Gene electrotransfer into murine skeletal muscle: A systematic analysis of parameters for long-term gene expression

Skeletal muscle is an attractive target tissue for delivery of therapeutic genes, since it is well vascularized, easily accessible, and has a high capacity for protein synthesis. For efficient transfection in skeletal muscle, several protocols have been described, including delivery of low voltage electric pulses and a combination of high and low voltage electric pulses. The aim of this study was to determine the influence of different parameters of electrotransfection on short-term and long-term transfection efficiency in murine skeletal muscle, and to evaluate histological changes in the treated tissue. Different parameters of electric pulses, different time lags between plasmid DNA injection and application of electric pulses, and different doses of plasmid DNA were tested for electrotransfection of tibialis cranialis muscle of C57BI/6 mice using DNA plasmid encoding green fluorescent protein (GFP). Transfection efficiency was assessed on frozen tissue sections one week after electrotransfection using a fluorescence microscope and also noninvasively, followed by an in vivo imaging system using a fluorescence stereo microscope over a period of several months. Histological changes in muscle were evaluated immediately or several months after electrotransfection by determining infiltration of inflammatory mononuclear cells and presence of necrotic muscle fibers. The most efficient electrotransfection into skeletal muscle of C57BI/6 mice in our experiments was achieved when one high voltage (HV) and four low voltage (LV) electric pulses were applied 5 seconds after the injection of 30 μg of plasmid DNA. This protocol resulted in the highest short-term as well as long-term transfection. The fluorescence intensity of the transfected area declined after 2-3 weeks, but GFP fluorescence was still detectable 18 months after electrotransfection. Extensive inflammatory mononuclear cell infiltration was observed immediately after the electrotransfection procedure using the described parameters, but no necrosis or late tissue damage was observed. This study showed that electric pulse parameters, time lag between the injection of DNA and application of electric pulses, and dose of plasmid DNA affected the duration of transgene expression in murine skeletal muscle. Therefore, transgene expression in muscle can be controlled by appropriate selection of electrotransfection protocol.

Serum 25-hydroxy vitamin D concentrations are more deficient/insufficient in peritoneal dialysis than haemodialysis patients in a sunny climate

Background Research has identified associations between serum 25(OH)D and a range of clinical outcomes in chronic kidney disease and wider populations. The present study aimed to investigate vitamin D deficiency/insufficiency in dialysis patients and the relationship with vitamin D intake and sun exposure. Methods A cross-sectional study was used. Participants included 30 peritoneal dialysis (PD) (43.3% male; 56.87 ± 16.16 years) and 26 haemodialysis (HD) (80.8% male; 63.58 ± 15.09 years) patients attending a department of renal medicine. Explanatory variables were usual vitamin D intake from diet/supplements (IU day−1) and sun exposure (min day−1). Vitamin D intake, sun exposure and ethnic background were assessed by questionnaire. Weight, malnutrition status and routine biochemistry were also assessed. Data were collected during usual department visits. The main outcome measure was serum 25(OH)D (nm). Results Prevalence of inadequate/insufficient vitamin D intake differed between dialysis modality, with 31% and 43% found to be insufficient (<50 nm) and 4% and 33% found to be deficient (<25 nm) in HD and PD patients, respectively (P < 0.001). In HD patients, there was a correlation between diet and supplemental vitamin D intake and 25(OH)D (ρ = 0.84, P < 0.001) and average sun exposure and 25(OH)D (ρ = 0.50, P < 0.02). There were no associations in PD patients. The results remained significant for vitamin D intake after multiple regression, adjusting for age, gender and sun exposure. Conclusions The results highlight a strong association between vitamin D intake and 25(OH)D in HD but not PD patients, with implications for replacement recommendations. The findings indicate that, even in a sunny climate, many dialysis patients are vitamin D deficient, highlighting the need for exploration of determinants and consequences.

Genome-wide analysis in a murine Dnmt1 knockdown model identifies epigenetically silenced genes in primary human pituitary tumors

DNA methylation at promoter CpG islands (CGI) is an epigenetic modification associated with inappropriate gene silencing in multiple tumor types. In the absence of a human pituitary tumor cell line, small interfering RNA-mediated knockdown of the maintenance methyltransferase DNA methyltransferase (cytosine 5)-1 (Dnmt1) was used in the murine pituitary adenoma cell line AtT-20. Sustained knockdown induced reexpression of the fully methylated and normally imprinted gene neuronatin (Nnat) in a time-dependent manner. Combined bisulfite restriction analysis (COBRA) revealed that reexpression of Nnat was associated with partial CGI demethylation, which was also observed at the H19 differentially methylated region. Subsequent genome-wide microarray analysis identified 91 genes that were significantly differentially expressed in Dnmt1 knockdown cells (10% false discovery rate). The analysis showed that genes associated with the induction of apoptosis, signal transduction, and developmental processes were significantly overrepresented in this list (P < 0.05). Following validation by reverse transcription-PCR and detection of inappropriate CGI methylation by COBRA, four genes (ICAM1, NNAT, RUNX1, and S100A10) were analyzed in primary human pituitary tumors, each displaying significantly reduced mRNA levels relative to normal pituitary (P < 0.05). For two of these genes, NNAT and S100A10, decreased expression was associated with increased promoter CGI methylation. Induced expression of Nnat in stable transfected AtT-20 cells inhibited cell proliferation. To our knowledge, this is the first report of array-based "epigenetic unmasking" in combination with Dnmt1 knockdown and reveals the potential of this strategy toward identifying genes silenced by epigenetic mechanisms across species boundaries.

Intramacrophage survival of uropathogenic Escherichia coli : differences between diverse clinical isolates and between mouse and human macrophages

Uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections. Recent studies have demonstrated that UPEC can invade and replicate within epithelial cells, suggesting that this bacterial pathogen may occupy an intracellular niche within the host. Given that many intracellular pathogens target macrophages, we assessed the interactions between UPEC and macrophages. Colonization of the mouse bladder by UPEC strain CFT073 resulted in increased expression of myeloid-restricted genes, consistent with the recruitment of inflammatory macrophages to the site of infection. In in vitro assays, CFT073 was able to survive within primary mouse bone marrow-derived macrophages (BMM) up to 24 h post-infection. Three additional well-characterized clinical UPEC isolates associated with distinct UTI symptomatologies displayed variable long-term survival within BMM. UPEC strains UTI89 and VR50, originally isolated from patients with cystitis and asymptomatic bacteriuria respectively, showed elevated bacterial loads in BMM at 24 h post-infection as compared to CFT073 and the asymptomatic bacteriuria strain 83972. These differences did not correlate with differential effects on macrophage survival or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1+ vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data suggest that some UPEC isolates may subvert macrophage anti-microbial pathways, and that host species differences may impact on intracellular UPEC survival.

Conserved signaling through vascular endothelial growth (VEGF) receptor family members in murine lymphatic endothelial cells

Lymphatic vessels guide interstitial fluid, modulate immune responses by regulating leukocyte and antigen trafficking to lymph nodes, and in a cancer setting enable tumor cells to track to regional lymph nodes. The aim of the study was to determine whether primary murine lymphatic endothelial cells (mLECs) show conserved vascular endothelial growth factor (VEGF) signaling pathways with human LECs (hLECs). LECs were successfully isolated from murine dermis and prostate. Similar to hLECs, vascular endothelial growth factor (VEGF) family ligands activated MAPK and pAkt intracellular signaling pathways in mLECs. We describe a robust protocol for isolation of mLECs which, by harnessing the power of transgenic and knockout mouse models, will be a useful tool to study how LEC phenotype contributes to alterations in lymphatic vessel formation and function.

Displaced granulosa cells in peritoneal washings : a rare diagnostic pitfall

Developing follicles and follicular cysts in the ovary are lined by granulosa cells. Approximately the size of histiocytes, non-neoplastic granulosa cells have scant granular to foamy cytoplasm and mildly atypical hyperchromatic nuclei, which may be mitotically active. 1 Displaced granulosa cells, derived from normal follicles and introduced into ovarian vascular channels, ovarian stroma and the fallopian tube, have been reported to cause diagnostic difficulty in histol- ogy, as they may mimic small cell carcinoma or other metastatic carcinomas. 2–4 The cells are thought to be displaced artefactually due to surgical trauma or during sectioning in the laboratory or during ovulation...

The co-transcriptome of uropathogenic Escherichia coli-infected mouse macrophages reveals new insights into host-pathogen interactions

Urinary tract infections (UTI) are among the most common infections in humans. Uropathogenic Escherichia coli (UPEC) can invade and replicate within bladder epithelial cells, and some UPEC strains can also survive within macrophages. To understand the UPEC transcriptional program associated with intramacrophage survival, we performed host–pathogen co-transcriptome analyses using RNA sequencing. Mouse bone marrow-derived macrophages (BMMs) were challenged over a 24 h time course with two UPEC reference strains that possess contrasting intramacrophage phenotypes: UTI89, which survives in BMMs, and 83972, which is killed by BMMs. Neither of these strains caused significant BMM cell death at the low multiplicity of infection that was used in this study. We developed an effective computational framework that simultaneously separated, annotated, and quantified the mammalian and bacterial transcriptomes. BMMs responded to the two UPEC strains with a broadly similar gene expression program. In contrast, the transcriptional responses of the UPEC strains diverged markedly from each other. We identified UTI89 genes upregulated at 24 h post-infection, and hypothesized that some may contribute to intramacrophage survival. Indeed, we showed that deletion of one such gene (pspA) significantly reduced UTI89 survival within BMMs. Our study provides a technological framework for simultaneously capturing global changes at the transcriptional level in co-cultures, and has generated new insights into the mechanisms that UPEC use to persist within the intramacrophage environment.

Cytokine secretion in macrophages : SNAREs, Rabs and membrane trafficking

Macrophages have the capacity to rapidly secrete a wide range of inflammatory mediators that influence the development and extent of an inflammatory response. Newly synthesized and/or preformed stored cytokines and other inflammatory mediators are released upon stimulation, the timing, and volume of which is highly regulated. To finely tune this process, secretion is regulated at many levels; at the level of transcription and translation and post-translationally at the endoplasmic reticulum (ER), Golgi, and at or near the cell surface. Here, we discuss recent advances in deciphering these cytokine pathways in macrophages, focusing on recent discoveries regarding the cellular machinery and mechanisms implicated in the synthesis, trafficking, and secretion of cytokines. The specific roles of trafficking machinery including chaperones, GTPases, cytoskeletal proteins, and SNARE membrane fusion proteins will be discussed.

Murine, but not human, ephrin-B2 can be efficiently cleaved by the serine protease kallikrein-4: Implications for xenograft models of human prostate cancer

Background Ephrin-B2 is the sole physiologically-relevant ligand of the receptor tyrosine kinase EphB4, which is over-expressed in many epithelial cancers, including 66% of prostate cancers, and contributes to cancer cell survival, invasion and migration. Crucially, however, the cancer-promoting EphB4 signalling pathways are independent of interaction with its ligand ephrin-B2, as activation of ligand-dependent signalling causes tumour suppression. Ephrin-B2, however, is often found on the surface of endothelial cells of the tumour vasculature, where it can regulate angiogenesis to support tumour growth. Proteolytic cleavage of endothelial cell ephrin-B2 has previously been suggested as one mechanism whereby the interaction between tumour cell-expressed EphB4 and endothelial cell ephrin-B2 is regulated to support both cancer promotion and angiogenesis. Methods An in silico approach was used to search accessible surfaces of 3D protein models for cleavage sites for the key prostate cancer serine protease, KLK4, and this identified murine ephrin-B2 as a potential KLK4 substrate. Mouse ephrin-B2 was then confirmed as a KLK4 substrate by in vitro incubation of recombinant mouse ephrin-B2 with active recombinant human KLK4. Cleavage products were visualised by SDS-PAGE, silver staining and Western blot and confirmed by N-terminal sequencing. Results At low molar ratios, KLK4 cleaved murine ephrin-B2 but other prostate-specific KLK family members (KLK2 and KLK3/PSA) were less efficient, suggesting cleavage was KLK4-selective. The primary KLK4 cleavage site in murine ephrin-B2 was verified and shown to correspond to one of the in silico predicted sites between extracellular domain residues arginine 178 and asparagine 179. Surprisingly, the highly homologous human ephrin-B2 was poorly cleaved by KLK4 at these low molar ratios, likely due to the 3 amino acid differences at this primary cleavage site. Conclusion These data suggest that in in vivo mouse xenograft models, endogenous mouse ephrin-B2, but not human tumour ephrin-B2, may be a downstream target of cancer cell secreted human KLK4. This is a critical consideration when interpreting data from murine explants of human EphB4+/KLK4+ cancer cells, such as prostate cancer cells, where differential effects may be seen in mouse models as opposed to human clinical situations.

Strain- and host species-specific inflammasome activation, IL-1β release, and cell death in macrophages infected with uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infections (UTIs). Little is known about interactions between UPEC and the inflammasome, a key innate immune pathway. Here we show that UPEC strains CFT073 and UTI89 trigger inflammasome activation and lytic cell death in human macrophages. Several other UPEC strains, including two multidrug-resistant ST131 isolates, did not kill macrophages. In mouse macrophages, UTI89 triggered cell death only at a high multiplicity of infection, and CFT073-mediated inflammasome responses were completely NLRP3-dependent. Surprisingly, CFT073- and UTI89-mediated responses only partially depended on NLRP3 in human macrophages. In these cells, NLRP3 was required for interleukin-1β (IL-1β) maturation, but contributed only marginally to cell death. Similarly, caspase-1 inhibition did not block cell death in human macrophages. In keeping with such differences, the pore-forming toxin α-hemolysin mediated a substantial proportion of CFT073-triggered IL-1β secretion in mouse but not human macrophages. There was also a more substantial α-hemolysin-independent cell death response in human vs. mouse macrophages. Thus, in mouse macrophages, CFT073-triggered inflammasome responses are completely NLRP3-dependent, and largely α-hemolysin-dependent. In contrast, UPEC activates an NLRP3-independent cell death pathway and an α-hemolysin-independent IL-1β secretion pathway in human macrophages. This has important implications for understanding UTI in humans.

Infection and cellular defense dynamics in a novel 17beta-estradiol murine model of chronic human group B streptococcus genital tract colonization reveal a role for hemolysin in persistence and neutrophil accumulation

Genital tract carriage of group B streptococcus (GBS) is prevalent among adult women; however, the dynamics of chronic GBS genital tract carriage, including how GBS persists in this immunologically active host niche long term, are not well defined. To our knowledge, in this study, we report the first animal model of chronic GBS genital tract colonization using female mice synchronized into estrus by delivery of 17β-estradiol prior to intravaginal challenge with wild-type GBS 874391. Cervicovaginal swabs, which were used to measure bacterial persistence, showed that GBS colonized the vaginal mucosa of mice at high numbers (106–107 CFU/swab) for at least 90 d. Cellular and histological analyses showed that chronic GBS colonization of the murine genital tract caused significant lymphocyte and PMN cell infiltrates, which were localized to the vaginal mucosal surface. Long-term colonization was independent of regular hormone cycling. Immunological analyses of 23 soluble proteins related to chemotaxis and inflammation showed that the host response to GBS in the genital tract comprised markers of innate immune activation including cytokines such as GM-CSF and TNF-α. A nonhemolytic isogenic mutant of GBS 874391, Δcyle9, was impaired for colonization and was associated with amplified local PMN responses. Induction of DNA neutrophil extracellular traps, which was observed in GBS-infected human PMNs in vitro in a hemolysin-dependent manner, appeared to be part of this response. Overall, this study defines key infection dynamics in a novel murine model of chronic GBS genital tract colonization and establishes previously unknown cellular and soluble defense responses to GBS in the female genital tract.

Effects of Tamoxifen and oestrogen on histology and radiographic density in high and low mammographic density human breast tissues maintained in murine tissue engineering chambers

Mammographic density (MD) is a strong risk factor for breast cancer. It is altered by exogenous endocrine treatments, including hormone replacement therapy and Tamoxifen. Such agents also modify breast cancer (BC) risk. However, the biomolecular basis of how systemic endocrine therapy modifies MD and MD-associated BC risk is poorly understood. This study aims to determine whether our xenograft biochamber model can be used to study the effectiveness of therapies aimed at modulating MD, by examine the effects of Tamoxifen and oestrogen on histologic and radiographic changes in high and low MD tissues maintained within the biochamber model. High and low MD human tissues were precisely sampled under radiographic guidance from prophylactic mastectomy fresh specimens of high-risk women, then inserted into separate vascularized murine biochambers. The murine hosts were concurrently implanted with Tamoxifen, oestrogen or placebo pellets, and the high and low MD biochamber tissues maintained in the murine host environment for 3 months, before the high and low MD biochamber tissues were harvested for histologic and radiographic analyses. The radiographic density of high MD tissue maintained in murine biochambers was decreased in Tamoxifen-treated mice compared to oestrogen-treated mice (p = 0.02). Tamoxifen treatment of high MD tissue in SCID mice led to a decrease in stromal (p = 0.009), and an increase in adipose (p = 0.023) percent areas, compared to placebo-treated mice. No histologic or radiographic differences were observed in low MD biochamber tissue with any treatment. High MD biochamber tissues maintained in mice implanted with Tamoxifen, oestrogen or placebo pellets had dynamic and measurable histologic compositional and radiographic changes. This further validates the dynamic nature of the MD xenograft model, and suggests the biochamber model may be useful for assessing the underlying molecular pathways of Tamoxifen-reduced MD, and in testing of other pharmacologic interventions in a preclinical model of high MD.

A clinical audit to compare peritonitis rates between peritoneal dialysis delivery systems

Peritonitis is a major problem for patients with end-stage kidney disease undergoing peritoneal dialysis (PD). It is the main cause of failure of PD. Two different PD delivery systems are used across Australia although there is inconsistent evidence comparing the systems. The aim of this retrospective audit is to compare the rates and risk of peritonitis in a cohort of incident patients using two PD delivery systems. All consecutive patients starting PD between 1 August 2010 and 31 March 2012 were included and followed until 30 June 2013. Data relating to accepted risk factors for peritonitis were collected and analysed. There were 50 patients (26 men; 24 women) aged between 30 and 87 years. There were 29 episodes of peritonitis in 17 patients. Rates of peritonitis were 1 episode per 69.19 patient-months compared with 1 episode per 18.67 patient-months. Mean times to first episode of peritonitis were 13.11 months compared to 7.13 months. The relative risk of PD-related peritonitis was twice as high (RR = 2.04, 95% CI = 0.85 to 4.94) for patients using the one system (44.4%) compared to a second system (21.7%). Since this is not a randomised trial no firm conclusions can be drawn. Centres should also monitor peritonitis rates for each system.