Design of an allosterically modulated doxycycline and doxorubicin drug-binding protein.

The allosteric interplay between distant functional sites present in a single protein provides for one of the most important regulatory mechanisms in biological systems. While the design of ligand-binding sites into proteins remains challenging, this holds even truer for the coupling of a newly engineered binding site to an allosteric mechanism that regulates the ligand affinity. Here it is shown how computational design algorithms enabled the introduction of doxycycline- and doxorubicin-binding sites into the serine proteinase inhibitor (serpin) family member α1-antichymotrypsin. Further engineering allowed exploitation of the proteinase-triggered serpin-typical S-to-R transition to modulate the ligand affinities. These design variants follow strategies observed in naturally occurring plasma globulins that allow for the targeted delivery of hormones in the blood. By analogy, we propose that the variants described in the present study could be further developed to allow for the delivery of the antibiotic doxycycline and the anticancer compound doxorubicin to tissues/locations that express specific proteinases, such as bacterial infection sites or tumor cells secreting matrix metalloproteinases.

[Changes of sex ratio and prevalence in transgender teenagers over the past 15 years]. / Transidentität in Jugend und Adoleszenz: Zur Veränderung der Sexratio und der Prävalenz in den letzten eineinhalb Jahrzehnten.

Changes of sex ratio and prevalence in transgender teenagers over the past 15 years Abstract. Evaluation of authors' 1434 expert opinions from 2005-2019 on transgender applicants (420 younger than 20 years old) for legal change of name and gender according to German "Law on Transsexuality" showed (1) in teenage applicants substantial changes of sex ratio from 2:1 to 10:1 in favour of transmales; (2) while prevalence of teenage transfemales during this period remained unchanged, prevalence of transmales rose significantly. According to our data, transgender teenagers are nowadays primarily natal females. Clinical and sociocultural aspects of these changes are discussed.

NewBG: A surrogate corticosteroid-binding globulin with an unprecedentedly high ligand release efficacy.

The introduction of ligand-binding sites into proteins and the engineering of molecular allosteric coupling pathways are topical issues in protein design. Here, we show that these issues can be addressed concurrently, using the serpin human α1-antichymotrypsin (ACT) as a model. We have introduced up to 15 amino acid substitutions into ACT, converting it into a surrogate corticosteroid-binding globulin (CBG), thereby creating a new binding globulin (NewBG). Human CBG and ACT share 46% sequence identity, and CBG served as the blue-print for our design, which was guided by side-chain-packing calculations, ITC measurements and crystal structure determinations. Upon transfer of ligand-interacting residues from CBG to ACT and mutation of specific second shell residues, a NewBG variant was obtained, which binds cortisol with 1.5 µM affinity. This novel serpin (NewBG-III) binds cortisol with a 33-fold lower affinity than CBG, but shares a similar ligand-binding profile and binding mode when probed with different steroid ligands and site-directed mutagenesis. An additional substitution, i.e. A349R, created NewBG-III-allo, which introduced an allosteric coupling between ligand binding and the serpin-like S-to-R transition in ACT. In NewBG-III-allo, the proteinase-triggered S-to-R transition leads to a greater than 200-fold reduction in ligand affinity, and crystal structures suggest that this is mediated by the L55V and A349R substitutions. This reduction significantly exceeds the 10-fold reduction in binding affinity observed in human CBG.

Fas expression by tumor stroma is required for cancer eradication.

The contribution of molecules such as perforin, IFN-γ (IFNγ), and particularly Fas ligand (FasL) by transferred CD8(+) effector T (T(E)) cells to rejection of large, established tumors is incompletely understood. Efficient attack against large tumors carrying a surrogate tumor antigen (mimicking a "passenger" mutation) by T(E) cells requires action of IFNγ on tumor stroma cells to avoid selection of antigen-loss variants. Because "cancer-driving" antigens (CDAs) are rarely counterselected, IFNγ may be expected to be dispensable in elimination of cancers by targeting a CDA. Here, initial regression of large, established tumors required neither IFNγ, FasL, nor perforin by transferred CD8(+) T(E) cells targeting Simian Virus (SV) 40 large T as CDA. However, cytotoxic T(E) cells lacking IFNγ or FasL could not prevent relapse despite retention of the rejection antigen by the cancer cells. Complete tumor rejection required IFNγ-regulated Fas by the tumor stroma. Therefore, T(E) cells lacking IFNγ or FasL cannot prevent progression of antigenic cancer because the tumor stroma escapes destruction if its Fas expression is down-regulated.

Differently immunogenic cancers in mice induce immature myeloid cells that suppress CTL in vitro but not in vivo following transfer.

Tumors frequently induce immature myeloid cells (iMC), which suppress specific and unrelated cytotoxic T lymphocyte (CTL) responses and are termed myeloid-derived suppressor cells (MDSC). Mainly analyzed by in vitro assays in tumor transplantation models, little is known about their function in autochthonous tumor models in vivo. We analyzed iMC in 3 SV40 large T (Tag)-driven conditional autochthonous cancer models with different immune status: (1) Early Tag-specific CTL competence and rare stochastic Tag activation leading to sporadic cancer, which induces an aberrant immune response and CTL tolerance; (2) Cre/LoxP recombinase-mediated hepatocellular carcinoma (HCC) development in neonatal Tag-tolerant mice; and (3) Tag-activation through Cre recombinase-encoding viruses in the liver and HCC development with systemic anti-Tag CTL immunity. In the first but not two latter models, tumors induced CTL hyporesponsiveness to tumor-unrelated antigens. Regardless of the model, tumors produced interleukin-6 and vascular endothelial growth factor but not granulocyte macrophage­colony-stimulating factor (GM-CSF) and induced iMC (CD11b(+)Gr-1(int)) that suppressed CTL responses in vitro. None of the iMC from the different tumor models suppressed CTL responses in adoptive cell transfer experiments unless GM-CSF was provided in vivo. Together, iMC expand independent of the type of antitumor response and are not immunosuppressive in a cell-autonomous fashion.

Prevalence of depressive symptoms and associated developmental disorders in preschool children: a population-based study.

Depression is an incapacitating disorder, which is often overlooked in preschool children. The aim of this study was to analyse the prevalence of depressive symptoms and co-occurring risk factors in a large, population-based sample of preschool children. All 653 children (of a total of 731) in a defined geographical area were assessed during the school-entry exam by community care paediatricians. In addition to clinical appraisal, parents filled out the Preschool Feelings Checklist, a 16-item screening instrument with good psychometric properties. The mean age was 6.2 years (range 5.0-7.6 years) and the sample included 344 boys and 305 girls. The prevalence of depressive symptoms of clinical relevance (total score ≥3) was 5.7% (37). There were no differences between boys and girls, and between younger (<6 years) and older (>6 years) children. Depressive symptoms were associated with parental separation and comorbid behavioural problems, but especially with developmental motor and speech problems and disorders. Migration to Germany had no influence. Depressive symptoms are common in preschool children and associated with developmental problems. Depression should be considered in children with speech and motor problems who are at special risk. Early detection and treatment are recommended.

ERAP2 supports TCR recognition of three immunotherapy targeted tumor epitopes.

The therapy of cancer by adoptive T cell transfer (ACT) requires T cell receptors (TCRs) with optimal affinity for HLA class I-bound peptides (pHLA-I). But not every patient responds to ACT. Therefore, it is critical to understand the individual factors influencing the recognition of HLA class I-bound peptides (pHLA-I) by TCRs. Focusing on three immunotherapy-targeted human HLA-A* 02:01-presented T cell epitopes we investigated the contribution of the ER-resident aminopeptidases ERAP1 and ERAP2 to TCR recognition of cancer cells. We found that ERAP2 on its own, when expressed in ERAP-deficient cells, elicited a strong CTL response towards the Tyrosinase368-376 epitope. In vitro generated TAP-dependent N-terminally extended epitope precursor peptides were differently customized by ERAP1 and ERAP2 and thus may serve as potential source for the Tyrosinase368-376 epitope. ERAP2 also influenced recognition of the gp100209-217 tumor epitope and enhanced T cell recognition of the MART-126/27-35 epitope in the absence of ERAP1 expression. Our results underline the relevance of ERAP2 for tumor epitope presentation and TCR recognition and may need to be considered when designing ACT in the future.

Migraine and weather: a prospective diary-based analysis.

AIMS: Weather is mentioned as a trigger factor by migraine patients most frequently. We examined the impact of meteorological factors and the impact of their day-to-day change on the risk of occurrence and persistence of headache and migraine and the correlation of subjective weather perception with objective weather data. METHODS: We performed a prospective, diary-based cohort study in 238 patients suffering from migraine with or without aura. Patients had to live within 25 km of the Vienna meteorological station and were required to keep a diary for 90 days. We analysed 11 meteorological parameters and 17 synoptic weather situations. For evaluating the hazard of occurrence and persistence of migraine and headache, we performed a univariate and a stepwise multivariate Cox regression analysis. We calculated correlations between subjective weather perception and meteorological data. RESULTS: In the uni- and multivariate analysis, a ridge of high pressure increased the risk of headache occurrence, lower mean daily wind speed increased the risk of migraine occurrence and a day-to-day change of daily sunshine duration increased the risk of migraine occurrence. A day-to-day change of the daily minimum temperature decreased the risk of migraine persistence. After correction for multiple testing, none of these findings remained statistically significant. Subjective weather perceptions did not correlate with the occurrence or persistence of migraine or headache. Subjective perception of cold and too-cold weather and of too-warm weather correlated with daily minimum, mean and maximum temperature. CONCLUSION: The influence of weather factors on migraine and headache is small and questionable.

Electronic and vibronic contributions to two-photon absorption in donor-acceptor-donor squaraine chromophores.

Many squaraines have been observed to exhibit two-photon absorption at transition energies close to those of the lowest energy one-photon electronic transitions. Here, the electronic and vibronic contributions to these low-energy two-photon absorptions are elucidated by performing correlated quantum-chemical calculations on model chromophores that differ in their terminal donor groups (diarylaminothienyl, indolenylidenemethyl, dimethylaminopolyenyl, or 4-(dimethylamino)phenylpolyenyl). For squaraines with diarylaminothienyl and dimethylaminopolyenyl donors and for the longer examples of 4-(dimethylamino)phenylpolyenyl donors, the calculated energies of the lowest two-photon active states approach those of the lowest energy one-photon active (1B(u)) states. This is consistent with the existence of purely electronic channels for low-energy two-photon absorption (TPA) in these types of chromophores. On the other hand, for all squaraines containing indolinylidenemethyl donors, the calculations indicate that there are no low-lying electronic states of appropriate symmetry for TPA. Actually, we find that the lowest energy TPA transitions can be explained through coupling of the one-photon absorption (OPA) active 1B(u) state with b(u) vibrational modes. Through implementation of Herzberg-Teller theory, we are able to identify the vibrational modes responsible for the low-energy TPA peak and to reproduce, at least qualitatively, the experimental TPA spectra of several squaraines of this type.

Gastric dilatation-volvulus associated with diaphragmatic hernia in three cats: clinical presentation, surgical treatment and presumptive aetiology.

Three cats were examined because of acute dyspnoea and sudden abdominal enlargement. In all cats, radiographs revealed gastric dilatation-volvulus (GDV) and diaphragmatic hernia. Cardiovascular shock and dyspnoea were treated by intravenous fluid-therapy, oxygen administration and relief of diaphragmatic pressure by means of stomach decompression and in one case placing the patient in an inclined position. Gastric decompression was performed by needle gastrocentesis, placement of a rhino-gastric tube, or a combination of these. Diaphragmatic herniorrhaphy was performed in either case; one cat also underwent gastropexy. The immediate postoperative period resolved uneventfully and the cats were doing well at follow-up. Feline GDV is a rare event in which diaphragmatic hernia may be a predisposing factor.

PRISEs (progesterone 5ß-reductase and/or iridoid synthase-like 1,4-enone reductases): Catalytic and substrate promiscuity allows for realization of multiple pathways in plant metabolism.

PRISEs (progesterone 5ß-reductase and/or iridoid synthase-like 1,4-enone reductases) are involved in cardenolide and iridoid biosynthesis. We here investigated a PRISE (rAtSt5ßR) from Arabidopsis thaliana, a plant producing neither cardenolides nor iridoids. The structure of rAtSt5ßR was elucidated with X-ray crystallography and compared to the known structures of PRISEs from Catharanthus roseus (rCrISY) and Digitalis lanata (rDlP5ßR). The three enzymes show a high degree of sequence and structure conservation in the active site. Amino acids previously considered to allow discrimination between progesterone 5ß-reductase and iridoid synthase were interchanged among rAtSt5ßR, rCrISY and rDlP5ßR applying site-directed mutagenesis. Structural homologous substitutions had different effects, and changes in progesterone 5ß-reductase and iridoid synthase activity were not correlated in all cases. Our results help to explain fortuitous emergence of metabolic pathways and product accumulation. The fact that PRISEs are found ubiquitously in spermatophytes insinuates that PRISEs might have a more general function in plant metabolism such as, for example, the detoxification of reactive carbonyl species.

ERAP1-Dependent Antigen Cross-Presentation Determines Efficacy of Adoptive T-cell Therapy in Mice.

Cytotoxic T lymphocytes can reject established tumors if their target peptide is efficiently presented by MHC class I molecules (pMHC-I) on the surface of cancerous cells. Therapeutic success upon adoptive T-cell transfer (ATT), however, requires additional cross-presentation of the same pMHC-I on noncancerous cells. Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that customizes the N-terminus of proteasome-generated peptides so they can be loaded onto MHC-I molecules in the endoplasmic reticulum (ER). We show here that ERAP1 is critically involved in the process of tumor rejection and assumes a dual role by independently operating on both sides. Direct presentation of two MHC-I-restricted epitopes of a cancer-driving transplantation rejection antigen through ERAP1 moderately affected tumor rejection by adoptively transferred T-cell receptor gene-modified T cells in each case. ERAP1 expression by antigen cross-presenting cells of the ATT recipients was critical for expansion of therapeutic monospecific T cells and correlated with tumor rejection. Specifically, lack of ERAP1 expression in the ATT recipient's noncancerous cells enabled progression of pMHC-I-positive, IFNγ-responsive tumors, despite the presence of antigen-specific functional cytotoxic T lymphocytes. These data reveal a decisive role for ERAP1 in T-cell-mediated tumor rejection and will enhance the choice of MHC-I-restricted epitopes targeted by adoptive T-cell transfer.Significance: This study demonstrates a role of ERAP1 in the efficacy of adoptive T-cell transfer and has potential to improve personalized T-cell therapy for solid tumors. Cancer Res; 78(12); 3243-54. ©2018 AACR.

Sex Differences in Percutaneous Coronary Intervention-Insights From the Coronary Angiography and PCI Registry of the German Society of Cardiology.

BACKGROUND: Several studies have suggested sex-related differences in diagnostic and invasive therapeutic coronary procedures. METHODS AND RESULTS: Data from consecutive patients who were enrolled in the Coronary Angiography and PCI Registry of the German Society of Cardiology were analyzed. We aimed to compare sex-related differences in in-hospital outcomes of patients undergoing percutaneous coronary intervention (PCI) for stable coronary artery disease, non-ST elevation acute coronary syndromes, ST elevation myocardial infarction, and cardiogenic shock. From 2007 until the end of 2009 data from 185 312 PCIs were prospectively registered: 27.9% of the PCIs were performed in women. Primary PCI success rate was identical between the sexes (94%). There were no sex-related differences in hospital mortality among patients undergoing PCI for stable coronary artery disease, non-ST elevation acute coronary syndromes, or cardiogenic shock except among ST elevation myocardial infarction patients. Compared to men, women undergoing primary PCI for ST elevation myocardial infarction have a higher risk of in-hospital death, age-adjusted odds ratio (1.19, 95% CI 1.06-1.33), and risk of ischemic cardiac and cerebrovascular events (death, myocardial infarction, transient ischemic attack/stroke), (age-adjusted odds ratio 1.19, 95% CI 1.16-1.29). Furthermore, access-related complications were twice as high in women, irrespective of the indication. CONCLUSIONS: Despite identical technical success rates of PCI between the 2 sexes, women with PCI for ST elevation myocardial infarction have a 20% higher age-adjusted risk of death and of ischemic cardiac and cerebrovascular events. Further research is needed to determine the reasons for these differences.

Preventing tumor escape by targeting a post-proteasomal trimming independent epitope.

Adoptive T cell therapy (ATT) can achieve regression of large tumors in mice and humans; however, tumors frequently recur. High target peptide-major histocompatibility complex-I (pMHC) affinity and T cell receptor (TCR)-pMHC affinity are thought to be critical to preventing relapse. Here, we show that targeting two epitopes of the same antigen in the same cancer cells via monospecific T cells, which have similar pMHC and pMHC-TCR affinity, results in eradication of large, established tumors when targeting the apparently subdominant but not the dominant epitope. Only the escape but not the rejection epitope required postproteasomal trimming, which was regulated by IFN-γ, allowing IFN-γ-unresponsive cancer variants to evade. The data describe a novel immune escape mechanism and better define suitable target epitopes for ATT.

Use and impact of thrombectomy in primary percutaneous coronary intervention for acute myocardial infarction with persistent ST-segment elevation: results of the prospective ALKK PCI-registry.

BACKGROUND: Data about the impact of thrombectomy in primary percutaneous coronary intervention (PCI) are inconsistent. The aim of our study was an evaluation of both the real-world use of thrombectomy and the impact of thrombectomy on outcome in unselected patients treated with primary PCI for ST-elevation myocardial infarction (STEMI). METHODS AND RESULTS: We used the data of the prospective ALKK PCI-registry of 35 hospitals from January 2010 to December 2013. A total of 10,755 patients receiving single-vessel primary PCI for acute STEMI were included. In 2176 patients (20.2 %) thrombectomy was performed. There was a wide range of use of thrombectomy in the different ALKK hospitals from 1.1 to 61.7 % (median 18.6 %, quartiles 6.0 and 40.3 %) with a general increase of use over the first years of the study period. In patients with and without thrombectomy there was TIMI 0 flow present before PCI in 6010 patients, TIMI 1 in 1338, TIMI 2 in 2002, and TIMI 3 in 1405. Patients with acute heart failure or cardiogenic shock received significantly more often thrombectomy. Fluoroscopy time (8.1 vs. 7.3 min, p < 0.0001) and dose area product (5373 cGy × cm(2) vs. 4802 cGy × cm(2), p < 0.0001) were significantly higher in patients treated with thrombectomy. The subgroup of patients with TIMI 0 flow before PCI had significantly higher rates of TIMI 3 flow after PCI when treated with thrombectomy (87.1 vs. 84.1 %, p < 0.01), while there was no difference in post-PCI TIMI 3 flow in patients with TIMI 1, 2 or 3 flow before PCI. Rates of major adverse cardiac and cerebrovascular events were similar in both groups in general and in all subgroups of TIMI flow. CONCLUSIONS: The use of thrombectomy in patients with STEMI is heterogenous between hospitals. Overall, there was no impact of thrombectomy on TIMI 3 patency or mortality after PCI. In the subgroup of STEMI patients with TIMI 0 flow before PCI individualized thrombectomy had a positive impact on restoration of normal blood flow.

Gender differences in therapeutic recommendation after diagnostic coronary angiography: insights from the Coronary Angiography and PCI Registry of the German Society of Cardiology.

BACKGROUND: There is information suggesting differences and underuse of invasive coronary diagnostic and therapeutic procedures in women compared to men. METHODS: Data from consecutive patients (pts) which were enrolled in the Coronary Angiography and PCI Registry of the German Society of Cardiology were analyzed. We compared gender-related differences in diagnosis and therapeutic recommendation of pts undergoing coronary angiography (XA) for stable coronary artery disease (CAD), non-ST elevation acute coronary syndromes (NSTE-ACS) and ST elevation myocardial infarction (STEMI). RESULTS: From 2004 until the end of 2009, data of 1,060,542 invasive procedures in 1,014,996 pts were prospectively registered. One-third (34.6%) of them were female. Women less often had significant CAD, irrespective of the indication for XA. In pts with relevant CAD, percutaneous coronary interventions (PCI) were recommended in 87.1% of women versus 89.1% of men with STEMI [age-adjusted OR (aOR) 0.98, 95% CI 0.93-1.04], in 67.1 vs. 66.8% in NSTE-ACS (aOR 1.10, 1.07-1.12), and in 50.3 vs 49.4% in stable CAD (aOR 1.07, 1.05-1.09). CONCLUSIONS: In pts with significant CAD, there was no difference in recommendation for PCI between the genders in stable CAD, whereas in STEMI and NSTE-ACS women were treated even more often with PCI. There were only minor differences in referral for CABG between women and men. Hence, our data provide strong evidence against a gender bias in use of invasive therapeutic procedures once the diagnosis of significant CAD has been confirmed.

Effects of racing and nontraining on plasma thyroid hormone concentrations in sled dogs.

OBJECTIVE: To determine the effects of racing and nontraining on plasma thyroxine (T4), free thyroxine (fT4), thyroid-stimulating hormone (TSH), and thyroglobulin autoantibody (TgAA) concentrations in sled dogs and compare results with reference ranges established for dogs of other breeds. DESIGN: Cross-sectional study. ANIMALS: 122 sled dogs. PROCEDURE: Plasma thyroid hormone concentrations were measured before dogs began and after they finished or were removed from the Iditarod Trail Sled Dog Race in Alaska and approximately 3 months after the race. RESULTS: Concentrations of T4 and fT4 before the race were less than the reference range for nonsled dogs in 26% and 18% of sled dogs, respectively. Immediately after racing, 92% of sled dogs had plasma T4 concentrations less than the reference range. Three months after the race, 25% of sled dogs had plasma T4 concentrations less than the reference range. For T4, fT4, TSH, and TgAA, significant differences were not detected in samples collected before the race versus 3 months later. CONCLUSIONS AND CLINICAL RELEVANCE: Plasma T4, fT4, and TSH concentrations decreased in dogs that complete a long distance sled dog race. Many clinically normal sled dogs have plasma T4 and fT4 values that are lower than the reference range for nonsled dogs. We suggest that the reference ranges for sled dogs are 5.3 to 40.3 nmol/L and 3.0 to 24.0 pmol/L for plasmaT4 and fT4 concentrations, respectively, and 8.0 to 370 mU/L for TSH.

Virus-induced hepatocellular carcinomas cause antigen-specific local tolerance.

T cell surveillance is often effective against virus-associated tumors because of their high immunogenicity. It is not clear why surveillance occasionally fails, particularly against hepatitis B virus- or hepatitis C virus-associated hepatocellular carcinoma (HCC). We established a transgenic murine model of virus-induced HCC by hepatocyte-specific adenovirus-induced activation of the oncogenic SV40 large T antigen (TAg). Adenovirus infection induced cytotoxic T lymphocytes (CTLs) targeted against the virus and TAg, leading to clearance of the infected cells. Despite the presence of functional, antigen-specific T cells, a few virus-infected cells escaped immune clearance and progressed to HCC. These cells expressed TAg at levels similar to HCC isolated from neonatal TAg-tolerant mice, suggesting that CTL clearance does not select for cells with low immunogenicity. Virus-infected mice revealed significantly greater T cell infiltration in early-stage HCC compared with that in late-stage HCC, demonstrating progressive local immune suppression through inefficient T cell infiltration. Programmed cell death protein-1 (PD-1) and its ligand PD-L1 were expressed in all TAg-specific CD8+ T cells and HCC, respectively, which contributed to local tumor-antigen-specific tolerance. Thus, we have developed a model of virus-induced HCC that may allow for a better understanding of human HCC.