Search

Non-human primate (NHP)-based model systems are highly relevant for biomedical research. However, only few NHP cell lines are available and the generation of additional cell lines is an urgent need to help in the refinement and replacement of these models. Using lentiviral transduction of c-Fos, we established cell lines from the brain of rhesus macaques (Macaca mulatta). Transcriptome analysis revealed that these cell lines are closely related to astrocytes, which was confirmed by immunoblot and immunofluorescence microscopy detecting expression of the astrocyte marker glial fibrillary acidic protein (GFAP). Quantitative real-time PCR (qRT-PCR) demonstrated that major pathways of the interferon (IFN) system are intact. Using retroviral pseudotypes we found that the cell lines are susceptible to entry driven by the glycoproteins of vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV) and to a lesser extent influenza A virus (IAV). Finally, these cells supported growth of Zika virus (ZIKV) and Papiine alphaherpesvirus 2 (PaHV2). In summary, we developed IFN-responsive cell lines from the rhesus macaque brain that allowed entry driven by several viral glycoproteins and were permissive to infection with ZIKV and a primate simplexvirus. These cell lines will be useful for efforts to analyze neurotropic viral infections in rhesus macaque models., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Reiter et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Non-human primate (NHP)-based model systems faithfully reproduce various viral diseases including Ebola, influenza, AIDS and Zika. However, only a small number of NHP cell lines are available and generation of additional cell lines could help to refine these models. We immortalized rhesus macaque kidney cells by lentiviral transduction with a vector encoding telomerase reverse transcriptase (TERT) and report the generation of three TERT-immortalized cell lines derived from rhesus macaque kidney. Expression of the kidney podocyte marker podoplanin on these cells was demonstrated by flow cytometry. Quantitative real-time PCR (qRT-PCR) was employed to demonstrate induction of MX1 expression upon stimulation with interferon (IFN) or viral infection, suggesting a functional IFN system. Further, the cell lines were susceptible to entry driven by the glycoproteins of vesicular stomatitis virus, influenza A virus, Ebola virus, Nipah virus and Lassa virus as assessed by infection with retroviral pseudotypes. Finally, these cells supported growth of Zika virus and the primate simplexviruses Cercopithecine alphaherpesvirus 2 and Papiine alphaherpesvirus 2. In summary, we developed IFN-responsive rhesus macaque kidney cell lines that allowed entry driven by diverse viral glycoproteins and were permissive to infection with Zika virus and primate simplexviruses. These cell lines will be useful for efforts to analyze viral infections of the kidney in macaque models., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Reiter et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Patients with bariatric surgery often show poor long-term compliance to recommendations for prevention of nutrient deficiency but it is unclear which factors contribute. We investigated the associations of age, sex, and socioeconomic status (SES) with adherence to guideline recommendations on protein intake and micronutrient supplementation., Methods: In a monocentric cross-sectional study we prospectively recruited patients with sleeve gastrectomy (SG) or Roux-en-Y gastric bypass (RYGB) and a minimum postoperative period of 6 months. Clinical and demographic data were obtained from the patients' medical files and by questionnaire. Patients reported on supplement usage, recorded their dietary intake for seven days and underwent physical examinations including blood testing., Results: We included 35 patients (SG: n = 25, RYGB: n = 10) with a mean (+SD) postoperative period of 20.2 (±10.4) months. Distributions of age, sex and SES were comparable between the SG and RYGB groups. Non-adherence to recommended protein intake was associated with age ≥ 50 years (p = 0.041) but not sex or SES. Protein intake inversely correlated with markers of obesity. There were no significant associations of age or sex with micronutrient supplementation. Only for vitamins A (p = 0.049) and B1 (p = 0.047) higher SES was associated with greater compliance. The only manifest deficiency associated with non-adherence to micronutrient supplementation was that for folic acid (p = 0.044)., Conclusion: In patients after bariatric surgery, those of older age and of lower SES might have a greater risk of unfavorable outcome and may require greater attention to micronutrient and protein supplementation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Wiese et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The experimental infection of rhesus macaques (rh) with simian immunodeficiency virus (SIV) is an important model for human immunodeficiency virus (HIV) infection of humans. The interferon-induced transmembrane protein 3 (IFITM3) inhibits HIV and SIV infection at the stage of host cell entry. However, it is still unclear to what extent the antiviral activity of IFITM3 observed in cell culture translates into inhibition of HIV/SIV spread in the infected host. We have shown previously that although rhIFITM3 inhibits SIV entry into cultured cells, polymorphisms in the rhIFITM3 gene are not strongly associated with viral load or disease progression in SIV infected macaques. Here, we examined whether rhIFITM3(2), which is closely related to rhIFITM3 at the sequence level, exerts antiviral activity and whether polymorphisms in the rhIFITM3(2) gene impact the course of SIV infection. We show that expression of rhIFITM3(2) is interferon-inducible and inhibits SIV entry into cells, although with reduced efficiency as compared to rhIFITM3. We further report the identification of 19 polymorphisms in the rhIFITM3(2) gene. However, analysis of a well characterized cohort of SIV infected macaques revealed that none of the polymorphisms had a significant impact upon the course of SIV infection. These results and our previous work suggest that polymorphisms in the rhIFITM3 and rhIFITM3(2) genes do not strongly modulate the course of SIV infection in macaques., Competing Interests: The authors have declared that no competing interests exist.

The global obesity epidemic constitutes a major cause of morbidity and mortality challenging public health care systems worldwide. Thus, a better understanding of its pathophysiology and the development of novel therapeutic options are urgently needed. Recently, alterations of the intestinal microbiome in the obese have been discussed as a promoting factor in the pathophysiology of obesity and as a contributing factor to related diseases such as type 2 diabetes and metabolic syndrome. The present pilot study investigated the effect of a structured weight loss program on fecal microbiota in obese type 2 diabetics. Twelve study subjects received a low-calorie formula diet for six weeks, followed by a nine week food reintroduction and stabilization period. Fecal microbiota were determined by 16S rRNA gene sequencing of stool samples at baseline, after six weeks and at the end of the study after fifteen weeks. All study subjects lost weight continuously throughout the program. Changes in fecal microbiota were most pronounced after six weeks of low-calorie formula diet, but reverted partially until the end of the study. However, the gut microbiota phylogenetic diversity increased persistently. The abundance of Collinsella, which has previously been associated with atherosclerosis, decreased significantly during the weight loss program. This study underlines the impact of dietary changes on the intestinal microbiome and further demonstrates the beneficial effects of weight loss on gut microbiota. Trial registration: ClinicalTrials.gov NCT02970838., Competing Interests: Nestlé Health Science Germany granted the study participants a 15% discount to the Optifast formula diet. LS received a Gerhard Domagk scholarship from University Medicine Greifswald made possible through an unrestricted educational grant from Baxter Deutschland GmbH (Unterschleissheim, Germany), Profusio GmbH (Greven, Germany) and Nutricia GmbH (Erlangen, Germany). This does not alter our adherence to all PLOS ONE policies on sharing data and materials.

Influenza A virus (IAV) infection poses a serious health threat and novel antiviral strategies are needed. Defective interfering particles (DIPs) can be generated in IAV infected cells due to errors of the viral polymerase and may suppress spread of wild type (wt) virus. The antiviral activity of DIPs is exerted by a DI genomic RNA segment that usually contains a large deletion and suppresses amplification of wt segments, potentially by competing for cellular and viral resources. DI-244 is a naturally occurring prototypic segment 1-derived DI RNA in which most of the PB2 open reading frame has been deleted and which is currently developed for antiviral therapy. At present, coinfection with wt virus is required for production of DI-244 particles which raises concerns regarding biosafety and may complicate interpretation of research results. Here, we show that cocultures of 293T and MDCK cell lines stably expressing codon optimized PB2 allow production of DI-244 particles solely from plasmids and in the absence of helper virus. Moreover, we demonstrate that infectivity of these particles can be quantified using MDCK-PB2 cells. Finally, we report that the DI-244 particles produced in this novel system exert potent antiviral activity against H1N1 and H3N2 IAV but not against the unrelated vesicular stomatitis virus. This is the first report of DIP production in the absence of infectious IAV and may spur efforts to develop DIPs for antiviral therapy., Competing Interests: The authors have declared that no competing interests exist.

Interferon-induced transmembrane proteins (IFITMs) have been recognized as important antiviral effectors of the innate immune system, both in cell culture and in infected humans. In particular, polymorphisms of the human IFITM3 gene have been shown to affect disease severity and progression in influenza A virus (FLUAV) and human immunodeficiency virus (HIV) infection, respectively. Rhesus macaques (Macaca mulatta) are commonly used to model human infections and the experimental inoculation of these animals with simian immunodeficiency virus (SIV) is one of the best models for HIV/AIDS in humans. However, information on the role of IFITM3 in SIV infection of rhesus macaques is currently lacking. We show that rhesus macaque (rh) IFITM3 inhibits SIV and FLUAV entry in cell culture, although with moderately reduced efficiency as compared to its human counterpart. We further report the identification of 16 polymorphisms in the rhIFITM3 gene, three of which were exonic and synonymous while the remainder was located in non-coding regions. Employing previously characterized samples from two cohorts of SIV-infected rhesus macaques, we investigated the relationship between these rhIFITM3 polymorphisms and both AIDS-free survival time and virus load. In cohort 1, several intronic polymorphisms were significantly associated with virus load or survival. However, an association with both parameters was not observed and significance was lost in most cases when animals were stratified for the presence of MHC allele Mamu-A1*001. Moreover, no significant genotype-phenotype associations were detected in cohort 2. These results suggest that, although IFITM3 can inhibit SIV infection in cell culture, genetic variation in rhIFITM3 might have only a minor impact on the course of SIV infection in experimentally infected animals.

Background: Among health care personnel working regular hours or rotating shifts can affect parameters of general health and nutrition. We have investigated physical activity, sleep quality, metabolic activity and stress levels in health care workers from both groups., Methods: We prospectively recruited 46 volunteer participants from the workforce of a University Medical Department of which 23 worked in rotating shifts (all nursing) and 21 non-shift regular hours (10 nursing, 13 clerical staff). All were investigated over 7 days by multisensory accelerometer (SenseWear Bodymedia® armband) and kept a detailed food diary. Physical activity and resting energy expenditure (REE) were measured in metabolic equivalents of task (METs). Quality of sleep was assessed as Pittsburgh Sleeping Quality Index and stress load using the Trier Inventory for Chronic Stress questionnaire (TICS)., Results: No significant differences were found for overall physical activity, steps per minute, time of exceeding the 3 METs level or sleep quality. A significant difference for physical activity during working hours was found between shift-workers vs. non-shift-workers (p<0.01) and for shift-working nurses (median = 2.1 METs SE = 0.1) vs. non-shift-working clerical personnel (median = 1.5 METs SE = 0.07, p<0.05). Non-shift-working nurses had a significantly lower REE than the other groups (p<0.05). The proportion of fat in the diet was significantly higher (p<0.05) in the office worker group (median = 42% SE = 1.2) whereas shift-working nurses consumed significantly more carbohydrates (median = 46% SE = 1.4) than clerical staff (median = 41% SE = 1.7). Stress assessment by TICS confirmed a significantly higher level of social overload in the shift working group (p<0.05)., Conclusion: In this prospective cohort study shift-working had no influence on overall physical activity. Lower physical activity during working hours appears to be compensated for during off-hours. Differences in nutritional habits and stress load warrant larger scale trials to determine the effect on implicit health-associated conditions., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Patients with biliopancreatic tumors frequently suffer from weight loss and cachexia. The in-hospital work-up to differentiate between benign and malignant biliopancreatic lesions requires repeated pre-interventional fasting periods that can aggravate this problem. We conducted a randomized intervention study to test whether routine in-hospital peripheral intravenous nutrition on fasting days (1000 ml/24 h, 700 kcal) has a beneficial effect on body weight and body composition., Material and Methods: 168 patients were screened and 100 enrolled in the trial, all undergoing in-hospital work-up for biliopancreatic mass lesions and randomized to either intravenous nutrition or control. Primary endpoint was weight loss at time of hospital discharge; secondary endpoints were parameters determined by bioelectric impedance analysis and quality of life recorded by the EORTC questionnaire., Results: Within three months prior to hospital admission patients had a median self-reported loss of 4.0 kg (25*th: -10.0 kg and 75*th* percentile: 0.0kg) of body weight. On a multivariate analysis nutritional intervention increased body weight by 1.7 kg (95% CI: 0.204; 3.210, p = 0.027), particularly in patients with malignant lesions (2.7 kg (95% CI: 0.71; 4.76, p < 0.01)., Conclusions: In a hospital setting, patients with suspected biliopancreatic mass lesions stabilized their body weight when receiving parenteral nutrition in fasting periods even when no total parenteral nutrition was required. Analysis showed that this effect was greatest in patients with malignant tumors. Further studies will be necessary to see whether patient outcome is affected as well., Trial Registration: ClinicalTrials.gov NCT02670265., Competing Interests: The authors have declared that no competing interests exist.

Objectives: To investigate changes in the fat content of abdominal compartments and muscle area during weight loss using confounder-adjusted chemical-shift-encoded magnetic resonance imaging (MRI) in overweight diabetics., Methods: Twenty-nine obese diabetics (10/19 men/women, median age: 59.0 years, median body mass index (BMI): 34.0 kg/m2) prospectively joined a standardized 15-week weight-loss program (six weeks of formula diet exclusively, followed by reintroduction of regular food with gradually increasing energy content over nine weeks) over 15 weeks. All subjects underwent a standardized MRI protocol including a confounder-adjusted chemical-shift-encoded MR sequence with water/fat separation before the program as well at the end of the six weeks of formula diet and at the end of the program at 15 weeks. Fat fractions of abdominal organs and vertebral bone marrow as well as volumes of visceral and subcutaneous fat were determined. Furthermore, muscle area was evaluated using the L4/L5 method. Data were compared using the Wilcoxon signed-rank test for paired samples., Results: Median BMI decreased significantly from 34.0 kg/m2 to 29.9 kg/m2 (p < 0.001) at 15 weeks. Liver fat content was normalized (14.2% to 4.1%, p < 0.001) and vertebral bone marrow fat (57.5% to 53.6%, p = 0.018) decreased significantly throughout the program, while fat content of pancreas (9.0%), spleen (0.0%), and psoas muscle (0.0%) did not (p > 0.15). Visceral fat volume (3.2 L to 1.6 L, p < 0.001) and subcutaneous fat diameter (3.0 cm to 2.2 cm, p < 0.001) also decreased significantly. Muscle area declined by 6.8% from 243.9 cm2 to 226.8 cm2., Conclusion: MRI allows noninvasive monitoring of changes in abdominal compartments during weight loss. In overweight diabetics, weight loss leads to fat reduction in abdominal compartments, such as visceral fat, as well as liver fat and vertebral bone marrow fat while pancreas fat remains unchanged.

Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI) zanamivir and the host cell interferon-inducible transmembrane (IFITM) proteins 1-3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels.

Protein Tyrosin Kinase 7 (PTK7) is upregulated in several human cancers; however, its clinical implication in breast cancer (BC) and lymph node (LN) is still unclear. In order to investigate the function of PTK7 in mediating BC cell motility and invasivity, PTK7 expression in BC cell lines was determined. PTK7 signaling in highly invasive breast cancer cells was inhibited by a dominant-negative PTK7 mutant, an antibody against the extracellular domain of PTK7, and siRNA knockdown of PTK7. This resulted in decreased motility and invasivity of BC cells. We further examined PTK7 expression in BC and LN tissue of 128 BC patients by RT-PCR and its correlation with BC related genes like HER2, HER3, PAI1, MMP1, K19, and CD44. Expression profiling in BC cell lines and primary tumors showed association of PTK7 with ER/PR/HER2-negative (TNBC-triple negative BC) cancer. Oncomine data analysis confirmed this observation and classified PTK7 in a cluster with genes associated with agressive behavior of primary BC. Furthermore PTK7 expression was significantly different with respect to tumor size (ANOVA, p = 0.033) in BC and nodal involvement (ANOVA, p = 0.007) in LN. PTK7 expression in metastatic LN was related to shorter DFS (Cox Regression, p = 0.041). Our observations confirmed the transforming potential of PTK7, as well as its involvement in motility and invasivity of BC cells. PTK7 is highly expressed in TNBC cell lines. It represents a novel prognostic marker for BC patients and has potential therapeutic significance.