Peptide-based microcapsules obtained by self-assembly and microfluidics as controlled environments for cell culture

Funding for this study was provided by the Portuguese Foundation for Science and Technology (FCT, grant PTDC/EBB-BIO/ 114523/2009). D. S. Ferreira gratefully acknowledges FCT for the PhD scholarship (SFRH/BD/44977/2008)

Necrosis binding of Ac-Lys⁰(IRDye800CW)-Tyr³-octreotate: a consequence from cyanine-labeling of small molecules.

There is a growing body of nuclear contrast agents that are repurposed for fluorescence-guided surgery. New contrast agents are obtained by substituting the radioactive tag with, or adding a fluorescent cyanine to the molecular structure of antibodies or peptides. This enables intra-operative fluorescent detection of cancerous tissue, leading to more complete tumor resection. However, these fluorescent cyanines can have a remarkable influence on pharmacokinetics and tumor uptake, especially when labeled to smaller targeting vectors such as peptides. Here we demonstrate the effect of cyanine-mediated dead cell-binding of Ac-Lys <sup>0</sup> (IRDye800CW)-Tyr <sup>3</sup> -octreotate (800CW-TATE) and how this can be used as an advantage for fluorescence-guided surgery. Binding of 800CW-TATE could be blocked with DOTA <sup>0</sup> -Tyr <sup>3</sup> -octreotate (DOTA-TATE) on cultured SSTR <sub>2</sub> -positive U2OS cells and was absent in SSTR <sub>2</sub> negative U2OS cells. However, strong binding was observed to dead cells, which could not be blocked with DOTA-TATE and was also present in dead SSTR <sub>2</sub> negative cells. No SSTR <sub>2</sub> -mediated binding was observed in frozen tumor sections, possibly due to disruption of the cells in the process of sectioning the tissue before exposure to the contrast agent. DOTA-TATE blocking resulted in an incomplete reduction of 61.5 ± 5.8% fluorescence uptake by NCI-H69-tumors in mice. Near-infrared imaging and dead cell staining on paraffin sections from resected tumors revealed that fluorescence uptake persisted in necrotic regions upon blocking with DOTA-TATE. This study shows that labeling peptides with cyanines can result in dead cell binding. This does not hamper the ultimate purpose of fluorescence-guided surgery, as necrotic tissue appears in most solid tumors. Hence, the necrosis binding can increase the overall tumor uptake. Moreover, necrotic tissue should be removed as much as possible: it cannot be salvaged, causes inflammation, and is tumorigenic. However, when performing binding experiments to cells with disrupted membrane integrity, which is routinely done with nuclear probes, this dead cell-binding can resemble non-specific binding. This study will benefit the development of fluorescent contrast agents

If, When, and How to Use Rifampin in Acute Staphylococcal Periprosthetic Joint Infections, a Multicentre Observational Study

Background: Rifampin is generally advised in the treatment of acute staphylococcal periprosthetic joint infections (PJI). However, if, when, and how to use rifampin remains a matter of debate. We evaluated the outcome of patients treated with and without rifampin, and analyzed the influence of timing, dose and co-antibiotic. Methods: Acute staphylococcal PJIs treated with surgical debridement between 1999 and 2017, and a minimal follow-up of 1 year were evaluated. Treatment failure was defined as the need for any further surgical procedure related to infection, PJI-related death or the need for suppressive antimicrobial treatment. Results: A total of 669 patients were analyzed. Treatment failure was 32.2% (131/407) in patients treated with rifampin and 54.2% (142/262) in whom rifampin was withheld (P < .001). The most prominent effect of rifampin was observed in knees (treatment failure 28.6% versus 63.9%, respectively, P < .001). The use of rifampin was an independent predictor of treatment success in the multi-variate analysis (OR 0.30, 95% CI 0.20 - 0.45). In the rifampin group, the use of a co-antibiotic other than a fluoroquinolone or clindamycin (OR 10.1, 95% CI 5.65 - 18.2) and the start of rifampin within 5 days after surgical debridement (OR 1.96, 95% CI 1.08 - 3.65) were predictors of treatment failure. The dosing of rifampin had no effect on outcome. Conclusions: Our data supports the use of rifampin in acute staphylococcal PJIs treated with surgical debridement, particularly in knees. Immediate start of rifampin after surgical debridement should probably be discouraged, but requires further investigation

A Second Surgical Debridement for Acute Periprosthetic Joint Infections Should Not Be Discarded

Background: In acute periprosthetic joint infections (PJIs), a second surgical debridement (debridement, antibiotics, and implant retention [DAIR]) is generally not recommended after a failed first one. We identified the failure rate of a second DAIR and aimed to identify patients in whom an additional debridement might still be beneficial. Methods: Patients with acute PJI of the hip or knee and treated with DAIR between 2006 and 2016 were retrospectively evaluated. A second DAIR was routinely performed provided that the soft tissue was intact. Failure of a second DAIR was described as (1) the need for additional surgical intervention to achieve infection control, (2) the need for antibiotic suppressive therapy due to persistent clinical and/or biochemical signs of infection, or (3) PJI related death. Results: From the 455 cases treated with DAIR, 144 cases underwent a second debridement (34.6%). Thirty-seven cases failed (37/144, 25.7%). The implant needed to be removed in 23 cases (23/144, 16%). Positive cultures during the second DAIR (odds ratio 3.16, 95% confidence interval 1.29-7.74) and chronic renal insufficiency (odds ratio 13.6, 95% confidence interval 2.03-91.33) were independent predictors for failure in the multivariate analysis. No difference in failure was observed between persistent infection with the same microorganism and reinfection with a new microorganism (failure rate 31.6% vs 34.6%, P =.83). Conclusion: A second DAIR had a low failure rate in our cohort of patients and the implant could be retained in the majority of them. Therefore, a second DAIR should not be discarded in acute PJIs

Focal segmental glomerulosclerosis, Coats’-like retinopathy, sensorineural deafness and chromosome 4 duplication: a new association

We describe the novel association in a girl of nephrotic syndrome due to focal segmental glomerulosclerosis, bilateral sensorineural deafness, basal ganglia calcification, bilateral retinopathy similar to that seen in Coats’ disease, with de novo duplication of a subtelomeric region of chromosome 4q35. The chromosomal duplication was identified during investigation of a possible association with features of fascio-scapulo-humeral dystrophy (FSHD). This duplication has not previously been reported with FSGS and adds to the expanding number of genetic associations with steroid-resistant nephrotic syndrome

Influence of ARHGEF3 and RHOA Knockdown on ACTA2 and Other Genes in Osteoblasts and Osteoclasts

Osteoporosis is a common bone disease that has a strong genetic component. Genome-wide linkage studies have identified the chromosomal region 3p14-p22 as a quantitative trait locus for bone mineral density (BMD). We have previously identified associations between variation in two related genes located in 3p14-p22, ARHGEF3 and RHOA, and BMD in women. In this study we performed knockdown of these genes using small interfering RNA (siRNA) in human osteoblast-like and osteoclast-like cells in culture, with subsequent microarray analysis to identify genes differentially regulated from a list of 264 candidate genes. Validation of selected findings was then carried out in additional human cell lines/cultures using quantitative real-time PCR (qRT-PCR). The qRT-PCR results showed significant down-regulation of the ACTA2 gene, encoding the cytoskeletal protein alpha 2 actin, in response to RHOA knockdown in both osteoblast-like (P<0.001) and osteoclast-like cells (P = 0.002). RHOA knockdown also caused up-regulation of the PTH1R gene, encoding the parathyroid hormone 1 receptor, in Saos-2 osteoblast-like cells (P<0.001). Other findings included down-regulation of the TNFRSF11B gene, encoding osteoprotegerin, in response to ARHGEF3 knockdown in the Saos-2 and hFOB 1.19 osteoblast-like cells (P = 0.003– 0.02), and down-regulation of ARHGDIA, encoding the Rho GDP dissociation inhibitor alpha, in response to RHOA knockdown in osteoclast-like cells (P<0.001). These studies identify ARHGEF3 and RHOA as potential regulators of a number of genes in bone cells, including TNFRSF11B, ARHGDIA, PTH1R and ACTA2, with influences on the latter evident in both osteoblast-like and osteoclast-like cells. This adds further evidence to previous studies suggesting a role for the ARHGEF3 and RHOA genes in bone metabolism

Activin Receptor-like Kinase 1 Ligand Trap Reduces Microvascular Density and Improves Chemotherapy Efficiency to Various Solid Tumors

Therapeutic cell differentiatio

Bisphosphonates induce apoptosis in human breast cancer cell lines

Breast cancer has a prodigious capacity to metastasize to bone. In women with advanced breast cancer and bone metastases, bisphosphonates reduce the incidence of hypercalcaemia and skeletal morbidity. Recent clinical findings suggest that some bisphosphonates reduce the tumour burden in bone with a consequent increase in survival, raising the possibility that bisphosphonates may have a direct effect on breast cancer cells. We have investigated the in vitro effects of bisphosphonates zoledronate, pamidronate, clodronate and EB 1053 on growth, viability and induction of apoptosis in three human breast cancer cell lines (MDA-MB-231, Hs 578T and MCF-7). Cell growth was monitored by crystal violet dye assay, and cell viability was quantitated by MTS dye reduction. Induction of apoptosis was determined by identification of morphological features of apoptosis using time-lapse videomicroscopy, identifying morphological changes in nucleis using Hoechst staining, quantitation of DNA fragmentation, level of expression of bcl-2 and bax proteins and identification of the proteolytic cleavage of Poly (ADP)-ribose polymerase (PARP). All four bisphosphonates significantly reduced cell viability in all three cell lines. Zoledronate was the most potent bisphosphonate with IC50values of 15, 20 and 3 μM respectively in MDA-MB-231, MCF-7 and Hs 578T cells. Corresponding values for pamidronate were 40, 35 and 25 μM, whereas clodronate and EB 1053 were more than two orders of magnitude less potent. An increase in the proportion of cells having morphological features characteristic of apoptosis, characteristic apoptotic changes in the nucleus, time-dependent increase in the percentage of fragmented chromosomal DNA, down-regulation in bcl-2 protein and proteolytic cleavage of PARP, all indicate that bisphosphonates have direct anti-tumour effects on human breast cancer cells. © 2000 Cancer Research Campaig

Bisphosphonates antagonise bone growth factors' effects on human breast cancer cells survival

Bone tissue constitutes a fertile 'soil' for metastatic tumours, notably breast cancer. High concentrations of growth factors in bone matrix favour cancer cell proliferation and survival, and a vicious cycle settles between bone matrix, osteoclasts and cancer cells. Classically, bisphosphonates interrupt this vicious cycle by inhibiting osteoclast-mediated bone resorption. We and others recently reported that bisphosphonates can also induce human breast cancer cell death in vitro, which could contribute to their beneficial clinical effects. We hypothesised that bisphosphonates could inhibit the favourable effects of 'bone-derived' growth factors, and indeed found that bisphosphonates reduced or abolished the stimulatory effects of growth factors (IGFs, FGF-2) on MCF-7 and T47D cell proliferation and inhibited their protective effects on apoptotic cell death in vitro under serum-free conditions. This could happen through an interaction with growth factors' intracellular phosphorylation transduction pathways, such as ERK1/2-MAPK. In conclusion, we report that bisphosphonates antagonised the stimulatory effects of growth factors on human breast cancer cell survival and reduced their protective effects against apoptotic cell death. Bisphosphonates and growth factors thus appear to be concurrent compounds for tumour cell growth and survival in bone tissue. This could represent a new mechanism of action of bisphosphonates in their protective effects against breast cancer-induced osteolysis.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe