Mild COVID-19 induces early, quantifiable, persistent troponin I elevations in elder men.

Importance: Elderly patients, especially men, are at risk of increased morbidity from coronavirus disease 2019 (COVID-19). Long-term data on troponin I levels in longitudinal observational studies of outpatients with mild to moderate COVID-19 are scarce. Objective: This controlled cohort study aimed to evaluate the course of troponin I concentrations over a long period in convalescent COVID-19 outpatients with mild to moderate symptoms. Setting and participants: In this cohort study, individuals with PCR-confirmed, mild to moderate SARS-CoV-2 infection as well as control individuals with confirmed negative PCR and negative SARS-CoV-2 serology were included. Study visits were performed from April 2020 through July 2021 (initialized during the first wave of the corona pandemic in Switzerland). A study visit in patients comprised blood draws every week in the first month and additionally after 8 weeks. This course was repeated in patients observed long-term. Results: This study enrolled 278 individuals from the Canton of St. Gallen, Switzerland, aged 12-92 years (59.5% women), who had mild to moderate COVID-19 symptoms (outpatients only) and a diagnosis confirmed by positive RT-PCR. Fifty-four of the participants with confirmed SARS-CoV-2 infection were followed for 14 months with repeat cycles of the testing protocol. In addition, 115 symptomatic patients that were PCR and serology negative were enrolled in the same time period as a control group. In COVID-19 patients, low-level troponin I concentrations (cTnI) were significantly increased from baseline until week 9 after positive RT-PCR diagnosis in men older than 54 years [ΔcTnI = 5.0 ng/L (median); 95% CI 4.1-6.0; p = 0.02]. The troponin I concentration remained elevated throughout 14 months in men older than 54 years within the cohort with a prolonged observation period. This statistically significant change in troponin I concentration was not dependent on co-morbidities in this group. ALT, Creatinine, BNP, and D-Dimer values after convalescence did not differ in comparison to the control cohort. Conclusion: In this analysis of individuals with confirmed SARS-CoV-2 infection, hs troponin I levels of men aged 54 or older significantly increased after infection. They remained elevated for at least 14 months after diagnosis. This suggests the possibility of an ongoing, long-term, low-grade myocardial injury. Further studies with focus on elderly patients and a prolonged observational period are necessary to elucidate whether the phenomenon observed is associated with detectable structural changes to the heart muscle or is without further clinical consequences.

Leptospirosis Followed by Kawasaki-Like Disease: Case Report From an Adult Swiss Patient and Review of the Literature.

Kawasaki disease (KD) is a vasculitis that mostly occurs in children, but rare cases in adults have been reported. We describe the case of a 43-year-old Swiss male who developed symptoms compatible with KD 7 weeks after leptospirosis, which was presumably acquired after swimming in a creek in the Swiss Alps. We performed a literature review and identified 10 other cases (all in children), in which Kawasaki-like disease was diagnosed in the context of leptospirosis. Outcome was favourable in most cases, including our patient. This exceptional case demonstrates both the possibility of autochthonous cases of leptospirosis in Switzerland as well as a possible association of leptospirosis with Kawasaki-like disease.

The pharmacokinetics of cell-penetrating peptides.

Cell-penetrating peptides (CPPs) are able to penetrate the cell membrane carrying cargoes such as peptides, proteins, oligonucleotides, siRNAs, radioisotopes, liposomes, and nanoparticles. Consequently, many delivery approaches have been developed to use CPPs as tools for drug delivery. However, until now a systematic analysis of their in vivo properties including potential tumor binding specificity for drug targeting purposes has not been conducted. Ten of the most commonly applied CPPs were obtained by solid phase peptide synthesis and labeled with (111)In or (68)Ga. Uptake studies were conducted using a panel of six tumor cell lines of different origin. The stability of the peptides was examined in human serum. Biodistribution experiments were conducted in nude mice bearing human prostate carcinoma. Finally, positron emission tomography (PET) measurements were performed in male Wistar rats. The in vitro uptake studies revealed high cellular uptake values, but no specificity toward any of the cell lines. The biodistribution in PC-3 tumor-bearing nude mice showed a high transient accumulation in well-perfused organs and a rapid clearance from the blood. All of the CPPs revealed a relatively low accumulation rate in the brain. The highest uptake values were observed in the liver (with a maximal uptake of 51 %ID/g observed for oligoarginine (R(9))) and the kidneys (with a maximal uptake of 94 %ID/g observed for NLS). The uptake values in the PC-3 tumor were low at all time points, indicating a lack of tumor specific accumulation for all peptides studied. A micro-PET imaging study with (68)Ga-labeled penetratin, Tat and transportan(10) (TP(10)) confirmed the organ distribution data. These data reveal that CPPs do not show evidence for application in tumor targeting purposes in vivo. However, CPPs readily penetrate into most organs and show rapid clearance from the circulation. The high uptake rates observed in vitro and the relatively low specificity in vivo imply that CPPs would be better suited for topical application in combination with cargoes which show passive targeting and dominate the pharmacokinetic behavior. In conclusion, CPPs are suitable as drug carriers for in vivo application provided that their pharmacokinetic properties are also considered in design of CPP drug delivery systems.

A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology.

UNLABELLED: Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy. METHODS: Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC). Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR. RESULTS: In vitro binding experiments of (125)I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney. CONCLUSIONS: These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.

Identification and characterization of a peptide with affinity to head and neck cancer.

UNLABELLED: Combination therapy has improved the quality of life for patients with squamous cell carcinomas of the head and neck (HNSCCs) but has not decisively changed prognosis. Targeted therapies, which enhance accumulation of the drug in the tumor, may be realized using tumor-specific binding peptides. This paper identifies and characterizes an HNSCC affine peptide. METHODS: From a phage library comprising 10(9) different displayed peptides, 1 peptide was enriched after 5 in vitro selection rounds on HNO223 tumor cells. Subsequently, the gained peptide sequence H(2)N-SPRGDLAVLGHKY-CONH(2) (HBP-1) was synthesized as an amide and labeled with (125)I. In vitro studies for binding kinetics and competition were performed with 5 different HNSCC cell lines. Furthermore, the stability of the peptide was evaluated in human serum. The in vivo biodistribution of (131)I-labeled peptide was determined in HNSCC tumor-bearing nude mice. The results were further validated in human HNSCC tumor tissue sections using fluorescence-labeled HBP-1. Competition experiments were performed to determine the binding sequence and validate the target. RESULTS: The HBP-1 motif was enriched in 62% of all phages sequenced. Labeled (125)I-HBP-1 showed binding to 5 different HNSCC cell lines and a maximum binding to HNO97 cells, with 11% of the applied dose per 10(6) cells and an inhibitory concentration of 50% of 38.9 nM. Stability experiments in human serum showed a half-life of 55 min. In 2 different HNSCC tumor xenografts, (131)I-HBP-1 accumulated rapidly, with stable uptake until 45 min after intravenous application. Peptide immunohistochemistry of HNSCC tissue sections exhibited tumor staining by HBP-1, whereas normal tissue remained negative. Sequence mutation and competition experiments revealed that the intrinsic RGD motif in combination with the intrinsic LXXL motif is responsible for the binding ability of HBP1. The RGDLXXL sequence within this peptide is known and indicates that binding occurs via the alpha(v)beta(6) rather than the alpha(v)beta(3) integrin. CONCLUSION: Within the sequence of HBP-1 is a RGDLXXL motif, and most likely it is targeting the alpha(v)beta(6) receptor of the integrin family of cell adhesion receptors. HBP-1 represents a promising lead structure for the development of targeted therapies or diagnostic procedures in patients with HNSCC.

Determination of morphine and 6-acetylmorphine in blood with use of dried blood spots.

The use of dried blood spots (DBS) which has successfully been introduced in neonatal metabolic screening is an appropriate method to reduce virus infection risk to a minimum, facilitating regular mailing and handling of samples in the laboratory. Injection diacetylmorphine use is notably associated with a prevalence of infection and a risk of transmission of blood-borne viruses. The aim of the present study was to establish a method to determine morphine and 6-acetylmorphine (6-AM) as accurately and sensitively from DBS as from whole blood. Analysis by liquid chromatography/tandem mass spectrometry was checked for carryover, ion suppression/enhancement, linearity of response, lower limits of detection and quantification, and the within-run and between-run assay imprecision for both whole blood and DBS after liquid/liquid extraction. DBS drying time and elution were optimized, and extraction efficiency from DBS was compared with that of blood and of a solution of the pure compounds. Short-term stability of morphine and 6-AM was determined at -20 degrees C, 4 degrees C, and 40 degrees C up to 7 days from both whole blood and DBS. Furthermore, it was tested whether analysis of DBS may be as reliable as that of whole blood investigating 50 authentic samples. The lower limit of detection was 0.4 ng of morphine per spot and 0.8 ng of 6-AM per spot using a DBS blood volume of 100 microL and was 0.3 and 0.7 ng/100 microL whole blood for morphine and 6-AM, respectively. Recovery rates of the analytes from DBS did not differ from those from whole blood and were > or =55% for 6-AM and > or =25% for morphine, and the within- and between-run coefficients of variation were always < or =9%. 6-AM degraded rapidly at both 4 degrees C and 40 degrees C in whole blood; however, it seemed to be much more stable in DBS. Significant correlations between real whole-blood samples and DBS were obtained. The DBS assay has potential as a precise and inexpensive option for the determination of morphine and 6-AM in small blood samples. Further, the DBS matrix proved to excellently stabilize 6-AM.

Enzymes/transporters.

Tracers that specifically target tumours are highly warranted for diagnosis and to monitor cancer chemotherapy response. However, as cancer cells arise from normal cells they do not substantially differ from the normal cells and therefore tumour specific targets are rare. Fortunately, the process of malignant transformation is associated with the up- or down-regulation of enzymes and transporters that play a crucial role in tumour growth. Consequently diagnostic imaging procedures have attained their major success with tracers that target enzymes and transporters that are over-expressed in tumours. The glucose transporters, the multi drug resistance transporters (MDRPs), several kinases and the family of cathepsins are prominent examples for enzymes and receptors that can be targeted for molecular imaging.

Xenobiotic geometry and media pH determine cytotoxicity through solubility.

Polychlorinated biphenyls (PCBs), a class of 209 individual congeners, have become persistent and ubiquitous environmental contaminants. The health impacts of PCBs, such as cancer, cardiovascular disease, developmental toxicity, and neurotoxicity, have been widely reported, but for many of these, the mechanisms of toxicity are still poorly understood. Many mechanistic studies involve cultured cells where the biological activity is dependent upon the solubility of the xenobiotic. In the present study, we investigated the factors that determine solubility as measured by diffraction spectroscopy and have modeled, with semiempirical and ab initio molecular orbital methods, the dihedral angle and calculated the dipole moment of a series of monofluorinated analogues (F-PCBs 3) of 4-chlorobiphenyl (PCB 3) as model compounds in vacuum and in water. We found a strong positive correlation between the dihedral angle, the rotation energy, the cavitation energy, the solubility, and the cytotoxicity in three human cell lines. The dipole moment was of minor influence. We also determined the influence of pH changes in a medium containing 10% fetal bovine serum (FBS), changes that could be expected when cells in culture are removed from a CO 2 incubator even for a short time. We found that the solubility is strongly affected by the pH and that this effect is not reversed by subsequent pH readjustment. In a study examining cytotoxicity, we showed that the actual pH and the pH history of a medium containing FBS were of major influence. We suggest that pH-driven changes in the tertiary and quaternary structure of albumin are responsible. These observations have implications for studies of the biological activity of semisoluble compounds, like PCBs and related compounds.

Synthesis and evaluation of intercalating somatostatin receptor binding peptide conjugates for endoradiotherapy.

PURPOSE: Intercalators, planar aromatic compounds, are able to interact with DNA by sandwiching themselves between the stacked bases at right angles to the long axis of the helix. Under certain circumstances, Auger-electron-emitting radionuclides can be extremely radiotoxic and produce extensive DNA damage. Auger electron-emitting radioisotopes, are known to be highly cytotoxic when localized in cell nuclei due to highly localized energy deposition by low energy Auger electrons. In addition binding to the DNA might increase the retention in the receptor expressing tissues. METHODS: In order to exploit the cytotoxic potential of intercalator-Auger-emitter conjugates, bis-benzimidazole dyes, Hoechst 33258 and 33342, were linked to a somatostatin receptor affine carrier peptide. For this purpose a bis-benzimidazole intercalating moiety was prepared using variations on the literature methods. The intercalating moieties were coupled under normal SPPS conditions to the carrier peptide, Tyr3-octreotate. To attach the chelating agent (DOTA) to the intercalating moiety, a free amine derivative was prepared and coupled in solution to DOTA tris-t-butyl ester. The resulting chelator-intercalator conjugate was then coupled to a Tyr3-octreotate carrying resin using SPPS. RESULTS: The peptide conjugates were obtained in good yields after HPLC chromatography. The cellular uptake of the novel conjugates was determined using fluorescence microscopy. All intercalator-peptide conjugates revealed somatostatin receptor binding affinities in the nanomolar range. CONCLUSIONS: The novel chelator-intercalator derivatives of the somatostatin receptor binding Tyr3-octreotate introduce a new scope to the range of tracers for therapeutic purposes.

Discovery of two novel, small-molecule inhibitors of DNA methylation.

DNA methyltransferases are promising targets for cancer therapy. In many cancer cells promoters of tumor suppressor genes are hypermethylated, which results in gene inactivation. It has been shown that DNA methyltransferase inhibitors can suppress tumor growth and have significant therapeutic value. However, the established inhibitors are limited in their application due to their substantial cytotoxicity. To discover novel compounds for the inhibition of human DNA methyltransferases, we have screened a set of small molecules available from the NCI database. Using a 3-dimensional model of the human DNA methyltransferase 1 and a modified docking and scoring procedure, we have identified a small list of molecules with high affinities for the active site of the enzyme. The two highest scoring structures were found to inhibit DNA methyltransferase activity in vitro and in vivo. The newly discovered inhibitors validate our screening procedure and also provide a useful basis for further rational drug development.

Epigenetic reactivation of tumor suppressor genes by a novel small-molecule inhibitor of human DNA methyltransferases.

DNA methylation regulates gene expression in normal and malignant cells. The possibility to reactivate epigenetically silenced genes has generated considerable interest in the development of DNA methyltransferase inhibitors. Here, we provide a detailed characterization of RG108, a novel small molecule that effectively blocked DNA methyltransferases in vitro and did not cause covalent enzyme trapping in human cell lines. Incubation of cells with low micromolar concentrations of the compound resulted in significant demethylation of genomic DNA without any detectable toxicity. Intriguingly, RG108 caused demethylation and reactivation of tumor suppressor genes, but it did not affect the methylation of centromeric satellite sequences. These results establish RG108 as a DNA methyltransferase inhibitor with fundamentally novel characteristics that will be particularly useful for the experimental modulation of epigenetic gene regulation.

Inhibition of O6-methylguanine-DNA methyltransferase by glucose-conjugated inhibitors: comparison with nonconjugated inhibitors and effect on fotemustine and temozolomide-induced cell death.

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is an important suicide enzyme involved in the defense against O(6)-alkylating mutagens. It also plays a role in the resistance of tumors to anticancer drugs targeting the O(6)-position of guanine, such as temozolomide and fotemustine. Several potent MGMT inhibitors have been developed sensitizing cells to O(6)-alkylating agents. Aimed at targeting MGMT inhibitors to tumor cells, we synthesized MGMT inhibitory compounds conjugated with glucose to improve uptake in tumor cells. Here, we compared O(6)-benzylguanine, O(6)-2-fluoropyridinylmethylguanine (O(6)FPG), O(6)-3-iodobenzylguanine, O(6)-4-bromothenylguanine, and O(6)-5-iodothenylguanine with the corresponding C8-linker beta-d-glucose derivatives. All glucose conjugated inhibitors were 3- to 5-fold less effective than the corresponding nonconjugated drugs as to MGMT inhibition that was measured in cell extracts (in vitro) and cultivated HeLaS3 cells (in vivo). Except for O(6)FPG, IC(50) values of the guanine derivatives applied in vitro and in vivo were correlated. A similar correlation was not obvious for the corresponding glucosides, indicating differences in cellular uptake. C8-alpha-d-glucosides were less effective than beta-glucosides. From the newly developed glucose-conjugated inhibitors tested, O(6)-4-bromothenylguanine-C8-beta-d-glucoside (O(6)BTG-C8-betaGlu) was most potent in inhibiting MGMT both in vitro and in vivo. At a concentration of 0.1 microM, it inhibited cellular MGMT to completion. It was not toxic, even when applied chronically to cells at high dose (up to 20 microM). O(6)BTG-C8-betaGlu strongly potentiated the killing effect of fotemustine and temozolomide, causing reversal from MGMT+ to MGMT- phenotype. Therefore, O(6)BTG-C8-betaGlu seems to be especially suitable for approaching MGMT inhibitor targeting in tumor therapy.

Establishment and functional validation of a structural homology model for human DNA methyltransferase 1.

Changes in DNA methylation patterns play an important role in tumorigenesis. The DNA methyltransferase 1 (DNMT1) protein represents a major DNA methyltransferase activity in human cells and is therefore a prominent target for experimental cancer therapies. However, there are only few available inhibitors and their high toxicity and low specificity have so far precluded their broad use in chemotherapy. Based on the strong conservation of catalytic DNA methyltransferase domains we have used a homology modeling approach to determine the three-dimensional structure of the DNMT1 catalytic domain. Our results suggest an overall structural conservation with other DNA methyltransferases but also indicate local conformational differences. To prove the validity of our model we used it as a template to design a novel derivative of the known DNA methyltransferase inhibitor 5-azacytidine. The resulting compound (N4-fluoroacetyl-5-azacytidine) functioned as an efficient inhibitor of DNA methylation in human tumor cell lines and also provides novel opportunities for pharmacological applications.