Advances in the Pathology and Molecular Biology of Sarcomas and the Impact on Treatment.

Sarcomas are a complex group of childhood and adult neoplasms with differentiation towards mesenchymal tissues that can occur at almost every anatomic site. Although pathologically diverse, they frequently show similar clinical presentations and radiological findings, such that correct histopathologic diagnosis, utilising the appropriate ancillary immunohistochemical and molecular techniques, underpins their management. This article gives an overview of the pathology, coupled with recent advances in molecular biology, of a selection of soft tissue sarcomas from a clinicopathological perspective, discussing histopathological diagnosis with developments in molecular diagnosis and the incorporation of these findings into diagnostic practice and current and potential targeted treatments.

Phosphoproteomics in translational research: a sarcoma perspective.

Phosphoproteomics has been extensively used as a preclinical research tool to characterize the phosphorylated components of the cancer proteome. Advances in the field have yielded insights into new drug targets, mechanisms of disease progression and drug resistance, and biomarker discovery. However, application of this technology to clinical research has been challenging because of practical issues relating to specimen integrity and tumour heterogeneity. Beyond these limitations, phosphoproteomics has the potential to play a pivotal role in translational studies and contribute to advances in different tumour groups, including rare disease sites like sarcoma. In this review, we propose that deploying phosphoproteomic technologies in translational research may facilitate the identification of better defined predictive biomarkers for patient stratification, inform drug selection in umbrella trials and identify new combinations to overcome drug resistance. We provide an overview of current phosphoproteomic technologies, such as affinity-based assays and mass spectrometry-based approaches, and discuss their advantages and limitations. We use sarcoma as an example to illustrate the current challenges in evaluating targeted kinase therapies in clinical trials. We then highlight useful lessons from preclinical studies in sarcoma biology to demonstrate how phosphoproteomics may address some of these challenges. Finally, we conclude by offering a perspective and list the key measures required to translate and benchmark a largely preclinical technology into a useful tool for translational research.

Detection of T lymphocytes in intestine of broiler chicks treated with Lactobacillus spp. and challenged with Salmonella enterica serovar Enteritidis.

The expression of immune response in the form of leukocytic infiltrate by CD3+, CD4+, and CD8+ cells in the epithelium and in the intestinal lamina propria of chicks was studied in the present work by means of immunohistochemical reaction. The chicks were treated with Lactobacillus spp. or cecal microflora (CM) and experimentally challenged or not with Salmonella enterica serovar Enteritidis. The 320 birds utilized were divided into 4 groups containing 80 chicks each and submitted to treatments with Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus acidophilus, and CM. Each group was subdivided into 4 subgroups of 20 birds each and classified into a subgroup that did not receive treatment (negative control), subgroup treated, subgroup treated and challenged with Salmonella Enteritidis, and subgroup only challenged with Salmonella Enteritidis (positive control). The results obtained show that the treatment with L. reuteri, L. salivarius, L. acidophilus, or CM and challenged or not with Salmonella Enteritidis determine immune response in the form of leukocytic infiltrate by CD3+ and CD8+ lymphocytes followed by CD4+ in the epithelium and in the lamina propria of the duodenum, jejunum, and cecum of chicks up to 12 d of age. The quantity of CD3+ lymphocytes was significantly higher (P < 0.05) in the intestine of chicks treated with L. acidophilus or CM and challenged or not with Salmonella Enteritidis; however, the higher quantity of CD8+ lymphocytes was in the intestine of chicks treated with CM and challenged with Salmonella Enteritidis. The duodenum was the segment in which the immune response by T cells (CD3+, CD4+, and CD8+) was stimulated with the greatest intensity, followed by, respectively, the jejunum and cecum. The quantity of CD3+ lymphocytes present in the duodenum, jejunum, and cecum increases with the age of chicks, independent of the stimulus determined by treatments or challenge.

Tissue distribution of calcineurin and its sensitivity to inhibition by cyclosporine.

The immunosuppressive activity of cyclosporine is mediated by inhibiting calcineurin phosphatase. However, calcineurin is widely distributed in other tissues. We examined the degree of calcineurin inhibition by cyclosporine in various tissues. In vitro, the cyclosporine concentration inhibiting 50% (IC50) of calcineurin was to approximately 10 ng/mL in human and mouse leukocytes suspensions. In vitro and in vivo IC50s of cyclosporine in homogenates of mouse kidney, heart, liver, testis, and spleen were also comparable (9-48 ng/mL). The maximum calcineurin inhibition by cyclosporine varied, from 83 to 95% of calcineurin activity in spleen, kidney, liver, and testis to 60% in heart and only 10% in brain. Maximum calcineurin inhibition was increased by the addition of cyclophilin A, indicating that cyclophilin concentrations were limiting in some tissues, at least in this assay. Western analysis of mouse tissues showed significantly less cyclophilin in heart than other tissues. cyclosporine concentrations per weight of tissue protein were highest in kidney and liver and lowest in brain and testis after oral dosing, with intermediate levels in spleen, heart, and whole blood. Thus each cyclosporine dose produces rapid and wide-spread inhibition of calcineurin in tissues, with differences in total susceptibility of each tissue.

The temporal profile of calcineurin inhibition by cyclosporine in vivo.

BACKGROUND: Cyclosporine (CsA) acts by inhibiting the phosphatase calcineurin (CN), but the time course and extent of inhibition in vivo are unknown. We examined the effect of single oral CsA doses on CN activity in humans and mice in vivo. METHODS: In humans, blood CsA levels were determined and CN activity was measured in whole blood and in blood leukocytes of patients up to 12 hr after CsA dosing (just before the second dose). Samples were collected from patients receiving a first single dose (2.5 mg/kg), and up to 14 days later after repeated dosing. In mice, after CsA dosing (12.5-200 mg/kg) by oral gavage, CsA levels in blood and tissue (spleen, kidney) were determined and CN activity was measured in spleen and kidney. RESULTS: In humans, peak CsA levels of 800-2285 microg/L at 1-2 hr produced 70-96% CN inhibition. Inhibition correlated closely with the rise and fall of CsA levels with no observable lag at the times sampled. Repeated doses showed similar CN inhibition to first dose, with no significant adaptation. In mice, CsA peaked at 1 hr in blood, spleen, and kidney, with higher concentrations in spleen and kidney than in blood. CN inhibition closely followed CsA concentrations/doses, and was greater in kidney than spleen. CONCLUSION: Thus CsA induces partial CN inhibition that varies directly with the blood and tissue levels, and may be greater in some tissues due to higher drug accumulation. The high CsA concentrations and CN inhibition in kidney may be relevant to nephrotoxicity.

Calcineurin and the biological effect of cyclosporine and tacrolimus.

The mechanism of the immunosuppressive effect of CyA and FK 506 can be monitored in vivo in humans. The picture emerging is of a close relationship between drug concentrations and CN inhibition. But many puzzles of the drugs remain. What is the role of CyA in the activation of transforming growth factor-beta (TGF-beta), particularly in relationship to nephrotoxicity and fibrogenesis? How important are the anti-inflammatory (non T) effects of CyA, and which cells do they operate in? Are there effects of CyA and FK 506 all attributable to CN inhibition, and how much of them are mediated through the NFATC family of transcription factors? Finally, it would be useful to know what the inhibitory effects of CyA are on tolerance and negative regulatory events.

Regulation of IFN-gamma expression in vivo. IFN-gamma up-regulates expression of its mRNA in normal and lipopolysaccharide-stimulated mice.

Immune responses to such stimuli as tissue injury, infection, and allografting result in localized IFN-gamma expression. Autoregulation of cytokine expression has been described for some cytokines in vitro, but whether this occurs in vivo is unknown. We therefore examined the role of murine IFN-gamma in the regulation of its own expression in vivo after stimulation with bacterial LPS. This agent is known to induce IFN-gamma expression in both spleen and kidney in a T cell-independent, cyclosporine-sensitive manner. We found that concomitant administration of a neutralizing mAb to IFN-gamma inhibited not only the MHC expression induced by LPS but also the increased IFN-gamma mRNA expression, suggesting an autoregulatory role for IFN-gamma. Inhibition was dose dependent and observed in both spleen and kidney. The effect was not seen with a neutralizing anti-IL-3 mAb, demonstrating specificity. The inhibition of IFN-gamma mRNA expression by the anti-IFN-gamma mAb occurred in both T cell-deficient athymic nude mice and their normal controls, suggesting that the autoamplification of IFN-gamma mRNA in vivo is T cell independent. Administration of rIFN-gamma to unstimulated mice induced IFN-gamma mRNA expression in spleen and kidney, supporting the conclusion that IFN-gamma up-regulates expression of its mRNA. Exposure of resting murine splenocytes to concentrations of rIFN-gamma as low as 10 U/ml in vitro induced expression of IFN-gamma mRNA. Thus, in vivo IFN-gamma may participate in an autoregulatory loop to amplify the amount of IFN-gamma expressed both at the site of local inflammation and at remote sites. This would have relevance in the mechanism by which the host defends itself against and prevents dissemination of an infectious agent.

[Malignant external otitis caused by Pseudomonas aeruginosa. Report of 3 cases]. / Otite externa maligna por Pseudomonas aeruginosa. Relato de três casos.

The cases of three patients aged over sixty, diabetic for a long time and having clinical, otological, neurological and microbiological findings of malignant external otitis by Pseudomonas aeruginosa are registered. The first patient, a 62 years old woman, had important neurological alterations, such as proven P. aeruginosa meningitis and involvement of the 6th, 7th, 8th, 9th and 10th left cranial nerves. Although mortality and morbidity rates of cases with these characteristics are reported to be high, this patient survived. The second was a 64 years old male with the classical complaints of this condition consisting of persistent and intensive ear pain, serous purulent discharge and an emerging tumourous lesion in the left external ear canal. Good results were obtained with the surgical treatment administered along with an antimicrobial plan which remained incomplete because the patient has requested his discharge from the hospital. In less than one month, he was again hospitalized already presenting neurological manifestations (paralysis of the 6th cranial nerve) following a fatal course in a few days. The third was also a male patient, 70 years old, whose initial complaints occurred after a month a butterfly penetrated his right ear during his sleep. In spite of the presence of neurological troubles (facial paralysis and stupor) he survived with a prolonged antibiotic therapy. After a literature revision on this pathology, considerations are made concerning its pathogeny, clinical findings, diagnosis, therapy and prognosis.

Criptococose: meningoencefalite subaguda com hidrocefalia e hidrocefalia oculta, como expressoes clinicas - a proposito de tres casos. / Cryptococcosis: subacute meningoencephalitis with hydrocephalus and occult hydrocephalus as clinical expressions - apropos of 3 cases

Os autores registram tres casos de hidrocefalia secundaria a meningoencefalite subaguda de etiologia criptococica. Nao tendo o fungo sido encontrado no liquido cefalorraquidiano, foi iniciado, em todos os tres enfermos, esquema triplice contra tuberculose associado a corticoide. Em dois destes pacientes, derivacao ventriculo-peritoneal precedeu o reconhecimento da etiologia fungica. No terceiro doente, foi realizada drenagem ventricular de emergencia, substituida posteriomente por derivacao ventriculo-atrial. As meningites que obedecem a esta etiologia sao muito mais frequentes do que se julga e, quando a puncao lombar nao revela o fungo, e importante colher liquor da cisterna magna ou dos ventriculos para exame direto e cultura. Estes casos e outros indicam a necessidade da implantacao, em nosso meio, do diagnostico sorologico da criptococose.Por outro lado, trazem o ensinamento de que, em presenca de meningoencefalites subagudas e/ou de hidrocefalia, devem ser esgotados todos os recursos disponiveis para a procura desta etiologia, o que representa reais vantagens, pois o tratameto com anfotericina B e 5-fluorocitosina pode eximir o paciente da insercao de derivacao e de suas complicacoes, bem como evitar a dsseminacao da micose