Management and outcomes of anorectal infection in the cancer patient

Background. The objective of this retrospective cohort study is to examine the presentation and outcomes for a contemporary series of cancer patients with anorectal infection. In addition, we seek to identify factors which are associated with surgical intervention. ^ Methods. The study cohort was identified from ICD-9 codes for diagnosis of infection of the anal and rectal region and patients who underwent a surgical oncology consultation between 1/2000 and 12/2006. Clinical presentation, treatment rendered, and outcomes were retrospectively recorded. ^ Results. Of the 100 patients evaluated by the surgical oncology service for anorectal infection, 42 were treated non-operatively and 58 underwent surgical intervention. Factors associated with surgical intervention based on logistic multivariable analysis included the diagnosis of an abscess (odds ratio [OR] 10.5; 95% confidence interval [CI] 2.9-38.5) and the documentation of erythema on physical examination (OR 3.1; 95% CI 1.1-8.4). Thrombocytopenia (platelets < 50,000) was associated with non-operative management (OR 0.3; 95% CI 0.1-0.7). Incision and drainage was the most common surgical procedure (79%) while a wide debridement for a necrotizing soft tissue infection was required in 2 patients. Infection-specific 90-day mortality was only 1% (N=1). However, the median overall survival for the entire cohort was only 14.4 months (95% CI 7.9-19.5). ^ Conclusions. Non-operative management is a reasonable treatment option for anorectal infection in patients with cancer. Identification of an abscess, erythema on physical exam, and thrombocytopenia were associated with management strategy. Although rare, necrotizing soft tissue infections are associated with significant mortality. ^

Characterization of Staphylococcus aureus adhesins important for respiratory tract infection

Staphylococcus aureus is a leading cause of lower respiratory tract infections in both adult and pediatric populations. In the past two decades, reports have described emergent incidence of severe necrotizing pneumonia in previously healthy individuals, frequently caused by antibiotic resistant strains. Additionally, S. aureus remains the most common cause of ventilator-associated pneumonia, contributing morbidity and mortality in intensive care units. As treatment of infection is made more difficult by the resistance to multiple antibiotics including vancomycin, there is a pressing need for novel strategies to prevent and treat S. aureus infections. Targeting essential mechanisms that promote infection such as adhesion, colonization, invasion, evasion of immune system and signaling may lead to inhibition of pathogenic surge. Staphylococcal adhesins of the MSCRAMM family (microbial surface components recognizing adherent matrix molecules) represent viable targets for such investigations. Understanding the molecular mechanism of binding is the first step toward the development of such therapies. Analysis of bacterial strains isolated from patients with staphylococcal pneumonia show increased expression of protein A, SdrD, SdrC and ClfB, cell surface proteins members of the MSCRAMM family. In this study the interaction of these MSCRAMMs with candidate ligands has been examined. We found that SdrD mediates S. aureus adherence to the lung epithelial cell line A549. Consistently, bacteria expressing SdrD have increased persistence in the lungs of infected mice after bronchoalveolar lavage in comparison with bacteria lacking this protein. Inhibition studies revealed that bacterial attachment can be abolished using neutralizing antibodies against SdrD. Using phage display, neurexin β isoforms were identified as SdrC binding partners. Previous reports postulated that MSCRAMMS bind their ligands by a 'dock, lock and latch' mechanism of interaction. Our data suggested that ClfB, an MSCRAMM responsible for nasal colonization, binds cytokeratin 10 by a 'dock and lock' variant of this model, in which the 'latching' event is not necessary. In summary, we have characterized aspects of molecular interaction between several MSCRAMMS and host components. We hope that continued delineation of these interactions will lead to identification of novel therapeutic targets or preventive strategies against S. aureus infections. ^

Direct visualization of antigen-specific T cells: HTLV-1 Tax11–19- specific CD8+ T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients

Human T lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropic spastic paraparesis is a demyelinating inflammatory neurologic disease associated with HTLV-1 infection. HTLV-1 Tax11–19-specific cytotoxic T cells have been isolated from HLA-A2-positive patients. We have used a peptide-loaded soluble HLA-A2–Ig complex to directly visualize HTLV-1 Tax11–19-specific T cells from peripheral blood and cerebrospinal fluid without in vitro stimulation. Five of six HTLV-1-associated myelopathy/tropic spastic paraparesis patients carried a significant number (up to 13.87%) of CD8+ lymphocytes specific for the HTLV-1 Tax11–19 peptide in their peripheral blood, which were not found in healthy controls. Simultaneous comparison of peripheral blood and cerebrospinal fluid from one patient revealed 2.5-fold more Tax11–19-specific T cells in the cerebrospinal fluid (23.7% vs. 9.4% in peripheral blood lymphocyte). Tax11–19-specific T cells were seen consistently over a 9-yr time course in one patient as far as 19 yrs after the onset of clinical symptoms. Further analysis of HTLV-1 Tax11–19-specific CD8+ T lymphocytes in HAM/TSP patients showed different expression patterns of activation markers, intracellular TNF-α and γ-interferon depending on the severity of the disease. Thus, visualization of antigen-specific T cells demonstrates that HTLV-1 Tax11–19-specific CD8+ T cells are activated, persist during the chronic phase of the disease, and accumulate in cerebrospinal fluid, showing their pivotal role in the pathogenesis of this neurologic disease.

Inhibition of Rho Is Required for cAMP-induced Melanoma Cell Differentiation

Up-regulation of the cAMP pathway by forskolin or α-melanocyte stimulating hormone induces melanocyte and melanoma cell differentiation characterized by stimulation of melanin synthesis and dendrite development. Here we show that forskolin-induced dendricity is associated to a disassembly of actin stress fibers. Since Rho controls actin organization, we studied the role of this guanosine triphosphate (GTP)-binding protein in cAMP-induced dendrite formation. Clostridium botulinum C3 exotransferase, which inhibits Rho, mimicked the effect of forskolin in promoting dendricity and stress fiber disruption, while the Escherichia coli toxin cytotoxic necrotizing factor-1 (CNF-1), which activates Rho and the expression of a constitutively active Rho mutant, blocked forskolin-induced dendrite outgrowth. In addition, overexpression of a constitutively active form of the Rho target p160 Rho-kinase (P160ROCK) prevented the dendritogenic effects of cAMP. Our results suggest that inhibition of Rho and of its target p160ROCK are required events for cAMP-induced dendrite outgrowth in B16 cells. Furthermore, we present evidence that Rho is involved in the regulation of melanogenesis. Indeed, Rho inactivation enhanced the cAMP stimulation of tyrosinase gene transcription and protein expression, while Rho constitutive activation impaired these cAMP-induced effects. This reveals that, in addition to controlling dendricity, Rho also participates in the regulation of melanin synthesis by cAMP.

Rho-dependent Regulation of Cell Spreading by the Tetraspan Membrane Protein Gas3/PMP22

Gas3/PMP22 plays a crucial role in regulating myelin formation and maintenance, and different genetic alterations in gas3/PMP22 are responsible for a set of human peripheral neuropathies. We have previously demonstrated that Gas3/PMP22 could regulate susceptibility to apoptosis in NIH3T3 cells but not in REF 52 cells. In this report we demonstrate that when the apoptotic response triggered by gas3/PMP22 was counteracted by Bcl-2 coexpression, morphological changes were observed. Time-lapse analysis confirmed that Gas3/PMP22 can modulate cell spreading, and this effect was strengthened after inhibition of phosphoinositide 3-kinase. Using the active form of the small GTPase RhoA, we have been able to dissect the different Gas3/PMP22 biological activities. RhoA counteracted the Gas3/PMP22-dependent morphological response but was unable to neutralize the apoptotic response. Treatment of NIH3T3 cells with cytotoxic necrotizing factor 1, which activates endogenous Rho, also counteracted Gas3/PMP22-mediated cell shape and spreading changes. Treatment of REF 52 cells, which are unresponsive to Gas3/PMP22 overexpression, with the C3 exoenzyme, inhibiting Rho activity, renders REF 52 cells responsive to Gas3/PMP22 overexpression for cell shape and spreading changes. Finally, assembly of stress fibers and focal adhesions complexes, in response to lysophosphatidic acid–induced endogenous Rho activation, was impaired in Gas3/PMP22-overexpressing cells. We hypothesize that cell shape and spreading regulated by Gas3/PMP22 through the Rho GTPase might have an important role during Schwann cells differentiation and myelinization.

Regulation of the Actin Cytoskeleton by Thrombin in Human Endothelial Cells: Role of Rho Proteins in Endothelial Barrier Function

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.

Sense- and antisense-mediated gene silencing in tobacco is inhibited by the same viral suppressors and is associated with accumulation of small RNAs

Antisense-mediated gene silencing (ASGS) and posttranscriptional gene silencing (PTGS) with sense transgenes markedly reduce the steady-state mRNA levels of endogenous genes similar in transcribed sequence. RNase protection assays established that silencing in tobacco plants transformed with plant-defense-related class I sense and antisense chitinase (CHN) transgenes is at the posttranscriptional level. Infection of tobacco plants with cucumber mosaic virus strain FN and a necrotizing strain of potato virus Y, but not with potato virus X, effectively suppressed PTGS and ASGS of both the transgenes and homologous endogenes. This suggests that ASGS and PTGS share components associated with initiation and maintenance of the silent state. Small, ca. 25-nt RNAs (smRNA) of both polarities were associated with PTGS and ASGS in CHN transformants as reported for PTGS in other transgenic plants and for RNA interference in Drosophila. Similar results were obtained with an antisense class I β-1,3-glucanase transformant showing that viral suppression and smRNAs are a more general feature of ASGS. Several current models hold that diverse signals lead to production of double-stranded RNAs, which are processed to smRNAs that then trigger PTGS. Our results provide direct evidence for mechanistic links between ASGS and PTGS and suggest that ASGS could join a common PTGS pathway at the double-stranded RNA step.

Differential activation of proliferation and cytotoxicity in human T-cell lymphotropic virus type I Tax-specific CD8 T cells by an altered peptide ligand.

Human T-cell leukemia virus type I (HTLV-I) gives rise to a neurologic disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the pathogenesis of the disease is unknown, the presence of a remarkably high frequency of Tax-specific, cytotoxic CD8 T cells may suggest a role of these cells in the development of HAM/TSP. Antigen-mediated signaling in a CD8 T-cell clone specific for the Tax(11-19) peptide of HTLV-I was studied using analog peptides substituted in their T-cell receptor contact residues defined by x-ray crystallographic data of the Tax(11-19) peptide in the groove of HLA-A2. CD8 T-cell stimulation with the wild-type peptide antigen led to activation of p56lck kinase activity, interleukin 2 secretion, cytotoxicity, and clonal expansion. A Tax analog peptide with an alanine substitution of the T-cell receptor contact residue tyrosine-15 induced T-cell-mediated cytolysis without activation of interleukin 2 secretion or proliferation. Induction of p56lck kinase activity correlated with T-cell-mediated cytotoxicity, whereas interleukin 2 secretion correlated with [3H]thymidine incorporation and proliferation. Moreover, Tax peptide analogs that activated the tyrosine kinase activity of p56lck could induce unresponsiveness to secondary stimulation with the wild-type peptide. These observations show that a single amino acid substitution in a T-cell receptor contact residue of Tax can differentially signal CD8 T cells and further demonstrate that primary activation has functional consequences for the secondary response of at least some Tax-specific CD8 T cells to HTLV-I-infected target cells.

A abordagem médica de seguimento das crianças com síndrome do intestino curto

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Systemic gene expression in arabidopsis during an incompatible interaction with Alternaria brassicicola

Pathogen challenge can trigger an integrated set of signal transduction pathways, which ultimately leads to a state of high alert, otherwise known as systemic or induced resistance in tissue remote to the initial infection. Although large-scale gene expression during systemic acquired resistance, which is induced by salicylic acid or necrotizing pathogens has been previously reported using a bacterial pathogen, the nature of systemic defense responses triggered by an incompatible necrotrophic fungal pathogen is not known. We examined transcriptional changes that occur during systemic defense responses in Arabidopsis plants inoculated with the incompatible fungal pathogen Alternaria brassicicola. Substantial changes (2.00-fold and statistically significant) were demonstrated in distal tissue of inoculated plants for 35 genes (25 up-regulated and 10 down-regulated), and expression of a selected subset of systemically expressed genes was confirmed using real-time quantitative polymerase chain reaction. Genes with altered expression in distal tissue included those with putative functions in cellular housekeeping, indicating that plants modify these vital processes to facilitate a coordinated response to pathogen attack. Transcriptional up-regulation of genes encoding enzymes functioning in the beta-oxidation pathway of fatty acids was particularly interesting. Transcriptional up-regulation was also observed for genes involved in cell wall synthesis and modification and genes putatively involved in signal transduction. The results of this study, therefore, confirm the notion that distal tissue of a pathogen-challenged plant has a heightened preparedness for subsequent pathogen attacks.

Initiation and progression of ossification of the posterior longitudinal ligament of the cervical spine in the hereditary spinal hyperostotic mouse (twy/twy)

Ossification of the posterior longitudinal ligament (OPLL) is a significantly critical pathology that can eventually cause serious myelopathy. Ossification commences in the vertebral posterior longitudinal ligaments, and intensifies and spreads with the progression of the disease, resulting in osseous projections and compression of the spinal cord. However, the paucity of histological studies the underlying mechanisms of calcification and ossification processes remain obscure. The pathological process could be simulated in the ossifying process of the ligament in mutant spinal hyperostotic mouse (twy/twy). The aim of this study is to observe that enlargement of the nucleus pulposus followed by herniation, disruption and regenerative proliferation of annulus fibrosus cartilaginous tissues participated in the initiation of ossification of the posterior longitudinal ligament of twy/twy mice.

Emergency emicolectomy for intestinal primary Aspergillosis in acute myeloid leukemia

ntestinal aspergillosis is an infection with a very high death rate especially in leukemic patients. Here we describe a case of a 46 years old woman with acute myeloid leukemia (LAM M5) who developed intestinal primary aspergillosis. This patient was diagnosed with LAM M5 through bone marrow aspiration and bone biopsy in March 2004. Symptoms of the disease were slight persistent fever, weight loss, asthenia, anemia, thrombocytopenia,and leukocytosis with high number of blasts in peripheral blood. After induction chemotherapy with ICE (Ifosfamide, Carboplatin, Etoposide), she developed neutropenia and high fever without apparent infective foci. She was treated with empiric antibiotic therapy, nevertheless she developed an intense diarrhea and ileo-cecal distention. Diagnostic exams didn’t show signs of a focal lesion. Despite the change in antibiotic treatment and the transfusions of granulocytes and blood cells, the patient developed extremely critical conditions with persistence of neutropenia and abdominal distention. A surgical treatment was decided at the time. We treated the patient with a two steps surgical procedure. The first step was a right abdominal ileostomy followed by improvement of general conditions and then the second step a right colectomy. The histological morphology confirmed necrotizing colitis with Aspergillus ife. At that time , treatment with voriconazole was started. The general conditions of the patient improved rapidly and we were able to treat the patient with other medical anti-leukemic therapies. The patient is now cured and in healthy state. We obtained a good clinical result as only in other few cases described in literature.

Evaluation of negative pressure vacuum-assisted system in acute and chronic wounds closure. Our experience

Negative-pressure therapy or vacuum-assisted closure (VAC) has been used in clinical applications since the 1940’s and has increased in popularity over the past decade. This dressing technique consists of an open cell foam dressing put into the wound cavity, a vacuum pump produces a negative pressure and an adhesive drape. A controlled sub atmospheric pressure from 75 to 150 mmHg is applied. The vacuum-assisted closure has been applied by many clinicians to chronic wounds in humans; however it cannot be used as a replacement for surgical debridement. The initial treatment for every contaminated wound should be the necrosectomy. The VAC therapy has a complementary function and the range of its indications includes pressure sores, stasis ulcers, chronic wounds such as diabetic foot ulcers, post traumatic and post operative wounds, infected wounds such as necrotizing fasciitis or sternal wounds, soft-tissue injuries, bone exposed injuries, abdominal open wounds and for securing a skin graft. We describe our experience with the VAC dressing used to manage acute and chronic wounds in a series of 135 patients, with excellent results together with satisfaction of the patients.

Pancreatic pseudocyst: case report and short literature review

We report a case of pancreatic pseudocyst secondary to acute necrotizing pancreatitis treated with open cystogastrostomy. Following a literature review, we stress the enormous benefits offered by modern diagnostic techniques, and especially imaging techniques, for the diagnosis and monitoring of this disease. Treatment should be delayed for at least six weeks, following which the drainage by open surgery offers the best results and lowest morbidity and mortality, followed by laparoscopy and endoscopy, indicated in particular cases and in patients where open surgery is contraindicated.

New clinical practice guideline on enteral feeding in very low birth weight infants: first part

Introduction: The nutrition of very low birth weight (VLBW) infants is aimed at promoting a similar growth to that occurring in the uterus. However, in practice this is difficult to achieve and extrauterine growth restriction is frequent. The current tendency is to avoid this restriction by means of early parenteral and enteral nutrition. Nonetheless, uncertainty about many of the practices related with nutrition has resulted in a great variation in the way it is undertaken. In 2009 and 2011 in our hospital there was an unexpected increase in necrotizing enterocolitis. To check to see whether our nutrition policy was involved, we undertook a systematic review and drew up clinical practice guidelines (CPG) about enteral feeding in VLBW infants. New considerations about the duration of the fortification and the use of probiotics have led to an update of these CPG. Methods: A total of 21 clinical questions were designed dealing with the type of milk, starting age, mode of administration, rate and volume of the increments, fortification, use of probiotics and protocol. After conducting a systematic search of the available evidence, the information was contrasted and summarized in order to draw up the recommendations. The quality of the evidence and the strength of the recommendations were determined from the SIGN scale Comment: These CPG aim to help physicians in their decision making. The protocolized application of well-proven measurements reduces the variation in clinical practice and improves results.