Replicative senescence of human dermal fibroblasts affects structural and functional aspects of the Golgi apparatus

It is well recognized that the world population is ageing rapidly. Therefore, it is important to understand ageing processes at the cellular and molecular levels to predict the onset of age‐related diseases and prevent them. Recent research has focused on the identification of ageing biomarkers, including those associated with the properties of the Golgi apparatus. In this context, Golgi‐mediated glycosylation of proteins has been well characterized. Additionally, other studies show that the secretion of many compounds, including pro‐inflammatory cytokines and extracellular matrix–degrading enzymes, is modified during ageing, resulting in physical and functional skin degradation. Since the Golgi apparatus is a central organelle of the secretory pathway, we investigated its structural organization in senescent primary human dermal fibroblasts using confocal and electron microscopy. In addition, we monitored the expression of Golgi‐related genes in the same cells. Our data showed a marked alteration in the Golgi morphology during replicative senescence. In contrast to its small and compact structure in non‐senescent cells, the Golgi apparatus exhibited a large and expanded morphology in senescent fibroblasts. Our data also demonstrated that the expression of many genes related to Golgi structural integrity and function was significantly modified in senescent cells, suggesting a relationship between Golgi apparatus function and ageing.     

Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C‐terminal domain

Melatonin receptors play important roles in the regulation of circadian and seasonal rhythms, sleep, retinal functions, the immune system, depression, and type 2 diabetes development. Melatonin receptors are approved drug targets for insomnia, non‐24‐hour sleep‐wake disorders, and major depressive disorders. In mammals, two melatonin receptors (MTRs) exist, MT₁ and MT₂, belonging to the G protein‐coupled receptor (GPCR) superfamily. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors proved to be difficult to obtain. Here, we describe the development of the first monoclonal antibodies (mABs) for mouse MT₁ and MT₂. Purified antibodies were extensively characterized for specific reactivity with mouse, rat, and human MT₁ and MT₂ by Western blot, immunoprecipitation, immunofluorescence, and proximity ligation assay. Several mABs were specific for either mouse MT₁ or MT₂. None of the mABs cross‐reacted with rat MTRs, and some were able to react with human MTRs. The specificity of the selected mABs was validated by immunofluorescence microscopy in three established locations (retina, suprachiasmatic nuclei, pituitary gland) for MTR expression in mice using MTR‐KO mice as control. MT₂ expression was not detected in mouse insulinoma MIN6 cells or pancreatic beta‐cells. Collectively, we report the first monoclonal antibodies recognizing recombinant and native mouse melatonin receptors that will be valuable tools for future studies.     

Tyrosinemia type 1 in pediatric nephrology: Not always straightforward

The main clinical features of tyrosinemia type 1 usually appear in the first months of life, including fever, diarrhea, vomiting, liver involvement, growth failure, and renal proximal tubulopathy with subsequent hypophosphatemic rickets. An early diagnosis is crucial in order to provide specific management and to prevent complications. Here, we report on two cases referred primarily to pediatric nephrologists for the diagnosis of “neonatal tubulopathy” and management of “X-linked hypophosphatemia (XLH),” respectively. Our aim is to emphasize that (1) even a mixed tubulopathy can reveal tyrosinemia, and (2) tyrosinemia is a classic differential diagnosis of XLH that should not be forgotten, especially in the era of the anti-FGF23 burosumab.     

Platelet receptor expression on three human megakaryoblast-like cell lines.

Platelets, the progeny of bone marrow megakaryocytes, are nonnucleated cells; many platelet proteins, including platelet membrane receptors, are believed to be derived from megakaryocytes. Several hematopoietic cell lines that exhibit megakaryocytic characteristics have been established as models for the study of megakaryocyte biology. We report here the screening of platelet receptor expression, in terms of functional coupling with the formation of two second messengers, calcium and cAMP, in three cell lines exhibiting megakaryoblastic properties: HEL, MEG-01, and DAMI. We show that all these cell lines respond to thrombin, ADP, epinephrine, and prostaglandin E1 (PGE1). However, transmembrane signaling pathways appear partly different from those present in mature platelets, because the action of thrombin was found to be positively coupled with the cAMP pathway, in addition to that of calcium, and because PGE1, which interacts with the cAMP pathway, also raises intracellular calcium levels in the three cell lines studied. Furthermore, an endothelin-1-induced increase in intracellular calcium level was observed in MEG-01 cells, strongly suggesting the expression of endothelin receptors on platelet precursors cells, whereas the presence of such receptors is controversial on platelets. These cell lines should prove useful in further studies of the expression and molecular pharmacology of platelet receptors on platelet precursor cells, as well as for the investigation of functional roles for platelet receptors on megakaryoblastic cells.     

Prostaglandin E2 level in tissue surrounding aseptic failed total hips

Summary Production of inflammatory mediators (IM) by cells and specifically macrophages around loosened implants may be responsible for their loosening. Our hypothesis was that different materials give rise to different amounts of these IM. It is thought that alumina/alumina for total hip replacement (THR), which has been used for 15 years in our orthopedic department, may produce less IM than other systems. We initiated a clinical prospective study to measure the level of prostaglandin E₂ (PGE₂) in tissue surrounding loosened prostheses to quantify PGE₂ production regarding the types of material involved in the friction couple, i.e., alumina/alumina versus metal/polyethylene, and the type of fixation, i.e., cemented versus cementless. A total of 29 THR revisions were performed in 28 patients. Four implant groups were identified: alumina/alumina cemented, alumina/alumina cementless, metal/polyethylene cemented, and metal/polyethylene cementless. For each revision, tissues surrounding the failed implants were harvested and processed, and the PGE₂ was measured in a blind manner using an immunoassay technique. As the measuring technique was difficult, at least three determinations for each sample were necessary. Some samples were excluded from the analysis for various reasons, for example, second or further revisions involving many different materials in the past, conjunction of metallic and alumina debris and samples taken from nonloosened components. Finally, 15 samples were considered adequate for inclusion in this study. Two groups were analyzed and compared: the alumina/alumina couple and the metal/polyethylene couple. Tissue surrounding the first group demonstrated a PGE₂ level of 69 ± 56 fmol/mg wet weight compared to 202 ± 156 fmol/mg for the second. The Wilcoxon test was applied, and there was a statistical difference (P < 0.05). When the alumina/alumina cemented group was compared to the alumina/alumina cementless group, there was a tendency to less PGE₂ with the cemented, but the difference was not statistically significant. Results thus demonstrate that alumina/alumina produces less IM than metal/polyethylene. However, these results do not indicate whether this is related to the type of debris, the amount, or both. This remains a preliminary study. Further studies with more samples, other mediators and growth factors, and a precise comparison to histological findings are still necessary.     

Botulinum toxin injection in patients with hereditary spastic paraparesis

In an open label study, we analyzed the efficacy of botulinum toxin injection at the lower limbs of patients with hereditary spastic paraparesis (HSP). Fifteen patients who showed disabling spasticity with no or poor effect of oral treatment were recruited consecutively. Botulinum toxin was injected (400 U; Botox^®) into the spastic muscles identified by clinical examination (equinus, varus, and pathological hip adduction). Patients were regularly assessed from the first day to the fifth month: spasticity (Ashworth), motor strength, range of movements, Functional Ambulation Categories (FAC), gait parameter, Rivermead Motor Assessment, self-analysis of benefit and satisfaction. We observed a moderate and significant ( P  < 0.05) reduction of ankle plantar flexor and hip adductor spasticity, with a partial increase in the range of the active and passive motion at the ankle and in gait velocity. At an individual level, six of 15 patients showed an increase in gait velocity. The FAC and RMA did not change. Patients often reported partial improvement in foot position and lower limb propulsion, and fair satisfaction. In conclusion, botulinum toxin injection can be effective in HSP patients with relatively ancient spasticity. This technique can be introduced into the therapeutic panel, which also includes physiotherapy, oral treatment and baclofen pump.