Bellmunt risk score as a survival predictor in patients with metastatic castration-resistant prostate cancer.

BACKGROUND: The prognosis of metastatic castration-resistant prostate cancer (mCRPC) is influenced by numerous individual factors. Despite various proposed prognostic models, the clinical application of these remains limited, probably due to complexity. Our study aimed to evaluate the predictive value of the Bellmunt risk score, which is well-known for urothelial carcinoma and easily assessed, in mCRPC patients. METHODS: The Bellmunt risk score was calculated from three risk factors (Eastern Cooperative Oncology Group Performance Status (ECOG PS) ≥1, serum hemoglobin <10 g/dL, presence of liver metastases) in 125 patients who received first-line mCRPC treatment between 2005 and 2023. In addition, a modified score was established (one point each for hemoglobin <10 g/dL and the presence of liver metastases added to the ECOG PS). Associations with overall survival (OS) under first- and second-line therapy were tested using Cox regression analyzes, log-rank tests, concordance index (C-index) and time-dependent receiver operating characteristic. RESULTS: There is a significant correlation between the level of the Bellmunt risk score and shorter OS (hazard ratio: 3.23, 95% confidence interval: 2.06-5.05; log-rank p < 0.001; C-index: 0.724). The semi-quantitative modified risk score showed even better prognostic discrimination (log-rank p < 0.001, C-index: 0.764). The score and its dynamics were also predictive in the second-line setting (log-rank p < 0.001 and = 0.01; C-index: 0.742 and 0.595). CONCLUSIONS: The Bellmunt risk score is easy to assess and provides useful prognostic information in mCRPC, and can support physicians in their treatment decisions.

Bi-allelic loss-of-function variants in BCAS3 cause a syndromic neurodevelopmental disorder.

BCAS3 microtubule-associated cell migration factor (BCAS3) is a large, highly conserved cytoskeletal protein previously proposed to be critical in angiogenesis and implicated in human embryogenesis and tumorigenesis. Here, we established BCAS3 loss-of-function variants as causative for a neurodevelopmental disorder. We report 15 individuals from eight unrelated families with germline bi-allelic loss-of-function variants in BCAS3. All probands share a global developmental delay accompanied by pyramidal tract involvement, microcephaly, short stature, strabismus, dysmorphic facial features, and seizures. The human phenotype is less severe compared with the Bcas3 knockout mouse model and cannot be explained by angiogenic defects alone. Consistent with being loss-of-function alleles, we observed absence of BCAS3 in probands' primary fibroblasts. By comparing the transcriptomic and proteomic data based on probands' fibroblasts with those of the knockout mouse model, we identified similar dysregulated pathways resulting from over-representation analysis, while the dysregulation of some proposed key interactors could not be confirmed. Together with the results from a tissue-specific Drosophila loss-of-function model, we demonstrate a vital role for BCAS3 in neural tissue development.

Multiple taxa inoculants of arbuscular mycorrhizal fungi enhanced colonization frequency, biomass production, and water use efficiency of cassava (Manihot esculenta).

Increasing water use efficiency (WUE) in crops is critical to maintaining agricultural production under climate change-exacerbated drought. One of these approaches may consist of leveraging on the beneficial interactions between crops and arbuscular mycorrhizal fungi (AMF). In this study, we investigated how inoculation with AMF from three different taxa (Claroideoglomus etunicatum (T1), Gigaspora margarita (T2), and Rhizophagus irregularis (T3)) and their combination (T123) and a non-inoculated "control" treatment in a greenhouse could achieve increased biomass production and water use efficiency in cassava under three levels of water availability (100% PC, 60%-moderate stress, and 30%-severe stress). Whereas T1 and T2 resulted in a lower growth rate for the plants than the control, T123 enhanced cassava height and the number of petioles and leaves. T123 and T3 increased the total plant dry biomass in comparison with uninoculated plants by 30% and 26%, respectively. The T123 and plants inoculated with T3 significantly increased cassava above-ground biomass by 19% as compared to T1 (8.68 ± 2.44 g) and T2 (8.68 ± 2.44 g) inoculated plants. T123 resulted in higher WUE, which was validated by the leaf carbon (δ13C) isotopic signature, significantly outperforming cassava with T1 and T2, yet there was no difference between the control and T3. Overall, this study demonstrated that the use of multiple AMF from different taxa can increase cassava growth and WUE under greenhouse conditions.

Pathophysiological interplay between O-GlcNAc transferase and the Machado-Joseph disease protein ataxin-3.

Aberrant O-GlcNAcylation, a protein posttranslational modification defined by the O-linked attachment of the monosaccharide N-acetylglucosamine (O-GlcNAc), has been implicated in neurodegenerative diseases. However, although many neuronal proteins are substrates for O-GlcNAcylation, this process has not been extensively investigated in polyglutamine disorders. We aimed to evaluate the enzyme O-GlcNAc transferase (OGT), which attaches O-GlcNAc to target proteins, in Machado-Joseph disease (MJD). MJD is a neurodegenerative condition characterized by ataxia and caused by the expansion of a polyglutamine stretch within the deubiquitinase ataxin-3, which then present increased propensity to aggregate. By analyzing MJD cell and animal models, we provide evidence that OGT is dysregulated in MJD, therefore compromising the O-GlcNAc cycle. Moreover, we demonstrate that wild-type ataxin-3 modulates OGT protein levels in a proteasome-dependent manner, and we present OGT as a substrate for ataxin-3. Targeting OGT levels and activity reduced ataxin-3 aggregates, improved protein clearance and cell viability, and alleviated motor impairment reminiscent of ataxia of MJD patients in zebrafish model of the disease. Taken together, our results point to a direct interaction between OGT and ataxin-3 in health and disease and propose the O-GlcNAc cycle as a promising target for the development of therapeutics in the yet incurable MJD.

KPNB1 modulates the Machado-Joseph disease protein ataxin-3 through activation of the mitochondrial protease CLPP.

Machado-Joseph disease (MJD) is characterized by a pathological expansion of the polyglutamine (polyQ) tract within the ataxin-3 protein. Despite its primarily cytoplasmic localization, polyQ-expanded ataxin-3 accumulates in the nucleus and forms intranuclear aggregates in the affected neurons. Due to these histopathological hallmarks, the nucleocytoplasmic transport machinery has garnered attention as an important disease relevant mechanism. Here, we report on MJD cell model-based analysis of the nuclear transport receptor karyopherin subunit beta-1 (KPNB1) and its implications in the molecular pathogenesis of MJD. Although directly interacting with both wild-type and polyQ-expanded ataxin-3, modulating KPNB1 did not alter the intracellular localization of ataxin-3. Instead, overexpression of KPNB1 reduced ataxin-3 protein levels and the aggregate load, thereby improving cell viability. On the other hand, its knockdown and inhibition resulted in the accumulation of soluble and insoluble ataxin-3. Interestingly, the reduction of ataxin-3 was apparently based on protein fragmentation independent of the classical MJD-associated proteolytic pathways. Label-free quantitative proteomics and knockdown experiments identified mitochondrial protease CLPP as a potential mediator of the ataxin-3-degrading effect induced by KPNB1. We confirmed reduction of KPNB1 protein levels in MJD by analyzing two MJD transgenic mouse models and induced pluripotent stem cells (iPSCs) derived from MJD patients. Our results reveal a yet undescribed regulatory function of KPNB1 in controlling the turnover of ataxin-3, thereby highlighting a new potential target of therapeutic value for MJD.

Calpain-1 ablation partially rescues disease-associated hallmarks in models of Machado-Joseph disease.

Proteolytic fragmentation of polyglutamine-expanded ataxin-3 is a concomitant and modifier of the molecular pathogenesis of Machado-Joseph disease (MJD), the most common autosomal dominant cerebellar ataxia. Calpains, a group of calcium-dependent cysteine proteases, are important mediators of ataxin-3 cleavage and implicated in multiple neurodegenerative conditions. Pharmacologic and genetic approaches lowering calpain activity showed beneficial effects on molecular and behavioural disease characteristics in MJD model organisms. However, specifically targeting one of the calpain isoforms by genetic means has not yet been evaluated as a potential therapeutic strategy. In our study, we tested whether calpains are overactivated in the MJD context and if reduction or ablation of calpain-1 expression ameliorates the disease-associated phenotype in MJD cells and mice. In all analysed MJD models, we detected an elevated calpain activity at baseline. Lowering or removal of calpain-1 in cells or mice counteracted calpain system overactivation and led to reduced cleavage of ataxin-3 without affecting its aggregation. Moreover, calpain-1 knockout in YAC84Q mice alleviated excessive fragmentation of important synaptic proteins. Despite worsening some motor characteristics, YAC84Q mice showed a rescue of body weight loss and extended survival upon calpain-1 knockout. Together, our findings emphasize the general potential of calpains as a therapeutic target in MJD and other neurodegenerative diseases.

Spatial variability and environmental drivers of cassava-arbuscular mycorrhiza fungi (AMF) associations across Southern Nigeria.

Cassava, forming starch-rich, tuberous roots, is an important staple crop in smallholder farming systems in sub-Saharan Africa. Its relatively good tolerance to drought and nutrient-poor soils may be partly attributed to the crop's association with arbuscular mycorrhiza fungi (AMF). Yet insights into AMF-community composition and richness of cassava, and knowledge of its environmental drivers are still limited. Here, we sampled 60 cassava fields across three major cassava-growing agro-ecological zones in Nigeria and used a DNA meta-barcoding approach to quantify large-scale spatial variation and evaluate the effects of soil characteristics and common agricultural practices on AMF community composition, richness and Shannon diversity. We identified 515 AMF operational taxonomic units (OTUs), dominated by Glomus, with large variation across agro-ecological zones, and with soil pH explaining most of the variation in AMF community composition. High levels of soil available phosphorus reduced OTU richness without affecting Shannon diversity. Long fallow periods (> 5 years) reduced AMF richness compared with short fallows, whereas both zero tillage and tractor tillage reduced AMF diversity compared with hoe tillage. This study reveals that the symbiotic relationship between cassava and AMF is strongly influenced by soil characteristics and agricultural management and that it is possible to adjust cassava cultivation practices to modify AMF diversity and community structure.

Cassava-maize intercropping systems in southern Nigeria: Radiation use efficiency, soil moisture dynamics, and yields of component crops.

Efficient utilization of incident solar radiation and rainwater conservation in rain-fed smallholder cropping systems require the development and adoption of cropping systems with high resource use efficiency. Due to the popularity of cassava-maize intercropping and the food security and economic importance of both crops in Nigeria, we investigated options to improve interception of photosynthetically active radiation (IPAR), radiation use efficiency (RUE), soil moisture retention, and yields of cassava and maize in cassava-maize intercropping systems in 8 on-farm researcher-managed multi-location trials between 2017 and 2019 in different agro-ecologies of southern Nigeria. Treatments were a combination of (1) maize planting density (low density at 20,000 maize plants ha-1 versus high density at 40,000 maize plants ha-1, intercropped with 12,500 cassava plants ha-1); (2) fertilizer application and management targeting either the maize crop (90 kg N, 20 kg P and 37 kg K ha-1) or the cassava crop (75 kg N, 20 kg P and 90 kg K ha-1), compared with control without fertilizer application. Cassava and maize development parameters were highest in the maize fertilizer regime, resulting in the highest IPAR at high maize density. The combined intercrop biomass yield was highest at high maize density in the maize fertilizer regime. Without fertilizer application, RUE was highest at low maize density. However, the application of the maize fertilizer regime at high maize density resulted in the highest RUE, soil moisture content, and maize grain yield. Cassava storage root yield was higher in the cassava fertilizer regime than in the maize fertilizer regime. We conclude that improved IPAR, RUE, soil moisture retention, and grain yield on nutrient-limited soils of southern Nigeria, or in similar environments, can be achieved by intercropping 40,000 maize plants ha-1 with 12,500 cassava plants ha-1 and managing the system with the maize fertilizer regime. However, for higher cassava storage root yield, the system should be managed with the cassava fertilizer regime.

Metabolic profiling in serum, cerebrospinal fluid, and brain of patients with cerebrotendinous xanthomatosis.

Cerebrotendinous xanthomatosis (CTX) is caused by autosomal recessive loss-of-function mutations in CYP27A1, a gene encoding cytochrome p450 oxidase essential for bile acid synthesis, resulting in altered bile acid and lipid metabolism. Here, we aimed to identify metabolic aberrations that drive ongoing neurodegeneration in some patients with CTX despite chenodeoxycholic acid (CDCA) supplementation, the standard treatment in CTX. Using chromatographic separation techniques coupled to mass spectrometry, we analyzed 26 sterol metabolites in serum and cerebrospinal fluid (CSF) of patients with CTX and in one CTX brain. Comparing samples of drug naive patients to patients treated with CDCA and healthy controls, we identified 7α,12α-dihydroxycholest-4-en-3-one as the most prominently elevated metabolite in serum and CSF of drug naive patients. CDCA treatment substantially reduced or even normalized levels of all metabolites increased in untreated patients with CTX. Independent of CDCA treatment, metabolites of the 27-hydroxylation pathway were nearly absent in all patients with CTX. 27-hydroxylated metabolites accounted for ∼45% of total free sterol content in CSF of healthy controls but <2% in patients with CTX. Metabolic changes in brain tissue corresponded well with findings in CSF. Interestingly, 7α,12α-dihydroxycholest-4-en-3-one and 5α-cholestanol did not exert toxicity in neuronal cell culture. In conclusion, we propose that increased 7α,12α-dihydroxycholest-4-en-3-one and lack of 27-hydroxycholesterol may be highly sensitive metabolic biomarkers of CTX. As CDCA cannot reliably prevent disease progression despite reduction of most accumulated metabolites, supplementation of 27-hydroxylated bile acid intermediates or replacement of CYP27A1 might be required to counter neurodegeneration in patients with progressive disease despite CDCA treatment.

Developing recommendations for increased productivity in cassava-maize intercropping systems in Southern Nigeria.

Cassava-maize intercropping is a common practice among smallholder farmers in Southern Nigeria. It provides food security and early access to income from the maize component. However, yields of both crops are commonly low in farmers' fields. Multi-locational trials were conducted in Southern Nigeria in 2016 and 2017 to investigate options to increase productivity and profitability through increased cassava and maize plant densities and fertilizer application. Trials with 4 and 6 treatments in 2016 and 2017, respectively were established on 126 farmers' fields over two seasons with a set of different designs, including combinations of two levels of crop density and three levels of fertilizer rates. The maize crop was tested at low density (LM) with 20,000 plants ha-1 versus high density (HM) with 40,000 plants ha-1. For cassava, low density (LC) had had 10,000 plants ha-1 versus the high density (HC) with 12,500 plants ha-1.; The fertilizer application followed a regime favouring either the maize crop (FM: 90 kg N, 20 kg P and 37 kg K ha-1) or the cassava crop (FC: 75 kg N, 20 kg P and 90 kg K ha-1), next to control without fertilizer application (F0). Higher maize density (HM) increased marketable maize cob yield by 14 % (3700 cobs ha-1) in the first cycle and by 8% (2100 cobs ha-1) in the second cycle, relative to the LM treatment. Across both cropping cycles, fertilizer application increased cob yield by 15 % (5000 cobs ha-1) and 19 % (6700 cobs ha-1) in the FC and FM regime, respectively. Cassava storage root yield increased by 16 % (4 Mg ha-1) due to increased cassava plant density, and by 14 % (4 Mg ha-1) due to fertilizer application (i.e., with both fertilizer regimes) but only in the first cropping cycle. In the second cycle, increased maize plant density (HM) reduced cassava storage root yield by 7% (1.5 Mg ha-1) relative to the LM treatment. However, the negative effect of high maize density on storage root yield was counteracted by fertilizer application. Fresh storage root yield increased by 8% (2 Mg ha-1) in both fertilizer regimes compared to the control without fertilizer application. Responses to fertilizer by cassava and maize varied between fields. Positive responses tended to decline with increasing yields in the control treatment. The average value-to-cost ratio (VCR) of fertilizer use for the FM regime was 3.6 and higher than for the FC regime (VCR = 1.6), resulting from higher maize yields when FM than when FC was applied. Revenue generated by maize constituted 84-91% of the total revenue of the cropping system. The highest profits were achieved with the FM regime when both cassava and maize were grown at high density. However, fertilizer application was not always advisable as 34 % of farmers did not realize a profit. For higher yields and profitability, fertilizer recommendations should be targeted to responsive fields based on soil fertility knowledge.

Increasing cassava root yield on farmers' fields in Nigeria through appropriate weed management.

Weed competition is the major biological stress affecting cassava production in smallholder farms in West and Central Africa, where yields are low compared with those in Asia and Latin America. Options for improved weed management are crucial in increasing productivity. Selected pre- and post-emergence herbicides, integrated with appropriate tillage and plant spacing, were tested in 96 sites in four locations in Nigeria, 24 in 2016 and 72 in 2017. Trials were split plots with six pre-emergence herbicides and no post-emergence treatment as main plots. Subplot treatments were four post-emergence herbicides, weeding with a motorized rotary weeder, short- and long-handled hoes, and no post-emergence weed control, i.e., regardless of pre-emergence treatments. Indaziflam-based treatments, irrespective of post-emergence treatment, and flumioxazin + pyroxasulfone applied pre-emergence followed by one weeding with a long-handled hoe provided >80% control of major broadleaf and grass weeds. Compared with herbicide use, farmer control practices (53%) were not efficient in controlling weeds. The highest root yield was produced where (1) s-metolachlor was combined with atrazine, and one weeding with a long-handled hoe or clethodim with lactofen, and (2) indaziflam + isoxaflutole was combined with glyphosate. An increase in root yield from 3.41 to 14.2 t ha-1 and from 3.0 to 11.99 t ha-1 was obtained where herbicides were used compared with farmers' practice and manual hoe weeding. Our results showed that integrating good agronomic practices with safe and effective use of appropriate herbicides can result in root yield >20 t ha-1. i.e., twice the national average root yield of 8-12 t ha-1, with >50% net profit. The use of appropriate herbicides can reduce the amount of manual labor required and improve livelihoods, specifically for women and children. Smallholder cassava farmers would require continuous training on the safe use and handling of herbicides to improve efficiency and prevent adverse effects on humans and the environment.

The value of tumor markers in men with metastatic prostate cancer undergoing [177 Lu]Lu-PSMA therapy.

BACKGROUND: Currently, prostate-specific membrane antigen-radioligand therapy (PSMA-RLT) is considered a last-line treatment option in advanced castration-resistant prostate cancer. Despite these patients' poor prognosis, accurate estimation of their overall survival (OS) is essential to determine whether benefits exist from the treatment and whether the loss of valuable time and unnecessary side effects can be avoided. The aim of the present study is to evaluate whether various biochemical markers can predict OS in men undergoing PSMA-RLT and whether the changes assessed after PSMA-RLT correlate with the OS. METHODS: The tested tumor markers in this retrospective analysis were alkaline phosphatase (ALP), bone-specific alkaline phosphatase (BAP), prostate-specific antigen (PSA), lactate dehydrogenase (LDH), chromogranin A, and pro-gastrin-releasing peptide (pro-GRP). For the evaluation, we performed blood tests before each PSMA-RLT cycle and during follow-up visits (which were 2-3 months apart). All patients were followed up until their deaths. To test the correlations between the tumor markers and survival, we conducted the logrank tests and the multivariate Cox proportional-hazards regression model. The significance level was set at P < .05. RESULTS: The study included 137 patients who received a total of 487 PSMA-RLT cycles between January 2015 and November 2017. Of the tested biochemical tumor markers, baseline ALP (120 U/L cut-off), LDH (248 U/L cut-off), and PSA (first quartile cut-off) correlated significantly with survival post-PSMA-RLT (P < .001 for ALP and LDH, and P = .007 for PSA). Stable and/or decreased values in most of the initially abnormal parameters were associated with significantly better OS; these parameters were ALP (P = .009), LDH (P = .005), PSA (P < .001), and pro-GRP (P = .013). The BAP and ALP responses also correlated significantly with survival in patients with bone metastases (P = .002 and P < .001, respectively). Furthermore, there was a strong correlation of the kinetic patterns of PSA, ALP, BAP, and LDH with the survival, showing that patients with steadily increasing markers had the shortest OS. CONCLUSION: Along with the established tumor marker PSA, ALP, LDH, BAP, and pro-GRP were correlated with the OS post-PSMA-RLT in the univariate and multivariate analyses.

Higher number of multidisciplinary tumor board meetings per case leads to improved clinical outcome.

BACKGROUND: This analysis aims at evaluating the impact of multidisciplinary tumor boards on clinical outcome of multiple tumor entities, the effect of the specific number of multidisciplinary tumor boards and potential differences between the tumor entities. METHODS: By a matched-pair analysis we compared the response to treatment, overall survival, relapse or disease free survival and progression free survival of patients whose cases were discussed in a tumor board meeting with patients whose cases were not. It was performed with patients registered in the cancer registry of the University of Bonn and diagnosed between 2010 and 2016. After the matching process with a pool of 7262 patients a total of 454 patients with 66 different tumor types were included in this study. RESULTS: First, patients with three or more multidisciplinary tumor board meetings in their history show a significantly better overall survival than patients with no tumor board meeting. Second, response to treatment, relapse free survival and time to progression were not found to be significantly different. Third, there was no significant difference for a specific tumor entity. CONCLUSION: This study revealed a positive impact of a higher number of multidisciplinary tumor boards on the clinical outcome. Also, our analysis hints towards a positive effect of multidisciplinary tumor boards on overall survival.

Apelin and apelin receptor expression in renal cell carcinoma.

BACKGROUND: The APLNR (apelin receptor) has been shown to be an essential gene for cancer immunotherapy, with deficiency in APLNR leading to immunotherapy failure. The aim of this study is to investigate the expression of APLN (apelin) and APLNR in patients with renal cell carcinoma (RCC), and its association with clinicopathological parameters and survival. METHODS: Three well-characterised patient cohorts with RCC were used: Study cohort 1 (clear-cell RCC; APLN/APLNR mRNA expression; n = 166); TCGA validation cohort (clear-cell RCC; APLN/APLNR mRNA expression; n = 481); Study cohort 2 (all RCC subtypes; APLNR protein expression/immunohistochemistry; n = 300). Associations between mRNA/protein expression and clinicopathological variables/patients' survival were tested statistically. RESULTS: While APLN showed only very weak association with tumour histological grade (TCGA cohort), APLNR/mRNA protein expression correlate significantly with ccRCC aggressiveness. APLNR is expressed in tumour vasculature and tumour cells at different levels, and these expression levels associate with tumour aggressiveness in opposing directions. APLNR expression was negatively correlated with PD-L1 expression by tumour cells in a subset of patients with ccRCC. APLNR expression in either compartment is an independent prognostic factor for survival of patients with ccRCC. CONCLUSION: The APLNR/APLN-system appears to play an important role in ccRCC, warranting further clinical investigation.

Outcome and safety of rechallenge [177Lu]Lu-PSMA-617 in patients with metastatic prostate cancer.

BACKGROUND: Data are sparse regarding the feasibility of radioligand therapy (RLT) with [177Lu]Lu-PSMA-617 as a retreatment. We aimed to assess the outcome and safety of rechallenge PSMA-RLT in patients with progressive prostatic cancer who previously benefited from this therapy. MATERIALS AND METHODS: Patients who received rechallenge therapy at our department from January 2015 to March 2018 were assessed. Non-haematological and haematological adverse events were evaluated from laboratory data and clinical reports and were graded according to the Common Terminology Criteria for Adverse Events (CTCAE v. 5.0). Time to prostate-specific-antigen (PSA) progression and the overall survival (OS) rate of the study patients were calculated from the date of the first rechallenge cycle. Furthermore, the OS calculated from the first cycle baseline PSMA-RLT was compared with the survival of patients who received only baseline PSMA-RLT. The response data were determined using [68Ga]Ga-PSMA-PET/CT and measurements of the tumour marker PSA. RESULTS: Included in this retrospective study were 30 patients who were initially treated with a median of 3 cycles (range 1-5) of PSMA-RLT and were eventually retreated after a median of 6 months (range 2-26). Each patient received a median of 3 (range 1-6) rechallenge cycles. None of the patients experienced a disabling or life-threatening grade 4 adverse event according to the Common Toxicity Criteria (CTC). Grade 3 toxicity occurred in 8 patients (27%). Serious adverse events included leucopoenia (n = 2), neutropoenia (n = 1), anaemia (n = 4), thrombopenia (n = 4) and elevated renal parameters (n = 1). Irreversible adverse events occurred in 21 patients (70%). The permanent adverse events were mild/moderate (CTC grade 1/2) in 19 patients and serious (CTC grade 3) in two patients, respectively. According to PSA measurements, 75-90% of patients showed a benefit (response/stable) from the first 4 rechallenge cycles. The median OS was 12 months calculated from the first rechallenge cycle and 25 months calculated from the first cycle baseline PSMA-RLT. For comparison, the median OS in patients who received only baseline PSMA-RLT was 9 months. The difference according to the logrank test was significant: p value <0.001. Patients with a PSA decrease after the first cycle of rechallenge PSMA-RLT survived a median of 19 months, while patients with a PSA increase survived only 6 months. CONCLUSION: Rechallenge prostate-specific membrane antigen (PSMA) therapy has an acceptable safety profile. The majority of the retreated patients benefited from the rechallenge therapy. Patients who showed a biochemical response achieved a longer OS compared to patients who did not respond. The median OS was significantly longer in patients after rechallenge PSMA-RLT than in patients who received only baseline PSMA-RLT.

Clinical Trials with Combination of Cytokine-Induced Killer Cells and Dendritic Cells for Cancer Therapy.

Adoptive cellular immunotherapy (ACI) is a promising treatment for a number of cancers. Cytokine-induced killer cells (CIKs) are considered to be major cytotoxic immunologic effector cells. Usually cancer cells are able to suppress antitumor responses by secreting immunosuppressive factors. CIKs have significant antitumor activity and are capable of eradicating tumors with few side effects. They are a very encouraging cell population used against hematological and solid tumors, with an inexpensive expansion protocol which could yield to superior clinical outcome in clinical trials employing adoptive cellular therapy combination. In the last decade, clinical protocols have been modified by enriching lymphocytes with CIK cells. They are a subpopulation of lymphocytes characterized by the expression of CD3+ and CD56+ wich are surface markers common to T lymphocytes and natural killer NK cells. CIK cells are mainly used in two diseases: in hematological patients who suffer relapse after allogeneic transplantation and in patients with hepatic carcinoma after surgical ablation to eliminate residual tumor cells. Dendritic cells DCs could play a pivotal role in enhancing the antitumor efficacy of CIKs.

Inhibition of Lithium Sensitive Orai1/ STIM1 Expression and Store Operated Ca2+ Entry in Chorea-Acanthocytosis Neurons by NF-κB Inhibitor Wogonin.

BACKGROUND/AIMS: The neurodegenerative disease Chorea-Acanthocytosis (ChAc) is caused by loss-of-function-mutations of the chorein-encoding gene VPS13A. In ChAc neurons transcript levels and protein abundance of Ca2+ release activated channel moiety (CRAC) Orai1 as well as its regulator STIM1/2 are decreased, resulting in blunted store operated Ca2+-entry (SOCE) and enhanced suicidal cell death. SOCE is up-regulated and cell death decreased by lithium. The effects of lithium are paralleled by upregulation of serum & glucocorticoid inducible kinase SGK1 and abrogated by pharmacological SGK1 inhibition. In other cell types SGK1 has been shown to be partially effective by upregulation of NFκB, a transcription factor stimulating the expression of Orai1 and STIM. The present study explored whether pharmacological inhibition of NFκB interferes with Orai1/STIM1/2 expression and SOCE and their upregulation by lithium in ChAc neurons. METHODS: Cortical neurons were differentiated from induced pluripotent stem cells generated from fibroblasts of ChAc patients and healthy volunteers. Orai1 and STIM1 transcript levels and protein abundance were estimated from qRT-PCR and Western blotting, respectively, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarco-endoplasmatic Ca2+-ATPase inhibitor thapsigargin (1µM), as well as CRAC current utilizing whole cell patch clamp recording. RESULTS: Orai1 and STIM1 transcript levels and protein abundance as well as SOCE and CRAC current were significantly enhanced by lithium treatment (2 mM, 24 hours). These effects were reversed by NFκB inhibitor wogonin (50 µM). CONCLUSION: The stimulation of expression and function of Orai1/STIM1/2 by lithium in ChAc neurons are disrupted by pharmacological NFκB inhibition.

Comprehensive Evaluation of Prostate Specific Membrane Antigen Expression in the Vasculature of Renal Tumors: Implications for Imaging Studies and Prognostic Role.

PURPOSE: Prostate specific membrane antigen is expressed by the endothelium of many tumors. The aim of the study was to find a rationale for prostate specific membrane antigen based imaging and investigate the prognostic role of vascular prostate specific membrane antigen expression in patients with renal cell carcinoma. MATERIALS AND METHODS: A total of 257 patients with renal cell carcinoma were included in study with a median followup exceeding 10.0 years. Prostate specific membrane antigen expression on tumor vessels was detected by immunohistochemistry. Vascular expression of FOLH1 gene (prostate specific membrane antigen) mRNA was investigated in clear cell carcinoma and papillary renal cell carcinoma using TCGA (The Cancer Genome Atlas) data. RESULTS: Endothelial prostate specific membrane antigen protein expression was higher in clear cell than in papillary and chromophobe renal cell carcinoma. Higher grade and stage, metastatic and lethal clear cell renal cell carcinoma showed higher prostate specific membrane antigen expression in tumor vessels. On univariate and multivariate analysis the intensity of positive vs negative endothelial prostate specific membrane antigen protein expression was significantly associated with overall survival. TCGA based analyses confirmed the prognostic role of vascular expression of FOLH1 mRNA. The analyses also supported the usefulness of prostate specific membrane antigen based imaging in cases of clear cell but not papillary renal cell carcinoma. CONCLUSIONS: We provide a rationale for further development of prostate specific membrane antigen targeted imaging in patients with clear cell renal cell carcinoma. The prognostic role of prostate specific membrane antigen was determined at the protein level in clear cell renal cell carcinoma and at the mRNA level in clear cell and papillary renal cell carcinoma.

NFAT5-sensitive Orai1 expression and store-operated Ca2+ entry in megakaryocytes.

The transcription factor nuclear factor of activated T cells 5 (NFAT5) is up-regulated in several clinical disorders, including dehydration. NFAT5-sensitive genes include serum and glucocorticoid-inducible kinase 1 (SGK1). The kinase is a powerful regulator of Orai1, a Ca2+ channel accomplishing store-operated Ca2+ entry (SOCE). Orai1 is stimulated after intracellular store depletion by the Ca2+ sensors stromal interaction molecule 1 (STIM1), or STIM2, or both. In the present study, we explored whether nuclear factor of activated T cell (NFAT)-5 influences Ca2+ signaling in megakaryocytes. To this end, human megakaryocytic (MEG-01) cells were transfected with NFAT5 or with siNFAT5. Platelets and megakaryocytes were isolated from wild-type mice with either access to water ad libitum or dehydration by 36 h of water deprivation. Transcript levels were determined with quantitative RT-PCR and protein abundance by Western blot analysis and flow cytometry, cytosolic (intracellular) Ca2+ concentration ([Ca2+]i) by fura-2-fluorescence. SOCE was estimated from the increase of [Ca2+]i following readdition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). Platelet degranulation was estimated from P-selectin abundance and integrin activation from αIIbß3 integrin abundance determined by flow cytometry. As a result, NFAT5 transfection or exposure to hypertonicity (+40 mM NaCl) of MEG-01 cells increased Orai1, Orai2, STIM1, and STIM2 transcript levels. Orai1 transcript levels were decreased by NFAT5 silencing. NFAT5 transfection and IκB inhibitor BMS 345541 (5 µM) increased SOCE, whereas NFAT5 silencing and SGK1 inhibitor GSK650394 (10 µM) decreased SOCE. In the mice, dehydration increased NFAT5 and Orai1 protein abundance in megakaryocytes and NFAT5, Orai1, and Orai2 abundance in platelets. Dehydration further augmented the degranulation and integrin activation by thrombin and collagen-related peptide. In summary, NFAT5 is a powerful regulator of Orai1-expression and SOCE in megakaryocytes.-Sahu, I., Pelzl, L., Sukkar, B., Fakhri, H., al-Maghout, T., Cao, H., Hauser, S., Gutti, R., Gawaz, M., Lang, F. NFAT5-sensitive Orai1 expression and store-operated Ca2+ entry in megakaryocytes.

A combinatorial approach to identify calpain cleavage sites in the Machado-Joseph disease protein ataxin-3.

Ataxin-3, the disease protein in Machado-Joseph disease, is known to be proteolytically modified by various enzymes including two major families of proteases, caspases and calpains. This processing results in the generation of toxic fragments of the polyglutamine-expanded protein. Although various approaches were undertaken to identify cleavage sites within ataxin-3 and to evaluate the impact of fragments on the molecular pathogenesis of Machado-Joseph disease, calpain-mediated cleavage of the disease protein and the localization of cleavage sites remained unclear. Here, we report on the first precise localization of calpain cleavage sites in ataxin-3 and on the characterization of the resulting breakdown products. After confirming the occurrence of calpain-derived fragmentation of ataxin-3 in patient-derived cell lines and post-mortem brain tissue, we combined in silico prediction tools, western blot analysis, mass spectrometry, and peptide overlay assays to identify calpain cleavage sites. We found that ataxin-3 is primarily cleaved at two sites, namely at amino acid positions D208 and S256 and mutating amino acids at both cleavage sites to tryptophan nearly abolished ataxin-3 fragmentation. Furthermore, analysis of calpain cleavage-derived fragments showed distinct aggregation propensities and toxicities of C-terminal polyglutamine-containing breakdown products. Our data elucidate the important role of ataxin-3 proteolysis in the pathogenesis of Machado-Joseph disease and further emphasize the relevance of targeting this disease pathway as a treatment strategy in neurodegenerative disorders.