limited by its detrimental effect on the Meibomian glands. Androgens have been reported to regulate the Meibomian gland function. Dehydroepiandrosterone (DHEA) is an androgen precursor considered as an efficient and physiological anti-ageing skin agent. This study aimed to investigate the role of the topical DHEA in ameliorating Meibomian gland dysfunction caused by isotretinoin employing different histological and immunohistochemical techniques. Twenty-four adult male albino rats were divided into four equal groups; control group, DHEA-treated group (1% twice daily for 3 months), isotretinoin-treated group (0.5mg/kg/day for 3 months), and both isotretinoin and DHEA-treated group. Meibomian gland specimens were processed for light microscopy. Immunohistochemical study was carried out using antibodies for proliferating cell nuclear antigen (PCNA), androgen receptor (AR) and estrogen receptor-alpha (ER-α). Sections from the isotretinoin-treated group revealed a reduction in the number and size of acini. Thickening and keratinization of the epithelial lining of the ducts were observed. Multiple degenerated acini and casts of acinar cells in the ducts of Meibomian glands were detected. Some dilated congested blood vessels and mononuclear cells were occasionally seen. A significant decrease in PAS reaction and a significant increase in collagen fiber content were detected. Immunohistochemical study revealed a significant increase in immunoexpression of PCNA in basal ductal cells coupled with decreased expression in basal acinar cells. Both AR and ER-α immunoexpression was significantly decreased. Minimal alterations were observed upon concomitant treatment with isotretinoin and DHEA as compared to the control group. Topical DHEA could prove to be beneficial in ameliorating isotretinoin-induced Meibomian gland dysfunction most probably through its androgenic effect.

Can vitamin E protect the pancreas against ethanol-induced alterations in adult male albino rats? A histomorphometric and immunohistochemical study

Background Ethanol has a destructive effect on the pancreas by inducing oxidative damage on pancreatic cells. This injurious effect could be ameliorated by the administration of vitamin E. Aim The aim of the study was to investigate the possible protective role of vitamin E on ethanol-induced alterations in the pancreas in adult male albino rats and also the effect of ethanol withdrawal. Materials and methods Forty adult male albino rats were divided into four equal groups (10 rats each): group I (control group), group II (ethanol-treated group), group III (ethanol and vitamin E-treated group), and group IV (recovery group). At the end of the experiment, the rats were sacrificed by cervical dislocation and their pancreas were excised and prepared for light microscopic and histochemical study. Detection of interleukin (IL)-1B and IL-6 by real-time PCR and proliferating cell nuclear antigen immunoexpression in the pancreatic tissue was carried out. Results Group II showed loss of acinar tissue, distorted degenerated pancreatic acini, dilated distorted interlobular ducts, distorted islets of Langerhans, and dilatation and congestion of the blood vessels. Interlobular inflammatory cellular infiltration was also detected. There was an increased collagen fiber deposition and diminution of the proliferating stem cells. There was a marked increase in IL-1B and IL-6 levels. Group III showed an obvious improvement in the structure of the pancreas. The recovery group showed improvement in the structure of the pancreas and minimal collagen deposition. Conclusion Vitamin E could protect the pancreas against ethanol-induced alteration in adult male albino rats.

Imunoexpressão de PCNA e α-SMA em tecidos de crescimento gengival

Purpose: This study aimed to investigate proliferating cell nuclear antigen (PCNA) and isoform-α of the smooth muscle actin (α-SMA) immunoexpression, as well as the epithelial thickness in druginduced gingival overgrowth (DIGO) and idiopathic gingival fibromatosis (IGF) patients. Methods: Gingival samples were obtained from 11 users of nifedipine and phenytoin, 6 of patients with IGF and 4 fibrous inflammatory hyperplasia. The specimens were submitted to immunohistochemical and morphological analysis. Results: Our results show that PCNA-epithelial positive cells were slightly more common among IGF patients, but there were not statistically significant differences among the groups (p>0.05). Comparing the vessels counts with α SMA-positive pericyte or smooth muscular cells, there was not statistical differences, although the control group presented a discrete higher count (p>0.05). The epithelial thickness analysis revealed that the DIGO group presented the higher mean, evidencing statistically significant differences in relation to the control group (p=0,002). Conclusion: In conclusion, our results demonstrated similarities between DIGO and IGF according to PCNA and α-SMA immunoexpression, although the epithelial thickness was higher in DIGO group.

Immunoexpression of proliferating cell nuclear antigen (PCNA) in spleen of splenectomized rats with preservation of inferior pole, submitted to hyperbaric oxygenation

To analyze PCNA immunoexpression on the inferior pole of the spleen of splenectomized rats submitted to hyperbaric oxygenation (HBO). Were analyzed fragments of the inferior pole of the spleen of 20 male Wistar rats submitted to splenectomy with preservation of the inferior pole. The rats were divided in two groups: group A (n=10) without HBO and group B (n=10) submitted to HBO at 2, 5 atmospheres per 120 minutes, twice a day for three days and once a day for seven days. The groups were then subdivided in four subgroups: A15 (n=5), with euthanasia on the 15th day; A45 (n=5), with euthanasia on the 45th day; B15 (n=5) with euthanasia on the 15th day and B45 with euthanasia on the 45th day. Respectively on these days, fragments of the inferior pole of the spleen of all animals were collected and analyzed with the immunohistochemistry technique in order to evaluate PCNA expression. There was an expressive increase in PCNA immunoreactivity in the group B. The 45 day postoperative period resulted in a higher level of positivity than the 15 day postoperative period (p<0.01). The quantitative analysis of proliferating cell nuclear antigen positive suggests that hyperbaric oxygenation increases cellular proliferation, contributing to splenic regeneration.

Chemopreventive Efficacy of Naringenin-Loaded Nanoparticles in 7,12-dimethylbenz(a)anthracene Induced Experimental Oral Carcinogenesis

Nanochemoprevention has been introduced recently as a novel approach for improving phytochemicals bioavailability and anti-tumor effect. The present study is designed to evaluate the chemopreventive efficacy of prepared naringenin-loaded nanoparticles (NARNPs) relative to efficacy of free naringenin (NAR) against 7,12-dimethyl benz(a)anthracene (DMBA)-induced oral carcinogenesis by evaluating the status of lipid peroxidation, antioxidants and immunoexpression patterns of proliferating cell nuclear antigen (PCNA) and p53 proteins. Transmission electron microscope (TEM) and dynamic light scattering (DLS) investigations have confirmed a narrow size distribution of the prepared nanoparticles (40-90nm) with ~88% encapsulation efficiency. Oral squamous cell carcinoma (OSCC) was developed in the buccal pouch of golden Syrian hamsters by painting with 0.5% DMBA in liquid paraffin three times a week for 14weeks. DMBA painted animals revealed the morphological changes, hyperplasia, dysplasia and well-differentiated squamous cell carcinoma. Moreover, the status of lipid peroxidation, antioxidants and immunoexpression of PCNA and p53 were significantly altered during DMBA-induced oral carcinogenesis. Oral administration of NARNPs (50mg NAR/kg body weight/day) to DMBA-treated animals completely prevented the tumor formation as compared to the free NAR and significantly reduced the degree of histological lesions, in addition to restoration of the status of biochemical and molecular markers during oral carcinogenesis. In addition, NARNPs have more potent anti-lipid peroxidative, antiproliferative effect and antioxidant potentials compared to free NAR in DMBA-induced oral carcinogenesis. In conclusion, the present study suggests that NARNPs could be a potentially useful drug carrier system for targeted delivery of naringenin for cancer chemoprevention.

Avaliação da proliferação celular como indicador prognóstico para mastocitomas cutâneos caninos

Este estudo teve como objetivo avaliar o valor prognóstico de marcadores de proliferação celular em casos de mastocitomas cutâneos caninos. Vinte e três casos foram analisados quanto à expressão imuno-histoquímica de Ki67 e do Antígeno Nuclear de Proliferação Celular (PCNA), sendo subsequentemente acompanhados clinicamente. Observou-se que a expressão de Ki67 mantém relação negativa com a tradicional graduação histopatológica (p= 0,0418; p&lt;0,05 entre os graus I e III), sendo um indicador confiável para o tempo de sobrevida pós-cirúrgica (p=0,0089). A imunoexpressão de PCNA, apesar de estar correlacionada à marcação por Ki67, não apresentou valores estatisticamente significantes na predição da mortalidade em função da doença e do tempo de sobrevida pós-cirúrgico. Os resultados obtidos confirmam que informações sobre a atividade proliferativa tumoral pela detecção imuno-histoquímica de Ki67 podem incrementar a classificação de mastocitomas cutâneos caninos quanto à malignidade.

Immunohistochemical evidence for an endocrine/paracrine role for ghrelin in the reproductive tissues of sheep

BackgroundThe gut hormone, ghrelin, is involved in the neuroendocrine and metabolic responses to hunger. In monogastric species, circulating ghrelin levels show clear meal-related and body weight-related changes. The pattern of secretion and its role in ruminant species is less clear. Ghrelin acts via growth hormone secretagogue receptors (GHSR-1a) to alter food intake, fat utilization, and cellular proliferation. There is also evidence that ghrelin is involved in reproductive function. In the present study we used immunohistochemistry to investigate the presence of ghrelin and GHSR-1a in sheep reproductive tissues. In addition, we examined whether ghrelin and GHSR-1a protein expression is developmentally regulated in the adult and fetal ovine testis, and whether there is an association with markers of cellular proliferation, i.e. stem cell factor (SCF) and proliferating cell nuclear antigen (PCNA).MethodsAntibodies raised against ghrelin and its functional receptor, GHSR-type 1a, were used in standard immunohistochemical protocols on various reproductive tissues collected from adult and fetal sheep. GHSR-1a mRNA presence was also confirmed by in situ hybridisation. SCF and PCNA immunoexpression was investigated in fetal testicular samples. Adult and fetal testicular immunostaining for ghrelin, GHSR-1a, SCF and PCNA was analysed using computer-aided image analysis. Image analysis data were subjected to one-way ANOVA, with differences in immunostaining between time-points determined by Fisher's least significant difference.ResultsIn adult sheep tissue, ghrelin and GHSR-1a immunostaining was detected in the stomach (abomasum), anterior pituitary gland, testis, ovary, and hypothalamic and hindbrain regions of the brain. In the adult testis, there was a significant effect of season (photoperiod) on the level of immunostaining for ghrelin (p < 0.01) and GHSR-1a (p < 0.05). In the fetal sheep testis, there was a significant effect of gestational age on the level of immunostaining for ghrelin (p < 0.001), GHSR-1a (p < 0.05), SCF (p < 0.05) and PCNA (p < 0.01).ConclusionEvidence is presented for the presence of ghrelin and its receptor in various reproductive tissues of the adult and fetal sheep. In addition, the data indicate that testicular expression of ghrelin and its receptor is physiologically regulated in the adult and developmentally regulated in the fetus. Therefore, the ghrelin ligand/receptor system may have a role (endocrine and/or paracrine) in the development (cellular proliferation) and function of the reproductive axis of the sheep.

CLINICAL FEATURES AND IMMUNOEXPRESSION OF p53, MIB-1 AND PROLIFERATING CELL NUCLEAR ANTIGEN IN ADRENAL NEOPLASMS

We evaluated the clinical features and immunoreactivity of p53 protein, MIB-1 antigen and proliferating cell nuclear antigen (PCNA) in adrenal neoplasms. A total of 26 patients with adrenocortical adenoma and 24 patients with carcinoma were treated with adrenalectomy. Clinical features and immunohistochemical reactions were compared in adult vs pediatric tumors. There was a bimodal age distribution of carcinomas and adenomas, with a first peak occurring before age 5 years. The proportion of carcinomas in children (18 of 29) was higher than in adults (6 of 21). Carcinoma and adenoma occurring in children presented more commonly as the virilizing syndrome, while in adults Cushing's syndrome was more common. All adenomas in adults were p53 negative, while in children 4 of 11 adenomas (36%) were p53 positive. Histological Weiss criteria were the most reliable pathological features to distinguish adenoma from carcinoma. Other pathological features, including tumor weight, rate of mitotic figures and immunoexpression of p53 protein, MIB-1 antigen and PCNA, exhibited a striking difference in adenomas and carcinomas but none demonstrated sensitivity or specificity of 100%. Of all the computerized tomographic characteristics analyzed, including tumor size, shape, necrosis/hemorrhage, attenuation and contrast enhancement, only tumor size (greater than 5 cm) showed sensitivity and specificity of 100% in the differential diagnosis. Children and adults with carcinoma had similar curves of survival (p = 0.76). Carcinoma stage and PCNA immunoexpression displayed an association with outcome. Endocrine syndromes differed in adults and children but other clinical features were similar in both groups. The role of p53 protein, MIB-1 antigen and proliferating cell nuclear antigen in discrimination of adenomas from carcinomas is unclear.