Incorporating selected non-communicable diseases into facility-based surveillance systems from a resource-limited setting in Africa.

BACKGROUND: As Mozambique faces a double burden of diseases, with a rise of Non Communicable Diseases (NCD) superimposed to uncontrolled communicable diseases (CD), routine disease surveillance system does not include NCD. The objectives of our study were to i) upgrade of the current surveillance system by adapting the data collection tools to NCD; ii) describe the occurrence and profile of selected NCD using these data collection tools. METHODS: Workshops were implemented in a first referral urban hospital of Mozambique to train clinical staff, administrative workers and nurses on NCD surveillance, as well as select conditions to be prioritized. Based on the WHO Global Action Plan and Brazaville Declaration for NCD prevention and control, we selected arterial hypertension, diabetes, stroke, chronic respiratory diseases, mental illness and cancers. Data collection tools used for CD were changed to include age, gender, outcome and visit type. Between February/2014 and January/2015 we collected data at an urban hospital in Mozambique's capital. RESULTS: Over 12 months 92,018 new patients were assisted in this hospital. Data was missing or diagnosis was unreadable in 2637 (2.9%) thus only 89,381 were used for analysis; of these 6423 (median age 27 years; 58.4% female) had at least one selected NCD as their primary diagnosis: arterial hypertension (2397;37.31%), mental illness (1497;23.30%), asthma (1495;23.28%), diabetes (628;9.78%), stroke (299;4.66%), chronic obstructive pulmonary disease 61 (0.95%) and cancers 46 (0.72%). Emergency transfers were needed for 76 patients (1.2%), mainly due to hypertensive emergencies (31; 40.8%) and stroke (18;23.7%). Twenty-four patients died at entry points (0.3%); 10 of them had hypertensive emergencies. CONCLUSION: Changes in existing surveillance tools for communicable diseases provided important data on the burden and outcomes of the selected NCD helping to identify priority areas for training and health care improvement. This information can be used to design the local NCD clinics and to strengthen the health information system in resource-limited settings in a progressive and sustainable way.

In vitro antimicrobial susceptibility testing methods: agar dilution to 3D tissue-engineered models.

In the field of orthopaedic surgery, bacterial invasion of implants and the resulting periprosthetic infections are a common and unresolved problem. Antimicrobial susceptibility testing methods help to define the optimal treatment and identify antimicrobial resistance. This review discusses proven gold-standard techniques and recently developed models for antimicrobial susceptibility testing, while also providing a future outlook. Conventional, gold-standard methods, such as broth microdilution, are still widely applied in clinical settings. Although recently developed methods based on microfluidics and microdroplets have shown advantages over conventional methods in terms of testing speed, safety and the potential to provide a deeper insight into resistance mechanisms, extensive validation is required to translate this research to clinical practice. Recent optical and mechanical methods are complex and expensive and, therefore, not immediately clinically applicable. Novel osteoblast infection and tissue models best resemble infections in vivo. However, the integration of biomaterials into these models remains challenging and they require a long tissue culture, making their rapid clinical implementation unlikely. A method applicable for both clinical and research environments is difficult to realise. With a continuous increase in antimicrobial resistance, there is an urgent need for methods that analyse recurrent infections to identify the optimal treatment approaches. Graphical abstract Timeline of published and partly applied antimicrobial susceptibility testing methods, listed according to their underlying mechanism, complexity and application in research or clinics.

[Screening for 4MRGN in German emergency departments]. / Screening auf 4MRGN in deutschen Notaufnahmen.

BACKGROUND: Multiresistant Gram-negative bacteria (MRGN) are a growing clinical problem. The practical implementation of the recommendation of the Commission for Hospital Hygiene and Infection Prevention (KRINKO) for screening according to 4MRGN (MRGN resistant to all four categories of antibiotics), however, varies considerably between emergency departments. OBJECTIVES: This study is intended to give an overview of the status quo and the quality assurance of 4MRGN screening and to show possibilities for process optimization. MATERIALS AND METHODS: In 2018, a web-based survey was conducted among emergency room directors and directors of clinics in the Association of Hospital Directors in Germany (VKD). RESULTS: The response rate of the 267 clinics surveyed was 31.1%. In all, 83.4% of the emergency rooms surveyed routinely screen for multiresistant pathogens. In 71.8% a standard procedure (SOP) is defined and 82.0% of the test criteria refer to the KRINKO recommendation. Only 39.7% of the clinics follow it without in-house adaptation. No clinic can give an exact number of actual risk patients per year. According to the median, 55 patients in an emergency room met the KRINKO screening criteria in 2017. Only 40 patients were screened for suspected 4MRGN. Quality assurance of the screening was performed by 41.0% of emergency departments. The responsibility lies mainly with the hygiene department. CONCLUSIONS: Even if screenings are carried out as far as possible, there is a lack of standardization in the recording of case numbers and quality assurance. Therefore, it can be assumed that there are numerous individuals with undetected 4MRGN. As a quality indicator, SOPs could clearly assign responsibilities and improve infection hygiene.

Defining the variety of cell types in developing and adult human kidneys by single-cell RNA sequencing.

The kidney is among the most complex organs in terms of the variety of cell types. The cellular complexity of human kidneys is not fully unraveled and this challenge is further complicated by the existence of multiple progenitor pools and differentiation pathways. Researchers disagree on the variety of renal cell types due to a lack of research providing a comprehensive picture and the challenge to translate findings between species. To find an answer to the number of human renal cell types, we discuss research that used single-cell RNA sequencing on developing and adult human kidney tissue and compares these findings to the literature of the pre-single-cell RNA sequencing era. We find that these publications show major steps towards the discovery of novel cell types and intermediate cell stages as well as complex molecular signatures and lineage pathways throughout development. The variety of cell types remains variable in the single-cell literature, which is due to the limitations of the technique. Nevertheless, our analysis approaches an accumulated number of 41 identified cell populations of renal lineage and 32 of non-renal lineage in the adult kidney, and there is certainly much more to discover. There is still a need for a consensus on a variety of definitions and standards in single-cell RNA sequencing research, such as the definition of what is a cell type. Nevertheless, this early-stage research already proves to be of significant impact for both clinical and regenerative medicine, and shows potential to enhance the generation of sophisticated in vitro kidney tissue.

Evaluation of a lateral-flow nanoparticle fluorescence assay for TB infection diagnosis.

BACKGROUND: Programmatic management of TB infection is a critical component of the WHO End TB Strategy. Interferon-gamma release assays (IGRAs) overcome some limitations of the tuberculin skin test, but implementation of IGRA testing in low-resource settings is challenging.METHODS: In this feasibility study, we evaluated performance of a novel digital lateral-flow assay, the QIAreach® QuantiFERON® TB (QIAreach-QFT) test, against the QuantiFERON®-TB Gold Plus (QFT-Plus) assay. A population with a mix of risk factors for TB infection (111 donors) were sampled over multiple days. A total of 207 individual blood samples were tested according to the manufacturer´s instructions.RESULTS: The overall percentage agreement was 95.6% (two-sided 95% CI 91.8-98), with a positive percentage agreement (i.e., sensitivity) of 100% (95% CI 94.7-100) and a negative percentage agreement (i.e., specificity) of 95.6% (95% CI 90.6-98.4). All QFT-Plus positive specimens with TB1-Nil and TB2-Nil values less than 1 IU/ml tested positive on QIAreach-QFT.CONCLUSIONS: QIAreach QFT is a deployable, accurate testing solution for decentralised testing. It has the potential to overcome key hurdles for TB infection screening in high-burden settings thus helping to achieve the WHO End TB programme goals.

Co-expression of p21(Waf1/Cip1) in adenovirus vectors improves expression of a second transgene.

First-generation adenoviral (Ad) vectors are frequently used vectors for experimental and clinical gene transfer. Earlier it has been shown that parallel overexpression of the cell cycle regulator p21(Waf1/Cip1) (p21) or antiapoptotic bcl-2 from a second vector reduces cytotoxicity and improves transgene expression. Here, we investigate whether the co-expression of p21 and alpha(1)-antitrypsin from a single vector improves vector safety and alpha(1)-antitrypsin expression. Cell lines (A549 and HeLa) and primary cells (small airway epithelial cells and hepatocytes) were infected with adenovirus vectors transducing alpha(1)-antitrypsin with (AdCMV.p21-RSV.hAAT) or without (AdRSV.hAAT) p21. alpha(1)-Antitrypsin expression and cytotoxicity were analyzed using western blot/ELISA and LDH/ALT/AST assays, respectively. Cell cycle profiles were determined by flow cytometry. Co-expression of p21 strongly increased the alpha(1)-antitrypsin expression in all cell types and at all doses tested. No changes in ALT/AST from hepatocytes and only minor increases in the LDH release in A549 and HeLa were observed with either vector. Cell cycle profiles were also not affected adversely. Incorporation of p21 in Ad vectors together with a gene of interest improves the vector performance; such vectors will allow the application of lower doses and thereby reduce immunological side effects.

Camostat attenuates airway epithelial sodium channel function in vivo through the inhibition of a channel-activating protease.

Inhibition of airway epithelial sodium channel (ENaC) function enhances mucociliary clearance (MCC). ENaC is positively regulated by channel-activating proteases (CAPs), and CAP inhibitors are therefore predicted to be beneficial in diseases associated with impaired MCC. The aims of the present study were to 1) identify low-molecular-weight inhibitors of airway CAPs and 2) to establish whether such CAP inhibitors would translate into a negative regulation of ENaC function in vivo, with a consequent enhancement of MCC. To this end, camostat, a trypsin-like protease inhibitor, provided a potent (IC(50) approximately 50 nM) and prolonged attenuation of ENaC function in human airway epithelial cell models that was reversible upon the addition of excess trypsin. In primary human bronchial epithelial cells, a potency order of placental bikunin > camostat > 4-guanidinobenzoic acid 4-carboxymethyl-phenyl ester > aprotinin >> soybean trypsin inhibitor = alpha1-antitrypsin, was largely consistent with that observed for inhibition of prostasin, a molecular candidate for the airway CAP. In vivo, topical airway administration of camostat induced a potent and prolonged attenuation of ENaC activity in the guinea pig trachea (ED(50) = 3 microg/kg). When administered by aerosol inhalation in conscious sheep, camostat enhanced MCC out to at least 5 h after inhaled dosing. In summary, camostat attenuates ENaC function and enhances MCC, providing an opportunity for this approach toward the negative regulation of ENaC function to be tested therapeutically.

Human visual acuity measured with colored test objects.

Visual acuity was measured with a grating test object in which alternating bars were matched in brightness but differed in wavelength. If the wavelength difference between adjacent bars was great enough, acuity scores were obtained which were as high as those obtained with test objects in which there was a large brightness difference between adjacent bars.

Controlled formation of biological tubule systems in extracellular matrix gels in vitro.

Organs such as the lung and the kidney are composed of epithelial and endothelial tubule-forming networks. To engineer such organs, it would be desirable to control the shape, spatial orientation and interconnectedness of the forming tubules. To study this, channels were formed in extracellular matrix (ECM) gels and were subsequently filled with Madin-Darby canine kidney epithelial cells or human microvascular endothelial cells. After 3-5 days, the epithelial cells self-assembled into tubular structures of up to 1 cm, with a lumen lined by a monolayer of polarized epithelial cells at 10 days. In contrast, endothelial cells assembled into tubules with multiple fine branches. We found that a complex pattern of tubular networks of significant length and regular anatomical shape was achieved by molding ECM gels through microfabricated grooved templates.

Epigenetics of complex diseases: from general theory to laboratory experiments.

Despite significant effort, understanding the causes and mechanisms of complex non-Mendelian diseases remains a key challenge. Although numerous molecular genetic linkage and association studies have been conducted in order to explain the heritable predisposition to complex diseases, the resulting data are quite often inconsistent and even controversial. In a similar way, identification of environmental factors causal to a disease is difficult. In this article, a new interpretation of the paradigm of "genes plus environment" is presented in which the emphasis is shifted to epigenetic misregulation as a major etiopathogenic factor. Epigenetic mechanisms are consistent with various non-Mendelian irregularities of complex diseases, such as the existence of clinically indistinguishable sporadic and familial cases, sexual dimorphism, relatively late age of onset and peaks of susceptibility to some diseases, discordance of monozygotic twins and major fluctuations on the course of disease severity. It is also suggested that a substantial portion of phenotypic variance that traditionally has been attributed to environmental effects may result from stochastic epigenetic events in the cell. It is argued that epigenetic strategies, when applied in parallel with the traditional genetic ones, may significantly advance the discovery of etiopathogenic mechanisms of complex diseases. The second part of this chapter is dedicated to a review of laboratory methods for DNA methylation analysis, which may be useful in the study of complex diseases. In this context, epigenetic microarray technologies are emphasized, as it is evident that such technologies will significantly advance epigenetic analyses in complex diseases.

Interaction of mouse polycomb-group (Pc-G) proteins Enx1 and Enx2 with Eed: indication for separate Pc-G complexes.

The Polycomb group (Pc-G) constitutes an important, functionally conserved group of proteins, required to stably maintain inactive homeobox genes repressed during development. Drosophila extra sex combs (esc) and its mammalian homolog embryonic ectoderm development (eed) are special Pc-G members, in that they are required early during development when Pc-G repression is initiated, a process that is still poorly understood. To get insight in the molecular function of Eed, we searched for Eed-interacting proteins, using the yeast two-hybrid method. Here we describe the specific in vivo binding of Eed to Enx1 and Enx2, two mammalian homologs of the essential Drosophila Pc-G gene Enhancer-of-zeste [E(z)]. No direct biochemical interactions were found between Eed/Enx and a previously characterized mouse Pc-G protein complex, containing several mouse Pc-G proteins including mouse polyhomeotic (Mph1). This suggests that different Pc-G complexes with distinct functions may exist. However, partial colocalization of Enx1 and Mph1 to subnuclear domains may point to more transient interactions between these complexes, in support of a bridging role for Enx1.

Effects of light intensity on membrane differentiation in Rhodopseudomonas capsulata.

Cells of Rhodopseudomonas capsulata, strain 37b4, leu-, precultivated anaerobically under low light intensity, were exposed to high light intensity (2000 W.m-2). The cells grew with a mass doubling time of 3 h. The synthesis of bacteriochlorophyll (BChl) began after two doublings of cell mass. Reaction center and light-harvesting BChl I (B-875) were the main constituents of the photosynthetic apparatus incorporated into the membrane. The size of the photosynthetic unit (total BChl/reaction center) decreased and light-harvesting BChl I became the dominating BChl species. Concomitant with the appearance of the different spectral forms of BChl the respective proteins were incorporated into the membrane, i.e. the three reaction center polypeptides, the polypeptide associated with light-harvesting BChl I, the two polypeptides associated with BChl II. A polypeptide of an apparent molecular weight of 45 000 was also incorporated. A lowering of the light intensity to 7 W.m-2 resulted in a lag phase of growth for 6 h. Afterwards, the time for doubling of cell mass was 11 h. The concentration of all three BChl complexes (reaction center, light-harvesting BChl I and II complexes)/cell and per membrane protein increased immediately. Also the size of the photosynthetic unit and the amount of intracytoplasmic membranes/cell increased. The activities of photophosphorylation, succinate dehydrogenase, NADH dehydrogenase and NADH oxidation (respiratory chain)/membrane protein are higher in membrane preparations isolated from cells grown at high light intensities than in such preparations from cells grown at low light intensities.

Murine cyclophilin-S1: a variant peptidyl-prolyl isomerase with a putative signal sequence expressed in differentiating F9 cells.

Fractionation of differentiating murine teratocarcinoma F9 cells and extraction of the nuclear/microsomal pellets with ethidium bromide led to the purification and microsequencing of the protein mCyP-S1, a novel cyclosporin A-sensitive peptidyl-prolyl cis-trans isomerase (PPIase). mCyP-S1 is a new member of the cyclophilin class of proteins. Cloning and sequencing of the mCyP-S1 cDNA revealed extended coding capacity for a putative N-terminal signal sequence, suggesting processing of mCyP-S1 during intracellular translocation across the membrane of the endoplasmic reticulum. mCyP-S1 is abundantly expressed in a variety of mouse organ tissues and its mRNA levels increase during F9 cell differentiation. Specific subcellular localization of PPIases is postulated to contribute to functional specificities of this class of enzymes.

Quality of life in adult patients with acute myeloid leukemia receiving intensive and prolonged chemotherapy -- a longitudinal study.

Intensification of treatment for acute myeloid leukemia (AML) in adult patients resulted in a substantial improvement in long-term prognosis. Therefore, the assessment of quality of life (QL) of patients undergoing treatment is of growing interest. This study was designed to evaluate QL in patients with AML treated according to the protocol of the German AML-Cooperative Group (Münster, Germany). The EORTC QLQ-C 30 questionnaire was used to analyze QL throughout therapy, evaluating defined specific parameters at 12 different time-points. Sixty-one patients were recruited within the first 30 months of the study. Those 28 patients who have completed the course of inpatient treatment (n=28) are evaluated for changes in the conceptually distinct QL domains: Physical Functioning (P<0.001), Role Functioning (P=0.001), Emotional Functioning (P < 0.001) and Social Functioning (P=0.007) improve significantly from beginning of chemotherapy to the end of inpatient treatment. Individual assessment of Global Health Status and Subjective QL improves significantly over the same time (P< 0.001). At the end of inpatient treatment patients suffer significantly less from fatigue, nausea/emesis, loss of appetite and sleep disturbance (P < 0.001). Although most patients with AML eventually relapse, the evaluation of QL in patients undergoing treatment shows that subjective benefit outweighs the adverse effects of antileukemic therapy.

NVP-QBE170: an inhaled blocker of the epithelial sodium channel with a reduced potential to induce hyperkalaemia.

BACKGROUND AND PURPOSE: Inhaled amiloride, a blocker of the epithelial sodium channel (ENaC), enhances mucociliary clearance (MCC) in cystic fibrosis (CF) patients. However, the dose of amiloride is limited by the mechanism-based side effect of hyperkalaemia resulting from renal ENaC blockade. Inhaled ENaC blockers with a reduced potential to induce hyperkalaemia provide a therapeutic strategy to improve mucosal hydration and MCC in the lungs of CF patients. The present study describes the preclinical profile of a novel ENaC blocker, NVP-QBE170, designed for inhaled delivery, with a reduced potential to induce hyperkalaemia. EXPERIMENTAL APPROACH: The in vitro potency and duration of action of NVP-QBE170 were compared with amiloride and a newer ENaC blocker, P552-02, in primary human bronchial epithelial cells (HBECs) by short-circuit current. In vivo efficacy and safety were assessed in guinea pig (tracheal potential difference/hyperkalaemia), rat (hyperkalaemia) and sheep (MCC). KEY RESULTS: In vitro, NVP-QBE170 potently inhibited ENaC function in HBEC and showed a longer duration of action to comparator molecules. In vivo, intratracheal (i.t.) instillation of NVP-QBE170 attenuated ENaC activity in the guinea pig airways with greater potency and duration of action than that of amiloride without inducing hyperkalaemia in either guinea pig or rat. Dry powder inhalation of NVP-QBE170 by conscious sheep increased MCC and was better than inhaled hypertonic saline in terms of efficacy and duration of action. CONCLUSIONS AND IMPLICATIONS: NVP-QBE170 highlights the potential for inhaled ENaC blockers to exhibit efficacy in the airways with a reduced risk of hyperkalaemia, relative to existing compounds.

Ex vivo breast cancer cell purging by adenovirus-mediated cytosine deaminase gene transfer and short-term incubation with 5-fluorocytosine completely prevents tumor growth after transplantation.

Peripheral blood progenitor harvests of breast cancer patients are contaminated with tumor cells, suggesting a potential role for these cells in the relapse after high-dose chemotherapy. Whereas physical purging methods do not eliminate contaminating tumor cells completely, pharmacological purging, although highly efficient, is hampered by a strong nonspecific toxicity toward hematopoietic progenitor cells. Taking advantage of the high efficiency of adenovirus-mediated gene transfer to epithelial cells, we selectively loaded breast cancer cells in vitro with a cytotoxic drug by gene transfer of the prodrug-converting enzyme cytosine deaminase (AdCMV.CD) and 5-fluorocytosine (5-FC). Despite the low dose of vector administered, limited exposure to 5-FC, and transplantation only of viable tumor cells into SCID mice, all animals that received cells treated in vitro with AdCMV.CD plus 5-FC were completely free of tumor development. These data show that the selective loading of tumor cells with AdCMV.CD/5-FC might be useful for purging of autografts.

The N-terminal signal peptide of the murine cyclophilin mCyP-S1 is required in vivo for ER localization.

Cyclophilins are a class of enzymes that are thought to be involved in protein folding by accelerating the isomerization of Xaa-Pro peptide bonds and that mediate the immunosuppressive effect of cyclosporin A. We described previously a murine cyclophilin, mCyP-S1, whose cDNA encoded a putative NH2-terminal signal sequence which was not present in the mature protein. Here we investigate the intracellular localization of mCyP-S1. We show by overexpression of the wild-type and an NH2-terminally truncated derivative that its signal sequence is necessary and functional in vivo for the translocation into the endoplasmic reticulum (ER). Immunocytochemistry and cell fractionations demonstrate the preferential localization of endogenous mCyP-S1 in the ER, or subcompartments thereof. In addition, the results indicate the presence of this cyclophilin in the nucleus.

The differentiation of the photosynthetic apparatus and the intracytoplasmic membrane in cells of Rhodopseudomonas capsulata upon variation of light intensity.

In cells of Rhodopseudomonas capsulata, grown at growth limiting light intensity (7 W x m-2), the cellular bacteriochlorophyll (Bchl) content increased 13-fold, the Bchl concentration of membranes 3.4 fold, the concentration of reaction centers in membranes 1.6-fold, the size of the photosynthetic unit twofold, the concentration of carotenoids in membranes 2.4 fold and the number of intracytoplasmic membrane vesicles, bearing the photosynthetic apparatus, 6..3 fold in comparison with cells grown at 2000 W x m-2 light intensity. Thus, the variation of incident caused mainly a variation of the amount of intracytoplasmic membrane vesicles per cell, but also a variation of the size and concentration of photosynthetic units in the membrane system.