Using single-cell RNA sequencing to unravel cell lineage relationships in the respiratory tract.

The respiratory tract is lined by a pseudo-stratified epithelium from the nose to terminal bronchioles. This first line of defense of the lung against external stress includes five main cell types: basal, suprabasal, club, goblet and multiciliated cells, as well as rare cells such as ionocytes, neuroendocrine and tuft/brush cells. At homeostasis, this epithelium self-renews at low rate but is able of fast regeneration upon damage. Airway epithelial cell lineages during regeneration have been investigated in the mouse by genetic labeling, mainly after injuring the epithelium with noxious agents. From these approaches, basal cells have been identified as progenitors of club, goblet and multiciliated cells, but also of ionocytes and neuroendocrine cells. Single-cell RNA sequencing, coupled to lineage inference algorithms, has independently allowed the establishment of comprehensive pictures of cell lineage relationships in both mouse and human. In line with genetic tracing experiments in mouse trachea, studies using single-cell RNA sequencing (RNAseq) have shown that basal cells first differentiate into club cells, which in turn mature into goblet cells or differentiate into multiciliated cells. In the human airway epithelium, single-cell RNAseq has identified novel intermediate populations such as deuterosomal cells, 'hybrid' mucous-multiciliated cells and progenitors of rare cells. Novel differentiation dynamics, such as a transition from goblet to multiciliated cells have also been discovered. The future of cell lineage relationships in the respiratory tract now resides in the combination of genetic labeling approaches with single-cell RNAseq to establish, in a definitive manner, the hallmarks of cellular lineages in normal and pathological situations.

Human cytomegalovirus and primary intracranial tumours: frequency of tumour infection and lack of correlation with systemic immune anti-viral responses.

AIMS: Human cytomegalovirus (HCMV) is a ubiquitous beta human herpesvirus able to influence infected cell survival and proliferation and to modulate the host immune response. As there is accumulating evidence that HCMV is detected in primary intracranial astrocytic tumours, in this study we looked for the presence of HCMV in intracranial tumours and tried to correlate this eventual presence with the anti-HCMV systemic immunoreactivity and with the detection of HCMV in peripheral blood. METHODS: In this study, we analysed 43 glioblastomas (GBM), 14 oligodendrogliomas (OL) and 20 meningiomas (MG) by immunofluorescence (IF) targeting HCMV immediate early antigen (IE1) and by nested PCR (nPCR) amplifying HCMV glycoprotein B (gB). RESULTS: Detection of IE1 by IF showed the presence of HCMV in 70% of GBM, 57% of OL and 85% of MG, in contrast to gB nPCR, which detected HCMV in only 50% of GBM, 38% of OL and 46% of MG. Unexpectedly, HCMV DNA and antigens were detected within GBM, OL and MG of patients that exhibit negative viral serology. More surprisingly, PCR on the peripheral blood did not detect HCMV in patients with a HCMV-positive tumour. CONCLUSIONS: Our results are in agreement with previous observations demonstrating HCMV in glial tumours and highlight the presence of HCMV in meningiomas. We also showed that anti-HCMV specific systemic immunoreactivity and detection of HCMV in peripheral blood are not predictive of HCMV presence in primary intracranial tumours.

Giant cell ependymoma of the thoracic spinal cord.

We report a new case of giant cell ependymoma (GCE) of the thoracic spinal cord. Ependymomas predominate in children and young adults and are frequently intracranial and infra-tentorial. However, a second age peak at 30-40 years is reported for spinal tumours. Microscopically, ependymomas show a large variety of histological features, among which a rare variant with giant cells. This 59-year-old woman presented with a 6-month history of numbness and burning sensation affecting the left lower limb and hemi-trunk. A cervico-thoracic MRI revealed a solid intra-medullary tumour at the level of T1-T3, slightly T1-hypointense, T2-hyperintense and contrast enhancing. A complete surgical resection was carried out through a C7 to T4 laminectomy. Recovery was complete with no sign of recurrence at 18-month follow-up. The initial histological diagnosis of glioblastoma was challenged on the basis of the imaging and operative findings of a well-circumscribed tumour. The case was sent to us for second opinion and we diagnosed a GCE, WHO grade II, with a biphasic pattern including a predominant giant cell component (>90%), with genetic evidence of polyploidy, and a very limited classic component, showing a characteristic loss of chromosome 22. Our report adds to the clinical, imaging, pathological and genetic characterisation of GCE and brings the first genetic evidence that these rare tumours are at least bi-clonal. It also suggests that GCE have a good prognosis after complete surgical resection.

[Third cranial nerve palsies in childhood. A case report of sellar germ cell tumor]. / Les paralysies du nerf moteur oculaire commun chez l'enfant: a propos d'une observation de tumeur germinale hypophysaire.

PURPOSE: Third cranial nerve palsies are unfrequent in childhood and adolescence and are most often congenital. The association of sellar germ cell tumor and ophthalmoplegia is considered as being very rare at this age. CASE REPORT: A 11-year-old young girl was examined in emergency with a third left cranial nerve partial palsy associated with one- year duration history of hypopituitarism with insipid diabetes and growth retardation. Cerebral IRM revealed a tumor of the pituitary gland. In histopathological examination of pituitary gland biopsies, lesions were compatibles with a sellar germ cell tumor. CONCLUSION: Although they are most often of a congenital nature, third cranial nerve palsies in childhood may be secondary to other causes that should be always taken in mind. When they are secondary to a sellar tumor and according to the clinical presentation and the IRM, the histopahological examination of biopsies is mandatory to have a precise diagnosis.

[Breast cancer: the interest of pathological classification]. / Cancer du sein: importance de la nomenclature anatomo-pathologique.

The breast pathology includes a large array of entities for which macroscopic and microscopic analysis remains fundamental. Tissue and cell morphology allows in most cases the distinction between benign or malignant tumours and therefore provides the clinicians with essential information for the therapeutic strategy. In the Pathology laboratory, immunohistochemistry and molecular biology have improved the specificity of the diagnosis and have introduced new prognostic and predictive markers for tumour management. The last edition of the WHO classification, released in 2003, distinguishes 21 varieties of invasive carcinoma and 2 categories of intraepithelial neoplasia based on the morphology and immunohistochemical profile. Other diseases can affect the breast, although much less frequently, such as Paget's disease of the nipple, phyllode tumours, sarcomas, lymphomas... These diseases will not be reviewed here.

[Fish and chips]. / Fish and chips.

Academic hospital laboratories should offer patients the possibility to have the most accurate diagnosis by the development of new analyses, such as molecular biology tests including FISH (Fluorescent In Situ Hybridization) and chips (microarrays,...). The purpose of this article is to describe the principles and the potential applications of these techniques.

Primary diffuse leptomeningeal gliomatosis: an autopsy case and review of the literature.

We report a case of primary diffuse leptomeningeal gliomatosis (PDLG) in a 76-year-old male presenting with confusion, dysarthria, diplopia, lumbal pain and headaches of recent onset. Neurological examination revealed nuchal rigidity and bilateral sixth cranial nerve palsy. The cerebrospinal fluid showed a marked hyperproteinorachia (4711 mg/L) and mild cytorachia (5-10 leucocytes/mm3) with a few atypical lymphoid cells. On admission, brain CT scan and MRI demonstrated diffuse and nodular leptomeningeal contrast enhancement predominant at the skull base and several osteolytic lesions in the right parietal bone. Extensive serological studies for infectious, autoimmune or neoplastic diseases were negative. The work-up diagnosis was neurosarcoidosis or multiple meningeal and osseous metastases of an unknown primary cancer. Surgical biopsy of the right parietal bone lesion showed only fibrous tissue with no evidence of tumour or inflammation. The patient was treated with high dose corticosteroids but its neurological status progressively worsened and he died of aspiration pneumonia 35 days after admission. Post-mortem examination revealed a PDLG, a rare fatal tumour with about 60 cases reported. PDGL is characterized by the diffusion of neoplastic glial cells throughout the leptomeninges without evidence of a primary intra-parenchymal lesion. Recognition of this rare brain tumour is important as recent reports suggest that radiotherapy and chemotherapy can improve patient survival.

[Glioblastoma, an example of translational research?]. / Les glioblastomes, un exemple de recherche translationnelle?

Among patients which develop glioblastoma multiform (GBM), recurrence is the rule despite continuous progress in surgery, radiotherapy and chemotherapy. In the adult, GBM is the most frequent and most aggressive tumour of the Central Nervous System. A better understanding of the mechanisms by which these tumours relapse could promote the use of preventive therapy and could increase patients' survival. GBM stem cells have been recently described and it was demonstrated that they are specifically implied in the experimental tumorigenesis. It is thus very attractive to speculate on a possible relationship between these GBM stem cells and the neural stem cells which are persisting in the neurogenic zones of the adult brain. In this review, we formulate and discuss the hypothesis by which, in a patient with GBM, malignant stem cells might be present in the neurogenic zones, away from the tumour mass. This hypothesis could explain the tumour relapse observed after the first treatments.

[Invasive pulmonary mucormycosis with invasion of the thoracic spine in a patient with myelodysplastic syndrome]. / Mucormycose invasive du poumon et du rachis dorsal.

We report the case of a 67-year-old patient who presented with a myelodysplastic syndrome and who developed a pulmonary mucormycosis with a rare extension to the dorsal spine. A decompressive laminectomy was attempted after failure of broad-spectrum antifungal treatment (Cancidas, V-Fend). The diagnosis was obtained after surgical biopsy. The scheduled lobectomy could not be performed because of altered clinical condition. The patient eventually died despite adapted antifungal treatment (Abelcet, Posaconazole). Pulmonary mucormycosis is a rare cause of mycotic infection that reaches most of the time immunocompromised patients. The pathogenic agent is part of zygomyces that have angio-invasive ability. Perineural propagation was recently described. Immunodepression, late diagnosis and lack of response to new generation antifungal drugs (V-Fend, Cancidas) are responsible for therapeutic failure in this disease. This case emphasizes the risk inherent to empirical antifungal treatment and the need of early biopsy in cases that do not respond to treatment.

[How I explore. . . vasculitis of the peripheral nervous system]. / Comment j'explore. . . une vascularite du système nerveux périphérique.

Vasculitic neuropathies require early diagnosis and therapy since their prognosis is potentially severe. The following features are usually suggestive of a vasculitic neuropathy: painful multiple mononeuropathy with an acute/subacute course, signs and symptoms of multisystem involvement (arthritis, asthma, renal and skin involvement), biological evidence of an inflammatory syndrome. In one third of patients however, vasculitic neuropathies present as a polyneuropathy. For other patients, peripheral neuropathy is the presentation mode of a necrotizing vasculitis, in the absence of biological inflammatory syndrome. A combined muscle and nerve biopsy, will usually demonstrate the vasculitic process. Vasculitic neuropathies often have a favourable prognosis when immunosuppressive therapy (corticosteroids and/or cyclophosphamide) is initiated at an early stage.

Macrophage migration inhibitory factor (MIF) expression in human glioblastomas correlates with vascular endothelial growth factor (VEGF) expression.

Macrophage migration inhibitory factor (MIF) is a peptide released upon hypothalamo-pituitary stimulation that acts as a potent endogenous antagonist of the glucocorticoid inhibition of acute inflammatory response and subsequent antigen-specific response. MIF also sustains tumour growth as it promotes angiogenesis, overcomes p53-mediated cell growth arrest and inhibits tumour-specific immune responses. Using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry, we studied MIF expression in 35 human glioblastomas and two normal brains. We compared these results with the expression of vascular endothelial growth factor (VEGF), the most potent angiogenic factor in glioblastomas. We detected MIF in normal cortical neurons and glial cells. All glioblastomas were positive for MIF mRNA with expression levels similar to or higher than those of normal brain. MIF immunoreactivity was seen mainly in tumour cells and less frequently in hyperplastic endothelial cells. The expressions of MIF and VEGF mRNA were strongly correlated (P < 0.0001). Our results demonstrate the expression of MIF in human glioblastomas, and indicate a close relationship with VEGF expression. This is of particular interest given the potential modulation of MIF by glucocorticosteroids.

[Scapulo-peroneal weakness with tardive symptoms of facio-scapulo-humeral muscular dystrophy, connected to 4q35 chromosomal deletion]. / Forme scapulo-péronière à révélation tardive d'une dystrophie musculaire facio-scapulo-humérale, liée à une délétion chromosomique 4q35.

We report the case of a 57-year-old woman, who presented with progressive weakness of ankle's dorsiflexors. Electromyography showed bilateral myogenic patterns in the anterior tibialis predominantly in the left side. Muscle biopsy of the right tibialis anterior showed non specific dystrophic changes. The familial evaluation revealed a son showing scapuloperoneal amyotrophy and facial involvement. Analysis of the propositus' DNA showed a mutation at locus 4q35, characteristic of facioscapulohumeral muscular dystrophy. This case illustrates the wide clinical spectrum of FSH dystrophy and the difficulty to diagnose unusual facial-sparing forms.

Meningeal inflammatory pseudotumour: a case report.

We report the case of a meningeal inflammatory pseudotumour occurring in a 23-year-old male presenting with focal seizures and headaches. Brain imaging techniques showed a 3.5 cm left parietal meningeal tumour. Histology of the surgical specimen showed a dense lymphoid infiltrate permeating the dura mater and leptomeninges, consisting of a predominant polyclonal B cell population as confirmed by immunophenotyping and genotyping. Cultures of serum, CSF, and surgical specimen were negative and there was no serological evidence of a systemic dysimmune disease. The postoperative course was complicated by an episode of brain oedema resolving under steroid therapy. The patient, free from all medication, is asymptomatic at 3 years of follow-up. We discuss previously published cases and the nosology of intracranial inflammatory pseudotumours.

Presence of oestrogen receptor type beta in human retina.

BACKGROUND/AIMS: Recent studies have demonstrated the existence of two oestrogen receptor subtypes alpha (ORalpha) and beta (ORbeta) with significant differences of expression among organs. Since important pathologies of human eye could be linked to hormonal status, the expression of ORbeta in ocular posterior segment was sought. METHODS: Immunohistochemical localisation of ORbeta and ORalpha protein and detection of OR mRNAs by reverse transcription-polymerase chain reaction (RT-PCR) were performed in macular and extramacular regions of the retina and in the choroid on male and female donors eyes. RESULTS: ORbeta protein was localised in the ganglion cell layer and in the choroid. At the transcriptional level, mRNA for ORbeta and for ORalpha were both present. Local differences in the expression level were observed, however, suggesting the possibility of variation in the ratio of ORalpha v ORbeta. CONCLUSIONS: The coexistence of two oestrogen receptor subtypes in the human ocular posterior segment raises acute questions about their potential physiological role, but offers a perspective for preferential targeting of a specific receptor subtype.

Detection of cytokines in human sural nerve biopsies: an immunohistochemical and molecular study.

In vitro and in vivo models have implicated numerous cytokines as major modulators of inflammation, destruction and repair in the peripheral nervous system (PNS). The in situ production of cytokines in human peripheral nerve disorders is still poorly documented. We studied the expression of interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha, IL-6, IL-10, IL-4, IL-3 and nerve growth factor (NGF) in 35 human sural nerve biopsies using immunohistochemistry; additional reverse transcription-polymerase chain reaction and mRNA in situ hybridization were performed for IL-4 and NGF. Expression of IL-1 beta and TNF-alpha was shown in both morphologically normal nerves and various neuropathies, and macrophages appeared as their predominant source. Levels of IL-1 beta and TNF-alpha expression were significantly correlated (P < 0.01) with each other and with expression of NGF. Multiple endoneurial sources were suggested for IL-6 and IL-10 with low immunoreactivity in the vast majority of cases. Conversely, IL-4 and IL-3 expression were found in neuropathies of various etiologies and Schwann cells appeared to be a predominant source of IL-4 in double-labeling immunofluorescence studies. IL-3 immunoreactivity correlated with IL-1 beta, TNF-alpha and IL-6. In this retrospective study, no specific cytokine profile of expression could be assigned to a precise subgroup of neuropathies. This is the first report of IL-4 and IL-3 expression in human neuropathies, and it may be important given the potential role of these cytokines in modulating macrophage activity in the PNS.

[How I investigate...a myopathy with a muscle biopsy]. / Comment j'explore ... une myopathie par biopsie musculaire.

Muscle biopsy is a valuable tool in the diagnosis of many neuromuscular disorders. It is an invasive investigation that will be considered after careful clinical examination, supplemented by biological and electrophysiological studies. The biopsy procedure and the subsequent processing of specimen in the laboratory significantly influence the information provided by the microscopic examination of muscle tissue. They are reviewed in details in this article. Close collaboration between clinicians and neuropathologists in the setting of an optimized procedure will further increase the diagnostic yield of muscle biopsy.

Nerve biopsy: indications and contribution to the diagnosis of peripheral neuropathy. The experience of the Born Bunge Foundation University of Antwerp and University of Liege between 1987 and 1997.

We reviewed 355 nerve biopsies analysed at the Laboratories of Neuropathology of the Born-Bunge Foundation/University of Antwerp (BBF/UIA) and University of Liège (ULg) between 1987 and 1997. We examined the indications for nerve biopsy, the yield of the procedure, and the influence of clinical and neuropathological parameters. Contributory biopsies accounted for 35.5% and 47.3% respectively at ULg and BBF/UIA laboratories: of these, one third showed specific histological findings, the majority being informative only when combined with the relevant clinical data. The profile of indications for nerve biopsy was roughly comparable in both laboratories. The search for an inflammatory neuropathy prompted 35-40% of all biopsies with more than 50% of specimens being informative in this indication. The lowest yield (20%) was obtained among the nerve biopsies performed in the absence of any presumptive aetiology. These accounted for 22-33% of all cases. Inadequate surgical resection, delays in transport or processing errors precluded histological study of 4% (BBF/UIA) to 8% (ULg) of the specimens. We conclude that nerve biopsies should be performed by experienced surgeons and handled in specialised laboratories. Only a relatively small number of causes of neuropathy can be diagnosed on the basis of histology alone. More often, contributory biopsies will result from the combination of non-specific suggestive histological features with relevant clinical information. The diagnostic yield of nerve biopsy is function of careful patient selection and close collaboration between the clinician and the neuropathologist.

[Report of a case of Acanthamoeba keratitis]. / A propos d'un cas de kératite à Acanthamoeba.

CLINICAL REPORT: A clinical report of a contact lenses wearer with Acanthamoeba keratitis pointed out the diagnosis problem. The medical treatment is needed previously to any surgery. Finally the patient underwent enucleation. DISCUSSION: The authors are considering the microbiological aspects and laboratory techniques are described. CONCLUSION: For this very severe but hopefully rare pathology, the sooner the treatment the best. A therapeutic approach is described.

Clinical and neuropathological parameters affecting the diagnostic yield of nerve biopsy.

The value of nerve biopsy in the investigation of peripheral neuropathies is an important and controversial issue, partially obscured by the large variations in the diagnostic yield routinely reported for this procedure. The aim of this study was to evaluate the clinical and neuropathological parameters affecting the yield of nerve biopsy. We compared the experience of two independent neuropathology laboratories with different patient recruitment and neuropathological methods over 11 years (01/1987-12/1997). Clinicopathological correlations were studied retrospectively in 355 patients. Using the same criteria of evaluation, contributive biopsies accounted for 35.5% in one laboratory, and 47.3% in the other. Clinical parameters affecting the yield of nerve biopsy were: (a) the presumptive diagnosis at time of referral for biopsy; (b) the distribution of symptoms; and (c) the interval between disease onset and biopsy. Greater yield was associated with clinically suspected vasculitis, inflammatory demyelinating neuropathy or hereditary sensorimotor neuropathies. Contributive findings were more often reported with multifocal or asymmetrical presentations, and onset-to-biopsy interval of less than 6 months. The contribution of nerve biopsy varied according to neuropathological techniques: (a) serial sections on frozen. paraffin-embedded and resin-embedded material improved sensitivity for interstitial pathology: (b) combined muscle biopsy increased sensitivity in the detection of vasculitis; and (c) teasing of nerve fibers added critical information to other classical techniques in only 4/102 cases.