Histomorphological Evaluation Research Articles

Effect of Low-intensity Pulsed Ultrasound (LIPUS) with Different Frequency on Bone Defect Healing

Low-intensity pulsed ultrasound (LIPUS) is known to promote bone defect healing. The frequency of the LIPUS is known to influence directivity and depth of penetration, but the differences on bone healing remains unknown. This study was to investigate the effect of LIPUS with different frequencies on bone defect healing. Bone defects of 1.6 mm in diameter were created in femurs of ten-week-old male Long-Evans rats (n=36). Experimetal groups were exposed LIPUS (intensity: 30 mW/cm2, burst width: 200 μs, time: 15 min/day) and divided into a low frequency (LF, 1.5 MHz) group and a high frequency (HF, 3.0 MHz) group. Non-exposed LIPUS group were used as control. After 3, 5, 7, and 10 days, femurs were removed and radiological, histomorphological and molecular biological evaluations were conducted. Micro-CT images showed that the depression in cortical bone was reduced in LF and HF groups. 3D bone morphological analysis at 10 days revealed that LIPUS increased cortical bone volume / tissue volume (BV/TV) and decreased BV/TV in the lower layer of cancellous bone (P<0.05). Histomorphologically, clot retraction was seen in the both LIPUS groups but not in the control group at 3 days. These effects were observed at a deeper layer in the LF group than in the HF group. No significant difference in osteopontin (OPN) gene expression was observed. However, osteocalcin (OCN) gene expression was significantly elevated in the HF group relative to the control group at 10 days (P<0.05). Immunohistochemical staining revealed that newly formed bone exhibited a positive reaction to OPN and OCN in both LIPUS groups. Healing of the bone defect area was noted in both LIPUS groups, but there was no clear difference in histomorphology between the LF and HF groups. LIPUS frequencies of 1.5 MHz and 3.0 MHz promote increased cortical bone mass and remodeling of cancellous bone in rat femurs with bone defects.

Prognosis according to histochemical analysis of liver metastases removed at liver resection

Liver metastases occur in 40-50 per cent of patients with colorectal cancer and determine long-term survival. The aim of this study was to examine the immunological architecture of colorectal liver metastases and its impact on patient survival. Specimens from patients with colorectal liver metastases were stained with haematoxylin and eosin and Masson trichrome, immunostained for α-smooth muscle actin, CD4, CD45RO and CD8, and analysed by flow cytometry. In addition to histomorphological evaluation, immunohistochemically stained sections were analysed for cell numbers in the tumour area, infiltrative margin and distant liver stroma separately. These findings were correlated with clinical data and patient outcome. Tumour containment by a fibrotic capsule around liver metastases was observed in 37·8 per cent of 201 patients and was prognostic for improved survival (median (s.e.) survival 64 (6) and 31 (4) months for patients with capsule and no capsule respectively; P < 0·001) and independently led to higher R0 resection rates (P = 0·040). In multivariable analysis, CD45RO(+) cell infiltration at the peritumoral margin with low CD45RO(+) cell infiltration in the distant liver stroma (P = 0·001) and fibrotic capsule formation (P = 0·008) both independently prolonged patient survival. Using these two factors, a cellular immune score was designed and shown to stratify patient survival in test and validation samples (both P < 0·001). Fibrotic capsule formation and localized cell infiltration of colorectal liver metastases by CD45RO(+) cells were related to prolonged patient survival. Based on these immunological criteria a cellular immune score was developed to stratify patients according to prognosis.

English

A postmenopausal 50yrs old para3 presented with abdominal pain and distension; decreased appetite and breathlessness. She was provisionally diagnosed as malignant ovarian tumor with secondaries. On USG 16x14 cm size well defined heterogeneous mass lesion was seen in left adnexa with internal vascularity; mild ascitis; right sided pleural effusion. MRI abdomen and pelvis showed large 19.1 x 12.7 cm mass with multiple septations and peripheral nodules. USG guided FNAC showed scanty inflammatory cells with proteinaceous fluid background, malignant cells were not seen. Pleural and ascitic fluid tap showed inflammatory and mesothelial cell but no malignant cell. Clinical and investigation profile suggest the Meigs's syndrome. Left side salpingo-ovariectomy done and the desired section were sent for histomorphological evaluation. Histology reported ovarian fibroma and thus confirm the diagnosis.

13IN - Diagnostic and Therapeutic Implications of Tumour-Infiltrating Lymphocytes in Breast Cancer

ABSTRACT In several types of tumors, approaches to modulate tumor-associated inflammation have resulted in comparably high response rates in first clinical evaluations. These targeted therapies included immune checkpoint inhibitors and have stimulated interest in the interaction between immune cells and tumor cells. Neoadjuvant therapy approaches in breast cancer have been used as a strategy to characterize subgroups of tumors with increased tumor-infiltrating lymphocytes (TILs). It has been shown that increased TILs are linked to an increased response rate to neoadjuvant therapy and improved prognosis after adjuvant therapy, in particular in triple-negative and HER2 positive breast cancer. We have recently described tumor-infiltrating lymphocytes (TILs) as predictors of pathological complete response to neoadjuvant chemotherapy in the GeparTrio, GeparQuinto and GeparSixto breast cancer trials. To further investigate the immunological status in tumor tissue we have evaluated a total of 12 immunologically relevant genes, including T-cell markers, B-cell markers, chemokines and immunoregulatory factors, in 481 pretherapeutic FFPE samples in the European FP7 project Responsify. All immune mRNAs had a strong positive correlation with each other and with stromal TILs. Hierarchical cluster analysis showed three different immune-subtypes of tumors with different expression of immunological genes and different amounts of tumor infiltrating lymphocytes. The results suggest that expression of immune marker mRNAs in breast cancer is predictive for response to neoadjuvant chemotherapy. In GeparSixto, these immunological parameters can be used in addition to TILs to identify patients with increased response rates to carboplatin. The results should be validated in other breast cancer trials evaluating carboplatin therapy. In addition, the studies investigating TILS and immune mRNA markers suggest that a subgroup of breast carcinomas is immunogenic. The presentation will provide an overview on recent results on immune markers for prediction of response to neoadjuvant chemotherapy and prognosis with a focus in standardized histomorphological evaluation of tumor-infiltrating lymphocytes. The project has been funded within the EU-FP7 project RESPONSIFY No 278659. Disclosure: C. Denkert: Sividon Diagnostics: research funding, co-founder and shareholder.

The protective role of thymoquinone in the prevention of gentamicin ototoxicity

ObjectiveTo investigate the potential protective effect of thymoquinone in gentamicin-induced ototoxicity through auditory brain stem responses (ABR) testing and histomorphological evaluation of the cochlea. MethodsThis study was conducted on 48 adult female Sprague–Dawley rats that were randomized into 4 groups. Group 1 received intraperitoneal gentamicin; group 2 received intraperitoneal gentamicin plus corn oil solution; group 3 received intraperitoneal thymoquinone; and group 4 received intraperitoneal gentamicin plus thymoquinone. All groups received the drugs (once daily) in the above-mentioned protocols over 15days. After conducting repeated ABR measurements, the rats were sacrificed, and their cochleae were isolated. ResultsABR thresholds were preserved in the gentamicin plus thymoquinone group when compared with the group receiving gentamicin alone. There were fewer TUNEL-positive cells and caspase-3 and caspase-9 expressions were weaker in the inner and outer hairy cells of the organ of Corti in the gentamicin plus thymoquinone group compared with the group receiving gentamicin alone. ConclusionThe ABR values and number of apoptotic cells did not significantly increase in the group receiving gentamicin plus thymoquinone when compared to the group receiving gentamicin alone. Again, the cochlear histomorphological findings were supportive of the auditory findings. In light of these findings, we conclude that gentamicin-induced ototoxicity may be prevented by thymoquinone use in rats.

Bioabsorbable Implant as a Tracheal Wall Substitute in Young Developing Canines

This study evaluated a polylactic and polyglycolic acid (PLA/PGA) implant as a partial tracheal substitute in young developing canines. This experimental and longitudinal study included local stray pups that received substitution of a short cervical tracheal segment with a PLA 85%/PGA 15% plaque. We measured clinical, endoscopic, and tomographic variables for 1 year, at which time we performed histomorphological evaluations of the implant using light and electron microscopy. There were no adverse events throughout the clinical progression. On endoscopic evaluation, the implant was covered with mucosal tissue beginning in the first month, without granulation or stenosis, and the circular shape of the trachea was altered. Tomographic images of the tracheal area at the implant site were similar to adjacent healthy areas (p = 0.423). At the end of the follow-up period, the plaque had biodegraded, and the space was covered by pseudostratified epithelium and ciliated cells similar to the neighboring tissue. Implantation of a PLA/PGA plaque constituted an effective (functional) replacement of a short semicircular cervical tracheal segment without limiting the growth of the recipient. Additional studies are required to prove the efficacy of these implants for larger tracheal segment replacements and in subjects at different stages of development.

Bt-maize (MON810) and non-GM soybean meal in diets for Atlantic salmon (Salmo salar L.) juveniles--impact on survival, growth performance, development, digestive function, and transcriptional expression of intestinal immune and stress responses.

Responses in Atlantic salmon (Salmo salar L.) juveniles (fry) fed diets containing genetically modified maize (Bt-maize, MON810) expressing Cry1Ab protein from first-feeding were investigated during a 99-day feeding trial. Four experimental diets were made; each diet contained ∼20% maize, either Bt-maize or its near-isogenic maternal line (non-GM maize). One pair was fishmeal-based while the other pair included standard (extracted) soybean meal (SBM; 16.7% inclusion level), with the intention of investigating responses to the maize varieties in healthy fish as well as in immunologically challenged fish with SBM-induced distal intestinal inflammation, respectively. Three replicate tanks of fry (0.17±0.01 g; initial mean weight ± SEM) were fed one of the four diets and samples were taken on days 15, 36, 48 and 99. Survival, growth performance, whole body composition, digestive function, morphology of intestine, liver and skeleton, and mRNA expression of some immune and stress response parameters in the distal intestine were evaluated. After 99 days of feeding, survival was enhanced and the intended SBM-induced inflammatory response in the distal intestine of the two groups of SBM-fed fish was absent, indicating that the juvenile salmon were tolerant to SBM. Mortality, growth performance and body composition were similar in fish fed the two maize varieties. The Bt-maize fed fish, however, displayed minor but significantly decreased digestive enzyme activities of leucine aminopeptidase and maltase, as well as decreased concentration of gut bile salts, but significantly increased amylase activity at some sampling points. Histomorphological, radiographic and mRNA expression evaluations did not reveal any biologically relevant effects of Bt-maize in the gastrointestinal tract, liver or skeleton. The results suggest that the Cry1Ab protein or other compositional differences in GM Bt-maize may cause minor alterations in intestinal responses in juvenile salmon, but without affecting overall survival, growth performance, development or health.

Effects of dietary phytosterols and soy saponins on growth, feed utilization efficiency and intestinal integrity of gilthead sea bream (Sparus aurata) juveniles

The use of plant ingredients in aquafeeds for piscivorous fish species is a reality that exposes fish to a number of antinutritional factors present in plants. The present study is the first to evaluate the effect of two purified antinutrients, saponins and phytosterols, in sea bream juveniles. For that purpose, seven diets were formulated: a control diet (fishmeal and fish oil based) and six experimental diets containing low (1 g kg− 1, SapL) or high (2 g kg− 1, SapH) levels of purified soya saponins, low (5 g kg− 1, PhytL) or high (10 g kg− 1, PhytH) levels of purified phytosterols or a combination of 1 g kg− 1 saponins + 5 g kg− 1 phytosterols (SapPhytL) or 2 g kg− 1 saponins + 10 g kg− 1 phytosterols (SapPhytH). Fish were fed for 48 days in order to evaluate growth performance, feed utilization, plasma cholesterol, and gut health as assessed by histomorphological evaluation and gene expression profiling of immune and functional markers. Fish fed the diets PhytH, SapPhytL and SapPhytH showed better feed utilization and PhytH and SapPhytH showed higher protein utilization than the other groups, although this was not reflected in improved growth performance. Histomorphological analysis of the distal intestine revealed increased variation in supranuclear vacuole sizes after 48 days of feeding diets SapH, SapPhytL and SapPhytH and increased number of intraepithelial leukocytes in response to all dietary treatments except SapL and SapPhytL. Although juvenile sea bream growth was not affected by dietary inclusion of saponins and phytosterols, the results indicated some disturbances of the intestinal mucosal structure that could compromise function and/or protection from potential dietary antigens or opportunistic pathogens.

Detection of subtle neurological alterations by the Catwalk XT gait analysis system

BackgroundA new version of the CatWalk XT system was evaluated as a tool for detecting very subtle alteration in gait based on higher speed sample rate; the system could also demonstrate minor changes in neurological function. In this study, we evaluated the neurological outcome of sciatic nerve injury intervened by local injection of hyaluronic acid. Using the CatWalk XT system, we looked for differences between treated and untreated groups and differences within the same group as a function of time so as to assess the power of the Catwalk XT system for detecting subtle neurological change.MethodsPeripheral nerve injury was induced in 36 Sprague–Dawley rats by crushing the left sciatic nerve using a vessel clamp. The animals were randomized into one of two groups: Group I: crush injury as the control; Group II: crush injury and local application with hyaluronic acid. These animals were subjected to neurobehavior assessment, histomorphology evaluation, and electrophysiology study periodically. These data were retrieved for statistical analysis.ResultsThe density of neurofilament and S-100 over the distal end of crushed nerve showed significant differences either in inter-group comparison at various time points or intra-group comparison from 7 to 28 days. Neuronal structure architecture, axon counts, intensity of myelination, electrophysiology, and collagen deposition demonstrate significant differences between the two groups. There was significant difference of SFI and angle of ankle in inter- group analysis from 7 to 28 days, but there were no significant differences in SFI and angle of ankle at time points of 7 and 14 days. In the Cat Walk XT analysis, the intensity, print area, stance duration, and swing duration all showed detectable differences at 7, 14, 21, and 28 days, whereas there were no significant difference at 7 and 14 days with CatWalk 7 testing. In addition, there were no significant differences of step sequence or regularity index between the two versions.ConclusionHyaluronic acid augmented nerve regeneration as early as 7 days after crush injury. This subtle neurological alteration could be detected through the CatWalk XT gait analysis but not the SFI, angle of ankle, or CatWalk 7 methods.

Hepatoid adenocarcinoma of the lung: report of five cases and review of the literature

The current diagnostic criteria for hepatoid adenocarcinoma of lung include typical acinar or papillary adenocarcinoma and a component resembling hepatocellular carcinoma and expressing α-fetoprotein (AFP). Distinguishing hepatoid adenocarcinoma of lung from hepatocellular carcinoma metastatic to lung is difficult in patients with both lung and liver masses and in patients at risk for lung and liver cancer because of smoking and viral hepatitis, respectively. We studied morphologic features of hepatoid adenocarcinoma of lung and established an immunohistochemical panel to facilitate distinction of hepatoid adenocarcinoma of lung from hepatocellular carcinoma metastatic to lung. Five cases of hepatoid adenocarcinoma of lung were stained with hematoxylin and eosin and mucicarmine for histomorphologic evaluation. The 14-marker immunohistochemical profile was established for hepatoid adenocarcinoma of lung and compared with that of hepatocellular carcinoma. Two cases of hepatoid adenocarcinoma of lung had signet-ring cell components. Three cases were pure hepatoid adenocarcinoma without components of acinar or papillary adenocarcinoma, signet-ring cells or neuroendocrine carcinoma. Like hepatocellular carcinoma, hepatoid adenocarcinoma of lung expresses CK8 (5/5), CK18 (5/5), AFP (3/5) and HepPar1 (5/5), shows cytoplasmic staining with TTF-1 (5/5) and does not express CK14 (0/5). Unlike hepatocellular carcinoma, it expresses CK5/6 (1/5), CK7 (3/5), CK19 (4/5), CK20 (1/5), HEA125 (5/5), MOC31 (5/5), monoclonal CEA (3/5) and napsin A (1/5). An immunohistochemical panel that includes a variety of cytokeratins, monoclonal CEA and EpCAM markers (HEA125 and MOC31) facilitates distinction of hepatoid adenocarcinoma of lung from hepatocellular carcinoma metastatic to lung, especially when correlated with clinical and radiologic findings. We propose modification of the current diagnostic criteria for hepatoid adenocarcinoma of lung. Tumor composition can be either pure hepatoid adenocarcinoma or hepatoid adenocarcinoma with components of typical acinar or papillary adenocarcinoma, signet-ring cells or neuroendocrine carcinoma. AFP expression is not requisite for diagnosis as long as other markers of hepatic differentiation are expressed.

A Novel Chitosan-γPGA Polyelectrolyte Complex Hydrogel Promotes Early New Bone Formation in the Alveolar Socket Following Tooth Extraction

A novel chitosan-γPGA polyelectrolyte complex hydrogel (C-PGA) has been developed and proven to be an effective dressing for wound healing. The purpose of this study was to evaluate if C-PGA could promote new bone formation in the alveolar socket following tooth extraction. An animal model was proposed using radiography and histomorphology simultaneously to analyze the symmetrical sections of Wistar rats. The upper incisors of Wistar rats were extracted and the extraction sockets were randomly treated with gelatin sponge, neat chitosan, C-PGA, or received no treatment. The extraction sockets of selected rats from each group were evaluated at 1, 2, 4, or 6 wk post-extraction. The results of radiography and histopathology indicated that the extraction sockets treated with C-PGA exhibited lamellar bone formation (6.5%) as early as 2 wk after the extraction was performed. Moreover, the degree of new bone formation was significantly higher (P < 0.05) in the extraction sockets treated with C-PGA at 6 wk post-extraction than that in the other study groups. In this study, we demonstrated that the proposed animal model involving symmetrical sections and simultaneous radiography and histomorphology evaluation is feasible. We also conclude that the novel C-PGA has great potential for new bone formation in the alveolar socket following tooth extraction.

Molecular characterization and xenogenic application of wharton’s jelly derived caprine mesenchymal stem cells

Aim of the present study was in vitro expansion and characterization of caprine wharton's jelly derived mesenchymal stem cells (cWJ-MSCs) to investigate their tissue healing potential in xenogenic animal model. Plastic adherent fibroblastoid cell populations with distinctive homogeneous morphology were isolated from caprine Wharton's jelly explants. These Wharton's jelly derived cells were found positive for the surface markers CD-73, STRO-1 and CD-105, whereas they were negative for hematopoetic stem cell marker CD-34. In vitro cultured cWJ-MSCs also showed differentiation properties into osteogenic, adipogenic and chondrogenic lineages as demonstrated by von Kossa, Oil Red-O and Alcian blue staining respectively, which was further confirmed and quantified by flow cytometric analysis. Furthermore, these well characterized cWJ-MSCs were evaluated for the wound-healing potential in full-thickness skin wounds in rabbit model for 28 days. Caprine WJ- MSCs treated skin wounds showed significantly (P < 0.05) higher percentage of wound contraction especially at the 21(st) day post transplantation when compared to PBS treated control group animals. Further, we observed better healing potential of cWJ-MSCs in terms of histo-morphological evaluation, epithelialisation and collagenization with matured vascularization stage by day 28 as compared to control. In conclusion, cWJ- MSCs provide an alternative inexhaustible source of mesenchymal stem cells and also unravel new perspectives pertaining to the therapeutic use of these cells in different species.

Spectrum of fungal keratitis: clinicopathologic study of 44 cases.

To determine the causative agents of fungal keratitis and study the predisposing factors over a period of ten years in a single tertiary care hospital. A retrospective analysis of fungal corneal ulcers was done from 2003-2012. Patients' clinical data were noted from the file records. Correlation of histopathological diagnosis was done with the report on fungal culture. Mycotic keratitis was established in 44 cases by a positive fungal culture. Direct microscopic examination of potassium hydroxide (KOH) mounts revealed fungal elements in 39 cases while 40 cases showed fungus on Gram stained smears. Males (54.55%) were more commonly affected than the females (45.45%). The age ranged from 18 to 82 years. Most common age group to be involved was 41-60 years. Predisposing risk factors were seen in 34 (77.27%) cases. Most common findings on clinical examination were anterior chamber reaction and conjunctival injection seen in all the cases. Other common findings were stromal infiltration and hypopyon seen in 20 (45.45%) and 18 (40.91%) cases respectively. On histopathological examination the fungus was typed, as aspergillus in 34 cases while no definite typing was possible in 10 cases. The predominant isolate was aspergillus flavus (59.09%) followed by fusarium (15.91%). Mixed fungal and bacterial infection was seen in 3 (6.82%) cases. Although culture is the gold standard for definitive diagnosis of fungal keratitis, direct microscopic examination of corneal scrapings or histomorphological evaluation of biopsies allow a rapid preliminary diagnosis. Early administration of antifungal treatment helps in preventing dreadful complications.

The effects of resveratrol on chronic constriction injury of sciatic nerve in rats

The aim of this study was to investigate the effects of resveratrol (RVT) in chronic constriction injury (CCI) of sciatic nerve by behavioral, histomorphological and immunohistochemical evaluations in rats. In this study, male Wistar rats were divided into three groups: sham (n=7), CCI+saline (n=7) and CCI+RVT (n=7). After inducing CCI, treatment with 10mg/kg/day of RVT or saline for 14 days was given. Locomotor function was assessed with rota-rod and open field tests. Morphologic alterations of sciatic nerve were assessed histologically by light and electron microscopy. Immunohistochemistry for insulin-like growth factor-1 (IGF-1) were performed. RVT treatment prevented motor impairment and histomorphological alterations caused by chronic constriction injury of sciatic nerve. IGF-1 immunoreactivity was significantly higher in RVT treated group then CCI induced group and positive correlated with morphometric parameters. These results indicate that RVT may reduce CCI induced damage and this effect may be mediated through the restoration of IGF-1 immunoreactivity.

Zirconia Implant with Rough Surface Produced by YAG Laser Treatment: Evaluation of Histomorphology and Strength of Osseointegration

Zirconia Implant with Rough Surface Produced by YAG Laser Treatment: Evaluation of Histomorphology and Strength of Osseointegration

Anti-ulcerogenic properties of Albuca setosa

Albuca setosa is a medicinal plant used by Xhosa tribe in the rural area of South Africa for the treatment of wounds, articulation problems, rheumatoid arthritis and digestive disorders. This study aimed to evaluate the healing effect of A. setosa on experimental induced gastric ulcer. The antiulcerogenic effects of A. setosa were investigated in male Wistar rats. Gastric ulcer was induced per os using indomethacin (50 mg/kg) and ethanol (2 ml/animal). The ulceration lesion index was calculated for each one of the ulcerated stomach; the macroscopic and histomorphology evaluation were made. In indomethacin-induced gastric ulcers, oral administration of A. setosa significantly inhibited (P<0.01) gastric ulcer formation by 82 and 83% at the dose of 100 and 200 mg/kg, respectively. In ethanolinduced gastric ulcers, A. setosa significantly inhibited (P<0.05) gastric ulcer formation by 39 and 35% at the dose of 100 and 200 mg/kg, respectively. Macroscopic evaluation of ulcerated stomachs of A. setosa treated groups showed a reduced area of gastric lesion, with moderate disruption of the gastric epithelium as well as the mucosa stromal cell. The results obtained in this study suggest that the A. setosa possesses some anti-ulcerogenic properties, which may support evidence for its traditional use.

Neurotrophic and neuroprotective actions of Achyranthes bidentata polypeptides on cultured dorsal root ganglia of rats and on crushed common peroneal nerve of rabbits

We have isolated Achyranthes bidentata Blume polypeptides (ABPP) from the aqueous extract of A. bidentata Blume, a traditional Chinese medicine with multiple therapeutic applications. In this study, we aimed to investigate neurotrophic effects of ABPP on cultured dorsal root ganglia (DRGs) of rats and neuroprotective effects on crushed common peroneal nerve of rabbits. Immunochemistry and Western blot analysis indicated that ABPP (0.01, 0.1, and 1.0μg/ml) encouraged neurite outgrowth from cultured DRG explants/neurons in a concentration-dependent manner through activation of ERK1/2. After crush injury to rabbit common peroneal nerve, animals received daily administration of ABPP for 5 weeks. Electrophysiological assessments and histomorphological evaluation showed that 6.0mg/kg ABPP significantly enhanced nerve regeneration and function restoration. Our findings suggest that ABPP could be used as a neurotrophic and neuroprotective agent to treat peripheral nerve crush injury.

Histomorphological evaluation of maternal and neonatal distal airspaces after maternal intake of nanoparticulate titanium dioxide: an experimental study in Wistar rats

This study was performed to determine the histomorphological alterations occurring in maternal and neonatal pulmonary distal airspaces of Wistar rats after maternal administration of titanium dioxide nanoparticles (TiO2 NPs). Thirty adult pregnant rats (150-250g) and their offspring were used in this study. Pregnant rats were randomly divided into control (n=15) and TiO2 NP-treated (n=15) groups. A suspension of TiO2 NPs in phosphate-buffered saline was given orally to the treated group (0.1ml/10g body weight once daily) from days 6 to 12 of gestation. At term, maternal and neonatal lungs were collected and processed for energy-dispersive X-ray (EDX) and histological analysis. The mean linear intercept (MLI) and airspace wall thickness were measured by a stereological procedure with image analysis to assess alveolarization. EDX analysis demonstrated the presence of TiO2 in maternal and neonatal lungs. The lungs of TiO2 NP-treated mothers revealed evidence of pneumocytic apoptosis, abnormal lamellar inclusions, and macrophage and inflammatory cell infiltrates. Significant thinning of alveolar septa was detected in the treated rats (p<0.001), but the MLI was constant in both groups (p=0.207). Neonatal lungs from treated mothers revealed deficient septation, thickened mesenchyme between the saccules, pneumocytic apoptosis, atypical lamellar inclusions, and macrophage infiltration. The thickness of the primary septa was significantly increased (p=0.001) with no significant change in MLI (p=0.579) compared with the control group. In conclusion, TiO2 NPs were detected in maternal and neonatal lungs after oral intake by pregnant rats. The pulmonary response manifested as inflammatory lesions and delayed saccular development in neonates.

Follicular-derived neoplasms: morphometric and genetic differences.

The distinction between follicular adenomas (FAs) and well differentiated follicular and papillary carcinomas is often a demanding task and sometimes only intuitive. We report an histomorphological evaluation of follicular neoplasms [FAs, follicular carcinomas (FCs), and follicular variant of papillary carcinomas (FVPTCs)], supported by a qualitative and quantitative image analysis and by a molecular characterization. Tumor fibrosis and haemorrhage, neoplastic capsule thickness, follicle diameter, number of neoplastic cells, nuclear diameter of neoplastic cells, vessels density, vessels area and intratumoral distribution were evaluated. Ras and BRAF mutations, RET/PTC1, RET/PTC3, and PAX8/PPARγ rearrangements were analyzed. Correlations with clinico-pathological features have been studied. We found that FAs had a more extensive intratumoral haemorrhage, while malignant neoplasms were characterized by an evident fibrosis, higher cellularity and larger size. FVPTCs had higher nuclear diameter; cells count was higher in the minimally invasive follicular thyroid carcinomas, as well as a thickener neoplastic capsule. The CD34 stain showed a higher microvessel density in the FVPTCs group. A higher peripheral vessels distribution was observed only in malignant neoplasms. We observed overall Ras mutations in 2.4% of adenomas, in 41.5% of FVPTCs, and in 44.8% of FCs. It is outstanding that there is a marked difference in the Ras mutation distribution between the benign and malignant tumors in our series. We found that genotyping of Ras gene family together with an accurate analysis of selected morphological features could help in the differential diagnosis of follicular-derived thyroid neoplasms.

Cross-generational feeding of Bt (Bacillus thuringiensis)-maize to zebrafish (Danio rerio) showed no adverse effects on the parental or offspring generations

In the present study, zebrafish (Danio rerio) were fed casein/gelatin-based diets containing either 19% Bt (Bacillus thuringiensis)-maize or its parental non-Bt (nBt)-maize control for two generations (F0: sixty fish; F1: forty-two to seventy fish per treatment). The study focused on growth and reproductive performance, liver CuZn superoxide dismutase (SOD) enzyme activity, gene transcript levels targeting important cellular pathways in the liver and mid-intestine, histomorphological evaluation of the intestine, differential leucocyte counts, offspring larva swimming activity and global DNA methylation in offspring embryos. No significant effects were observed in the parental generation. The offspring were either fed the same diets as those fed to their parents (Bt-Bt or nBt-nBt) or switched from the Bt diet to the nBt diet (Bt-nBt). The Bt-Bt offspring exhibited a significantly higher body mass increase, specific growth rate and feed utilisation than fish fed the nBt-nBt diet and/or fish fed the Bt-nBt diet. Liver and mid-intestinal gene transcript levels of CuZn SOD were significantly higher in fish fed the nBt-nBt diet than in those fed the Bt-Bt diet. Liver gene transcript levels of caspase 6 were significantly lower for the nBt-nBt group than for the Bt-Bt group. Overall, enhanced growth performance was observed in fish fed the Bt diet for two generations than in those fed the nBt diet for one and two generations. Effects observed on gene biomarkers for oxidative stress and the cell cycle (apoptosis) may be related to the contamination of nBt-maize with fumonisin B1 and aflatoxin B1. In conclusion, it is suggested that Bt-maize is as safe and nutritious as its nBt control when fed to zebrafish for two generations.