Competing Actions of Type 1 Angiotensin II Receptors Expressed on T Lymphocytes and Kidney Epithelium during Cisplatin-Induced AKI.

Inappropriate activation of the renin-angiotensin system (RAS) contributes to many CKDs. However, the role of the RAS in modulating AKI requires elucidation, particularly because stimulating type 1 angiotensin II (AT1) receptors in the kidney or circulating inflammatory cells can have opposing effects on the generation of inflammatory mediators that underpin the pathogenesis of AKI. For example, TNF-α is a fundamental driver of cisplatin nephrotoxicity, and generation of TNF-α is suppressed or enhanced by AT1 receptor signaling in T lymphocytes or the distal nephron, respectively. In this study, cell tracking experiments with CD4-Cre mT/mG reporter mice revealed robust infiltration of T lymphocytes into the kidney after cisplatin injection. Notably, knockout of AT1 receptors on T lymphocytes exacerbated the severity of cisplatin-induced AKI and enhanced the cisplatin-induced increase in TNF-α levels locally within the kidney and in the systemic circulation. In contrast, knockout of AT1 receptors on kidney epithelial cells ameliorated the severity of AKI and suppressed local and systemic TNF-α production induced by cisplatin. Finally, disrupting TNF-α production specifically within the renal tubular epithelium attenuated the AKI and the increase in circulating TNF-α levels induced by cisplatin. These results illustrate discrepant tissue-specific effects of RAS stimulation on cisplatin nephrotoxicity and raise the concern that inflammatory mediators produced by renal parenchymal cells may influence the function of remote organs by altering systemic cytokine levels. Our findings suggest selective inhibition of AT1 receptors within the nephron as a promising intervention for protecting patients from cisplatin-induced nephrotoxicity.

The influence of transforming growth factor-beta1 gene polymorphisms on the severity of gingival overgrowth associated with concomitant use of cyclosporin A and a calcium channel blocker

The purpose of this study was to determine whether the prevalence and severity of gingival overgrowth in renal transplant recipients concomitantly treated with cyclosporin and a calcium channel blocker was associated with functional polymorphisms within the signal sequence of the transforming growth factor-(TGF)beta1 gene.

Efficacy and mechanism of hypoxic postconditioning in salvage of ex vivo human rectus abdominis muscle from hypoxia/reoxygenation injury

In reconstructive surgery, skeletal muscle may endure protracted ischemia before reperfusion, which can lead to significant ischemia/reperfusion injury. Ischemic postconditioning induced by brief cycles of reperfusion/reocclusion at the end of ischemia has been shown to salvage skeletal muscle from ischemia/reperfusion injury in several animal models. However, ischemic postconditioning has not been confirmed in human skeletal muscle. Using an established in vitro human skeletal muscle hypoxic conditioning model, we tested our hypothesis that hypoxic postconditioning salvages ex vivo human skeletal muscle from hypoxia/reoxygenation injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP) and preservation of ATP synthesis. Muscle strips (~0.5×0.5×15mm) from human rectus abdominis muscle biopsies were cultured in Krebs-Henseleit-HEPES buffer, bubbled with 95%N(2)/5%CO(2) (hypoxia) or 95%O(2)/5%CO(2) (reoxygenation). Samples were subjected to 3h hypoxia/2h reoxygenation. Hypoxic postconditioning was induced by one or two cycles of 5min reoxygenation/5min hypoxia after 3h hypoxia. Muscle injury, viability and ATP synthesis after 2h of reoxygenation were assessed by measuring lactate dehydrogenase (LDH) release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction and ATP content, respectively. Hypoxic postconditioning or treatment with the mPTP-opening inhibitors Cyclosporine A (CsA, 5×10(-6)M) or N-Methyl-4-isoleucine Cyclosporine (NIM811, 5×10(-6)M) 10min before reoxygenation decreased LDH release, increased MTT reduction and increased muscle ATP content (n=7 patients; P

Mechanisms of modified LDL-induced pericyte loss and retinal injury in diabetic retinopathy

Aims/hypothesis: In previous studies we have shown that extravasated, modified LDL is associated with pericyte loss, an early feature of diabetic retinopathy (DR). Here we sought to determine detailed mechanisms of this LDLinduced pericyte loss.

Methods: Human retinal capillary pericytes (HRCP) were exposed to ‘highly-oxidised glycated’ LDL (HOG-LDL) (a model of extravasated and modified LDL) and to 4-hydroxynonenal or 7-ketocholesterol (components of oxidised LDL), or to native LDL for 1 to 24 h with or without 1 h of pretreatment with inhibitors of the following: (1) the scavenger receptor (polyinosinic acid); (2) oxidative stress (N-acetyl cysteine); (3) endoplasmic reticulum (ER) stress (4-phenyl butyric acid); and (4) mitochondrial dysfunction (cyclosporin A). Oxidative stress, ER stress, mitochondrial dysfunction, apoptosis and autophagy were assessed using techniques including western blotting, immunofluorescence, RT-PCR, flow cytometry and TUNEL assay. To assess the relevance of the results in vivo, immunohistochemistry was used to detect the ER stress chaperon, 78 kDa glucose-regulated protein, and the ER sensor, activating transcription factor 6, in retinas from a mouse model of DR that mimics exposure of the retina to elevated glucose and elevated LDL levels, and in retinas from human participants with and without diabetes and DR.

Results: Compared with native LDL, HOG-LDL activated oxidative and ER stress in HRCP, resulting in mitochondrial dysfunction, apoptosis and autophagy. In a mouse model of diabetes and hyperlipidaemia (vs mouse models of either condition alone), retinal ER stress was enhanced. ER stress was also enhanced in diabetic human retina and correlated with the severity of DR.

Conclusions/interpretation: Cell culture, animal, and human data suggest that oxidative stress and ER stress are induced by modified LDL, and are implicated in pericyte loss in DR.

Favorable effect on acute and chronic graft-versus-host disease with cyclophosphamide and in vivo anti-CD52 monoclonal antibodies for marrow transplantation from HLA-identical sibling donors for acquired aplastic anemia

Between August 1989 and November 2003, 33 patients at our center with acquired aplastic anemia underwent bone marrow transplantation (BMT) from HLA-identical sibling donors with cyclophosphamide and in vivo anti-CD52 monoclonal antibodies (MoAb) for conditioning. The median age at BMT was 17 years (range, 4-46 years). Before BMT, 58% were heavily transfused (>50 transfusions), and 42% had previously experienced treatment failure with antithymocyte globulin-based immunosuppressive therapy. Unmanipulated bone marrow was used as the source of stem cells in all patients except 1. Graft-versus-host disease (GVHD) prophylaxis was with cyclosporine alone in 19 (58%) patients; 14 received anti-CD52 MoAb in addition to cyclosporine. The conditioning regimen was well tolerated without significant acute toxicity. Graft failure was seen in 8 patients (primary, n = 4; secondary, n = 4). Of those whose grafts failed, 4 survived long-term (complete autologous recovery, n = 2; rescue with previously stored marrow, n = 1; second allograft, n = 1). The cumulative incidence of graft failure and grade II to IV acute and chronic GVHD was 24%, 14%, and 4%, respectively. None developed extensive chronic GVHD. With a median follow-up of 59 months, the 5-year survival was 81% (95% confidence interval, 68%-96%). No unexpected early or late infectious or noninfectious complications were observed. We conclude that the conditioning regimen containing cyclophosphamide and anti-CD52 MoAb is well tolerated and effective for acquired aplastic anemia with HLA-matched sibling donors. The favorable effect on the incidence and severity of GVHD is noteworthy in this study and warrants further investigation.

The influence of cyclosporin alone, or cyclosporin and methotrexate, on the incidence of mixed haematopoietic chimaerism following allogeneic sibling bone marrow transplantation for severe aplastic anaemia

We previously reported a randomized trial comparing Cyclosporin-A (CsA) and short-term methotrexate versus CsA alone for graft-versus-host disease (GvHD) prophylaxis in 71 patients undergoing allogeneic haematopoietic stem cell transplantation (HSCT) from a human leucocyte antigen-identical sibling for severe aplastic anaemia (SAA). We found a better survival in the group receiving the two-drug prophylaxis regimen with no significant difference in the probability of developing GvHD between the two groups. The present study details chimaeric analysis and its influence on survival and GvHD occurrence in 45 of the original 71 patients in whom serial samples were available. Analysis was carried out in a blinded prospective manner. Seventy-two per cent achieved complete donor chimaerism (DC), 11% stable mixed chimaerism (SMC) and 17% progressive mixed chimaerism (PMC). The overall 5-year survival probability was 82% (+/-11%) with a significant survival advantage (P = 0.0009) in DC or SMC compared to those with PMC. Chronic GvHD was more frequent in DC patients, whereas no patient with SMC developed chronic GvHD. Graft failure occurred in 50% of the PMC group. This study demonstrates the relevance of chimaerism analysis in patients receiving HSCT for SAA and confirms the occurrence of mixed chimaerism in a significant proportion of recipients.

Cyclosporin A and short-term methotrexate versus cyclosporin A as graft versus host disease prophylaxis in patients with severe aplastic anemia given allogeneic bone marrow transplantation from an HLA-identical sibling:results of a GITMO/EBMT randomized trial

A randomized trial was carried out comparing cyclosporin A (CsA) and short-term methotrexate (MTX) versus CsA alone for graft versus host disease (GVHD) prophylaxis in patients with severe aplastic anemia (SAA) undergoing allogeneic bone marrow transplantation (BMT) from a compatible sibling. Seventy-one patients (median age, 19 years; range, 4-46 years) were randomized to receive either CsA and MTX or CsA alone for the first 3 weeks after BMT. Subsequently, both groups received CsA orally, with gradual drug reduction until discontinuation 8 to 12 months after BMT. Patients randomized in both arms had comparable characteristics and received the same preparative regimen (ie, cyclophosphamide 200 mg/kg over 4 days). The median time for neutrophil engraftment was 17 days (range, 11-31 days) and 12 days (range, 4-45 days) for patients in the CsA/MTX group and the CsA alone group, respectively (P =.01). No significant difference was observed in the probability of either grade 2, grade 3, or grade 4 acute GVHD or chronic GVHD developing in the 2 groups. The Kaplan-Meier estimates of 1-year transplantation-related mortality rates for patients given either CsA/MTX or CsA alone were 3% and 15%, respectively (P =.07). With a median follow-up of 48 months from BMT, the 5-year survival probability is 94% for patients in the CsA/MTX group and 78% for those in the CsA alone group (P =. 05). These data indicate that the use of CsA with MTX is associated with improved survival in patients with SAA who receive transplants from compatible siblings. (Blood. 2000;96:1690-1697)

Donor chimaerism is a strong indicator of disease free survival following bone marrow transplantation for chronic myeloid leukaemia

Although Chronic Myeloid Leukaemia (CML) can be treated successfully with allogeneic bone marrow transplantation (BMT), leukaemia relapse remains a significant clinical problem. Molecular monitoring of the post transplant marrow can be useful in predicting relapse particularly in CML patients where the Philadelphia chromosome or its molecular counterpart, the BCR-ABL fusion messenger RNA can be used as a leukaemia specific marker of minimal residual disease (MRD). We have investigated chimaerism (using polymerase chain reaction of short tandem repeat sequences (STR-PCR)) and MRD status (using reverse transcriptase PCR of the BCR-ABL fusion mRNA) in a serial fashion in 18 patients who were in clinical and haematological remission post allogeneic BMT for chronic phase CML. Eleven patients exhibited complete donor chimaerism with no evidence of minimal residual disease. Five patients had transient or low level stable MC. Late MC and MRD was observed in two patients who relapsed > 6 years after T cell depleted BMT for CML. Thus STR-PCR is an appropriate screening test in the post transplant setting for CML patients, but those patients exhibiting mixed haemopoietic chimaerism should also be monitored using a leukaemia specific sensitive molecular assay.

Rejection of the second allogeneic graft in a patient with severe aplastic anemia reversed by antilymphocyte globulin and donor lymphocyte infusions

Rejection after allogeneic BMT for aplastic anemia is a complication with a high risk of mortality. We describe a patient who, following a second episode of rejection after a second BMT entered a third durable remission subsequent to treatment with ALG, donor lymphocyte infusions, GM-CSF, and erythropoietin. Therapy was well tolerated. At 5 years after rejection treatment, his hematopoiesis is of complete donor origin as determined by analyses of short tandem repeats. Thus, donor lymphocyte infusions can be considered as a therapy option for marrow rejection after allogeneic BMT for aplastic anemia.

Drug induced gingival overgrowth: A role for Protease Activated Receptors?

Background: Protease activated receptors (PAR) belong to a subfamily of G protein coupled receptors. They consist of seven transmembrane domains but are not classical receptors as their agonist is a circulating serine proteinase. This proteinase cleaves an N-terminal extracellular domain of the receptor to reveal a new N-terminal tethered ligand which binds intramolecularly, thus converting an extracellular proteolytic event into a transmembrane signal. Therefore, the cleavage and activation of PARs provide a mechanism whereby proteinases can directly influence the inflammatory response. Gingival hyperplasia or gingival enlargement is a side effect of some drugs such as cyclosporine, a potent immunosuppressant. To date, the potential role of PAR in the inflammation associated with the pathogenesis of gingival overgrowth has not been studied. Objectives: The present study was designed to determine whether proteinases derived from extracts of cyclosporine induced hyperplasia were capable of activating PAR in vitro. Methods: Cell lysates were derived from tissue obtained from gingival overgrowth of patients requiring surgical excision. Cell lines over-expressing PARs were maintained in Dulbecco's modified Eagle's medium (DMEM), containing 10% foetal calf serum (FCS) in 5% CO2. The cells were treated with gingival overgrowth lysates and agonist stimulated calcium release from the cells was recorded using the Fluo-4-Direct™ Calcium Assay Kit from Invitrogen, according to manufacturer's instructions. Results: Calcium release by activated PAR on tumour cells was detected in those treated with gingival hyperplasia lysates. Samples from healthy gingival fibroblasts did not elicit this response. Conclusions: The identification of mediators of the molecular events central to the inflammatory phenotype elicited by gingival hyperplasia is important. To this end, our experiments show that in vitro, enzymes derived from overgrown gingival tissue are capable of activating PAR and thereby provide evidence for the potential role of PAR in sustaining gingival hyperplasia.

ANTIOXIDANT, ANTIMICROBIAL AND TOXICOLOGICAL PROPERTIES OF SCHINUS MOLLE L. ESSENTIAL OILS

Schinus molle L. is commonly known as pink pepper or American pepper, of Anacardiaceae family, from subtropical regions of South America, introduced and naturalized in South Europe, including Portugal. In folk medicine, plant extracts and essential oil has related as having antibacterial, antiviral, antifungal, anti-inflammatory, antitumoral, antispasmodic, analgesic and antidepressive properties. The aim of present study was to evaluate the chemical composition and biological activities of essential oil extracted from leaves and fruits of S. molle. For this purpose, the essential oils were analyzed by gas chromatography (GC/FID) and antioxidant properties were evaluated by the free radical DPPH and by system β-carotene/linoleic acid methods. The antimicrobial activities were screened against pathogenic bacteria and fungi and food spoiling fungi by the disc diffusion assay and minimal inhibitory concentration (MIC) was determined for sensitive strains. Toxicity of essential oils were carried out by the brine shrimp mortality test (EC50) and acute lethal dose (DL50) determination after oral administration in Swiss mice The major components in leaf essential oil were α-phellandrene, β-phellandrene and limonene, while myrcene, α-phellandrene and 1,8-cineole are the main components in the fruit essential oil. The essential oils of leaf and fruit of S. molle showed antioxidant activity through the two mechanisms: the ability to capture free radicals and protection of lipid peroxidation. These oils exhibited also a broad microbial activity spectrum, against pathogenic bacteria Gram-positive and Gram-negative and Candida spp. The fruit essential oil showed high cytotoxicity against Artemia salina. Essential oils of leaves and fruits of S. molle showed significant antioxidant and microbial properties, so the studies continue to clarify more in deep its toxicity, including hepatotoxicity and nephrotoxicity, and to evaluate its medicinal or nutraceutical potential.

Evaluation and Potential Cost-Effectiveness of Active Surveillance Pharmacovigilance for First-Line HAART in Namibia

Thesis (Ph.D.)--University of Washington, 2016-02

In Vitro Assessment of the Synergy between Polymyxin B (PMB) and Polymyxin B Nonapeptide (PMBN) and Antibiotics on Biofilms from Diabetic Foot Infections

Background: The increasing resistance of Gram-negative bacteria isolated from nosocomial infections and chronic wounds, such as diabetic foot ulcers has renewed research interests in the use of polymyxins in the treatment of multidrug resistant infections. The added resistance conferred by biofilm development in such infections and the absence of novel antibiotics presuppose that polymyxins are the likely drugs of choice in spite of their nephrotoxicity. The effects of PMB and PMBN have been previously assessed on planktonic bacteria isolated from various infections. Methods: This current study assessed the synergy between a PMB/PMBN and two antibiotics (ceftazidime and levofloxacin) in an attempt to develop a strategy for biofilm disruption using the Minimum Biofilm Eradication Concentration Physiology and Genetic assay (MBEC™ P & G, Innovotech Inc, Edmonton, Alberta, Canada) according to manufacturer’s instructions. Klebsiella pneumoniae (K. pneumoniae) and Proteus mirabilis (P. mirabilis) biofilms of initial broth suspensions of 108 colony forming units per mL, cultivated on the pegs of the MBEC device were challenged with 5120 µg/mL of both ceftazidime and levofloxacin in a ten-fold dilution assay and in the presence of 100 and 500 µg/mL PMB and PMBN. Results: From table of results (Table 1), it can be deduced that both ceftazidime and levofloxacin are very effective in inhibiting biofilm development (as shown by percentage inhibition (PI)) when augmented with PMB and PMBN. This is about 100-fold increase in efficacy when compared to the antibiotics used on their own. The percentage reduction (PR) in biofilm was also increased considerably when PMB and PMBN concentrations were increased to 500 µg/mL. PMB was more effective than its less antibacterial derivative PMBN. Levofloxacin was also found to be more effective than ceftazidime when combined with both PMB and PMBN due to its enhanced cell-membrane permeability and as an anti-DNA replication uncoupling agent. Conclusion: The above results indicate that the synergy between antibiotics and cell membrane permeabilising agents may provide alternate strategies towards biofilm eradication

Anti-Tumor Necrosis Factor Alpha-Induced Drug Eruptions: One Patient, More Than a Pattern

Background: Tumor necrosis factor alpha (TNFα) antagonists are effective in treating several immune-inflammatory diseases, including psoriasis and inflammatory bowel disease. The paradoxical and unpredictable induction of psoriasis and psoriasiform skin lesions is a recognized adverse event, although of unclear aetiology. However, histological analysis of these eruptions remains insufficient, yet suggesting that some might constitute a new pattern of adverse drug reaction, rather than true psoriasis. Case report: The authors report the case of a 43-year-old woman with severe recalcitrant Crohn disease who started treatment with infliximab. There was also a personal history of mild plaque psoriasis without clinical expression for the past eight years. She developed a heterogeneous cutaneous eruption of psoriasiform morphology with pustules and crusts after the third infliximab infusion. The histopathological diagnosis was of a Sweet-like dermatosis. The patient was successfully treated with cyclosporine in association with both topical corticosteroid and vitamin D3 analogue. Three weeks after switching to adalimumab a new psoriasiform eruption was observed, histologically compatible with a psoriasiform drug eruption. Despite this, and considering the beneficial effect on the inflammatory bowel disease, it was decided to maintain treatment with adalimumab and to treat through with topicals, with progressive control of skin disease. Discussion: Not much is known about the pathogenesis of psoriasiform eruptions induced by biological therapies, but genetic predisposition and Koebner phenomenon may contribute to it. Histopathology can add new facets to the comprehension of psoriasiform reactions. In fact, histopathologic patterns of such skin lesions appear to be varied, in a clear asymmetry with clinical findings. Conclusion: The sequential identification in the same patient of two clinical and histopathologic patterns of drug reaction to TNFα antagonists is rare. Additionally, to the authors’ knowledge, there is only one other description in literature of a TNFα antagonist-induced Sweet-like dermatosis, emphasizing the singularity of this case report.