Chemically defined culture media: rational recipes or witches' brew?

A rational approach to study cells, tissues or even organs is to isolate them from the body and bring them into a controlled, and therefore reproducible, environment. In vivo, cells are surrounded by the extracellular matrix, and the body fluids nourish them. In vitro, these fluids are replaced by culture media. In the early days of tissue culture, tissue was cultured in a drop of clotted lymph. The early-day natural nutrient media have gradually become replaced by media of a more defined composition, culminating in the advent of completely defined culture media.Biomedical Reviews 1996; 6: 111-119

Low gonorrhoea antimicrobial resistance and culture positivity rates in general practice: a pilot study

Objective In the Netherlands, the Gonococcal Resistance to Antimicrobials Surveillance (GRAS) programme is carried out at Centres for Sexual Health (CSH), which provide care for sexual high-risk populations. However, half of gonorrhoea infections are diagnosed in general practice (GP). We performed a pilot study to explore expanding GRAS to GPs using laboratory-based surveillance. Additionally, antimicrobial resistance patterns of GP and CSH patients were compared. Methods Three laboratories from different regions were included, which all perform gonorrhoea diagnostics for GPs and used ESwab for patient sampling. Additional culturing for all GP patients with gonorrhoea took place from February to July 2018. After positive PCR-nucleic acid amplification test, residual ESwab material was used for culture. In positive cultures, susceptibility testing was performed for azithromycin, ciprofloxacin, cefotaxime and ceftriaxone using Etest. Results During the study period, 484 samples were put in culture. 16.5% of cultures were positive (n=80). Antimicrobial resistance levels were low, with 2.6% resistance to azithromycin, 21.5% to ciprofloxacin and 0.0% to cefotaxime and ceftriaxone. Resistance levels in CSH GRAS data (first half of 2018) were 19.2% for azithromycin, 31.5% for ciprofloxacin, 1.9% for cefotaxime and 0.0% for ceftriaxone. Conclusions Culture positivity rates for GP patients were low, probably due to long transportation times and awaiting PCR test results before attempting culture. Positivity rates might be improved by making changes in sampling and/or transportation methods, but that would require involvement of GPs and patients instead of keeping the surveillance lab based. Resistance levels appeared to be lower at GPs than at the CSH, indicating that resistance might emerge first in more high-risk populations. It is important to consider all potentially relevant patient populations when establishing a gonococcal antimicrobial resistance surveillance programme. However, based on the findings from this study the current GRAS programme will not be extended to GPs

A small protein probe for correlated microscopy of endogenous proteins

Probes are essential to visualize proteins in their cellular environment, both using light microscopy as well as electron microscopy (EM). Correlated light microscopy and electron microscopy (CLEM) requires probes that can be imaged simultaneously by both optical and electron-dense signals. Existing combinatorial probes often have impaired efficiency, need ectopic expression as a fusion protein, or do not target endogenous proteins. Here, we present FLIPPER-bodies to label endogenous proteins for CLEM. Fluorescent Indicator and Peroxidase for Precipitation with EM Resolution (FLIPPER), the combination of a fluorescent protein and a peroxidase, is fused to a nanobody against a target of interest. The modular nature of these probes allows an easy exchange of components to change its target or color. A general FLIPPER-body targeting GFP highlights histone2B-GFP both in fluorescence and in EM. Similarly, endogenous EGF receptors and HER2 are visualized at nm-scale resolution in ultrastructural context. The small and flexible FLIPPER-body outperforms IgG-based immuno-labeling, likely by better reaching the epitopes. Given the modular domains and possibilities of nanobody generation for other targets, FLIPPER-bodies have high potential to become a universal tool to identify proteins in immuno-CLEM with increased sensitivity compared to current approaches

A New Type of Metal Recognition by Human T Cells: Contact Residues for Peptide-independent Bridging of T Cell Receptor and Major Histocompatibility Complex by Nickel

In spite of high frequencies of metal allergies, the structural basis for major histocompatibility complex (MHC)-restricted metal recognition is among the unanswered questions in the field of T cell activation. For the human T cell clone SE9, we have identified potential Ni contact sites in the T cell receptor (TCR) and the restricting human histocompatibility leukocyte antigen (HLA)-DR structure. The specificity of this HLA-DR–promiscuous VA22/VB17+ TCR is primarily harbored in its α chain. Ni reactivity is neither dependent on protein processing in antigen-presenting cells nor affected by the nature of HLA-DR–associated peptides. However, SE9 activation by Ni crucially depends on Tyr29 in CDR1α, an N-nucleotide–encoded Tyr94 in CDR3α, and a conserved His81 in the HLA-DR β chain. These data indicate that labile, nonactivating complexes between the SE9 TCR and most HLA-DR/peptide conjugates might supply sterically optimized coordination sites for Ni ions, three of which were identified in this study. In such complexes Ni may effectively bridge the TCR α chain to His81 of most DR molecules. Thus, in analogy to superantigens, Ni may directly link TCR and MHC in a peptide-independent manner. However, unlike superantigens, Ni requires idiotypic, i.e., CDR3α-determined TCR amino acids. This new type of TCR–MHC linkage might explain the high frequency of Ni-reactive T cells in the human population

Usage of TCRAV and TCRBV gene families in human fetal and adult TCR rearrangements

We have investigated fetal and adult T-cell receptor (TCR) A and B V-gene repertoires both by fluorescence-activated cell sorter (FACS) analysis with the avialable TCR V region-specific mAbs and by the polymerase chain reaction (PRC) with TRC V gene family-specific oligonucleotides. Among the low number of CD3+ T cells, most of the TRC V region tested for could be detected by FACS analysis in liver, bone marrow, and spleen derived from a 14-week-old fetus and two 15-weeks-old fetuses. Similarly, the PCR analysis showed that the majority of the TCRAV and TCRBV families were expressed in the peripheral organs of the 13-week-old fetus, although an apparent absence of particular TCR V families was found in liver and bone marrow. This was most probably the consequence of the low number of CD3+ T cells in these organs. In 17-week-old week-old fetal thymi the level of expression of some TCRAV and TCRBV gene families, in particular those that contain single member, was lower compared to post-partum thymi and adult peripheral blood mononuclear cells. The combined data of FACS and PCR analysis demonstrate that TCR genes belonging to the majority of TCR V gene families can be used in TCR α and β chain rearrngements during early human fetal life. Our data also suggest that the expression levels of some of the single member TCR V gene families may be influenced by the development stage

Stargardt disease:monitoring incidence and diagnostic trends in the Netherlands using a nationwide disease registry

PURPOSE: To assess the incidence of Stargardt disease (STGD1) and to evaluate demographics of incident cases. METHODS: For this retrospective cohort study, demographic, clinical and genetic data of patients with a clinical diagnosis of STGD1 were registered between September 2010 and January 2020 in a nationwide disease registry. Annual incidence (2014-2018) and point prevalence (2018) were assessed on the basis of this registry. RESULTS: A total of 800 patients were registered, 56% were female and 83% were of European ancestry. The incidence was 1.67-1.95:1,000,000 per year and the point prevalence in 2018 was approximately 1:22,000-1:19,000 (with and without 10% of potentially unregistered cases). Age at onset was associated with sex (p = 0.027, Fisher's exact); 1.9x more women than men were observed (140 versus 74) amongst patients with an age at onset between 10 and 19 years, while the sex ratio in other age-at-onset categories approximated one. Late-onset STGD1 (≥45 years) constituted 33% of the diagnoses in 2014-2018 compared to 19% in 2004-2008. Diagnostic delay (≥2 years between the first documentation of macular abnormalities and diagnosis) was associated with older age of onset (p = 0.001, Mann-Whitney). Misdiagnosis for age-related macular degeneration (22%) and incidental STGD1 findings (14%) was common in patients with late-onset STGD1. CONCLUSION: The observed prevalence of STGD1 in real-world data was lower than expected on the basis of population ABCA4 allele frequencies. Late-onset STGD1 was more frequently diagnosed in recent years, likely due to higher awareness of its phenotype. In this pretherapeutic era, mis- and underdiagnosis of especially late-onset STGD1 and the role of sex in STGD1 should receive special attention

Exercise training in pediatric patients with end-stage renal disease

The objective of this study was to determine the feasibility and efficacy of an exercise training program to improve exercise capacity and fatigue level in pediatric patients with end-stage renal disease (ESRD). Twenty children on dialysis intended to perform a 12-week graded community-based exercise program. Exercise capacity and fatigue level were studied; muscle force and health-related quality of life were secondary outcomes. All outcomes were measured at baseline (T = 0) and after intervention (T = 1). Fourteen of the 20 patients (70%) either did not start the program or did not complete the program. Of these patients, seven did not complete or even start the exercise program because of a combination of lack of time and motivational problems. Six patients were not able to continue the program or were unable to do the follow-up measurements because of medical problems. Exercise capacity and muscle strength was higher after the exercise program in the children who completed the training. In conclusion, exercise training is difficult to perform in children with ESRD and is not always feasible in real-life situations for many children with ESRD

Relation between Chlamydia trachomatis infection and pelvic inflammatory disease, ectopic pregnancy and tubal factor infertility in a Dutch cohort of women previously tested for chlamydia in a chlamydia screening trial

Objectives A better understanding of Chlamydia trachomatis infection (chlamydia)–related sequelae can provide a framework for effective chlamydia control strategies. The objective of this study was to estimate risks and risk factors of pelvic inflammatory disease (PID), ectopic pregnancy and tubal factor infertility (TFI) with a follow-up time of up until 8 years in women previously tested for chlamydia in the Chlamydia Screening Implementation study (CSI) and participating in the Netherlands Chlamydia Cohort Study (NECCST). Methods Women who participated in the CSI 2008–2011 (n=13 498) were invited in 2015–2016 for NECCST. Chlamydia positive was defined as a positive CSI-PCR test, positive chlamydia serology and/or selfreported infection (time dependent). Data on PID, ectopic pregnancy and TFI were collected by self-complete