GIANT CELL TUMOR OF BONE: A MULTICENTER EPIDEMIOLOGICAL STUDY IN BRAZIL.

Introduction: Giant cell tumor of bone (GCTB) mainly affects young adults' long bone epiphyses, threatening bone strength and joint function. Surgery is the primary treatment, although post-surgery recurrence is significant. This study analyzes patient profiles, treatments, and outcomes for GCTB in Brazil. Methods: We retrospectively assessed local recurrence, metastasis, and treatment approaches in 643 GCTB patients across 16 Brazilian centers (1989-2021), considering regional differences. Results: 5.1% (n=33) developed pulmonary metastases, 14.3% (n=92) had pathological fractures, and the local recurrence rate was 18.2% (n=114). Higher rates of pulmonary metastases (12.1%) and advanced tumors (Campanacci III, 88.9%) were noted in lower-income North and Northeast regions. The North also had more pathological fractures (33.3%), extensive resections (61.1%), and amputations (27.8%). These regions faced longer surgical delays (36-39 days) than the South and Southeast (27-33 days). Conclusions: Our findings corroborate international data, underscoring regional disparities in Brazil that may lead to worse outcomes in disadvantaged areas. This highlights the need for improved orthopedic oncology care in Brazil's economically and structurally challenged regions. Level of Evidence III; Retrospective Cohort.

Introdução: O tumor de células gigantes do osso (TCG) atinge principalmente epífises de ossos longos em adultos jovens, impactando a resistência óssea e a funcionalidade articular. O tratamento principal é cirúrgico, mas há significativa recorrência pós-operatória. Este estudo analisa o perfil de pacientes e tumores de TCG no Brasil, abordagens de tratamento e resultados. Métodos: Avaliamos retrospectivamente taxas de recorrência, metástase e tratamentos em 643 pacientes tratados em 16 centros brasileiros de 1989 a 2021, considerando a distribuição geopolítica. Resultados: 5,1% desenvolveram metástases pulmonares e 14,3% tiveram fraturas patológicas. A recorrência local foi de 18,2%. Regiões economicamente menos favorecidas, como Norte e Nordeste, mostraram maiores incidências de metástases pulmonares (12,1%) e tumores avançados (Campanacci III, 88,9%). O Norte teve alta ocorrência de fraturas patológicas (33,3%), cirurgias extensas (61,1%) e amputações (27,8%). Nessas regiões, o tempo pré-cirúrgico foi mais longo (médias de 36 e 39 dias) comparado ao Sul e Sudeste (27 e 33 dias, respectivamente). Conclusões: Os resultados refletem disparidades regionais no Brasil, sugerindo que condições socioeconômicas influenciam os desfechos clínicos. Estes achados são importantes para melhorar o cuidado oncológico ortopédico em regiões desfavorecidas do país. Nível de Evidência III; Coorte Retrospectiva.

Giant cell tumor of bone: a multicenter epidemiological study in brazil / Tumor de células gigantes ósseas: estudo epidemiológico multicêntrico no brasil

ABSTRACT Introduction: Giant cell tumor of bone (GCTB) mainly affects young adults' long bone epiphyses, threatening bone strength and joint function. Surgery is the primary treatment, although post-surgery recurrence is significant. This study analyzes patient profiles, treatments, and outcomes for GCTB in Brazil. Methods: We retrospectively assessed local recurrence, metastasis, and treatment approaches in 643 GCTB patients across 16 Brazilian centers (1989-2021), considering regional differences. Results: 5.1% (n=33) developed pulmonary metastases, 14.3% (n=92) had pathological fractures, and the local recurrence rate was 18.2% (n=114). Higher rates of pulmonary metastases (12.1%) and advanced tumors (Campanacci III, 88.9%) were noted in lower-income North and Northeast regions. The North also had more pathological fractures (33.3%), extensive resections (61.1%), and amputations (27.8%). These regions faced longer surgical delays (36-39 days) than the South and Southeast (27-33 days). Conclusions: Our findings corroborate international data, underscoring regional disparities in Brazil that may lead to worse outcomes in disadvantaged areas. This highlights the need for improved orthopedic oncology care in Brazil's economically and structurally challenged regions. Level of Evidence III; Retrospective Cohort.

RESUMO Introdução: O tumor de células gigantes do osso (TCG) atinge principalmente epífises de ossos longos em adultos jovens, impactando a resistência óssea e a funcionalidade articular. O tratamento principal é cirúrgico, mas há significativa recorrência pós-operatória. Este estudo analisa o perfil de pacientes e tumores de TCG no Brasil, abordagens de tratamento e resultados. Métodos: Avaliamos retrospectivamente taxas de recorrência, metástase e tratamentos em 643 pacientes tratados em 16 centros brasileiros de 1989 a 2021, considerando a distribuição geopolítica. Resultados: 5,1% desenvolveram metástases pulmonares e 14,3% tiveram fraturas patológicas. A recorrência local foi de 18,2%. Regiões economicamente menos favorecidas, como Norte e Nordeste, mostraram maiores incidências de metástases pulmonares (12,1%) e tumores avançados (Campanacci III, 88,9%). O Norte teve alta ocorrência de fraturas patológicas (33,3%), cirurgias extensas (61,1%) e amputações (27,8%). Nessas regiões, o tempo pré-cirúrgico foi mais longo (médias de 36 e 39 dias) comparado ao Sul e Sudeste (27 e 33 dias, respectivamente). Conclusões: Os resultados refletem disparidades regionais no Brasil, sugerindo que condições socioeconômicas influenciam os desfechos clínicos. Estes achados são importantes para melhorar o cuidado oncológico ortopédico em regiões desfavorecidas do país. Nível de Evidência III; Coorte Retrospectiva.

Critically ill patients with COVID-19-associated acute kidney injury treated with kidney replacement therapy: Comparison between the first and second pandemic waves in São Paulo, Brazil.

INTRODUCTION: This study aimed to compare the characteristics and outcomes of critically ill patients with COVID-19-associated acute kidney injury (AKI) who were treated with kidney replacement therapy (KRT) in the first and second waves of the pandemic in the megalopolis of Sao Paulo, Brazil. METHODS: A multicenter retrospective study was conducted in 10 intensive care units (ICUs). Patients aged ≥18 years, and treated with KRT due to COVID-19-associated AKI were included. We compared demographic, laboratory and clinical data, KRT parameters and patient outcomes in the first and second COVID-19 waves. RESULTS: We assessed 656 patients (327 in the first wave and 329 in the second one). Second-wave patients were admitted later (7.1±5.0 vs. 5.6±3.9 days after the onset of symptoms, p<0.001), were younger (61.4±13.7 vs. 63.8±13.6 years, p = 0.023), had a lower frequency of diabetes (37.1% vs. 47.1%, p = 0.009) and obesity (29.5% vs. 40.0%, p = 0.007), had a greater need for vasopressors (93.3% vs. 84.6%, p<0.001) and mechanical ventilation (95.7% vs. 87.8%, p<0.001), and had higher lethality (84.8% vs. 72.7%, p<0.001) than first-wave patients. KRT quality markers were independently associated with a reduction in the OR for death in both pandemic waves. CONCLUSIONS: In the Sao Paulo megalopolis, the lethality of critically ill patients with COVID-19-associated AKI treated with KRT was higher in the second wave of the pandemic, despite these patients being younger and having fewer comorbidities. Potential factors related to this poor outcome were difficulties in health care access, lack of intra-hospital resources, delay vaccination and virus variants.

The Health Status of Horses Used for at Least Six Complete Cycles of Loxoscelic Antivenom Production.

Antivenom production against Loxosceles venom relies on horses being immunized and bled for plasma harvest. One horse can partake in several cycles of antivenom production, which will require years of constant venom and adjuvant inoculation and bleeding. The actual impact on the health of horses that participate in several antivenom-producing cycles is unknown. Therefore, this study aimed to evaluate the general health status of horses that underwent at least six cycles of loxoscelic antivenom production. Seven crossbred horses that had partaken in six to eight complete antivenom-producing cycles were used and established as the immunized group (IG). Under the same handling and general management, eleven horses were established as the control group (CG). The horses were evaluated regarding their general clinical status and had their blood sampled, and an ECG recorded. The IG presented lower RBC and PCV, despite keeping values within inferior limits for the species. Renal function was not impaired, and liver-related enzymes were higher than those in the CG, probably due to liver exertion from immunoglobulin synthesis. ECG showed some abnormalities in the IG, such as atrioventricular block and a wandering atrial pacemaker, corroborated by an increase in CK-MB. The cardiovascular abnormalities were mainly found in the horses that participated in several antivenom-producing cycles. The overall results indicate that these horses had some impairment of their general health status. Once available, some alternative, less toxic antigens should replace the venom for immunization of horses used for antivenom production.

Bovine Grafting: An Effective Alternative after Curettage of Benign Bone Tumors.

We retrospectively reviewed 28 patients (15 women and 13 men) with benign bone tumors or pseudotumors treated with curettage and filling with freeze-dried bovine bone graft Orthogen (Baumer S/A, São Paulo, Brazil). The aim of the study was to evaluate the rate of incorporation of Orthogen into the host bone, as well as to describe the outcomes of bone healing (quality, time, and complications). General characteristics, tumor volume, size, site, complications, percent filled, and healing quality at 6 and 12 months were assessed through radiographs. Mean patient age was 20.5 (range 4.7-75.1) years. The most common lesion type was simple bone cyst (12/28), and the most common sites were the tibia (7/28) and humerus (7/28). There were no postoperative pathologic fractures. Two cases (7.1%) of serous fluid leakage through the wound occurred. Mean cavity volume was 20.1 (range 2.7-101.4) cm3. At 6 and 12 months, 75% and 77.8% of cavities, respectively, showed complete bone healing. At 12 months, 81% of cavities filled >90% with graft showed complete bone healing vs. only 19% of those filled <90%. Filling with bovine bone graft resulted in few complications and excellent healing after curettage of benign bone tumors or pseudotumors. Complete healing occurred in most cases by 12 months. Cavities with a higher percentage of filling had a higher rate of complete radiographic incorporation.

The major role of junctional diversity in the horse antibody repertoire.

The antibody repertoire (Rep-seq) sequencing revolutionized the diversity of antigen B cell receptor studies, allowing deep and quantitative analysis to decipher the role of adaptive immunity in health and disease. Particularly, horse (Equus caballus) polyclonal antibodies have been produced and used since the century XIX to treat and prophylaxis diphtheria, tuberculosis, tetanus, pneumonia, and, more recently, COVID-19. However, our knowledge about the horse B cell receptors repertories is minimal. We present a deep horse antibody heavy chain repertoire (IGH) characterization of non-infected horses using NGS (Next generation sequencing). This study obtained a mean of 248,169 unique IgM clones and 66,141 unique IgG clones from four domestic adult horses. Rarefaction analysis showed sequence coverage was between 52 % and 82 % in IgM and IgG isotypes. We observed that besides horses antibody can use all functional IGHV genes, around 80 % of their antibodies use only three IGHV gene segments, and around 55 % use only one IGHJ gene segment. This limited VJ diversity seems to be compensated by the junctional diversity of these antibodies. We observed that the junctional diversity in horse antibodies is widespread, present in more than 90 % of horse antibodies. Besides this, the length of this region seems to be higher in horse antibodies than in other species. N1 and N2 nucleotides addition range from 0 to 111 nucleotides. In addition, around 45 % of the antibody clones have more than ten nucleotides in both the N1 and N2 junction regions. This diversity mechanism may be one of the most important in providing variability to the equine antibody repertoire. This study provides new insights regarding horse antibody composition, diversity generation, and particularities compared to other species, such as the frequency and length of N nucleotide addition. This study also points out the urgent need to better characterize TdT in horses and other species to better understand antibody repertoire characteristics.

Clinical Effects of the Immunization Protocol Using Loxosceles Venom in Naïve Horses.

Bites of brown spiders (Loxosceles spp.) are responsible for dermonecrotic lesions and potentially systemic envenoming that can lead to death. The only effective therapy is the use of the antivenom, usually produced in horses. However, little is known about the consequences of the systematic use of the Loxosceles venom and adjuvants and of the bleedings on antivenom-producing horses. Thus, the aim of this study was to evaluate the clinical changes in horses in their first immunization protocol for Loxosceles antivenom production. Eleven healthy horses, never immunized, were evaluated in three different periods: T0 (before immunization); T1 (after their first venom immunization); and T2 (after their first bleeding). Horses were clinically evaluated, sampled for blood, and underwent electrocardiographic (ECG) recordings. Several suppurated subcutaneous abscesses occurred due to the use of Freund's adjuvants and thrombophlebitis due to systematic venipunctures for the bleeding procedures. ECG showed arrhythmias in few horses in T2, such as an increase in T and R waves. In summary, the immunization protocol impacted on horses' health, especially after bleeding for antivenom procurement.

Production and Functional Evaluation of Anti-Loxosceles Sera Raised by Immunizations of Rabbits with Mutated Recombinant Phospholipases-D.

Loxoscelism is the clinical condition triggered after the bite of spiders of the genus Loxosceles. The main species involved in accidents in South America are L. intermedia, L. laeta, and L. gaucho. The only specific treatment is the anti-Loxosceles serum produced with crude venoms. As phospholipases D (PLDs) trigger most of the effects observed in accidents, we developed and evaluated second-generation sera using mutated PLDs as antigens. Three isoforms of PLDs with site-directed mutations without biological activities were used for rabbit immunizations: D32A-E34A (L. gaucho), W230A (L. intermedia), and H12A-H47A (L. laeta). Sera were produced using crude venoms of three species of Loxosceles enriched with mutated recombinant PLDs (MIX) or using only mutated PLDs (REC). Immunizations stimulated the immune system from the second immunization with higher antibody production in the REC group. In vivo neutralization assays demonstrated that both sera reduced edema and dermonecrosis caused by Loxosceles intermedia crude venom. Follow-up of animals during the immunization protocols and in the neutralization assays demonstrated that the mutated proteins and the sera are safe. Results demonstrate the potential of using mutated recombinant PLDs in total or partial replacement of Loxosceles venoms in animal immunizations to produce anti-Loxosceles sera for treatments of Loxoscelism.

A protective vaccine against the toxic activities following Brown spider accidents based on recombinant mutated phospholipases D as antigens.

Accidents involving Brown spiders are reported throughout the world. In the venom, the major toxins involved in the deleterious effects are phospholipases D (PLDs). In this work, recombinant mutated phospholipases D from three endemic species medically relevant in South America (Loxosceles intermedia, L. laeta and L. gaucho) were tested as antigens in a vaccination protocol. In such isoforms, key amino acid residues involved in catalysis, magnesium-ion coordination, and binding to substrates were replaced by Alanine (H12A-H47A, E32A-D34A and W230A). These mutations eliminated the phospholipase activity and reduced the generation of skin necrosis and edema to residual levels. Molecular modeling of mutated isoforms indicated that the three-dimensional structures, topologies, and surface charges did not undergo significant changes. Mutated isoforms were recognized by sera against the crude venoms. Vaccination protocols in rabbits using mutated isoforms generated a serum that recognized the native PLDs of crude venoms and neutralized dermonecrosis and edema induced by L. intermedia venom. Vaccination of mice prevented the lethal effects of L. intermedia crude venom. Furthermore, vaccination of rabbits prevented the cutaneous lesion triggered by the three venoms. These results indicate a great potential for mutated recombinant PLDs to be employed as antigens in developing protective vaccines for Loxoscelism.

Prospective Use of Brown Spider Venom Toxins as Therapeutic and Biotechnological Inputs.

Brown spider (genus Loxosceles) venoms are mainly composed of protein toxins used for predation and defense. Bites of these spiders most commonly produce a local dermonecrotic lesion with gravitational spread, edema and hemorrhage, which together are defined as cutaneous loxoscelism. Systemic loxoscelism, such as hematological abnormalities and renal injury, are less frequent but more lethal. Some Loxosceles venom toxins have already been isolated and extensively studied, such as phospholipases D (PLDs), which have been recombinantly expressed and were proven to reproduce toxic activities associated to the whole venom. PLDs have a notable potential to be engineered and converted in non-toxic antigens to produce a new generation of antivenoms or vaccines. PLDs also can serve as tools to discover inhibitors to be used as therapeutic agents. Other Loxosceles toxins have been identified and functionally characterized, such as hyaluronidases, allergen factor, serpin, TCTP and knottins (ICK peptides). All these toxins were produced as recombinant molecules and are biologically active molecules that can be used as tools for the potential development of chemical candidates to tackle many medical and biological threats, acting, for instance, as antitumoral, insecticides, analgesic, antigens for allergy tests and biochemical reagents for cell studies. In addition, these recombinant toxins may be useful to develop a rational therapy for loxoscelism. This review summarizes the main candidates for the development of drugs and biotechnological inputs that have been described in Brown spider venoms.

Clinical impact of brachyury expression in Ewing sarcoma patients.

PURPOSE: The T-box transcription factor brachyury has been demonstrated as a prognostic factor in a variety of cancer types and considered a novel oncotarget in solid tumors. Brachyury acts as a regulator of the epithelial-mesenchymal transition (EMT) process, leading to more aggressive behavior and poorer prognosis. However, recent literature evidence suggests a tumor suppressor role in other neoplasms. In the present study, we aimed to study brachyury expression and its prognostic impact in Ewing sarcoma, an aggressive neoplasm of young individuals. METHODS: We analyzed the expression of brachyury by immunohistochemistry in a series of 96 Ewing sarcomas in a tissue microarray and investigated the association of the protein expression with the clinical parameters and overall survival. RESULTS: More than half of the cases (51%, n â€‹= â€‹49) depicted positive nuclear brachyury expression, while a lack of expression was observed in 49% (n â€‹= â€‹47) of cases. Nuclear brachyury staining was significantly associated with non-white ethnicity (p â€‹= â€‹0.04) and axial localization (p â€‹= â€‹0.025). Importantly, lack of brachyury expression was significantly associated with lower overall survival in multivariate analyses (hazard ratio - HR: 2.227, p â€‹= â€‹0.008). CONCLUSIONS: Our findings indicate, that brachyury is an independent prognostic biomarker in Ewing sarcoma, which might suggest a tumor suppressor role and which yet to be fully elucidated.

Production of a novel recombinant brown spider hyaluronidase in baculovirus-infected insect cells.

Hyaluronidases are low expressed toxins of brown spider venoms, but, as highly active molecules, they present an important role as spreading factors. By degrading extracellular matrix components, these enzymes favor the diffusion of toxins in the affected tissue and at systemic level. Here, a novel isoform of hyaluronidase of Loxosceles intermedia Mello-Leitão (1934) venom was cloned, expressed in a baculovirus-insect cell expression system and fully active purified. This recombinant enzyme, named LiHyal2 (Loxosceles intermedia Hyaluronidase isoform 2), shares high identity with hyaluronidases of other spiders and scorpions. The catalytic and sugar binding amino acid residues are conserved in LiHyal2, human, and honeybee venom hyaluronidases and the molecular model of LiHyal2 shares major similarities with their crystal structures, including the active site. LiHyal2 was expressed as a 45 kDa protein and degraded hyaluronic acid (HA) and chondroitin sulphate as demonstrated by HA zymography and agarose gel electrophoresis. Lectin blot analysis revealed that LiHyal2 is post-translationally modified by the addition of high mannose N-linked carbohydrates. In vivo experiments showed that LiHyal2 potentialize dermonecrosis and edema induced by a recombinant phospholipase-D (PLD) of L. intermedia venom, as well as enhance the increase in capillary permeability triggered by this PLD, indicating that these toxins act synergistically during envenomation. Altogether, these results introduce a novel approach to express spider recombinant toxins, contribute to the elucidation of brown spider venom mechanisms and add to the development of a more specific treatment of envenomation victims.

Brown Spiders' Phospholipases-D with Potential Therapeutic Applications: Functional Assessment of Mutant Isoforms.

Phospholipases-D (PLDs) found in Loxosceles spiders' venoms are responsible for the dermonecrosis triggered by envenomation. PLDs can also induce other local and systemic effects, such as massive inflammatory response, edema, and hemolysis. Recombinant PLDs reproduce all of the deleterious effects induced by Loxosceles whole venoms. Herein, wild type and mutant PLDs of two species involved in accidents-L. gaucho and L. laeta-were recombinantly expressed and characterized. The mutations are related to amino acid residues relevant for catalysis (H12-H47), magnesium ion coordination (E32-D34) and binding to phospholipid substrates (Y228 and Y228-Y229-W230). Circular dichroism and structural data demonstrated that the mutant isoforms did not undergo significant structural changes. Immunoassays showed that mutant PLDs exhibit conserved epitopes and kept their antigenic properties despite the mutations. Both in vitro (sphingomyelinase activity and hemolysis) and in vivo (capillary permeability, dermonecrotic activity, and histopathological analysis) assays showed that the PLDs with mutations H12-H47, E32-D34, and Y228-Y229-W230 displayed only residual activities. Results indicate that these mutant toxins are suitable for use as antigens to obtain neutralizing antisera with enhanced properties since they will be based on the most deleterious toxins in the venom and without causing severe harmful effects to the animals in which these sera are produced.

Signs of impending rupture in abdominal aortic and iliac artery aneurysms by computed tomography: Outcomes in 41 patients.

OBJECTIVES: This study aimed to determine the prevalence of signs of impending rupture (SIR) in asymptomatic patients with abdominal aortic and iliac artery aneurysms, and to evaluate whether these signs were associated with rupture in asymptomatic patients. METHODS: This was a retrospective study of patients with abdominal aortic and iliac artery aneurysms identified on computed tomography (CT) over a 10-year period in a single center. The CT scans were reviewed by two reviewers, and patients with SIR were assigned to one of three groups: (1) early symptomatic (ES), (2) late symptomatic (LS), and (3) always asymptomatic (AA). The four main SIR described in the literature were investigated: 1) crescent sign, 2) focal wall discontinuity of circumferential calcifications, 3) aortic bulges or blebs, and 4) aortic draping. RESULTS: From a total of 759 aortic and iliac aneurysm reports on 2226 CT scans, we identified 41 patients with at least one SIR, and a prevalence of 4.14% in asymptomatic patients. Focal wall discontinuity of circumferential calcifications was the most common sign, and it was present in 46.3% of these patients (19/41); among these, 26 were repaired (ES: 9, LS: 2, AA: 15). Eleven asymptomatic patients underwent follow-up CT. The aneurysm increased in size in 6 of the 11 (54.5%) patients, and three ruptured (all with discontinuity of calcifications), one of which had no increase in diameter. CONCLUSIONS: The presence of focal wall discontinuity of circumferential calcifications was the most common SIR. There was a prevalence of all signs in less than 5% of asymptomatic patients. In unrepaired patients, the signs could be observed on follow-up CT scans with an increase in aneurysm size, indicating that the presence of SIR alone in the absence of other clinical factors or aneurysm characteristics is an insufficient indication for surgery.

Signs of impending rupture in abdominal aortic and iliac artery aneurysms by computed tomography: Outcomes in 41 patients

OBJECTIVES: This study aimed to determine the prevalence of signs of impending rupture (SIR) in asymptomatic patients with abdominal aortic and iliac artery aneurysms, and to evaluate whether these signs were associated with rupture in asymptomatic patients. METHODS: This was a retrospective study of patients with abdominal aortic and iliac artery aneurysms identified on computed tomography (CT) over a 10-year period in a single center. The CT scans were reviewed by two reviewers, and patients with SIR were assigned to one of three groups: (1) early symptomatic (ES), (2) late symptomatic (LS), and (3) always asymptomatic (AA). The four main SIR described in the literature were investigated: 1) crescent sign, 2) focal wall discontinuity of circumferential calcifications, 3) aortic bulges or blebs, and 4) aortic draping. RESULTS: From a total of 759 aortic and iliac aneurysm reports on 2226 CT scans, we identified 41 patients with at least one SIR, and a prevalence of 4.14% in asymptomatic patients. Focal wall discontinuity of circumferential calcifications was the most common sign, and it was present in 46.3% of these patients (19/41); among these, 26 were repaired (ES: 9, LS: 2, AA: 15). Eleven asymptomatic patients underwent follow-up CT. The aneurysm increased in size in 6 of the 11 (54.5%) patients, and three ruptured (all with discontinuity of calcifications), one of which had no increase in diameter. CONCLUSIONS: The presence of focal wall discontinuity of circumferential calcifications was the most common SIR. There was a prevalence of all signs in less than 5% of asymptomatic patients. In unrepaired patients, the signs could be observed on follow-up CT scans with an increase in aneurysm size, indicating that the presence of SIR alone in the absence of other clinical factors or aneurysm characteristics is an insufficient indication for surgery.

Forty Years of the Description of Brown Spider Venom Phospholipases-D.

Spiders of the genus Loxosceles, popularly known as Brown spiders, are considered a serious public health issue, especially in regions of hot or temperate climates, such as parts of North and South America. Although the venoms of these arachnids are complex in molecular composition, often containing proteins with distinct biochemical characteristics, the literature has primarily described a family of toxins, the Phospholipases-D (PLDs), which are highly conserved in all Loxosceles species. PLDs trigger most of the major clinical symptoms of loxoscelism i.e., dermonecrosis, thrombocytopenia, hemolysis, and acute renal failure. The key role played by PLDs in the symptomatology of loxoscelism was first described 40 years ago, when researches purified a hemolytic toxin that cleaved sphingomyelin and generated choline, and was referred to as a Sphingomyelinase-D, which was subsequently changed to Phospholipase-D when it was demonstrated that the enzyme also cleaved other cellular phospholipids. In this review, we present the information gleaned over the last 40 years about PLDs from Loxosceles venoms especially with regard to the production and characterization of recombinant isoforms. The history of obtaining these toxins is discussed, as well as their molecular organization and mechanisms of interaction with their substrates. We will address cellular biology aspects of these toxins and how they can be used in the development of drugs to address inflammatory processes and loxoscelism. Present and future aspects of loxoscelism diagnosis will be discussed, as well as their biotechnological applications and actions expected for the future in this field.

Expression of neurotrophins and their receptors in primary osteosarcoma. / Expressão de neurotrofinas e de seus receptores no osteossarcoma primário.

OBJECTIVE: to determine the expression of neurotrophins and their tyrosine-kinase receptors in patients with osteosarcoma (OS) and their correlation with clinical outcomes. METHODS: we applied immunohistochemistry to biopsy specimens of patients consecutively treated for primary OS at a single institution between 2002 and 2015, analyzing them for expression receptors of tyrosine kinase A and B (TrKA and TrKB), neural growth factor (NGF) and brain derived neurotrophic factor (BDNF). Independently, two pathologists classified the immunohistochemical markers as negative (negative or weak focal) or positive (moderate focal/diffuse or strong focal/diffuse). RESULTS: we analyzed data from 19 patients (10 females and 9 males), with median age of 12 years (5 to 17.3). Tumors' location were 83.3% in the lower limbs, and 63.2% of patients had metastases at diagnosis. Five-year overall survival was 55.3%. BDNF was positive in 16 patients (84%) and NGF in 14 (73%). TrKA and TrKB presented positive staining in four (21,1%) and eight (42,1%) patients, respectively. Survival analysis showed no significant difference between TrK receptors and neurotrophins. CONCLUSION: primary OS samples express neurotrophins and TrK receptors by immunohistochemistry. Future studies should explore their role in OS pathogenesis and determine their prognostic significance in larger cohorts.

OBJETIVO: determinar a expressão de neurotrofinas e seus receptores tirosina quinases em pacientes com osteossarcoma (OS) e sua correlação com desfechos clínicos. MÉTODOS: biópsias de tumores primários de pacientes com OS tratados em uma única instituição, consecutivamente, entre 2002 e 2015, foram analisados através de imuno-histoquímica para expressão de receptores de tirosina quinase A e B (TrKA e TrKB), fator de crescimento neural (NGF) e fator neurotrófico derivado do cérebro (BDNF). De forma independente, dois patologistas classificaram os marcadores de imuno-histoquímica como negativos (negativos e focais fracos) ou positivos (moderado focal/difuso ou forte focal/difuso). RESULTADOS: foram analisados dados de 19 pacientes (10 do sexo feminino e 9 do masculino) com mediana de idade de 12 anos (5 a 17,3 anos). Dos tumores, 83,3% estavam localizados em membros inferiores e 63,2% dos pacientes eram metastáticos ao diagnóstico. A sobrevida global em cinco anos foi de 55,3%. BDNF foi positivo em 16 pacientes (84%) e NGF em 14 pacientes (73%). TrKA e TrKB apresentaram coloração positiva em quatro (21,1%) e oito (42,1%) pacientes, respectivamente. A análise de sobrevida não demonstrou diferença significativa entre receptores TrK e neurotrofinas. CONCLUSÃO: amostras de OS primário expressam neurotrofinas e receptores TrK através de imuno-histoquímica. Estudos futuros podem auxiliar na identificação do papel das mesmas na patogênese do OS e determinar se há possível correlação prognóstica.

Late neurological deficit after attempted PICC insertion in the arm.

INTRODUCTION:: Insertion of central catheters in peripheral vessels is a common procedure performed for a variety of indications, including parenteral nutrition. Hyperemesis gravidarum may require parenteral nutrition in exceptional situations. Although globally safe, insertion of peripherally inserted central catheters (PICCs) can lead to complications. CASE REPORT:: We describe a case of a pregnant woman who required parenteral nutrition and underwent an unsuccessful PICC insertion attempt resulting in arterial puncture, and who 34 days later presented with right upper limb paresthesia. A pseudoaneurysm with nerve compression was diagnosed and treated by open surgery, without maternal or fetal complications. CONCLUSION:: We recommend active surveillance ultrasound (e.g. in the first 24-48 h) of unsuccessful PICC insertion attempts, because late complications may occur and require invasive procedures for treatment.

Next-generation sequencing reveals new insights about gene usage and CDR-H3 composition in the horse antibody repertoire.

Horse serum antibodies have been used for greater than a century for the treatment and prophylaxis of infectious diseases and envenomations. Little is known, however, about the immunogenetic diversity that produces horse serum antibodies. Here, we employed next-generation sequencing for a first-in-kind comprehensive analysis of the equine B-cell repertoire. Nearly 45,000 and 30,000 clonotypes were obtained for the heavy-chain (IGH) and lambda light-chain (IGL) loci, respectively. We observed skewed use of the common subgroups IGHV2 (92.49%) and IGLV8 (82.50%), consistent with previous reports, but also novel use of the rare genes IGHV6S1 and IGLV4S2. CDR-H3 amino acid composition revealed different amino acid patterns at positions 106 and 116 compared to human, rabbit, and mouse, suggesting that an extended conformation predominates among horse CDR-H3 loops. Our analysis provides new insights regarding the mechanisms employed to generate antibody diversity in the horse, and could be applicable to the optimized design of synthetic antibodies intended for future therapeutic use.

Expressão de neurotrofinas e de seus receptores no osteossarcoma primário / Expression of neurotrophins and their receptors in primary osteosarcoma

RESUMO Objetivo: determinar a expressão de neurotrofinas e seus receptores tirosina quinases em pacientes com osteossarcoma (OS) e sua correlação com desfechos clínicos. Métodos: biópsias de tumores primários de pacientes com OS tratados em uma única instituição, consecutivamente, entre 2002 e 2015, foram analisados através de imuno-histoquímica para expressão de receptores de tirosina quinase A e B (TrKA e TrKB), fator de crescimento neural (NGF) e fator neurotrófico derivado do cérebro (BDNF). De forma independente, dois patologistas classificaram os marcadores de imuno-histoquímica como negativos (negativos e focais fracos) ou positivos (moderado focal/difuso ou forte focal/difuso). Resultados: foram analisados dados de 19 pacientes (10 do sexo feminino e 9 do masculino) com mediana de idade de 12 anos (5 a 17,3 anos). Dos tumores, 83,3% estavam localizados em membros inferiores e 63,2% dos pacientes eram metastáticos ao diagnóstico. A sobrevida global em cinco anos foi de 55,3%. BDNF foi positivo em 16 pacientes (84%) e NGF em 14 pacientes (73%). TrKA e TrKB apresentaram coloração positiva em quatro (21,1%) e oito (42,1%) pacientes, respectivamente. A análise de sobrevida não demonstrou diferença significativa entre receptores TrK e neurotrofinas. Conclusão: amostras de OS primário expressam neurotrofinas e receptores TrK através de imuno-histoquímica. Estudos futuros podem auxiliar na identificação do papel das mesmas na patogênese do OS e determinar se há possível correlação prognóstica.

ABSTRACT Objective: to determine the expression of neurotrophins and their tyrosine-kinase receptors in patients with osteosarcoma (OS) and their correlation with clinical outcomes. Methods: we applied immunohistochemistry to biopsy specimens of patients consecutively treated for primary OS at a single institution between 2002 and 2015, analyzing them for expression receptors of tyrosine kinase A and B (TrKA and TrKB), neural growth factor (NGF) and brain derived neurotrophic factor (BDNF). Independently, two pathologists classified the immunohistochemical markers as negative (negative or weak focal) or positive (moderate focal/diffuse or strong focal/diffuse). Results: we analyzed data from 19 patients (10 females and 9 males), with median age of 12 years (5 to 17.3). Tumors' location were 83.3% in the lower limbs, and 63.2% of patients had metastases at diagnosis. Five-year overall survival was 55.3%. BDNF was positive in 16 patients (84%) and NGF in 14 (73%). TrKA and TrKB presented positive staining in four (21,1%) and eight (42,1%) patients, respectively. Survival analysis showed no significant difference between TrK receptors and neurotrophins. Conclusion: primary OS samples express neurotrophins and TrK receptors by immunohistochemistry. Future studies should explore their role in OS pathogenesis and determine their prognostic significance in larger cohorts.