Endocytosis and Transport of Growth Factor Receptors in Peripheral Axon Regeneration: Novel Lessons from Neurons Expressing Lysine-Deficient FGF Receptor Type 1 in vitro

In the course of peripheral nerve regeneration, axons encounter different extracellular growth factors secreted by non-neuronal cells at the injury site and retrogradely transported after binding to neuronal membrane receptor tyrosine kinases. The present study reviews the role of receptor transport in peripheral axon outgrowth and provides novel data on trafficking of fibroblast growth factor receptor type 1 (FGFR1). Differences in receptor transport are determined by different numbers of lysine residues acting as ubiquitination sites in the intracellular receptor domain. We previously demonstrated that overexpression of mutant FGFR1-25R (25 out of 29 intracellular lysines replaced with arginine) results in enhanced receptor recycling as compared to wild-type FGFR1 followed by strong stimulation of elongative axon growth in vitro. Here, the effects of lysine-deficient FGFR1 (FGFR1-29R lacking all 29 cytoplasmic lysine residues) or of only 15 lysine mutations (FGFR1-15R) on axon outgrowth and concomitant changes in signal pathway activation were investigated by immunocytochemistry and morphometry of cultured primary neurons. Overexpression of FGFR1-15R in adult sensory neurons resulted in enhanced receptor recycling, which was accompanied by increased axon elongation without stimulating axon branching. By contrast, FGFR1-29R was neither endocytosed nor axon outgrowth affected. Although overexpression of FGFR1-15R or FGFR1-25Ra strongly promoted elongation, we did not detect increased signal pathway activation (ERK, AKT, PLC, or STAT3) in neurons expressing mutant FGFR1 as compared with wild-type neurons raising the possibility that other signaling pathways or signaling independent mechanisms may be involved in the axon outgrowth effects of recycled FGF receptors. Anat Rec, 302:1268–1275, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.     

Successful Outcome in Patients with Fanconi Anemia Undergoing T Cell-Replete Mismatched Related Donor Hematopoietic Cell Transplantation Using Reduced-Dose Cyclophosphamide Post-Transplantation

Allogeneic hematopoietic cell transplantation (HCT) has been shown to restore normal hematopoiesis in patients with Fanconi anemia (FA), with excellent results in matched related donor HCT. Outcomes of alternative donor HCT are less favorable, however. In patients without FA, several reports have documented stable engraftment and/or a low risk of graft-versus-host disease (GVHD) using unmanipulated HLA-mismatched related donors and post-HCT cyclophosphamide (PT-CY) for GVHD prophylaxis. Data on the use of this approach in patients with FA are scarce, and thus we launched a study of HLA-mismatched related donor HCT in these patient. Here we report our findings in 19 patients. The conditioning was fludarabine 30 mg/m²/day for 5 days, antithymocyte globulin 5 mg/kg/day for 4 days, and total body irradiation (total dose, 200 cGy). GVHD prophylaxis was cyclosporine and mycophenolate and reduced doses of PT-CY, 25 mg/kg, on days +3 and +5. All patients exhibited absolute neutrophil count recovery. Grade III-IV acute GVHD occurred in 3 patients, and chronic GVHD occurred in 1 patient. At a mean follow-up of 38.3 ± 5.8 months, the 5-year probability of overall survival for our patients was 89.2% ± 7.2%. The regimen was well tolerated; hemorrhagic cystitis occurred in 7 patients, and severe mucositis occurred in 5 patients. There were 2 deaths; the primary cause of death was severe GVHD in 1 patient and leukemia recurrence in the other. We conclude that in patients with FA lacking a matched related donor, the use of mismatched related HCT with low-dose PT-CY is a viable option; it is well tolerated, with a high rate of engraftment and an acceptable incidence of GVHD.     

CAPN5 genetic inactivation phenotype supports therapeutic inhibition trials

Small molecule pharmacological inhibition of dominant human genetic disease is a feasible treatment that does not rely on the development of individual, patient‐specific gene therapy vectors. However, the consequences of protein inhibition as a clinical therapeutic are not well‐studied. In advance of human therapeutic trials for CAPN5 vitreoretinopathy, genetic inactivation can be used to infer the effect of protein inhibition in vivo. We created a photoreceptor‐specific knockout (KO) mouse for Capn5 and compared the retinal phenotype to both wild‐type and an existing Capn5 KO mouse model. In humans, CAPN5 loss‐of‐function (LOF) gene variants were ascertained in large exome databases from 60,706 unrelated subjects without severe disease phenotypes. Ocular examination of the retina of Capn5 KO mice by histology and electroretinography showed no significant abnormalities. In humans, there were 22 LOF CAPN5 variants located throughout the gene and in all major protein domains. Structural modeling of coding variants showed these LOF variants were nearby known disease‐causing variants within the proteolytic core and in regions of high homology between human CAPN5 and 150 homologs, yet the LOF of CAPN5 was tolerated as opposed to gain‐of‐function disease‐causing variants. These results indicate that localized inhibition of CAPN5 is a viable strategy for hyperactivating disease alleles.     

Double-Negative T Cell Levels Correlate with Chronic Graft-versus-Host Disease Severity

Chronic graft-versus-host disease (cGVHD) is a major complication, affecting 50% to 80% of long-term survivors of allogeneic hematopoietic stem cell transplantation. Current cGVHD therapies are neither specific nor curative, and patients are typically maintained for several months to years under immunosuppressive regimens that are associated with important side effects and increased susceptibility to life-threatening infections. As a result, continued investigation into the pathology of the disease and the search for novel diagnostic and therapeutic strategies to treat cGVHD remains a high priority. We report that the cellular dynamics of various immune cell subsets are related to cGVHD onset and severity in a cohort of allogeneic hematopoietic stem cell transplantation recipients. We document a decrease in the proportion of CD45RO⁺ CD4^?CD8^? (double-negative [DN]) T cells at the onset of cGVHD, a time at which serum levels of B cell activating factor and B cells are increased. We also find that DN T cell levels are correlated with cGVHD severity. Our present findings are in line with the view that activated DN T cells exhibit their immunoregulatory potential by eliminating B cells in vivo. Taken together, these findings suggest that maintaining elevated DN T cell numbers before the onset of cGVHD may prevent pathological B cell responses.     

Assessing computational predictions of the phenotypic effect of cystathionine‐beta‐synthase variants

Accurate prediction of the impact of genomic variation on phenotype is a major goal of computational biology and an important contributor to personalized medicine. Computational predictions can lead to a better understanding of the mechanisms underlying genetic diseases, including cancer, but their adoption requires thorough and unbiased assessment. Cystathionine‐beta‐synthase (CBS) is an enzyme that catalyzes the first step of the transsulfuration pathway, from homocysteine to cystathionine, and in which variations are associated with human hyperhomocysteinemia and homocystinuria. We have created a computational challenge under the CAGI framework to evaluate how well different methods can predict the phenotypic effect(s) of CBS single amino acid substitutions using a blinded experimental data set. CAGI participants were asked to predict yeast growth based on the identity of the mutations. The performance of the methods was evaluated using several metrics. The CBS challenge highlighted the difficulty of predicting the phenotype of an ex vivo system in a model organism when classification models were trained on human disease data. We also discuss the variations in difficulty of prediction for known benign and deleterious variants, as well as identify methodological and experimental constraints with lessons to be learned for future challenges.     

Triazole resistance in Aspergillus fumigatus: recent insights and challenges for patient management

BackgroundTriazole resistance in Aspergillus fumigatus is widespread and threatens first-line triazole therapy in patients with Aspergillus diseases.ObjectivesTo give an overview of the microbiology, epidemiology and clinical significance of triazole resistance in aspergillosis.SourcesPubMed search for articles on resistance in Aspergillus species.ContentTriazoles are not mutagenic but select resistance when spontaneous mutations occur that are better able to proliferate in the triazole-containing environment. The major target for resistance mutations involves the Cyp51A gene, encoding an enzyme involved in cell wall synthesis. Triazole-resistance selection environments include patient treatment and organic matter containing triazole fungicide residues. Reported resistance frequencies vary widely between countries and hospitals, and resistance significantly complicates the diagnosis and treatment of Aspergillus diseases. Cultures may harbour various resistance phenotypes and multiple colonies must be analysed to detect resistance. PCR tests have become available for resistance detection in culture-negative patients, but show limited sensitivity. Individuals with triazole-resistant invasive aspergillosis have a 21% higher day-42 mortality compared with triazole-susceptible infection, and to prevent excess mortality resistant cases require first-line therapy that covers resistance. The recent ESCMID-ECMM-ERS Aspergillus guideline recommends resistance testing in A. fumigatus and local resistance surveillance. If resistance rates exceed 10% liposomal amphotericin B or triazole and echinocandin first-line therapy should be considered.ImplicationsTriazole resistance significantly complicates the management of aspergillosis and multidisciplinary research from a ‘One-health’ perspective is required to retain the triazole class for medical use.     

‘Test n Treat’ (TnT): a cluster randomized feasibility trial of on-site rapid Chlamydia trachomatis tests and treatment in ethnically diverse,sexually active teenagers attending technical colleges

ObjectivesWe conducted a cluster-randomized feasibility trial of 90-minute Chlamydia trachomatis tests and same day on-site treatment (‘Test n Treat/TnT’) in six technical colleges in London, England, to assess TnT uptake rates; follow-up rates; prevalence of C. trachomatis at baseline and 7 months; time to treatment; acceptability of TnT.MethodsParticipants completed questionnaires and provided genitourinary samples at baseline and 7 months. Participants were informed that baseline samples would not be tested for 7 months and were advised to get screened independently. Colleges were randomly allocated 1:1 to intervention (TnT) or control (no TnT).One month and 4 months post recruitment, participants at intervention colleges were texted invitations for on-site free C. trachomatis tests. A purposive sample of students who did/did not attend for screening were interviewed (n = 26).ResultsFive hundred and nine sexually active students were recruited: median age 17.9 years, 47% male, 50% black ethnicity, 55% reporting two or more sexual partners in the previous year. TnT uptake was 13% (33/259; 95% CI 8.9–17.4%) at 1 month and 10% (26/259; 6.7–14.4%) at 4 months with overall C. trachomatis positivity 5.1% (3/59; 1.1–14.2%). Follow-up at 7 months was 62% (317/509) for questionnaires and 52% (264/509) for samples. C. trachomatis prevalence was 6.2% (31/503) at baseline and 6.1% (16/264) at 7 months. Median time from test to treatment was 15 h. Interviews suggested low test uptake was associated with not feeling at risk, perceptions of stigma, and little knowledge of sexually transmitted infections (STIs).ConclusionsDespite high C. trachomatis rates at baseline and follow-up, uptake of testing was low. Like many countries, England urgently needs better sex education, including making STI testing routine/normal.Trial registration ISRCTN58038795     

Changes in the incidence and bacterial aetiology of paediatric parapneumonic pleural effusions/empyema in Germany, 2010–2017: a nationwide surveillance study

ObjectivesParapneumonic pleural effusions/empyema (PPE/PE) are severe complications of community-acquired pneumonia. We investigated the bacterial aetiology and incidence of paediatric PPE/PE in Germany after the introduction of universal pneumococcal conjugate vaccine (PCV) immunization for infants.MethodsChildren <18 years of age hospitalized with pneumonia-associated PPE/PE necessitating pleural drainage or persisting >7 days were reported to the German Surveillance Unit for Rare Diseases in Childhood between October 2010 and June 2017. All bacteria detected in blood or pleural fluid (by culture/PCR) were included, with serotyping for Streptococcus pneumoniae.ResultsThe median age of all 1447 PPE/PE patients was 5 years (interquartile range 3–10). In 488 of the 1447 children with PPE/PE (34%), 541 bacteria (>40 species) were detected. Aerobic gram-positive cocci accounted for 469 of 541 bacteria detected (87%); these were most frequently Streptococcus pneumoniae (41%), Streptococcus pyogenes (19%) and Staphylococcus aureus (6%). Serotype 3 accounted for 45% of 78 serotyped S. pneumoniae strains. Annual PPE/PE incidence varied between 14 (95%CI 12–16) and 18 (95%CI 16–21) PPE/PE per million children. Incidence of S. pneumoniae PPE/PE decreased from 3.5 (95%CI 2.5–4.6) per million children in 2010/11 to 1.5 (95%CI 0.9–2.4) in 2013/14 (p 0.002), followed by a re-increase to 2.2 (95%CI 1.5–3.2) by 2016/17 (p 0.205).ConclusionsIn the era of widespread PCV immunization, cases of paediatric PPE/PE were still caused mainly by S. pneumoniae and, increasingly, by S. pyogenes. The re-increase in the incidence of PPE/PE overall and in S. pneumoniae-associated PPE/PE indicates ongoing changes in the bacterial aetiology and requires further surveillance.