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Purpose: This prospective Brazilian single-arm trial was conducted to determine response to chemotherapy and survival after response-based radiotherapy in children with intracranial germinomas, in the setting of a multi-institutional study in a middle-income country (MIC) with significant disparity of subspecialty care., Patients and Methods: Since 2013, 58 patients with histologic and/or serum and CSF tumor marker evaluations of primary intracranial germ cell tumors were diagnosed; 43 were germinoma with HCGβ levels ≤200 mIU/mL and five between 100 and 200 mIU/mL. The treatment plan consisted of four cycles of carboplatin and etoposide followed by 18 Gy whole-ventricular field irradiation (WVFI) and primary site(s) boost up to 30 Gy; 24 Gy craniospinal was prescribed for disseminated disease., Results: Mean age 13.2 years (range, 4.7-25.5 years); 29 were males. Diagnosis was made by tumor markers (n = 6), surgery (n = 25), or both (n = 10). Two bifocal cases with negative tumor markers were treated as germinoma. Primary tumor location was pineal (n = 18), suprasellar (n = 14), bifocal (n = 10), and basal ganglia/thalamus (n = 1). Fourteen had ventricular/spinal spread documented by imaging studies. Second-look surgery occurred in three patients after chemotherapy. Thirty-five patients achieved complete responses after chemotherapy, and eight showed residual teratoma/scar. Toxicity was mostly grade 3/4 neutropenia/thrombocytopenia during chemotherapy. At a median follow-up of 44.5 months, overall and event-free survivals were 100%., Conclusion: The treatment is tolerable, and WVFI dose reduction to 18 Gy preserves efficacy; we have demonstrated the feasibility of successfully conducting a prospective multicenter trial in a large MIC despite resource disparity.

There is general perception that elite athletes are highly susceptible to changes in immunohematological profile. The objective of this study was to compare immunohematological parameters of elite athletes of different aerobic and muscular strength sports and analyze changes over 2 months. Sixteen judoists and 14 swimmers were evaluated 2 months before (M1) and immediately prior to competition (M2). Hemogram and lymphocytes subpopulations were assessed with automatic counter and flow cytometry, respectively. Judoists had higher neutrophils and lower monocytes and eosinophils percentages than swimmers at M1 and M2. At M2 judoists had lower red blood cells (RBC), hemoglobin, and hematocrit than swimmers. At M2 judoists' hematocrit and CD16 decreased while swimmers' hemoglobin and hematocrit increased. In conclusion, neither sports characteristics nor intense training seem to displace the athletes' immunohematological profile out of the clinical range, despite the possibility of occurrence of microlesions that may stimulate production of leukocytes and reduction of RBC in judoists.

Background: Previous studies reported significant differences in the clinical presentation and outcomes of papillary thyroid carcinoma (PTC) in pediatric patients compared with adults. Previous studies have suggested that the clinicopathological differences observed between pediatric and adult PTCs may be due the existence of distinct genetic alterations. However, the knowledge of genetic events in pediatric PTCs is based primarily on studies in radiation-exposed PTCs or in the few studies that enrolled predominantly adolescent patients. The aim of this study was to characterize the known oncogenic alterations of the MAPK pathway found in adult and radiation-exposed PTCs in a cohort of predominantly sporadic pediatric PTC patients., Methods: Thirty-five pediatric PTCs were screened for the most prevalent fusions (RET/PTC1, RET/PTC2, RET/PTC3, ETV6-NTRK3, and AGK-BRAF) and point mutations (BRAF V600E and NRAS Q61 ) described in sporadic pediatric PTCs. The mutational status was correlated with clinicopathological data., Results: Mutations were found in 20 out of 35 (57%) PTC cases. Fusion oncogenes were the main genetic alterations found. RET/PTC1-3 rearrangements were found in 13 (37%), ETV6-NTRK3 in 3 (9%), AGK-BRAF in 4 (11%), and BRAF V600E in 3 (9%). No mutation was found in NRAS Q61 . BRAF V600E was associated with older age and larger tumor size (p < 0.05), and RET/PTC3 was associated with a larger tumor size and multifocality (p < 0.05)., Conclusions: The genetic signature in this cohort was remarkably different than that observed in adults. Although observed at a lower prevalence, the spectrum of mutations was quite similar to that described in radiation-exposed pediatric PTCs. As mutations were unidentifiable in over 40% of the PTC cases, more comprehensive studies conducted in these patients will help to decipher the genetic landscape of sporadic pediatric PTCs.

Taphonomic processes affecting bone post mortem are important in forensic, archaeological and palaeontological investigations. In this study, the application of tissue microarray (TMA) analysis to a sample of femoral bone specimens from 20 exhumed individuals of known period of burial and age at death is described. TMA allows multiplexing of subsamples, permitting standardized comparative analysis of adjacent sections in 3-D and of representative cross-sections of a large number of specimens. Standard hematoxylin and eosin, periodic acid-Schiff and silver methenamine, and picrosirius red staining, and CD31 and CD34 immunohistochemistry were applied to TMA sections. Osteocyte and osteocyte lacuna counts, percent bone matrix loss, and fungal spheroid element counts could be measured and collagen fibre bundles observed in all specimens. Decalcification with 7% nitric acid proceeded more rapidly than with 0.5 M EDTA and may offer better preservation of histological and cellular structure. No endothelial cells could be detected using CD31 and CD34 immunohistochemistry. Correlation between osteocytes per lacuna and age at death may reflect reported age-related responses to microdamage. Methodological limitations and caveats, and results of the TMA analysis of post mortem diagenesis in bone are discussed, and implications for DNA survival and recovery considered.

Background: Although numerous anatomic studies about the anterior cruciate ligament (ACL) structure and attachments have been performed, these studies have not reached consensus on the ACL footprint., Purpose: To investigate the existing controversy regarding the morphology of the tibial ACL insertion (footprint) and confirm histologically that the tibial ACL footprint is not completely filled with ligament tissue., Study Design: Descriptive laboratory study., Methods: The tibial ACL footprint was dissected from 20 different fresh-frozen cadaveric knees (all males; mean age, 68.8 ± 5.4 years [range, 55-80 years]; mean weight, 78 ± 6.6 kg [range, 45-93 kg]). Two knees, 1 with severe osteoarthritis and 1 with previous knee surgery, were excluded. The tibial ACL insertion was observed, and this area was longitudinally divided into 4 parallel slices (0%-25%, 25-50%, 50%-75%, and 75%-100%), embedded in paraffin wax, and stained with hematoxylin-eosin, alcian blue, and picrosirius-polarization. The specimens were measured using a microscope to determine the distances from the anterior to the posterior border of the ACL ligament tibial insertion and the distance from the posterior border to the end of the ligament fibers of the ACL ligament tibial insertions., Results: The 18 evaluated knee specimens confirmed the finding of a C-shaped tibial insertion of the ACL. The measurements showed that the ligament (vertical parallel collagen fibers) occupied only 30.8% of the complete insertion. The remaining area was filled with synovial tissue, demonstrating histologically the "C" shape., Conclusion: This study confirms macroscopically the C-shaped tibial insertion of the ACL and shows histologically that synovial tissue is an indirect insertion filling the major part of the footprint., Clinical Relevance: This anatomic study suggests a different shape of the ACL tibial footprint, which may be useful for new perspectives regarding ACL reconstruction surgery research., Competing Interests: The authors declared that they have no conflicts of interest in the authorship and publication of this contribution.

Understanding the impact of training sessions on the immune response is crucial for the adequate periodization of training, to prevent both a negative influence on health and a performance impairment of the athlete. This study evaluated acute systemic immune cell changes in response to an actual swimming session, during a 24-h recovery period, controlling for sex, menstrual cycle phases, maturity, and age group. Competitive swimmers (30 females, 15 ± 1.3 years old; and 35 males, 16.5 ± 2.1 years old) performed a high-intensity training session. Blood samples were collected before, immediately after, 2 h after, and 24 h after exercise. Standard procedures for the assessment of leukogram by automated counting (Coulter LH 750, Beckman) and lymphocytes subsets by flow cytometry (FACS Calibur BD, Biosciences) were used. Subjects were grouped according to competitive age groups and pubertal Tanner stages. Menstrual cycle phase was monitored. The training session induced neutrophilia, lymphopenia, and a low eosinophil count, lasting for at least 2 h, independent of sex and maturity. At 24 h postexercise, the acquired immunity of juniors (15-17 years old), expressed by total lymphocytes and total T lymphocytes (CD3(+)), was not fully recovered. This should be accounted for when planning a weekly training program. The observed lymphopenia suggests a lower immune surveillance at the end of the session that may depress the immunity of athletes, highlighting the need for extra care when athletes are exposed to aggressive environmental agents such as swimming pools.

Purpose: The aim of this prospective clinical study was to evaluate the clinical and histomorphometric data of newly formed bone tissue from fresh frozen human allograft in sinus lift surgery., Patients and Methods: Thirty-three sinus lift procedures were performed in 20 patients, divided into two groups. The control group (n = 8) received autogenous bone from the mandibular ramus, and the experimental group (n = 12) received fresh frozen bone (FFB) allograft in chips. After 6 months, 52 implants were placed and 50 biopsies were collected for histomorphometric analysis. Cone beam computed tomography scans were performed at preoperative, immediate postoperative, and delayed postoperative time intervals to assess the degree of graft volume loss., Results: There was no statistically significant difference between groups as regards degree of graft volume loss (p = .983), total bone area (p = .191), remaining particles (p = .348), and proportion of active osteoblasts (p = .867). There was a statistically significant difference in the vitality rate between the groups (p = .043). In both groups, all implants were clinically osseointegrated after 4 months., Conclusion: FFB allograft was shown to be a feasible substitute for autogenous bone graft in sinus lift surgery., (© 2014 Wiley Periodicals, Inc.)

This report describes a rare case of nodular fasciitis (NF) of the oral cavity, discussing the clinical, histological, and immunohistochemical characteristics. Histopathologic diagnosis of this type of lesion can be challenging due to its differential diagnosis, which principally includes sarcoma. The patient presented with a painless, well-defined nodule, reported as increasing in size, located at the apical aspect of the upper left buccal sulcus. Histologically, the lesion revealed spindle cell proliferation arranged in fascicles, while immunohistochemistry demonstrated positivity for smooth muscle actin. Eight months after complete surgical excision, no signs of local recurrence have been observed.

Despite a more advanced stage of disease at presentation, a better response to radioiodine (RAI) therapy and a reduced overall mortality have been reported in pediatric differentiated thyroid cancer (DTC) in comparison to adult DTC. Few studies suggested that the better response to RAI therapy in pediatric patients might be associated with an increased expression of NIS. However, a marked heterogeneity within the pediatric group has been recognized. Children (<10 years old) usually present a more aggressive disease than adolescents (≥10-18 years old). By analyzing the expression of thyroid-specific genes in 38 sporadic pediatric tumors, we show that the expression of NIS, PDS, and TSHR was lower in children than adolescents (P < 0.05). A linear regression confirmed the association between NIS expression and age. Most significantly, NIS was expressed at similar levels in DTC from children and adults, whereas PDS and TSHR expression was even lower in DTC from children, compared to adolescents and adults. Our data suggest that biological behaviors of DTC in adolescents might differ from those in children and adults. Therefore, the premise that the expression of thyroid-specific genes is higher in tumors from pediatric patients than in adults is not entirely true and might be too oversimplified.

Background/purpose: Pediatric adrenocortical tumor (ACT) remains a challenging disease. Tumor weight and disease stage are still the most used indicators to prognosis and guidance of clinical decisions. Histology has not added meaningful data for risk stratification and management. ACT is metabolically active, highly vascularized, locally invasive and has the propensity to produce distant metastasis. Our objective was to correlate the expression of vascular endothelial growth factor (VEGF) and intratumoral microvessel density (MVD) with clinical and prognostic aspects in pediatric ACT., Procedure: In 27 tumors, immunohistochemical expression of VEGF, CD105 (endoglin) and CD34 was analyzed. MVD was determined by CD34 and CD105 antibodies. MVD and VEGF expression was correlated with clinical characteristics and outcome. Normal pediatric glands were used as controls., Results: Endoglin MVD was significantly higher and CD34 MVD was significantly lower in ACT than control. The VEGF expression did not differ between groups. Cytoplasmic staining for endoglin was correlated with hypertension in ACT. Endoglin MVD greater than 1 mv/field, CD34 MVD less than 32 mv/field and VEGF expression levels above 4.8% were associated with clinical and biological indicators of poor prognosis., Conclusions: Endoglin and CD34 MVD values are potential histological markers to refine the histologic classification of pediatric ACT., (Copyright © 2015 Elsevier Inc. All rights reserved.)

This paper describes the surgical correction of ankylosis of the right temporomandibular joint and reconstruction of the middle and lower thirds of the right side of the face in a young man who had undergone treatment for hemangiopericytoma 10 years earlier, which led to serious functional, esthetic, and psychological problems and diminished his quality of life. The results of reconstruction surgery with alloplastic material (Medopor®) and surgical planning with fibrotic tissue due to radiation and the risk of osteoradionecrosis are discussed.

Purpose: Infant medulloblastoma (MB) is a malignant neuroepithelial embryonal tumor of the cerebellum, believed to derive from precursor granule cells with stem or progenitor cells appearance, and caused by a change in expression profile of genes related to the development. This work aims to study the expression profile of these genes in MB tumors, correlating with clinicopathological characteristics., Methods: We quantified, by qPCR in 40 MB tumor samples, the expression of genes in HH (PTCH1, PTCH2, and GLI1), WNT (APC, CTNNB1, WIF1, and DKK2), and NOTCH pathways (NOTCH2 and HES1), which have a crucial role in development, and genes as MYCC, MYCN, and TERT, correlating this findings to patient's clinicopathological characteristics., Results: Considering the universal RNA as our control sample, and considering the median of gene expression in the control samples as our cutoff, we observed that HES1 gene showed decreased expression compared to control (p = 0.0059), but patients with HES1 overexpression were directly related to a shorter survival (p = 0.0165). Individuals with higher GLI1 gene expression had significant shorter survival (p = 0.0469), and high expression was prevalent in patients up to 5 years old (p = 0.0479). Patients showing high PTCH2 expression were related to worse survival (p = 0.0426), and it was correlated with GLI1 high expression (p = 0.0094). We also observed a concomitant overexpression of WIF1 and DKK2 genes in a subgroup of MB samples (n = 11, p = 0.0118)., Conclusions: Our results suggest the presence of activated developmental signaling pathways in MB, which are important for cell proliferation and maintenance, and that may be targeted for novel therapeutic options.

Purpose: Little information is available regarding the tumor features, prognostic factors, and treatment results in children and adolescents and young adults (AYAs) with osteosarcoma diagnosed in developing countries. We reviewed the results of three observational cohorts of osteosarcoma patients treated in an emerging country., Methods: A total of 604 patients below the age of 30 years with high-grade osteosarcoma were prospectively enrolled in the Brazilian Osteosarcoma Treatment Group (BOTG) studies III, IV, and V. Gender, age, time from onset of symptoms to diagnosis, primary tumor site, presence or absence of metastases at diagnosis, tumor size, type of surgery (limb-sparing or amputation), treatment protocol, and histological response were correlated with survival., Results: The estimated 5-year overall survival and event-free survival (EFS) rates for the 553 eligible patients were 49% and 39% respectively; of the 390 non-metastatic patients included in the total, overall- and event-free survival were 59% and 48% respectively. Metastases at diagnosis, primary tumor site, type of surgery, and histological response were significant predictors of overall survival and EFS in univariate and multivariate analysis, whereas tumor size and treatment protocol lost prognostic significance in multivariate analysis., Conclusion: We report on the outcome of three consecutive studies for the treatment of osteosarcoma carried out in Brazil over 15 years. Although the survival rates presented are below those reported in current literature, it represents the result of a favorable experience gathered from the national collaborative work.

Osteosarcoma is a malignant bone tumor with high metastatic potential. Metastasis at diagnosis is the most significant prognostic factor in predicting the clinical outcome of osteosarcoma. We compared the gene expression of metastases that were present at the time of initial diagnosis to those developed later in the course of the disease. We used quantitative real-time polymerase chain reaction to evaluate the gene expression of MDM2, CXCR4, RANKL, RB1, and OSTERIX in 98 samples of osteosarcoma taken from 47 patients (74 metastases and 24 primary tumors) and 30 nonmalignant lung tissues surrounding osteosarcoma metastases. In addition, we investigated the copy number changes of RB1 and MDM2 genes in 12 primary cultures of pulmonary metastases of osteosarcoma, using interphase fluorescence in situ hybridization. Metastases from metastatic patients at diagnosis were characterized by low expression of RB1 and RANKL (P = .0009 and P = .0109, respectively) and overexpression of CXCR4 and MDM2 (P = .0389 and P = .0325, respectively). The loss of RANKL and gain of CXCR4 could also be detected in the primary tumors of metastatic patients at diagnosis (P = .0121 and P = .0264, respectively). Thus, some early genetic events such as the loss of RANKL and the gain of CXCR4 expressions probably facilitate the metastatic progression concomitant with the primary tumor establishment, supporting the role of the CXCR4 receptor in directing osteosarcoma metastases to the lung. On the other hand, late events such as the loss of RB1 and gain of MDM2, crucial regulators of cell cycle, appear to be related to the final mechanisms contributing to the metastatic establishment of osteosarcoma., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Introduction: The aim of this study was to evaluate the association between frequency of the posterior cruciate ligament (PCL) mechanoreceptors and age in men., Methods: Nineteen normal right knees harvested from human male cadavers were evaluated. Age ranged from 17 to 64 years with a mean of 35 years old. PCL was separated for sampling in femoral and tibial portions. Topographic distribution and frequency within the ligament texture were determined employing the Pro-Image digital analysis system. Mechanoreceptors were counted and classified according to the criteria proposed by Freeman & Wyke., Results: A total of 1820 mechanoreceptors were found, type II being the most frequent one. Analysis of the femoral portion of the ligament showed an equivalent predominance of Types II and IV mechanoreceptors. Tibial portion had a predominance of type II mechanoreceptors, followed by type IV. At this portion, receptors Types I and III were less commonly identified., Conclusion: In the tibial portion of the PCL, there is predominance of Type II mechanoreceptors followed by types IV, I and III mechanoreceptors, respectively. No relationship was found between the total number of mechanoreceptors and age in the femoral and tibial portions of the PCL.

The purpose of our study was to investigate the prevalence of the PAX3/7-FKHR fusion genes and quantify the IGF2 gene expression in rhabdomyosarcoma (RMS) samples. Soft tissue sarcomas account 5% of childhood cancers and 50% of them are RMS. Morphological evaluation of pediatric RMS has defined two histological subtypes, embryonal (ERMS) and alveolar (ARMS). Chromosomal analyses have demonstrated two translocations associated with ARMS, resulting in the PAX3/7-FKHR rearrangements. Reverse transcriptase-polymerase chain reaction (RT-PCR) is extremely useful in the diagnosis of ARMS positive for these rearrangements. Additionally, several studies have shown a significant involvement of IGF pathway in the pathogenesis of RMS. The presence of PAX3/7-FKHR gene fusions was studied in 25 RMS samples from patients attending the IOP-GRAACC/UNIFESP and three RMS cell lines by RT-PCR. IGF2 gene expression was quantified by qPCR and related with clinic pathological parameters. Of the 25 samples, nine (36%) were ARMS and 16 (64%) were ERMS. PAX3/7-FKHR gene fusions expression was detected in 56% of ARMS tumor samples. IGF2 overexpression was observed in 80% of samples and could indicate an important role of this pathway in RMS biology., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Purpose: Comparing the ionizing radiation effects on bone neoformation of rats tibiae previously submitted to radiotherapy with a single dosage of 30Gy with the contralateral tibiae that have received secondary radiation., Methods: In thirty male Wistar rats, 30 days before surgical procedure when round defects would be created on the bone, the right tibia was irradiated with 30Gy and the left tibia received a calculated secondary radiation dose of 7Gy. Sacrifices were performed after 4, 7, 14, 21, 56 and 84 postoperative days and both tibiae were removed for histological processing., Results: The left tibiae that received the dose of 7Gy has shown more bone neoformation from 14(th) postoperative days, giving evidences of less damage to cellular population responsible by bone neoformation. On the other hand, the dose of 30Gyon right tibiae did not exhibit significant differences among the periods, suggesting damage of long-lasting or even permanent duration., Conclusion: Tibiae submitted to radiation dose of 30Gy have shown more damage to bone cells than tibiae that received secondary radiation dose of 7Gy, especially observed on 14(th), 56(th) and 84(th) postoperative days.

Background: Osteosarcoma (OS) is the most frequent bone tumor in children and adolescents. Tumor antigens are encoded by genes that are expressed in many types of solid tumors but are silent in normal tissues, with the exception of placenta and male germ-line cells. It has been proposed that antigen tumors are potential tumor markers., Objectives: The premise of this study is that the identification of novel OS-associated transcripts will lead to a better understanding of the events involved in OS pathogenesis and biology., Methods: We analyzed the expression of a panel of seven tumor antigens in OS samples to identify possible tumor markers. After selecting the tumor antigen expressed in most samples of the panel, gene expression profiling was used to identify osteosarcoma-associated molecular alterations. A microarray was employed because of its ability to accurately produce comprehensive expression profiles., Results: PRAME was identified as the tumor antigen expressed in most OS samples; it was detected in 68% of the cases. Microarray results showed differences in expression for genes functioning in cell signaling and adhesion as well as extracellular matrix-related genes, implying that such tumors could indeed differ in regard to distinct patterns of tumorigenesis., Conclusions: The hypothesis inferred in this study was gathered mostly from available data concerning other kinds of tumors. There is circumstantial evidence that PRAME expression might be related to distinct patterns of tumorigenesis. Further investigation is needed to validate the differential expression of genes belonging to tumorigenesis-related pathways in PRAME-positive and PRAME-negative tumors.

Brainstem gliomas constitute 10% to 20% of all pediatric tumors of the central nervous system, and diffusely infiltrative brainstem gliomas are the most common brainstem tumors associated with a poor prognosis. A small subset of these tumors is benign, showing low-grade features on histology. The role of chemotherapy in the management of these tumors is ill defined, especially in the neonates. There are anecdotal reports of spontaneous remission, but the natural history of these tumors does not support a wait-and-see approach. Thus, we report a successful experience of chemotherapy in a 4-month-old girl with a diffuse brainstem fibrillary astrocytoma, treated with vinorelbine (30 mg/m/d on days 0, 8, and 22), a vinca alkaloid that has shown activity against glioma. Our experience suggests that vinorelbine may be effective in pediatric low-grade gliomas as this patient showed significant clinical improvement over a short period of time.

Globin genes, which encode the protein subunits of hemoglobin (Hb), are organized in two different gene clusters and present a coordinated and differential pattern of expression during development. Concerning the human alpha-globin gene cluster (located at chromosome region 16p13.3), four upstream highly conserved elements known as multispecies conserved sequences (MCS-R1-4) or DNase I hypersensitive sites (HSs) are implicated in the long-range regulation of downstream gene expression. However, only the absence of the MCS-R2 site (HS-40) has proven to drastically downregulate the expression of those genes, and consequently, it has been regarded as the major and crucial distal regulatory element. In this study, Multiplex Ligation-dependent Probe Amplification was used to screen for deletions in the telomeric region of the short arm of chromosome 16, in an attempt to explain the alpha-thalassemia or the HbH disease present in a group of Portuguese patients. We report four novel and five uncommon deletions that remove the alpha-globin distal regulatory elements and/or the complete alpha-globin gene cluster. Interestingly, one of them occurred de novo and removes all HSs except HS-10, while other eliminates only the HS-40 site, the latter being replaced by the insertion of a 39 nucleotide orphan sequence. Our results demonstrate that HS-10 alone does not significantly enhance the alpha-globin gene expression. The absence of HS-40 in homozygosity, found in a patient with Hb H disease, strongly downregulates the expression of alpha-globin genes but it is not associated with a complete absence of alpha-globin chain production. The study of naturally occurring deletions in this region is of great interest to understand the role of each upstream regulatory element in the native human erythroid environment., (2010 Elsevier Inc. All rights reserved.)

Background and Objectives: Several studies have suggested that low-level laser therapy (LLLT) can ameliorate oral mucositis; however, the mechanisms involved are not well understood. The aim of this study was to investigate the mechanisms of action of LLLT on chemotherapy-induced oral mucositis, as related to effects on collagen expression and inflammation., Materials and Methods: A hamster cheek pouch model of oral mucositis was used with all animals receiving intraperitoneal 5-fluorouracil, followed by surface irritation. Animals were randomly allocated into three groups, and treated with an InGaAIP diode laser at a wavelength of 660 nm and output power of 35 or 100 mW laser, or no laser. Clinical severity of mucositis was assessed at four time-points by a blinded examiner. Buccal pouch tissue was harvested from a subgroup of animals in each group at four time-points. Collagen was qualitatively and quantitatively evaluated after picrosirius staining. The density of the neutrophil infiltrate was also scored., Results: Peak clinical severity of mucositis was reduced in the 35 mW laser group as compared to the 100 mW and control groups. The reduced peak clinical severity of mucositis in the 35 mW laser group was accompanied by a decrease in the number of neutrophils and an increase in the proportion of mature collagen as compared to the other two groups. The total quantity of collagen was significantly higher in the control (no laser) group at the day 11 time-point, as compared to the 35 mW laser group, consistent with a more prolonged inflammatory response in the control group., Conclusion: This study supports two mechanisms of action for LLLT in reducing mucositis severity. The increase in collagen organization in response to the 35 mW laser indicates that LLLT promotes wound healing. In addition, LLLT also appears to have an anti-inflammatory effect, as evidenced by the reduction in neutrophil infiltrate., ((c) 2010 Wiley-Liss, Inc.)

Purpose: To evaluate the influence of PRP addition on bone repair of circular defects created in irradiated tibiae of rats by histometric analysis., Methods: Sixty male Wistar rats had the right tibiae irradiated with 30 Gy. After 30 days monocortical defects were created and platelet-rich plasma was applied in 30 rats. In the control group defects were created but not filled. The animals were desanguinated after 4, 7, 14, 21, 56 and 84 days and the tibiae removed for histological processing., Results: There was a tendency in the PRP group to increased bone neoformation from 14-days to 84-days; in the control group increased bone neoformation was not seen after 21 days or later., Conclusion: The addition of platelet-rich plasma had a beneficial effect in the initial cellular regeneration period and enhanced bone formation in later periods when compared to control.

Context and Objective: There is controversy regarding which imaging method is best for identifying early degenerative alterations in intervertebral discs. No correlations between such methods and histological finds are presented in the literature. The aim of this study was to correlate the thickness of intervertebral discs measured on simple radiographs with the degree of degeneration seen on magnetic resonance images and the histological findings relating to nerve ends inside the discs., Design and Setting: Cross-sectional correlation study on the lumbar spines of human cadavers, at Universidade Federal de São Paulo (Unifesp), São Paulo, Brazil., Methods: Ten lumbar spinal columns were extracted from human cadavers and subjected to magnetic resonance imaging and simple radiography. They were classified according to the degree of disc degeneration seen on magnetic resonance, and the thickness of the discs was measured on radiographs. The intervertebral discs were then extracted, embedded in paraffin and analyzed immunohistochemically with protein S100, and the nerve fibers were counted and classified., Results: No correlation was observed between the thickness of the intervertebral discs and the degree of degeneration seen on magnetic resonance images. Only the uppermost lumbar discs (L1/L2 and L2/L3) presented a correlation between their thickness and type I and IV nerve endings., Conclusion: Reduced disc thickness is unrelated to increased presence of nerve ends in intervertebral discs, or to the degree of disc degeneration.

Goal of Work: The aim of this study was to investigate the mechanisms whereby low-intensity laser therapy may affect the severity of oral mucositis., Materials and Methods: A hamster cheek pouch model of oral mucositis was used with all animals receiving intraperitoneal 5-fluorouracil followed by surface irritation. Animals were randomly allocated into three groups and treated with a 35 mW laser, 100 mW laser, or no laser. Clinical severity of mucositis was assessed at four time-points by a blinded examiner. Buccal pouch tissue was harvested from a subgroup of animals in each group at four time-points. This tissue was used for immunohistochemistry for cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), and factor VIII (marker of microvessel density) and the resulting staining was quantified., Main Results: Peak severity of mucositis was reduced in the 35 mW laser group as compared to the 100 mW laser and control groups. This reduced peak clinical severity of mucositis in the 35 mW laser group was accompanied by a significantly lower level of COX-2 staining. The 100 mW laser did not have an effect on the severity of clinical mucositis, but was associated with a decrease in VEGF levels at the later time-points, as compared to the other groups. There was no clear relationship of VEGF levels or microvessel density to clinical mucositis severity., Conclusion: The tissue response to laser therapy appears to vary by dose. Low-intensity laser therapy appears to reduce the severity of mucositis, at least in part, by reducing COX-2 levels and associated inhibition of the inflammatory response.

We aimed to evaluate the prognostic significance of traditional clinical predictors in osteosarcoma through an international collaboration of 10 teams of investigators (2680 patients) who participated. In multivariate models the mortality risk increased with older age, presence of metastatic disease at diagnosis, development of local recurrence when the patient was first seen, use of amputation instead of limb salvage/wide resection, employment of unusual treatments, use of chemotherapeutic regimens other than anthracycline and platinum and use of methotrexate. It was also influenced by the site of the tumour. The risk of metastasis increased when metastatic disease was present at the time the patient was first seen and also increased with use of amputation or unusual treatment combinations or chemotherapy regimens not including anthracycline and platinum. Local recurrence risk was higher in older patients, in those who had local recurrence when first seen and when no anthracycline and platinum were used in chemotherapy. Results were similar when limited to patients seen after 1990 and treated with surgery plus combination chemotherapy. This large-scale international collaboration identifies strong predictors of major clinical outcomes in osteosarcoma.

The phenotype of increased Hb A2 typical of beta-thalassaemia (beta-thal) carriers can be reduced to normal or borderline values because of the co-inheritance of a delta-globin gene (HBD, MIM #142000) mutation, which may lead to misinterpretation of diagnostic results. To know the spectrum of delta-globin mutations in the Portuguese population we performed a mutational analysis of the delta-globin gene in a group of 51 Portuguese beta-thal carriers presenting microcytosis, hypochromia and a normal/borderline Hb A2 level and in another group of 15 individuals suspected to have delta-globin structural abnormalities. The heterozygosity for the beta(+)IVS-I-6T-->C (HBB:c. 92+6T>C) mutation was the main cause for the mentioned atypical beta-thal carrier phenotype. Furthermore, eight individuals were double heterozygous for one common beta-thal mutation and the delta(+)Cd27G-->T mutation (Hb A2-Yialousa; HBD:c.82G>T). One of them also presented a novel delta-globin gene promoter mutation,-80G-->A (HBD:c.-130G>A), responsible for about 25% decrease of the promoter activity in transient expression assays. One the other hand, in the other group of 15 individuals suspected to have delta-globin structural abnormalities observed by biochemical methods, some known Hb A2 variants were identified - Hb A2' (HBD:c.49G>C), Hb A2-Babinga (HBD:c.410G>A), and Hb A2-Wrens (HBD:c.295G>A), and the novel Hb A2-Fogo [delta64(E8)(Gly-->Ser); (HBD:c.193G>A)]. This novel Hb A2 variant was observed segregating in linkage with Hb E (HBB:c.79G>A) in a three generation family. In conclusion, six different delta-globin mutations were found, being two of them new molecular defects. All delta-alleles identified were found linked to the expected beta-globin cluster haplotype. All mutations caused a low Hb A2 level and through this could lead to misdiagnosis when inherited together with a beta-thal allele.

Aim: Considering the roles of c-erB-2 and p53 oncoproteins in tumor progression, we aimed to evaluate their expression in hydatidiform moles, and the possible predictive value of this immunoexpression in postmolar follow-up., Methods: Group I comprised 35 patients with progression to gestational trophoblastic tumor, and group II included 32 patients with progression to spontaneous remission. Immunohistochemical tests were performed by streptavidin-peroxidase method. c-erbB-2 immunoexpression was evaluated according to quantitative and semiquantitative criteria; p53 according to percentage of cells with stained nuclei. Data were analyzed by Student t-test, Mann-Whitney test, ROC curve and logistic regression analysis., Results: c-erbB-2 and p-53 expressions were significantly increased in group I. Quantitative and semiquantitative analysis of c-erb-2 showed that its expression may be associated with mole hydatidiform progression to gestational trophoblastic tumor. Taking into account cells with complete membranous delineation we proposed a cut-off value of 10.8%. Similarly, considering the percentage of cells presenting nuclei marked by p53 we suggested a cut-off value of 40.1% for the prediction of malignant transformation of mole hydatidiform., Conclusions: c-erbB-2 and p53 immunoexpression in hydatidiform mole are usually increased with malignant transformation. In addition to beta-fraction of human chorionic gonadotropin, they could possibly help the establishment of a therapeutic protocol.

Hemoglobin (Hb) Loves Park [beta68 (E12) Leu-->Phe] was identified in a 2-year-old Portuguese boy with anemia, microcytosis, and hypochromia. This Hb variant was detected by isoelectric focusing and quantified by reverse-phase high-performance liquid chromatography (HPLC) (48.4%), and the DNA mutation was identified by HBB (beta-globin gene) sequencing. Hematological and biochemical analyses performed on his parents revealed normal hematological parameters and normal hemoglobin and globin chain profiles. DNA sequence analysis of the HBB gene of both parents showed the absence of the Hb Loves Park mutation. Study of the haplotypes in the beta-globin gene cluster confirmed parenthood. Moreover, paternity was confirmed by the study of nine short tandem repeats (STRs) and four variable-number tandem repeat (VNTRs) loci. The most likely explanation for these results is that the Hb Loves Park mutation has occurred de novo in this family. The original American cases of Hb Loves Park, from a family of Italian origin, which were never published, as well as two additional cases, are also included in this report. Functional studies revealed that Hb Loves Park is stable and has a decreased oxygen affinity., (Copyright 2006 Wiley-Liss, Inc.)

We describe a novel alpha-thalassaemia determinant in a 3-year-old girl presenting a mild microcytic and hypochromic anaemia, and normal haemoglobin A2 level. Molecular studies revealed heterozygosity for a novel microdeletion (-C) at codon 22 of the alpha2-globin gene. As the frameshift mutation generates a premature translation termination codon at position 48/49, we investigated the effect of the nonsense codon on the alpha2-globin gene expression. Although it does not affect RNA splicing, the premature nonsense codon induces accelerated mRNA degradation. To our knowledge, this is the first time the nonsense-mediated mRNA decay has been reported to occur in human alpha-globin mRNA.

Orbital granulocytic sarcoma is a localized tumor consisting of malignant cells of myeloid origin. This tumor may present in association with acute myelogenous leukemia. Granulocytic sarcoma may be found in a variety of locations throughout the body including the orbit and typically affects children and young adults. There is a slight male predominance in these cases. This is an uncommon case report of a 33-year-old Latin-American woman who was admitted to the Hospital for rapidly progressive orbital proptosis. There was no systemic manifestation of leukemia. The occurrence of orbital granulocytic sarcoma before the development of systemic leukemia in children and young adults is not uncommon and these cases frequently develop hematological evidence within 2 months after initial orbital disease. In this case report, there was no systemic manifestation of leukemia in the last 30 months, even in the presence of orbital tumors. Granulocytic sarcoma is most frequently confused with malignant lymphoma, rhabdomyosarcoma and neuroblastoma. The differential diagnosis of these cases can be challenging, particularly when there is no evidence of systemic leukemia, when imaging features are not sufficiently specific to distinguish granulocytic neoplasms from other tumors. To establish the diagnosis often a biopsy is required. The treatment in such cases (orbital granulocytic sarcoma) is not standardized. Orbital granulocytic sarcoma may be suspected in cases of orbital tumors even in the absence of systemic manifestations of leukemia at any age.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma. Although the presence of p53 gene mutations has been considered as a bad prognostic feature in DLBCL, its clinical significance is still controversial. The aims of this study were: detect the presence of mutations in exons 5 to 9 of the p53 gene and correlate it to prognosis in DLBCL. Fifty-one DLBCL patients were enrolled in this study. Expression of p53 was evaluated by immunohistochemistry. The screening of p53 mutations was performed using PCR-SSCP methods. Cases showing a mobility shift on SSCP electrophoresis were analyzed by automatic sequencing. We could identify 8 missense mutations in 6 of 48 cases (12.5%). In addition, we found a known polymorphism at codon 213 and 2 instances of silent mutations. Of all mutations/polymorphisms found, 7 (64%) were localized in codons previously described as p53 hot spots in NHL cases. Of the remaining alterations (4 or 36%), 2 mutations were localized in codons previously described as hot spots for p53 in other tumors and 2 (codon 142 of the exon 5 and codon 195 of the exon 6), in codons not described as hot spots for p53 up to now. The presence of missense mutations in exons 5 to 9 of p53 gene had adverse impact on overall survival (P = 0.020). Cox's Regression Model identified that high-risk International Prognostic Index (IPI) and p53 gene mutations have independent negative impact on OS. Therefore, the association of IPI with cellular factors, such as p53 mutation, can be very helpful in deciding when we should indicate more aggressive therapies in patients with DLBCL, to somehow increase the chance of cure in these patients.

Hb Yaoundé [beta134(H12)Val-->Ala] is a rare, silent and asymptomatic hemoglobin (Hb) variant. It was previously reported in a Black man from Cameroon, in association with Hb Kenitra [beta69(E13)Gly-->Arg], and was subsequently found and described as Hb Mataro in a sub-Saharan child. To date, Hb Yaoundé has not been described in Caucasian people and molecular studies have never been performed. Here we describe a three-generation Caucasian Portuguese family in whom Hb Yaoundé was found in association with Hb C [beta6(A3)Glu-->Lys] (in the proband) and in a heterozygous state (in the proband's mother). The Hb studies of the proband's hemolysate, performed by isoelectric focusing (IEF) and low-pressure cation exchange chromatography, only revealed an Hb variant identified as Hb C by comparison with a control. However, analysis performed by reversed-phase high-performance liquid chromatography (HPLC) showed two different beta chain variants and a complete absence of the normal beta chain. This uncommon Hb variant was identified as Hb Yaoundé by DNA sequencing analysis of the beta-globin gene (codon 134, GTG-->GCG). The beta-globin gene haplotypes were determined in all family members using polymerase chain reaction (PCR)-based methodology; Hb Yaoundé was found to be associated with the Mediterranean haplotype II. This study is the first description of Hb Yaoundé in Caucasian individuals, and its association with a Mediterranean haplotype supports the hypothesis of an independent genetic origin other than African.

For almost three decades, tamoxifen has been used in the adjuvant treatment of breast cancer. It has also proven effective in the chemoprophylaxis of this disease and in the treatment of cyclic mastalgia. Since a fibroadenoma is a benign hormone-dependent neoplasm which contains estrogen receptor (ER) levels higher than in the mammary lobule, an evaluation of the effect of this drug on the proliferative activity of both the epithelium and the stroma of fibroadenomas in premenopausal women following the administration of 10 or 20 mg/day over 22 days was proposed. Forty women with fibroadenoma were selected for a randomized double-blind trial. They had regular menstrual cycles and had received neither hormones nor become pregnant 12 months prior to this study. Patients were divided into three groups: A (n = 14; placebo), B (n = 13; tamoxifen 10 mg/day), and C (n = 13; tamoxifen 20 mg/day). The treatment was initiated on the first day of their menstrual cycle and the surgeries were performed on the 22nd day. Estradiol, progesterone, and steroid hormone binding globulin (SHBG) were measured twice. The first measurement was performed on the 22nd day of the previous menstrual cycle and the second one was performed on the day of surgery. The fibroadenoma was fixed in 10% formaldehyde solution and stained with hematoxylin and eosin and then processed through immunohistochemical reaction (PC-10, DAKO code number M879, Denmark A/S). The immunoexpression of the proliferative cell nuclear antigen (PCNA) of at least 500 epithelial and 500 stromal cells was evaluated. Such cells were interactively counted using the Kontron Imaging System KS-300 computerized analysis system, with x 400 magnification. As to PCNA expression in the fibroadenomas' epithelium, the average percentage of stained nuclei in groups A, B, and C was 25.2, 19.3, and 18.0, respectively. However, no significant difference was found in the variance analysis of these data (p = 0.168). As to the study of the fibroadenomas' stroma, the average percentage of stained nuclei found in groups A, B, and C was 32.4, 23.2, and 18.4, respectively. The variance analysis (p = 0.031) and Fisher's multiple comparison test (1.39; 26.67 confidence interval [CI]) confirmed that the number of PCNA-expressing nuclei in the stroma was significantly lower in group C (20 mg/day) compared to group A (control). However, there was no significant difference between group B (10 mg/day) and group C (20 mg/day). It was found that tamoxifen reduced the expression of PCNA in the epithelium and the stroma of the fibroadenoma. However, the effect was only statistically significant in the stroma when a 20 mg/day dose was administered.

Background: Gastroesophageal reflux disease is a frequent cause of morbidity in childhood, including esophagitis and recurrent respiratory symptoms; however histological and morphometric studies in esophageal biopsies of children are scarce., Aim: To study histological and morphometric findings in children with reflux esophagitis., Patients and Methods: We studied 26 esophageal biopsies of children (mean age: 4.1 years +/- 3.4) with reflux esophagitis, which prevailed in boys (84.6%); post-prandial vomiting (76.9%) and repetitive bronchopneumonia (38.5%) were the most frequent symptoms. The diagnosis was made by X-ray in 18, by pH evaluation in 5 and by scintilography in 3 patients. The control group was formed by seven children without reflux symptoms who died from meningococcemia or congenital heart malformation (mean age: 2.5 years +/- 2.3). Histological variables were studied by hematoxylin-eosin and periodic acid of Schiff stain, inflammatory infiltrate, epithelial and basal layer thickness, papillary length and its ratio with the thickness of the epithelium. Morphometry was performed at a digital system connected to pro-image software. Student's t test, Mann-Whitney test, Fisher and Pearson's correlation methods were used for the statistical analysis., Results: Epithelial and basal zone thickness, papillary length and its rate with thickness of epithelium, among the reflux group patients were higher than the control group. Eosinophils, neutrophils and "balloon cells" were not observed in the control group. Intraepithelial capillaries occurred in 11 cases in the reflux group (mean diameter: 59 mum)., Conclusion: Epithelial and basal zone thickness, papillary length and its ratio with thickness of epithelium, resulted greater in the gastroesophageal reflux group than in the control group. There was a direct correlation between thickness of epithelium, basal zone thickness and papillary length, showing increased epithelial cell turnover. Eosinophils, neutrophils and "balloon cells" were observed only in patients with gastroesophageal reflux, thus serving as specific markers of this disease.

The purpose of this study was to study the monoclonal antibody MIB-1 in the normal breast epithelium adjacent to a fibroadenoma in women in the luteal phase of the menstrual cycle who were treated with tamoxifen at doses of 10 and 20 mg for 22 days. The proliferative activity of the mammary epithelium adjacent to the fibroadenoma was studied by immunohistochemistry on the basis of the monoclonal antibody MIB-1 (Immunotech, catalog No. 0505, lot 001). The study was randomized and double blind and was conducted on 44 women with fibroadenomas divided into three groups: A (n=16, placebo), B (n=15, tamoxifen, 10 mg), and C (n=13, tamoxifen, 20 mg). Tamoxifen was administered for 22 days starting on the 2nd day of the menstrual cycle, and a biopsy was taken on the 23rd day. Serum estradiol, progesterone, sex hormone binding globulin, follicle-stimulating hormone, luteinizing hormone, and prolactin were measured before treatment (21st and 24th day of the previous menstrual cycle) and on the day of the biopsy. The mean percentage of stained nuclei per 1,000 cells was 9.2 in group A, 4.5 in group B, and 3.2 in group C. The Fisher's test revealed that tamoxifen significantly reduced MIB-1 at doses of 10 and 20 mg compared with the placebo group (p < 0.0001), with no significant differences between doses in terms of proliferative activity (p=0.21). Groups B and C presented a significant increase in progesterone (p=0.038), estradiol (p < 0.001), and sex hormone binding globulin (p=0.001) levels. Elevation of serum follicle-stimulating hormone concentration (p=0.0045) and a fall in prolactin levels (p=0.0055) were observed. We conclude that tamoxifen significantly reduced the proliferative activity of the mammary epithelium at the doses of 10 and 20 mg/day.

The authors present the results of treatment of 10 patients with Ollier's disease using the Ilizarov technique. The Ilizarov device was used to treat leg length discrepancy and to enhance the conversion of chondroma cartilage into normal mature bone, with no curettage and bone grafting. The mean duration of treatment was 9.4 months. This technique was highly efficient in treating the disease. It led to conversion of the abnormal cartilage into histologically mature bone in all patients. Some complications were seen, such as decreased knee mobility, which required prolonged use of the device. The Ilizarov technique is successful in treating patients with Ollier's disease despite some complications and the difficulty in using the technique.

Fourteen boys (56%) and 11 girls (44%) 4 to 17 years of age (mean, 12.2 years) who had osteosarcoma and open epiphyseal plates were studied. A possible correlation between transepiphyseal spread of osteosarcoma and radiologic and histopathologic findings was investigated. Epiphyseal plate invasion was detected radiologically in only 11 patients (44%), whereas histopathologic examination showed transepiphyseal extension in 21 patients (84%). The authors conclude that the epiphyseal plate is not a barrier against tumor growth and strongly recommend that limb salvage surgery preserving the epiphysis be planned carefully.

Neuroblastoma is the second most common solid tumor occurring in children. Amplification of the MYCN oncogene is associated with poor prognosis. To identify neuroblastoma tumors with MYCN amplification, we studied the number of copies of MYCN in interphase cells by fluorescence in situ hybridization in 20 neuroblastoma patients. MYCN amplification appeared in 7 tumor specimens. Interphase and metaphase studies showed a tumor cell population with both forms of amplification, double minutes and homogeneously staining regions, in two patients. These patients showed a smaller tumor cell subpopulation with the presence of more than one homogeneously staining region, suggesting that gene amplification was undergoing karyotype evolution.

Objectives: To investigate the proliferative activity of the mammary gland epithelium and plasma levels of progesterone, estradiol, prolactin, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and sex hormone-binding globulin (SHBG) in premenopausal women treated with 10 and 20 mg of tamoxifen (TAM) for 22 days., Patients and Methods: A randomized double-blind study was performed with 43 premenopausal women with a diagnosis of fibroadenoma of the breast. The patients were divided into three groups: A (n = 15, placebo); B (n = 15, TAM 10 mg/day) and C (n = 13, TAM 20 mg/day). They started taking an oral dose of TAM or placebo on the very first day of the menstrual cycle. Lumpectomy was performed on the 22nd day of therapy. Normal breast tissue samples were collected during surgery, immediately immersed in 10% buffered formalin, processed for routine histology and immunohistochemistry for proliferating cell nuclear antigen (PCNA) detection. Two peripheral blood samples were collected, both on the 22nd day of the menstrual cycle, in order to evaluate the hormone levels. PCNA expressing epithelial cells were quantified by using a digital program Kontron Image System KS-300 in 1000 cells (400 x )., Results: The percentage of cells expressing PCNA was significantly higher in the group receiving placebo (group A, 50.3%) when compared to groups receiving TAM 10 or 20 mg/day (group B, 24.1%; and group C, 23.2%, respectively) (P < 0.001). Differences between groups B and C were not significant. Levels of progesterone, estradiol and SHBG were significantly higher in B and C groups compared to group A. Increasing concentrations of FSH (P < 0.0045) and lower levels of prolactin (P < 0.0055) were only found in the group receiving 20 mg/day of TAM (group C)., Conclusions: A 22-day TAM therapy, either with 10 or 20 mg/day, significantly reduced the PCNA expression and therefore the proliferative activity of the normal human breast tissue. Increasing levels of estradiol, progesterone and SHBG were associated with TAM therapy at 10 or 20 mg/day. However, a significant change of the level of FSH and prolactin was reached only with a 20-mg/day dose.

We describe two patients who underwent cardiac transplantation for chronic cardiomyopathy of Chagas' disease, and in whom the disease was reactivated with the development of cutaneous lesions. In both cases, the skin lesions regressed completely after 2 months of therapy with allopurinol.

We report a case of malignant schwannoma of the duodenum, a neural tumor, which rarely occurs in the gastroduodenal tract. The diagnosis was done after an hemorragic episode of the upper gastrointestinal tract and the treatment done was a local resection. Two years later the patient had no symptoms, but the control endoscopy showed a recurrence of the tumor in the second portion of the duodenum and the patient was submitted to a gastroduodenopancreatectomy. The authors reported the case and make a revision on intestinal schwannomas.