Protease inhibitors prevent plasminogen-mediated, but not pemphigus vulgaris-induced, acantholysis in human epidermis

Pemphigus is an autoimmune blistering disease of the skin and mucous membranes. It is caused by autoantibodies directed against desmosomes, which are the principal adhesion structures between epidermal keratinocytes. Binding of autoantibodies leads to the destruction of desmosomes resulting in the loss of cell-cell adhesion (acantholysis) and epidermal blisters. The plasminogen activator system has been implicated as a proteolytic effector in pemphigus. We have tested inhibitors of the plasminogen activator system with regard to their potential to prevent pemphigus-induced cutaneous pathology. In a human split skin culture system, IgG preparations of sera from pemphigus vulgaris patients caused histopathologic changes (acantholysis) similar to those observed in the original pemphigus disease. All inhibitors that were tested (active site inhibitors directed against uPA, tPA, and/or plasmin; antibodies neutralizing the enzymatic activity of uPA or tPA; substances interfering with the binding of uPA to its specific cell surface receptor uPAR) failed to prevent pemphigus vulgaris IgG-mediated acantholysis. Plasminogen-mediated acantholysis, however, was effectively antagonized by the synthetic active site serine protease inhibitor WX-UK1 or by p-aminomethylbenzoic acid. Our data argue against applying anti-plasminogen activator/anti-plasmin strategies in the management of pemphigus

Novel bi- and trifunctional inhibitors of tumor-associated proteolytic systems

Serine proteases, cysteine proteases, and matrix metalloproteinases (MMPs) are involved in cancer cell invasion and metastasis. Recently, a recombinant bifunctional inhibitor (chCysuPA(19-31)) directed against cysteine proteases and the urokinasetype plasminogen activator (uPA)/plasmin serine protease system was generated by introducing the uPA receptor (uPAR)binding site of uPA into chicken cystatin (chCysWT). In the present study, we designed and recombinantly produced multifunctional inhibitors also targeting MMPs. The inhibitors comprise the Nterminal inhibitory domain of human TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) or TIMP-3, fused to chCysuPA(19-31) or chCysWT. As demonstrated by various techniques, these fusion proteins effectively interfere with all three targeted protease systems. In in vitro Matrigel invasion assays, the addition of recombinant inhibitors strongly reduced invasion of ovarian cancer cells (OVMZ-6\#8). Additionally, OVMZ 6\#8 cells were stably transfected with expression plasmids encoding the various inhibitors. Synthesis and secretion of the inhibitors was verified by a newly developed ELISA, which selectively detects the recombinant proteins. Invasive capacity of inhibitorproducing cells was significantly reduced compared to vectortransfected control cells. Thus, these novel, compact, and smallsize inhibitors directed against up to three different tumorassociated proteolytic systems may represent promising agents for prevention of tumor cell migration and metastasis

Recurrent mini-outbursts and a magnetic white dwarf in the symbiotic system FN Sgr

AIMS: We investigated the optical variability of the symbiotic binary FN Sgr, with photometric monitoring during $\simeq$55 years and with a high-cadence Kepler light curve lasting 81 days. METHODS: The data obtained in the V and I bands were reduced with standard photometric methods. The Kepler data were divided into subsamples and analyses with the Lomb-Scargle algorithm. RESULTS: The V and I band light curves showed a phenomenon never before observed with such recurrence in any symbiotic system, namely short outbursts, starting between orbital phase 0.3 and 0.5 and lasting about a month, with a fast rise and a slower decline, and amplitude of 0.5-1 mag. In the Kepler light curve we discovered three frequencies with sidebands. We attribute a stable frequency of 127.5 d$^{-1}$ (corresponding to an 11.3 minutes period) to the white dwarf rotation. We suggest that this detection probably implies that the white dwarf accretes through a magnetic stream, like in intermediate polars. The small outbursts may be ascribed to the stream-disc interaction. Another possibility is that they are due to localized thermonuclear burning, perhaps confined by the magnetic field, like recently inferred in intermediate polars, albeit on different timescales. We measured also a second frequency around 116.9 d$^{-1}$ (corresponding to about 137 minutes), which is much less stable and has a drift. It may be due to rocky detritus around the white dwarf, but it is more likely to be caused by an inhomogeneity in the accretion disk. Finally, there is a third frequency close to the first one that appears to correspond to the beating between the rotation and the second frequency.Comment: Accepted for publication in Astronomy and Astrophysic

NICER monitoring of supersoft X-ray sources

We monitored four supersoft sources - two persistent ones, CAL 83 and MR Vel, and the recent novae YZ Ret (Nova Ret 2020) and V1674 Her (Nova Her 2021) - with NICER. The two persistent SSS were observed with unvaried X-ray flux level and spectrum, respectively, 13 and 20 years after the last observations. Short period modulations of the supersoft X-ray source (SSS) appear where the spectrum of the luminous central source was fully visibl (in CAL 83 and V1674 Her) and were absent in YZ Ret and MR Vel, in which the flux originated in photoionized or shocked plasma, while the white dwarf (WD) was not observable. We thus suggest that the pulsations occur on, or very close to, the WD surface. The pulsations of CAL 83 were almost unvaried after 15 years, including an irregular drift of the $\simeq$67 s period by 2.1 s. Simulations, including previous XMM-Newton data, indicate actual variations in period length within hours, rather than an artifact of the variable amplitude of the pulsations. Large amplitude pulsations with a period of 501.53$\pm$0.30 s were always detected in V1674 Her, as long as the SSS was observable. This period seems to be due to rotation of a highly magnetized WD.We cannot confirm the maximum effective temperature of ($\simeq$145,000 K) previously inferred for this nova, and discuss the difficulty in interpreting its spectrum. The WD appears to present two surface zones, one of which does not emit SSS flux.Comment: in press in the Astrophysical Journa

The Predictive Value of PITX2 DNA Methylation for High-Risk Breast Cancer Therapy: Current Guidelines, Medical Needs, and Challenges

High-risk breast cancer comprises distinct tumor entities such as triple-negative breast cancer (TNBC) which is characterized by lack of estrogen (ER) and progesterone (PR) and the HER2 receptor and breast malignancies which have spread to more than three lymph nodes. For such patients, current (inter)national guidelines recommend anthracycline-based chemotherapy as the standard of care, but not all patients do equally benefit from such a chemotherapy. To further improve therapy decision-making, predictive biomarkers are of high, so far unmet, medical need. In this respect, predictive biomarkers would permit patient selection for a particular kind of chemotherapy and, by this, guide physicians to optimize the treatment plan for each patient individually. Besides DNA mutations, DNA methylation as a patient selection marker has received increasing clinical attention. For instance, significant evidence has accumulated that methylation of the PITX2 (paired-like homeodomain transcription factor 2) gene might serve as a novel predictive and prognostic biomarker, for a variety of cancer diseases. This review highlights the current understanding of treatment modalities of high-risk breast cancer patients with a focus on recommended treatment options, with special attention on the future clinical application of PITX2 as a predictive biomarker to personalize breast cancer management

Protein kinase Cδ expression in breast cancer as measured by real-time PCR, western blotting and ELISA

The protein kinase C (PKC) family of genes encode serine/threonine kinases that regulate proliferation, apoptosis, cell survival and migration. Multiple isoforms of PKC have been described, one of which is PKCδ. Currently, it is unclear whether PKCδ is involved in promoting or inhibiting cancer formation/progression. The aim of this study was therefore to investigate the expression of PKCδ in human breast cancer and relate its levels to multiple parameters of tumour progression. Protein kinase Cδ expression at the mRNA level was measured using real-time PCR (n=208) and at protein level by both immunoblotting (n=94) and ELISA (n=98). Following immunoblotting, two proteins were identified, migrating with molecular masses of 78 and 160 kDa. The 78 kDa protein is likely to be the mature form of PKCδ but the identity of the 160 kDa form is unknown. Levels of both these proteins correlated weakly but significantly with PKCδ concentrations determined by ELISA (for the 78 kDa form, r=0.444, P<0.005, n=91 and for the 160 kDa form, r=0.237, P=0.023, n=91) and with PKCδ mRNA levels (for the 78 kDa form, r=0.351, P=0.001, n=94 and for the 160 kDa form, r=0.216, P=0.037, n=94). Protein kinase Cδ mRNA expression was significantly higher in oestrogen receptor (ER)-positive compared with ER-negative tumours (P=0.007, Mann–Whitney U-test). Increasing concentrations of PKCδ mRNA were associated with reduced overall patient survival (P=0.004). Our results are consistent with a role for PKCδ in breast cancer progression

Switch-maintenance gemcitabine after first-line chemotherapy in patients with malignant mesothelioma (NVALT19):an investigator-initiated, randomised, open-label, phase 2 trial

Background Almost all patients with malignant mesothelioma eventually have disease progression after first-line therapy. Previous studies have investigated maintenance therapy, but none has shown a great effect. We aimed to assess the efficacy and safety of switch-maintenance gemcitabine in patients with malignant mesothelioma without disease progression after first-line chemotherapy. Methods We did a randomised, open-label, phase 2 trial in 18 hospitals in the Netherlands (NVALT19). We recruited patients aged older than 18 years with unresectable malignant mesothelioma with no evidence of disease progression after at least four cycles of first-line chemotherapy (with platinum and pemetrexed), who had a WHO performance status of 0-2, adequate organ function, and measurable or evaluable disease. Exclusion criteria were active uncontrolled infection or severe cardiac dysfunction, serious disabling conditions, symptomatic CNS metastases, radiotherapy within 2 weeks before enrolment, and concomitant use of any other drugs under investigation. Patients were randomly assigned (1:1), using the minimisation method, to maintenance intravenous gemcitabine (1250 mg/m(2) on days 1 and 8, in cycles of 21 days) plus supportive care, or to best supportive care alone, until disease progression, unacceptable toxicity, serious intercurrent illness, patient request for discontinuation, or need for any other anticancer agent, except for palliative radiotherapy. A CT scan of the thorax or abdomen (or both) and pulmonary function tests were done at baseline and repeated every 6 weeks. The primary outcome was progression-free survival in the intention-to-treat population. Safety was analysed in all participants who received one or more doses of the study drug or had at least one visit for supportive care. Recruitment is now closed; treatment and follow-up are ongoing. This study is registered with the Netherlands Trial Registry, NTR4132/NL3847. Findings Between March 20, 2014, and Feb 27, 2019, 130 patients were enrolled and randomly assigned to gemcitabine plus supportive care (65 patients [50%]) or supportive care alone (65 patients [50%]). No patients were lost to follow-up; median follow-up was 36.5 months (95% CI 34.2 to not reached), and one patient in the supportive care group withdrew consent. Progression-free survival was significantly longer in the gemcitabine group (median 6.2 months [95% CI 4.6-8.7]) than in the supportive care group (3.2 months [2.8-4.1]; hazard ratio [HR] 0.48 [95% CI 0.33-0.71]; p=0.0002). The benefit was confirmed by masked independent central review (HR 0.49 [0.33-0.72]; p=0.0002). Grade 3-4 adverse events occurred in 33 ( 52%) of 64 patients in the gemcitabine group and in ten (16%) of 62 patients in the supportive care group. The most frequent adverse events were anaemia, neutropenia, fatigue or asthenia, pain, and infection in the gemcitabine group, and pain, infection, and cough or dyspnoea in the supportive care group. One patient (2%) in the gemcitabine group died, due to a treatment-related infection. Interpretation Switch-maintenance gemcitabine, after first-line chemotherapy, significantly prolonged progression-free survival compared with best supportive care alone, among patients with malignant mesothelioma. This study confirms the activity of gemcitabine in treating malignant mesothelioma

High Resolution X-ray Spectra of RS Ophiuchi (2006 and 2021): Revealing the cause of SSS variability

Context. The ∼ 10 − 20 year recurrent symbiotic nova RS Oph exploded on 2021 August 9, the seventh confirmed recorded outburst since 1898. During the previous outburst in 2006, the current fleet of X-ray space observatories was already in operation, and thanks to the longevity of Swift, XMM-Newton, and Chandra, a direct comparison between these two outbursts is possible. The Swift monitoring campaign revealed similar behaviour during the early shock phase but very different behaviour during the super-soft source (SSS) phase. Two XMM-Newton⋆ observations were made during the 2021 SSS phase on days 37.1 and 55.6 after the 2021 optical peak. We focus in this work on the bright SSS observation on day 55.6 and compare to SSS Chandra and XMM-Newton grating observations made on days 39.7, 54, and 66.9 after the 2006 optical peak. Aims. By exploring the reasons for the differences between the 2006 and 2021 outbursts, we aim to obtain a better general understanding of the emission and absorption mechanisms. While the emission mechanisms hold the key to the physics of novae and nuclear burning, absorption processes may dominate what we observe, and we aim to explore the cause of the gross initial variability in the observed SSS emission. Methods. We present a novel approach to down-scaling the observed (brighter) 2006 SSS spectra to match the 2021 day 55.6 spectrum by parameter optimisation of: (1) a constant factor (representing fainter source emission, smaller radius, eclipses, etc.), (2) a multi-ionisation photoelectric absorption model (representing different line-of-sight absorption), and (3) scaling with a ratio of two blackbody models with different effective temperatures (representing different brightness and colours). This model approach does not depend on a source model assuming the intrinsic source to be the same. It is therefore more sensitive to incremental changes than modelling approaches where source and absorption are modelled simultaneously. Results. The 2021d55.6 spectrum can be reproduced remarkably well by multiplying the (brighter) 2006d39.7 and 2006d54 spectra with the absorption model, while the 2006d66.9 spectrum requires additional colour changes to match the 2021.d55.6 spectrum. The 2006d39.7 spectrum much more closely resembles the 2021d55.6 spectrum in shape and structure than the same-epoch 2006d54 spectrum: The spectra on days 2006d39.7 and 2021d55.6 are richer in absorption lines with a deeper O i absorption edge, and blueshifts are higher (∼ 1200 km s−1 ) than on day 2006d54 (∼ 700 km s−1 ). In the SSS light curves on days 2006d39.7, 2006d54, and 2021d55.6, brightness and hardness variations are correlated, indicating variations of the O i column density. Only on day 2006d39.7, a 1000s lag is observed. The 35s period was detected on day 2021d55.6 with lower significance compared to 2006d54. Conclusions. We conclude that the central radiation source is the same, while absorption is the principal reason for observing lower soft-X-ray emission in 2021 than in 2006. This is consistent with a similar 2006 and 2021 [Fe x] line-flux evolution. We explain the reduction in line blueshift, depth in O i edge, and number of absorption lines from day 2006d39.7 to 2006d54 by deceleration and heating of the ejecta within the stellar wind of the companion. In 2021, less such deceleration and heating was observed, which we interpret as due to viewing at different angles through an inhomogeneous density distribution of the stellar wind, allowing free expansion in some directions (probed in 2021) and a higher degree of deceleration in others (probed in 2006). The higher absorption in 2021 can then be explained by the lower-temperature absorbing plasma being more opaque to soft X-rays. Our approach of scaling observations against observations is free of ambiguities from imperfect source models and can be applied to other grating spectra with complex continuum sources

Clinical performance of an analytically validated assay in comparison to microarray technology to assess PITX2 DNA-methylation in breast cancer

Significant evidence has accumulated that DNA-methylation of the paired-like homeodomain transcription factor 2 (PITX2) gene can serve as a prognostic and predictive biomarker in breast cancer. PITX2 DNA-methylation data have been obtained so far from microarray and polymerase chain reaction (PCR)-based research tests. The availability of an analytically validated in vitro methylation-specific real-time PCR assay format (therascreen PITX2 RGQ PCR assay) intended for the determination of the percent methylation ratio (PMR) in the (PITX2) promoter 2 prompted us to investigate whether the clinical performance of these different assay systems generate comparable clinical outcome data. Mathematically converted microarray data of a previous breast cancer study (n = 204) into PMR values leads to a PITX2 cut-off value at PMR 14.73. Recalculation of the data to experimentally equivalent PMRs with the PCR PITX2 assay leads to a cut-off value at PMR 12 with the highest statistical significance. This cut-off predicts outcome of high-risk breast cancer patients to adjuvant anthracycline-based chemotherapy (n = 204; Hazard Ratio 2.48; p < 0.001) comparable to microarray generated results (n = 204; Hazard ratio 2.32; p < 0.0001). The therascreen PITX2 RGQ PCR assay is an analytically validated test with high reliability and robustness and predicts outcome of high-risk breast cancer patients to anthracycline-based chemotherapy