Review on treatment of pleural metastasis and malignant pleural effusion with Pressurized IntraThoracic Aerosol Chemotherapy (PITAC).

Background: Malignant pleural effusion (MPE) is a common and debilitating condition seen in advanced cancer disease, and life-expectancy is short. Symptoms include pain and severe shortness of breath. Current first-line treatment options include pleural drainage using catheters as well as pleurodesis. However, these treatment modalities are often inefficient and patients need repeated procedures. Pressurized IntraThoracic Aerosol Chemotherapy (PITAC) is a minimally invasive procedure, where antineoplastic agents are nebulized under pressure into the pleural space. Content: We present the preliminary safety, feasibility, and response assessment data for PITAC based on a comprehensive literature review. Summary: Five retrospective studies reported data on 38 PITACs in 21 patients. Data were heterogeneous and incomplete on several important aspects such as procedure, safety, local effect and long-term outcomes. PITAC seems technically feasible with a low risk of complications and may provide some reduction in MPE in selected cases. Outlook: PITAC seems feasible, but prospective phase I and II studies are needed to define safety, indications, and efficacy.

Inhibition of Interleukin-33 to Reduce Glomerular Endothelial Inflammation in Diabetic Kidney Disease.

Introduction: Inflammation is a significant contributor to cardiorenal morbidity and mortality in diabetic kidney disease (DKD). The pathophysiological mechanisms linking systemic, subacute inflammation and local, kidney injury-initiated immune maladaptation is partially understood. Methods: Here, we explored the expression of proinflammatory cytokines in patients with DKD; investigated mouse models of type 1 and type 2 diabetes (T2D); evaluated glomerular signaling in vitro; performed post hoc analyses of systemic and urinary markers of inflammation; and initiated a phase 2b clinical study (FRONTIER-1; NCT04170543). Results: Transcriptomic profiling of kidney biopsies from patients with DKD revealed significant glomerular upregulation of interleukin-33 (IL-33). Inhibition of IL-33 signaling reduced glomerular damage and albuminuria in the uninephrectomized db/db mouse model (T2D/DKD). On a cellular level, inhibiting IL-33 improved glomerular endothelial health by decreasing cellular inflammation and reducing release of proinflammatory cytokines. Therefore, FRONTIER-1 was designed to test the safety and efficacy of the IL-33-targeted monoclonal antibody tozorakimab in patients with DKD. So far, 578 patients are enrolled in FRONTIER-1. The baseline inflammation status of participants (N > 146) was assessed in blood and urine. Comparison to independent reference cohorts (N > 200) validated the distribution of urinary tumor necrosis factor receptor 1 (TNFR1) and C-C motif chemokine ligand 2 (CCL2). Treatment with dapagliflozin for 6 weeks did not alter these biomarkers significantly. Conclusion: We show that blocking the IL-33 pathway may mitigate glomerular endothelial inflammation in DKD. The findings from the FRONTIER-1 study will provide valuable insights into the therapeutic potential of IL-33 inhibition in DKD.

GDF-15 is associated with sarcopenia and frailty in acutely admitted older medical patients.

BACKGROUND: Growth differentiation factor-15 (GDF-15) has been associated with senescence, lower muscle strength, and physical performance in healthy older people. Still, it is not clear whether GDF-15 can be utilized as a biomarker of sarcopenia and frailty in the early stages of hospitalization. We investigated the association of plasma GDF-15 with sarcopenia and frailty in older, acutely admitted medical patients. METHODS: The present study is based on secondary analyses of cross-sectional data from the Copenhagen PROTECT study, a prospective cohort study including 1071 patients ≥65 years of age admitted to the acute medical ward at Copenhagen University Hospital, Bispebjerg, Denmark. Muscle strength was assessed using handgrip strength, and lean mass was assessed using direct segmental multifrequency bioelectrical impedance analyses and used to clarify the potential presence of sarcopenia defined according to guidelines from the European Working Group on Sarcopenia in Older People. Frailty was evaluated using the Clinical Frailty Scale. Plasma GDF-15 was measured using electrochemiluminescence assays from Meso Scale Discovery (MSD, Rockville, MD, USA). RESULTS: We included 1036 patients with completed blood samples (mean age 78.9 ± 7.8 years, 53% female). The median concentration of GDF-15 was 2669.3 pg/mL. Systemic GDF-15 was significantly higher in patients with either sarcopenia (P < 0.01) or frailty (P < 0.001) compared with patients without the conditions. Optimum cut-off points of GDF-15 relating to sarcopenia and frailty were 1541 and 2166 pg/mL, respectively. CONCLUSIONS: Systemic GDF-15 was higher in acutely admitted older medical patients with sarcopenia and frailty compared with patients without. The present study defined the optimum cut-off for GDF-15, related to the presence of sarcopenia and frailty, respectively. When elevated above the derived cutoffs, GDF-15 was strongly associated with frailty and sarcopenia in both crude and fully adjusted models.

Eight weeks of treatment with mineralocorticoid receptor blockade does not alter vascular function in individuals with and without type 2 diabetes.

Aldosterone has been suggested to be involved in the microvascular complications observed in type 2 diabetes. We aimed to investigate the effect of mineralocorticoid receptor (MR) blockade on endothelial function in individuals with type 2 diabetes compared to healthy controls. We included 12 participants with type 2 diabetes and 14 controls. We measured leg hemodynamics at baseline and during femoral arterial infusion of acetylcholine and sodium nitroprusside before and 8 weeks into treatment with MR blockade (eplerenone). Acetylcholine infusion was repeated with concomitant n-acetylcysteine (antioxidant) infusion. No difference in leg blood flow or vascular conductance was detected before or after the treatment with MR blockade in both groups and there was no difference between groups. Infusion of n-acetylcysteine increased baseline blood flow and vascular conductance, but did not change the vascular response to acetylcholine before or after treatment with MR blockade. Skeletal muscle eNOS content was unaltered by MR blockade and no difference between groups was detected. In conclusion, we found no effect of MR blockade endothelial function in individuals with and without type 2 diabetes. As the individuals with type 2 diabetes did not have vascular dysfunction, these results might not apply to individuals with vascular dysfunction.

Multi-omics and imaging mass cytometry characterization of human kidneys to identify pathways and phenotypes associated with impaired kidney function.

Despite the recent advances in our understanding of the role of lipids, metabolites, and related enzymes in mediating kidney injury, there is limited integrated multi-omics data identifying potential metabolic pathways driving impaired kidney function. The limited availability of kidney biopsies from living donors with acute kidney injury has remained a major constraint. Here, we validated the use of deceased transplant donor kidneys as a good model to study acute kidney injury in humans and characterized these kidneys using imaging and multi-omics approaches. We noted consistent changes in kidney injury and inflammatory markers in donors with reduced kidney function. Neighborhood and correlation analyses of imaging mass cytometry data showed that subsets of kidney cells (proximal tubular cells and fibroblasts) are associated with the expression profile of kidney immune cells, potentially linking these cells to kidney inflammation. Integrated transcriptomic and metabolomic analysis of human kidneys showed that kidney arachidonic acid metabolism and seven other metabolic pathways were upregulated following diminished kidney function. To validate the arachidonic acid pathway in impaired kidney function we demonstrated increased levels of cytosolic phospholipase A2 protein and related lipid mediators (prostaglandin E2) in the injured kidneys. Further, inhibition of cytosolic phospholipase A2 reduced injury and inflammation in human kidney proximal tubular epithelial cells in vitro. Thus, our study identified cell types and metabolic pathways that may be critical for controlling inflammation associated with impaired kidney function in humans.

The role of T-type calcium channels in elderly human vascular function: A pilot randomized controlled trial.

Endothelial dysfunction develops with age and may precede cardiovascular disease. Animal data suggest that T-type calcium channels play an important role in endothelial function, but data from humans are lacking. This study included 15 healthy, sedentary, elderly males for a double blinded, randomized controlled trial. For 8 weeks, they were given 40 mg/day of either efonidipine (L- and T-type calcium channel blocker (CCB)) or nifedipine (L-type CCB). Vascular function was evaluated by graded femoral arterial infusions of acetylcholine (ACh; endothelium-dependent vasodilator) and sodium nitroprusside (endothelium-independent vasodilator) both with and without co-infusion of N-acetylcysteine (NAC; antioxidant). We measured leg blood flow and mean arterial pressure and calculated leg vascular conductance to evaluate the leg vascular responses. Despite no significant change in blood pressure in either group, we observed higher leg blood flow responses (Δ 0.43 ± 0.45 l/min, P = 0.006) and leg vascular conductance (Δ 5.38 ± 5.67 ml/min/mmHg, P = 0.005) to intra-arterial ACh after efonidipine, whereas there was no change in the nifedipine group, and no differences between groups. We found no upregulation of endothelial nitric oxide synthase in vastus lateralis muscle biopsies within or between groups. Smooth muscle cell responsiveness was unaltered by efonidipine or nifedipine. Intravenous co-infusion of NAC did not affect endothelium-dependent vasodilatation in either of the CCB groups. These results suggest that 8 weeks' inhibition of T- and L-type calcium channels augments endothelium-dependent vasodilatory function in healthy elderly males. Further studies are required to elucidate if T-type calcium channel inhibition can counteract endothelial dysfunction.

Inhibition of cystathionine-gamma lyase dampens vasoconstriction in mouse and human intracerebral arterioles.

AIM: In extracerebral vascular beds cystathionine-gamma lyase (CSE) activity plays a vasodilatory role but the role of this hydrogen sulfide (H2 S) producing enzyme in the intracerebral arterioles remain poorly understood. We hypothesized a similar function in the intracerebral arterioles. METHODS: Intracerebral arterioles were isolated from wild type C57BL/6J mouse (9-12 months old) brains and from human brain biopsies. The function (contractility and secondary dilatation) of the intracerebral arterioles was tested ex vivo by pressure myography using a perfusion set-up. Reverse transcription polymerase chain reaction was used for detecting CSE expression. RESULTS: CSE is expressed in human and mouse intracerebral arterioles. CSE inhibition with L-propargylglycine (PAG) significantly dampened the K+ -induced vasoconstriction in intracerebral arterioles of both species (% of maximum contraction: in human control: 45.4 ± 2.7 versus PAG: 27 ± 5.2 and in mouse control: 50 ± 1.5 versus PAG: 33 ± 5.2) but did not affect the secondary dilatation. This effect of PAG was significantly reversed by the H2 S donor sodium hydrosulfide (NaSH) in human (PAG + NaSH: 38.8 ± 7.2) and mouse (PAG + NaSH: 41.7 ± 3.1) arterioles, respectively. The endothelial NO synthase (eNOS) inhibitor, Nω-Nitro-l-arginine methyl ester (L-NAME), and the inhibitor of soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reversed the effect of PAG on the K+ -induced vasoconstriction in the mouse arterioles and attenuated the K+ -induced secondary dilatation significantly. CONCLUSION: CSE contributes to the K+ -induced vasoconstriction via a mechanism involving H2 S, eNOS, and sGC whereas the secondary dilatation is regulated by eNOS and sGC but not by CSE.

3D vascularised proximal tubules-on-a-multiplexed chip model for enhanced cell phenotypes.

Modelling proximal tubule physiology and pharmacology is essential to understand tubular biology and guide drug discovery. To date, multiple models have been developed; however, their relevance to human disease has yet to be evaluated. Here, we report a 3D vascularized proximal tubule-on-a-multiplexed chip (3DvasPT-MC) device composed of co-localized cylindrical conduits lined with confluent epithelium and endothelium, embedded within a permeable matrix, and independently addressed by a closed-loop perfusion system. Each multiplexed chip contains six 3DvasPT models. We performed RNA-seq and compared the transcriptomic profile of proximal tubule epithelial cells (PTECs) and human glomerular endothelial cells (HGECs) seeded in our 3D vasPT-MCs and on 2D transwell controls with and without a gelatin-fibrin coating. Our results reveal that the transcriptional profile of PTECs is highly dependent on both the matrix and flow, while HGECs exhibit greater phenotypic plasticity and are affected by the matrix, PTECs, and flow. PTECs grown on non-coated Transwells display an enrichment of inflammatory markers, including TNF-a, IL-6, and CXCL6, resembling damaged tubules. However, this inflammatory response is not observed for 3D proximal tubules, which exhibit expression of kidney signature genes, including drug and solute transporters, akin to native tubular tissue. Likewise, the transcriptome of HGEC vessels resembled that of sc-RNAseq from glomerular endothelium when seeded on this matrix and subjected to flow. Our 3D vascularized tubule on chip model has utility for both renal physiology and pharmacology.

Generic benzalkonium chloride-preserved travoprost eye drops are not identical to the branded polyquarternium-1-preserved travoprost eye drop: Effect on cultured human conjunctival goblet cells and their physicochemical properties.

PURPOSE: To investigate the effect of polyquaternium-1 (PQ)-preserved and benzalkonium chloride (BAK)-preserved travoprost eye drops on viability of primary human conjunctival goblet cell (GC) cultures and on secretion of mucin and cytokines. Furthermore, to evaluate the physicochemical properties of the branded travoprost eye drop Travatan® and available generics. METHODS: The effect of travoprost eye drops was evaluated on GC cultures. Cell viability was assessed through lactate dehydrogenase (LDH) and tetrazolium dye (MTT) colorimetric assays. Mucin secretion was evaluated by immunohistochemical staining. Secretion of interleukin (IL)-6 and IL-8 was measured using BD Cytometric Bead Arrays. pH, viscosity, droplet mass, osmolality and surface tension were measured for all included eye drops. RESULTS: In the LDH assay, BAK travoprost caused significant GC loss after 2 hrs of incubation compared to the control. PQ travoprost caused no GC loss at any time point. Both PQ- and BAK travoprost caused secretion of mucin to the cytoplasma. No difference in IL-6 and IL-8 secretion was identified compared to controls. The pH values for the generics were lower (pH 6.0) than the pH value for Travatan (pH 6.7; p < 0.0001). The viscosity was lowest for Travatan, while the mean droplet mass was higher for Travatan (35 mg) than the generics (28-30 mg; p ≤ 0.0318). The osmolality and surface tension did not differ between the eye drops investigated. CONCLUSION: BAK travoprost caused GC loss, indicating that PQ preservation may be preferable in treatment of glaucoma. Furthermore, physicochemical properties of branded and generic travoprost eye drops can not be assumed to be identical.

Interleukin 17A infusion has no acute or long-term hypertensive action in conscious unrestrained male mice.

Interleukin 17A (IL-17A) is a candidate mediator of inflammation-driven hypertension, but its direct effect on blood pressure is obscure. The present study was designed to test the hypothesis that systemic IL-17A concentration-dependently increases blood pressure and amplifies ANGII-induced hypertension in mice. Blood pressure was measured by indwelling chronic femoral catheters before and during IL-17A infusion w/wo angiotensin II (ANGII, 60ng/kg/min) in male FVB/n mice. Baseline blood pressure was recorded, and three experimental series were conducted: (1) IL-17A infusion with increasing concentrations over 6 days (two series with IL-17A from two vendors, n = 11); (2) ANGII infusion with IL-17A or vehicle for 9 days (n = 11); and (3) acute bolus infusions with four different concentrations (n = 5). Plasma IL-17A and IL-6 concentrations were determined by ELISA. Mean arterial and systolic blood pressures (MAP, SBP) decreased significantly after IL-17A infusion while heart rate was unchanged. In these mice, plasma IL-17A and IL-6 concentrations increased up to 3500- and 2.4-fold, respectively, above baseline. ANGII infusion increased MAP (~ 25 mmHg) and co-infusion of IL-17A attenuated ANGII-induced hypertension by 4.0 mmHg. Here, plasma IL-17A increased 350-fold above baseline. Acute IL-17A bolus infusion did not change blood pressure or heart rate. IL-17A receptor and IL-6 mRNAs were detected in aorta, heart, and kidneys of mice after IL-17A infusion. Nonphysiologically high concentrations of IL-17A reduce baseline blood pressure and increase IL-6 formation in male FVB/n mice. It is concluded that IL-17A is less likely to drive hypertension as the sole cytokine mediator during inflammation in vivo.

Association of perceived work pace and physical work demands with occupational accidents: a cross-sectional study of ageing male construction workers in Denmark.

BACKGROUND: Occupational accidents continue to be a significant public health challenge worldwide. Construction workers in particular are at high risk of occupational accidents, and thus it is of major importance to identify possible predictors of occupational accidents among construction workers. We aimed to investigate the association between self-reported work pace and physical work demands and occupational accidents among ageing male construction workers in Denmark. METHODS: Data on perceived work pace, physical work demands, and occupational accidents was acquired from questionnaires sent to ageing construction workers in Denmark in 2016 as part of the ALFA project (ALdring og Fysisk Arbejde; Ageing and Physical Work). A sample of 1270 Danish male construction workers above 50 years of age was included in the present study. Multiple logistic regression models were applied, with adjustments for age, smoking, body mass index, musculoskeletal disorders, occupation, work experience, and support at work. RESULTS: Of 1270 construction workers, 166 (13.1%) reported an occupational accident within the last 12 months. There was no significant association between perceived work pace and occupational accidents, but physical work demands were associated with higher odds for occupational accidents, with an odds ratio of 2.27 (95% confidence interval 1.26-4.10) for medium physical work demands and 2.62 (95% confidence interval 1.50-4.57) for high physical work demands. CONCLUSIONS: Ageing male construction workers with high physical work demands had statistically significant higher odds of having an occupational accident. By contrast, perceived work pace was not associated with occupational accidents in this large cross-sectional study.

Intestinal sodium/glucose cotransporter 3 expression is epithelial and downregulated in obesity.

AIM: We aimed to determine whether the sodium/glucose cotransporter family member SGLT3, a proposed glucose sensor, is expressed in the intestine and/or kidney, and if its expression is altered in mouse models of obesity and in humans before and after weight-loss surgery. MAIN METHODS: We used in-situ hybridization and quantitative PCR to determine whether the Sglt3 isoforms 3a and 3b were expressed in the intestine and kidney of C57, leptin-deficient ob/ob, and diabetic BTBR ob/ob mice. Western blotting and immunohistochemistry were also used to assess SGLT3 protein levels in jejunal biopsies from obese patients before and after weight-loss Roux-en-Y gastric bypass surgery (RYGB), and in lean healthy controls. KEY FINDINGS: Sglt3a/3b mRNA was detected in the small intestine (duodenum, jejunum and ileum), but not in the large intestine or kidneys of mice. Both isoforms were detected in epithelial cells (confirmed using intestinal organoids). Expression of Sglt3a/3b mRNA in duodenum and jejunum was significantly lower in ob/ob and BTBR ob/ob mice than in normal-weight littermates. Jejunal SGLT3 protein levels in aged obese patients before RYGB were lower than in lean individuals, but substantially upregulated 6 months post-RYGB. SIGNIFICANCE: Our study shows that Sglt3a/3b is expressed primarily in epithelial cells of the small intestine in mice. Furthermore, we observed an association between intestinal mRNA Sglt3a/3b expression and obesity in mice, and between jejunal SGLT3 protein levels and obesity in humans. Further studies are required to determine the possible role of SGLT3 in obesity.

Past, present and future, the experience of time during examination for malignant brain tumor: a qualitative observational study.

BACKGROUND: Primary malignant brain tumor is a severe disease with a poor prognosis causing reduced life expectancy and possible alteration in the perception of time. The aim of this study was to gain deeper insight into the perception of time from the perspective of patients with brain cancer as they pass through the Danish Integrated Brain Cancer Pathway at a university hospital in Denmark. METHODS: Data were generated by shadowing six patients and relatives during their visit to and hospitalisation in a neurosurgical department. RESULTS: Through one constructed case, three perspectives of time were identified. The patient's perception of time during his illness, the healthcare system's perception of time and, finally, an ethical time perspective. The analysis showed a discrepancy between patients' and healthcare professionals' perception of time. Furthermore, the results revealed an ethical time dimension. CONCLUSIONS: The findings contribute to a better understanding of the perception of time among seriously ill patients and may further healthcare professionals' awareness of how to support patients in achieving a more meaningful use of their remaining lifetime.

Aldosterone Induces Vasoconstriction in Individuals with Type 2 Diabetes: Effect of Acute Antioxidant Administration.

CONTEXT: Individuals with type 2 diabetes have an increased risk of endothelial dysfunction and cardiovascular disease. Plasma aldosterone could contribute by reactive oxygen species-dependent mechanisms by inducing a shift in the balance between a vasoconstrictor and vasodilator response to aldosterone. OBJECTIVE: We aimed to investigate the acute vascular effects of aldosterone in individuals with type 2 diabetes compared with healthy controls and if infusion of an antioxidant (n-acetylcysteine [NAC]) would alter the vascular response. METHODS: In a case-control design, 12 participants with type 2 diabetes and 14 healthy controls, recruited from the general community, were studied. Leg hemodynamics were measured before and during aldosterone infusion (0.2 and 5 ng min-1 [L leg volume]-1) for 10 minutes into the femoral artery with and without coinfusion of NAC (125 mg kg-1 hour-1 followed by 25 mg kg-1 hour-1). Leg blood flow and arterial blood pressure was measured, and femoral arterial and venous blood samples were collected. RESULTS: Compared with the control group, leg blood flow and vascular conductance decreased during infusion of aldosterone at the high dose in individuals with type 2 diabetes, whereas coinfusion of NAC attenuated this response. Plasma aldosterone increased in both groups during aldosterone infusion and there was no difference between groups at baseline or during the infusions. CONCLUSION: These results suggests that type 2 diabetes is associated with a vasoconstrictor response to physiological levels of infused aldosterone and that the antioxidant NAC diminishes this response.

Immunohistochemical Localization of Deleted in Malignant Brain Tumors 1 in Normal Human Tissues.

Deleted in malignant brain tumors 1 (DMBT1) is part of the innate immune system and is expressed on mucosal surfaces in various tissues throughout the human body. However, to date, the localization of DMBT1 has not been investigated systematically and comprehensively in normal human tissues. In this study, we analyzed the mRNA expression of DMBT1 in human tissue by quantitative real-time PCR and examined its localization and distribution in the tissue by immunohistochemical staining using the monoclonal DMBT1 antibody HYB213-6. Anti-ovalbumin was used as an isotype control. The highest level of mRNA expression of DMBT1 was found in the small intestine, and the expression level was high throughout the luminal digestive tract. The expression of DMBT1 was especially high in the luminal digestive tract and salivary glands. The lowest expression level was found in the spleen. Immunohistochemical staining showed a high expression level of DMBT1 on mucosal surfaces throughout the body. There was a clear correlation between the mRNA expression and immunohistochemical expression of DMBT1 in the tissue. DMBT1 is strongly expressed on mucosal surfaces and in salivary glands.

CFP suppresses breast cancer cell growth by TES-mediated upregulation of the transcription factor DDIT3.

Breast cancer is a heterogeneous genetic disease driven by the accumulation of individual mutations per tumor. Whole-genome sequencing approaches have identified numerous genes with recurrent mutations in primary tumors. Although mutations in well characterized tumor suppressors and oncogenes are overrepresented in these sets, the majority of the genetically altered genes have so far unknown roles in breast cancer progression. To improve the basic understanding of the complex disease breast cancer and to potentially identify novel drug targets or regulators of known cancer-driving pathways, we analyzed 86 wild-type genes and 94 mutated variants for their effect on cell growth using a serially constructed panel of MCF7 cell lines. We demonstrate in subsequent experiments that the metal cation transporter CNNM4 regulates growth by induction of apoptosis and identified a tumor suppressive role of complement factor properdin (CFP) in vitro and in vivo. CFP appears to induce the intracellular upregulation of the pro-apoptotic transcription factor DDIT3 which is associated with endoplasmic reticulum-stress response.

Periarterial fat from two human vascular beds is not a source of aldosterone to promote vasoconstriction.

Mouse adipocytes have been reported to release aldosterone and reduce endothelium-dependent relaxation. It is unknown whether perivascular adipose tissue (PVAT) releases aldosterone in humans. The present experiments were designed to test the hypothesis that human PVAT releases aldosterone and induces endothelial dysfunction. Vascular reactivity was assessed in human internal mammary and renal segmental arteries obtained at surgery. The arteries were prepared with/without PVAT, and changes in isometric tension were measured in response to the vasoconstrictor thromboxane prostanoid receptor agonist U46619 and the endothelium-dependent vasodilator acetylcholine. The effects of exogenous aldosterone and of mineralocorticoid receptor (MR) antagonist eplerenone were determined. Aldosterone concentrations were measured by ELISA in conditioned media incubated with human adipose tissue with/without angiotensin II stimulation. Presence of aldosterone synthase and MR mRNA was examined in perirenal, abdominal, and mammary PVAT by PCR. U46619 -induced tension and acetylcholine-induced relaxation were unaffected by exogenous and endogenous aldosterone (addition of aldosterone and MR blocker) in mammary and renal segmental arteries, both in the presence and absence of PVAT. Aldosterone release from incubated perivascular fat was not detectable. Aldosterone synthase expression was not consistently observed in human adipose tissues in contrast to that of MR. Thus, exogenous aldosterone does not affect vascular reactivity and endothelial function in ex vivo human arterial segments, and the tested human adipose tissues have no capacity to synthesize/release aldosterone. In perspective, physiologically relevant effects of aldosterone on vascular function in humans are caused by systemic aldosterone originating from the adrenal gland.

One-step FPLC-size-exclusion chromatography procedure for purification of rDMBT1 6 kb with increased biological activity.

Deleted in Malignant Brain Tumor 1 (DMBT1, alias SAG or gp340) is a pattern recognition receptor involved in immune defense, cell polarization, differentiation and regeneration. To investigate the role of the protein in physiological and pathological processes, the protein has often been isolated from saliva or produced in vitro and purified by a multistep affinity purification procedure using bacteria, followed by FPLC. Here, we compared a simple, one-step FPLC-SEC protocol for purification of recombinant DMBT1 6 kb, with that of the standard bacteria affinity purification-based protocol. Our data suggest that our FPLC-SEC protocol yields DMBT1 in a more native conformation.

Human DMBT1-Derived Cell-Penetrating Peptides for Intracellular siRNA Delivery.

Small interfering RNA (siRNA) is a promising molecule for gene therapy, but its therapeutic administration remains problematic. Among the recently proposed vectors, cell-penetrating peptides show great promise in in vivo trials for siRNA delivery. Human protein DMBT1 (deleted in malignant brain tumor 1) is a pattern recognition molecule that interacts with polyanions and recognizes and aggregates bacteria. Taking advantage of these properties, we investigated whether specific synthetic DMBT1-derived peptides could be used to formulate nanoparticles for siRNA administration. Using an electrophoretic mobility shift assay and UV spectra, we identified two DMBT1 peptides that could encapsulate the siRNA with a self- and co-assembly mechanism. The complexes were stable for at least 2 hr in the presence of either fetal bovine serum (FBS) or RNase A, with peptide-dependent time span protection. ζ-potential, circular dichroism, dynamic light scattering, and transmission electron microscopy revealed negatively charged nanoparticles with an average diameter of 10-800 nm, depending on the reaction conditions, and a spherical or rice-shaped morphology, depending on the peptide and ß-helix conformation. We successfully transfected human MCF7 cells with fluorescein isothiocyanate (FITC)-DMBT1-peptide-Cy3-siRNA complexes. Finally, DMBT1 peptides encapsulating an siRNA targeting a fluorescent reporter gene showed efficient gene silencing in MCF7-recombinant cells. These results lay the foundation for a new research line to exploit DMBT1-peptide nanocomplexes for therapeutic siRNA delivery.