Cutting edge: Failure of antigen-specific CD4+ T cell recruitment to the kidney during systemic candidiasis.

Candida albicans is the leading cause of systemic candidiasis, a fungal disease associated with high mortality and poor treatment options. The kidney is the target organ during infection and whose control is largely dependent on innate immunity, because lymphocytes appear redundant for protection. In this article, we show that this apparent redundancy stems from a failure of Ag-specific CD4(+) T cells to migrate into infected kidneys. In contrast, Ag-specific CD8(+) T cells are recruited normally. Using Ag-loaded immunoliposomes to artificially reverse this defective migration, we show that recruited Ag-specific CD4(+) T cells polarize toward a Th17 phenotype in the kidney and are protective during fungal infection. Therefore, our data explain the redundancy of CD4(+) T cells for defense against systemic infection with C. albicans and have important implications for our understanding of antifungal immunity and the control of renal infections.

Histone H4K20/H3K9 demethylase PHF8 regulates zebrafish brain and craniofacial development.

X-linked mental retardation (XLMR) is a complex human disease that causes intellectual disability. Causal mutations have been found in approximately 90 X-linked genes; however, molecular and biological functions of many of these genetically defined XLMR genes remain unknown. PHF8 (PHD (plant homeo domain) finger protein 8) is a JmjC domain-containing protein and its mutations have been found in patients with XLMR and craniofacial deformities. Here we provide multiple lines of evidence establishing PHF8 as the first mono-methyl histone H4 lysine 20 (H4K20me1) demethylase, with additional activities towards histone H3K9me1 and me2. PHF8 is located around the transcription start sites (TSS) of approximately 7,000 RefSeq genes and in gene bodies and intergenic regions (non-TSS). PHF8 depletion resulted in upregulation of H4K20me1 and H3K9me1 at the TSS and H3K9me2 in the non-TSS sites, respectively, demonstrating differential substrate specificities at different target locations. PHF8 positively regulates gene expression, which is dependent on its H3K4me3-binding PHD and catalytic domains. Importantly, patient mutations significantly compromised PHF8 catalytic function. PHF8 regulates cell survival in the zebrafish brain and jaw development, thus providing a potentially relevant biological context for understanding the clinical symptoms associated with PHF8 patients. Lastly, genetic and molecular evidence supports a model whereby PHF8 regulates zebrafish neuronal cell survival and jaw development in part by directly regulating the expression of the homeodomain transcription factor MSX1/MSXB, which functions downstream of multiple signalling and developmental pathways. Our findings indicate that an imbalance of histone methylation dynamics has a critical role in XLMR.

An electronic alert to decrease Kayexalate ordering.

Important safety concerns have recently emerged regarding the use of sodium polystyrene sulfonate (Kayexalate), a cation-exchange resin commonly used for the treatment of hyperkalemia. We implemented an electronic alert system at a tertiary care academic medical center to warn providers of the safety concerns of Kayexalate. We assessed the number of Kayexalate prescriptions per month, as well as the number of grams of Kayexalate ordered per month, one year before versus one year after implementing the alert. The mean (±SD) number of Kayexalate orders decreased from 123 (±12) to 76 (±14) orders/month (38% absolute reduction, p < 0.001) after implementing the alert. Additionally, the mean (±SD) amount of Kayexalate prescribed decreased from 3332 (±329) to 1885 (±358) g/month (43% absolute reduction, p < 0.001). We conclude that an electronic alert is an effective tool to decrease Kayexalate ordering.

Nanoscale Plasma Coating Inhibits Formation of Staphylococcus aureus Biofilm.

Staphylococcus aureus commonly infects medical implants or devices, with devastating consequences for the patient. The infection begins with bacterial attachment to the device, followed by bacterial multiplication over the surface of the device, generating an adherent sheet of bacteria known as a biofilm. Biofilms resist antimicrobial therapy and promote persistent infection, making management difficult to futile. Infections might be prevented by engineering the surface of the device to discourage bacterial attachment and multiplication; however, progress in this area has been limited. We have developed a novel nanoscale plasma coating technology to inhibit the formation of Staphylococcus aureus biofilms. We used monomeric trimethylsilane (TMS) and oxygen to coat the surfaces of silicone rubber, a material often used in the fabrication of implantable medical devices. By quantitative and qualitative analysis, the TMS/O2 coating significantly decreased the in vitro formation of S. aureus biofilms; it also significantly decreased in vivo biofilm formation in a mouse model of foreign-body infection. Further analysis demonstrated TMS/O2 coating significantly changed the protein adsorption, which could lead to reduced bacterial adhesion and biofilm formation. These results suggest that TMS/O2 coating can be used to effectively prevent medical implant-related infections.

Evergrowing incisors of diprotodont marsupials record age and life history.

OBJECTIVE: Tooth growth and wear are commonly used tools for determining the age of mammals. The most speciose order of marsupials, Diprotodontia, is characterised by a pair of procumbent incisors within the lower jaw. This study examines the growth and wear of these incisors to understand their relationship with age and sex. DESIGN: Measurements of mandibular incisor crown and root length were made for two sister species of macropodid (kangaroos and wallabies); Macropus giganteus and Macropus fuliginosus. Histological analysis examined patterns of dentine and cementum deposition within these teeth. Broader generalisability within Diprotodontia was tested using dentally reduced Tarsipes rostratus - a species disparate in body size and incisor function to the studied macropodids. RESULTS: In the macropodid sample it is demonstrated that the hypsodont nature of these incisors makes measurements of their growth (root length) and wear (crown length) accurate indicators of age and sex. Model fitting finds that root growth proceeds according to a logarithmic function across the lifespan, while crown wear follows a pattern of exponential reduction for both macropodid species. Histological results find that secondary dentine deposition and cementum layering are further indicators of age. Incisor measurements are shown to correlate with age in the sample of T. rostratus. CONCLUSIONS: The diprotodontian incisor is a useful tool for examining chronological age and sex, both morphologically and microstructurally. This finding has implications for population ecology, palaeontology and marsupial evolution.

3D adaptive optics in a light sheet microscope.

We report on a single plane illumination microscope (SPIM) incorporating adaptive optics in the imaging arm. We show how aberrations can occur from the sample mounting tube and quantify the aberrations both experimentally and computationally. A wavefront sensorless approach was taken to imaging a green fluorescent protein (GFP) labelled transgenic zebrafish. We show improvements in image quality whilst recording a 3D "z-stack" and show how the aberrations come from varying depths in the fish.

Single centre experience with pegylated interferon and ribavirin for hepatitis C: looking back before moving forward.

BACKGROUND: Hepatitis C treatment is successful in 40-80% of patients in drug sponsored registration trials. However, few studies have examined treatment outcomes in non-trial, routine clinical practice settings. AIM: The aim of this study was to investigate the treatment outcomes and predictors of a sustained virological response in a routine clinical setting. METHODS: Data were collected retrospectively on patients treated for hepatitis C between January 2004 and March 2010 in a tertiary hospital setting. Demographics, treatment outcomes and potential predictors of outcome (viral genotype, viral load, virological response, platelet count, alanine transaminase level, glucose, ferritin, weight, fibrosis and cirrhosis, compliance, dose reductions, adverse events, psychiatric and alcohol history) were recorded. Univariate and multiple logistic regressions were performed. RESULTS: A total of 405 patients was treated during the study period. On an intention to treat basis, sustained virological response rates were 55%, 82% and 72% in genotypes 1, 2 and 3 respectively. Predictors of response were gender, age, genotype, weight, fibrosis, cirrhosis, platelet count and alanine transaminase on univariate analysis. Age, genotype, cirrhosis and platelet count were independently associated with sustained virological response on multiple logistic regression. CONCLUSION: In our cohort, treatment outcomes for genotype 1 and 2 were similar to results from clinical trials but results for genotype 3 were inferior. Clinicians should not assume that results from registration trials are transferable to their own clinical practice. This has particular relevance for the new era of triple therapy regimens containing direct antivirals.

Implications of paper vs stainless steel biological indicator substrates for formaldehyde gas decontamination.

AIMS: This study was undertaken to determine the effectiveness of biological indicators currently being employed during formaldehyde decontamination. Data suggest that detectable amounts of formaldehyde are absorbed into the paper strips contained in currently used biological indicators. Absorbed formaldehyde has the potential to inhibit the growth of indicator spores, thus leading to false negative results. Indicators composed of either stainless steel carriers or paper strips were investigated to determine whether stainless steel carriers can be used as an alternative to paper strip indicators. METHODS AND RESULTS: Biological indicators were exposed to formaldehyde gas and were tested for the presence of formaldehyde and any possible inhibition of spore growth. Absorbed formaldehyde was detected in the paper strip carriers while no formaldehyde was detected from any of the stainless steel carriers. Exposed paper strips were found to inhibit growth of up to 1 × 10(6) spores while the stainless steel carriers did not inhibit the growth of spores. CONCLUSIONS: During decontamination, biological indicators composed of paper spore strips absorb formaldehyde and inhibit growth of any surviving spores. Stainless steel carriers do not absorb formaldehyde and are an ideal alternative substrate for biological indicators. SIGNIFICANCE AND IMPACT OF THE STUDY: The popular paper strip biological indicator can lead to false negative results during decontamination and is unsuitable for validating formaldehyde decontamination.

Association of periodontal disease, oral procedures, and other clinical findings with bacterial endocarditis in dogs.

OBJECTIVE: To identify risk factors potentially associated with the development of bacterial endocarditis in dogs and determine whether periodontal disease and surgical procedures (oral and nonoral) were associated with bacterial endocarditis. DESIGN: Retrospective case-control study. ANIMALS: 76 dogs with (cases) and 80 dogs without (controls) bacterial endocarditis. PROCEDURES: Medical records were reviewed for information on signalment, physical examination findings, recent medical history, and results of echocardiography, clinicopathologic testing, and necropsy. RESULTS: None of the dogs with endocarditis had a history of undergoing any dental or oral procedure in the 3 months prior to the diagnosis of endocarditis, and no significant difference was found between groups with regard to the prevalence of oral infection. Dogs with endocarditis were significantly more likely to have undergone a nonoral surgical procedure that required general anesthesia in the preceding 3 months or to have developed a new heart murmur or a change in intensity of an existing heart murmur. Preexisting cardiac dis-ease (congenital or acquired) was not found to be a risk factor. CONCLUSIONS AND CLINICAL RELEVANCE: Results did not provide any evidence of an association between bacterial endocarditis in dogs and either dental or oral surgical procedures or oral infection. Findings suggested that the routine use of prophylactic antimicrobial administration in dogs undergoing oral procedures needs to be reevaluated.

Survival of viral pathogens in animal feed ingredients under transboundary shipping models.

The goal of this study was to evaluate survival of important viral pathogens of livestock in animal feed ingredients imported daily into the United States under simulated transboundary conditions. Eleven viruses were selected based on global significance and impact to the livestock industry, including Foot and Mouth Disease Virus (FMDV), Classical Swine Fever Virus (CSFV), African Swine Fever Virus (ASFV), Influenza A Virus of Swine (IAV-S), Pseudorabies virus (PRV), Nipah Virus (NiV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Swine Vesicular Disease Virus (SVDV), Vesicular Stomatitis Virus (VSV), Porcine Circovirus Type 2 (PCV2) and Vesicular Exanthema of Swine Virus (VESV). Surrogate viruses with similar genetic and physical properties were used for 6 viruses. Surrogates belonged to the same virus families as target pathogens, and included Senecavirus A (SVA) for FMDV, Bovine Viral Diarrhea Virus (BVDV) for CSFV, Bovine Herpesvirus Type 1 (BHV-1) for PRV, Canine Distemper Virus (CDV) for NiV, Porcine Sapelovirus (PSV) for SVDV and Feline Calicivirus (FCV) for VESV. For the remaining target viruses, actual pathogens were used. Virus survival was evaluated using Trans-Pacific or Trans-Atlantic transboundary models involving representative feed ingredients, transport times and environmental conditions, with samples tested by PCR, VI and/or swine bioassay. SVA (representing FMDV), FCV (representing VESV), BHV-1 (representing PRV), PRRSV, PSV (representing SVDV), ASFV and PCV2 maintained infectivity during transport, while BVDV (representing CSFV), VSV, CDV (representing NiV) and IAV-S did not. Notably, more viruses survived in conventional soybean meal, lysine hydrochloride, choline chloride, vitamin D and pork sausage casings. These results support published data on transboundary risk of PEDV in feed, demonstrate survival of certain viruses in specific feed ingredients ("high-risk combinations") under conditions simulating transport between continents and provide further evidence that contaminated feed ingredients may represent a risk for transport of pathogens at domestic and global levels.

Fibronectin silanized titanium alloy: a bioinductive and durable coating to enhance fibroblast attachment in vitro.

Long term success of percutaneous implants is dependent on soft tissue attachment to prevent infection and epithelial downgrowth, which leads to failure of the implant. Fibronectin coatings are known to enhance fibroblast attachment in vitro, but are subject to desorption from serum protein competition in vivo. This paper quantifies the binding of fibronectin to titanium alloy by silanization and the durability of this attachment when soaked in protein-rich fluid compared with adsorbed fibronectin. The biological activity of fibronectin bound to silanized titanium alloy was confirmed by analyzing cell area, morphology, immunolocalization of focal contacts, and metabolism of dermal fibroblasts. This was compared with both adsorbed fibronectin and uncoated titanium alloy. Silanized titanium alloy bound over twice the amount of fibronectin compared to untreated titanium alloy. On soaking in fetal calf serum there was no significant loss of fibronectin (p = 0.589) from the silanized surface but a significant 44% loss (p = 0.002) from untreated surfaces. Fibroblasts on silanized fibronectin had significantly larger cell areas and more vinculin focal contact markers when compared to untreated surfaces (p < 0.005). The results confirm the durability of silanized fibronectin from protein competition and bioactive effect on fibroblasts.

Gel-seq: whole-genome and transcriptome sequencing by simultaneous low-input DNA and RNA library preparation using semi-permeable hydrogel barriers.

The advent of next generation sequencing has fundamentally changed genomics research. Unfortunately, standard protocols for sequencing the genome and the transcriptome are incompatible. This forces researchers to choose between examining either the DNA or the RNA for a particular sample. Here we describe a new device and method, collectively dubbed Gel-seq, that enables researchers to simultaneously sequence both DNA and RNA from the same sample. This technology makes it possible to directly examine the ways that changes in the genome impact the transcriptome in as few as 100 cells. The heart of the Gel-seq protocol is the physical separation of DNA from RNA. This separation is achieved electrophoretically using a newly designed device that contains several different polyacrylamide membranes. Here we report on the development and validation of this device. We present both the manufacturing protocol for the device and the biological protocol for preparing genetic libraries. Using cell lines with uniform expression (PC3 and Hela), we show that the libraries generated with Gel-seq are similar to those developed using standard methods for either RNA or DNA. Furthermore, we demonstrate the power of Gel-seq by generating a matched genome and transcriptome library from a sample of 100 cells collected from a mouse liver tumor.

Spatial and functional modeling of carnivore and insectivore molariform teeth.

The interaction between the two main competing geometric determinants of teeth (the geometry of function and the geometry of occlusion) were investigated through the construction of three-dimensional spatial models of several mammalian tooth forms (carnassial, insectivore premolar, zalambdodont, dilambdodont, and tribosphenic). These models aim to emulate the shape and function of mammalian teeth. The geometric principles of occlusion relating to single- and double-crested teeth are reviewed. Function was considered using engineering principles that relate tooth shape to function. Substantial similarity between the models and mammalian teeth were achieved. Differences between the two indicate the influence of tooth strength, geometric relations between upper and lower teeth (including the presence of the protocone), and wear on tooth morphology. The concept of "autocclusion" is expanded to include any morphological features that ensure proper alignment of cusps on the same tooth and other teeth in the tooth row. It is concluded that the tooth forms examined are auto-aligning, and do not require additional morphological guides for correct alignment. The model of therian molars constructed by Crompton and Sita-Lumsden ([1970] Nature 227:197-199) is reconstructed in 3D space to show that their hypothesis of crest geometry is erroneous, and that their model is a special case of a more general class of models.

Gene transfer in vivo with DNA-liposome complexes: lack of autoimmunity and gonadal localization.

Direct gene transfer into localized arterial segments can be performed in vivo by transfection with DNA-liposome complexes. This technique holds promise for the treatment of human diseases, including malignancy and cardiovascular disorders. We have previously characterized the potential toxicity of this form of treatment in mice in vivo (Stewart et al., 1992). In this report, we examined two issues relevant to long-term expression of foreign recombinant genes: (i) the potential for autoimmune damage to major organs and (ii) DNA localization in gonadal tissue. Autoimmunity and toxicity of allogeneic major histocompatibility (MHC) gene transfer was assessed in mice after induction of an immune response to a recombinant murine class I MHC gene by direct gene transfer in vivo. Histological examination of brain, heart, lung, liver, kidney, spleen, and skeletal muscle revealed no clinically significant immunopathology or organ damage. The toxicity of gene delivery by DNA liposomes was also analyzed in pigs and rabbits in vivo. No histopathology was observed following the introduction of plasmids encoding several different gene products, and analysis of serum following DNA liposome delivery revealed no abnormalities of serum biochemical parameters. The potential for transfer of recombinant DNA into testes and ovary in animals was evaluated by the polymerase chain reaction. Although evidence of recombinant plasmid was consistently observed in transfected, but not untransfected, arterial sites and occasionally in lung, kidney, spleen, and liver, no plasmid DNA was detected in testes or ovary. These studies suggest that uptake of recombinant DNA following direct gene transfer by liposomal transfection in major organs is not associated with autoimmunity, toxicity, or gonadal localization.(ABSTRACT TRUNCATED AT 250 WORDS)

A new cationic liposome DNA complex enhances the efficiency of arterial gene transfer in vivo.

An important goal of gene therapy for cardiovascular diseases and cancer is the development of effective vectors for catheter-based gene delivery. Although adenoviral vectors have proven effective for this purpose in animal models, the ability to achieve comparable gene transfer with nonviral vectors would provide potentially desirable safety and toxicity features for clinical studies. In this report, we describe the use of a new cationic DNA-liposome complex using an improved expression vector and lipid, N-(3-aminopropyl)-N, N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminium bromide/dioleyl phosphatidylethanolamine (GAP-DL-RIE/DOPE) to optimize catheter-mediated gene transfer in porcine arteries. The efficiency of this vector was compared to DNA alone, DNA with a previously described cationic liposome complex, (+/-)-N-(2-hydroxyethyl)-N, N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE/DOPE), and a replication-defective adenoviral vector in a porcine artery gene transfer model. When used in optimal ratios, GAP-DL-RIE/DOPE liposomes provided a 15-fold higher level of gene expression in arteries compared to DNA alone or DMRIE/DOPE. Gene expression was observed in intimal and medial cells. However, when compared to adenoviral vectors (10(10) pfu/ml), gene expression following GAP-DLRIE/DOPE transfection was approximately 20-fold lower. Following intravenous injection of GAP-DLRIE/DOPE in mice, biochemical, hematological, and histopathological abnormalities were not observed. Significant improvements in the efficacy of arterial gene expression can be achieved by optimization of transfection condition with DNA-liposome complexes in vivo that may prove useful for arterial gene delivery in cardiovascular diseases and cancer.

Dietary and other correlates of changes in total and low density lipoprotein cholesterol in hypercholesterolemic men: the lipid research clinics coronary primary prevention trial.

Correlates of changes in total (TOTAL-C) and low density lipoprotein cholesterol (LDL-C) were examined in the 3806 hypercholesterolemic men of the Lipid Research Clinics Coronary Primary Prevention Trial. These correlates included changes in weight, dietary and alcohol intake, plasma glucose and thyroxine, cigarette smoking, packet count, lipid-lowering drugs other than cholestyramine, and antihypertensive drugs. In both placebo plus diet and cholestyramine plus diet treatment groups, decreases in Quetelet index and in saturated fat and cholesterol intake and increases in polyunsaturated fat intake were consistently associated with reductions in TOTAL-C and in LDL-C. In the cholestyramine group, plasma glucose and smoking were predictors of increased TOTAL-C and LDL-C; age and packet count were predictors of decreased TOTAL-C and LDL-C. Diuretic use was associated with increases in TOTAL-C in both groups and with increases in LDL-C in the cholestyramine group.

Direct gene transfer for the understanding and treatment of human disease.

Direct gene transfer has been used to develop molecular genetic interventions for acquired diseases in several animal models. Through the use of intravascular catheters or anatomically localized injection of DNA liposome complexes, specific tissues can be transduced with recombinant genes. Several promising applications of this method for the study of vascular biology have been demonstrated by direct gene transfer into arteries in vivo. Delivery, via catheter, of genes that modulate the thrombogenic or proliferative properties of vascular cells may someday provide therapy for stenotic lesions of atherosclerosis or following angioplasty. Cancer is another acquired disorder in which direct gene transfer may improve the efficacy of treatment. Introduction of class I MHC or cytokine genes with antitumor or immunostimulatory effects have demonstrated promise in animal models. Direct transfer of an allogeneic class I MHC gene into tumors in vivo induces a CD8+ CTL response against weak antigens on poorly immunogenic tumors. The efficacy of this antitumor response can be augmented to induce regression of actively growing established tumors. Additional strategies, such as intratumoral delivery of combinations of multiple cytokine and MHC genes, may serve to improve the antitumor response. A clinical gene therapy protocol is underway to analyze the safety and efficacy of DNA liposome-mediated gene transfer in humans. Development of improved gene delivery systems and introduction of recombinant genes into visceral tumors by intravascular catheter will extend the application of direct gene transfer to immunotherapy of malignancies. These clinical trials of direct gene transfer will help to develop new treatment strategies for human diseases.

Direct gene transfer for treatment of human cancer.

Genetic instability within malignant cells gives rise to mutant proteins which can be recognized by the immune system. Recognition of tumor-associated antigens by T lymphocytes could thus contribute to the elimination of neoplastic cells. We have developed a molecular genetic intervention for the treatment of malignancies based upon the knowledge that foreign major histocompatibility complex (MHC) proteins expressed on the cell surface are efficient at stimulating an immune response. Expression of this foreign MHC gene within tumors induced a cytotoxic T cell response to the introduced gene. More importantly, the immune system recognized tumor-specific antigens on unmodified tumor cells as foreign. Growth of the tumors diminished, and in many cases, there was complete regression. This research provides evidence that direct gene transfer in vivo can induce cell-mediated immunity against specific gene products, and offers the potential for effective immunotherapy for the treatment of cancer and infectious diseases in man. Our laboratory conducted a phase I clinical trial to determine the safety and efficacy of this treatment in humans. These studies suggest that direct gene transfer provides a safe and feasible approach for the treatment of human cancer. More recent developments using combination gene therapy and the initiation of a second human trial with improvements on this technology have been implemented. Finally, we have begun to define mechanisms of resistance to immune recognition by established malignancies.

Growth of tracheal anastomoses: advantage of absorbable interrupted sutures.

Growth of the trachea after complete transection and anastomosis was studied in four groups of 1-month-old New Zealand white rabbits. The trachea was transected at the fifth cartilaginous ring and then anastomosed with continuous 6-0 polypropylene (Prolene) (group 1), interrupted 6-0 polypropylene (group 2), continuous 6-0 polydioxanone (PDS) (group 3), or interrupted 6-0 PDS (group 4). The animals were followed up for 90 to 103 days (mean follow-up, 95 days). At the time the animals were killed, body weight had increased 125% (1.2 to 2.7 +/- 0.18 kg). Growth of the trachea was assessed at the time of death. Results from this study suggest that growth of a tracheal anastomosis is retarded in a growing animal model. The degree of resultant stenosis was significantly less when an absorbable suture material (PDS) and an interrupted suturing technique were used.