Sorting nexin 24 is required for α-granule biogenesis and cargo delivery in megakaryocytes.

Germline defects affecting the DNA-binding domain of the transcription factor FLI1 are associated with a bleeding disorder that is characterized by the presence of large, fused α-granules in platelets. We investigated whether the genes showing abnormal expression in FLI1-deficient platelets could be involved in platelet α-granule biogenesis by undertaking transcriptome analysis of control platelets and platelets harboring a DNA-binding variant of FLI1. Our analysis identified 2,276 transcripts that were differentially expressed in FLI1-deficient platelets. Functional annotation clustering of the coding transcripts revealed significant enrichment for gene annotations relating to protein transport, and identified Sorting nexin 24 (SNX24) as a candidate for further investigation. Using an induced pluripotent stem cell-derived megakaryocyte model, SNX24 expression was found to be increased during the early stages of megakaryocyte differentiation and downregulated during proplatelet formation, indicating tight regulatory control during megakaryopoiesis. CRISPR-Cas9 mediated knockout (KO) of SNX24 led to decreased expression of immature megakaryocyte markers, CD41 and CD61, and increased expression of the mature megakaryocyte marker CD42b (P=0.0001), without affecting megakaryocyte polyploidisation, or proplatelet formation. Electron microscopic analysis revealed an increase in empty membrane-bound organelles in SNX24 KO megakaryocytes, a reduction in α-granules and an absence of immature and mature multivesicular bodies, consistent with a defect in the intermediate stage of α-granule maturation. Co-localization studies showed that SNX24 associates with each compartment of α-granule maturation. Reduced expression of CD62P and VWF was observed in SNX24 KO megakaryocytes. We conclude that SNX24 is required for α-granule biogenesis and intracellular trafficking of α-granule cargo within megakaryocytes.

Lacrimal sac ciliated HPV16 positive, adenosquamous carcinoma-A case report of a unique histological variant at this site and a review of the literature.

A 47-year-old female developed a reddish swelling of the right medial canthus over 3 months. On examination, a red, firm mass, involving the right medial canthal and extending into the inferior fornix was present and the globe was displaced upwards and inwards. A staging MRI scan confirmed a lacrimal sac lesion with anterior orbit extension. After an equivocal biopsy, the patient underwent debulking surgery. Histology showed a lacrimal sac invasive adenosquamous carcinoma, comprising poorly differentiated squamous carcinoma and invasive adenocarcinoma areas arranged in a tubulo-glandular pattern. The adenocarcinoma harboured numerous cilia. p16 showed block positivity of both components and micro-dissected tissue from both areas showed the presence of HPV16 DNA by PCR. This is the first description of ciliated adenosquamous carcinoma of the lacrimal sac and this finding is placed into the context of what is known about ciliated head and neck adenosquamous carcinomas and the role of high-risk HPV.

Cholestasis Due to USP53 Deficiency.

OBJECTIVES: Although a number of genetic forms of cholestasis have been identified, the genetic etiology of disease remains unidentified in a subset of cholestasis patients. METHODS: Whole exome sequencing (WES) was performed in DNA from patients diagnosed with cholestasis, at different points on the continuum from progressive familial intrahepatic cholestasis to benign recurrent intrahepatic cholestasis, in whom no disease mutations in known cholestasis genes had been identified. Candidate genes were then assessed in a larger patient sample, by targeted next-generation sequencing (NGS). Disease features at presentation and follow-up were collected from available medical records. RESULTS: By WES, we identified 3 patients with homozygous mutations in USP53. Screening of USP53 in a larger set of patients identified 4 additional patients with homozygous mutations in USP53. Six of the 7 patients had deletion mutations, and 1 had a missense mutation; 3 of the patients were siblings, all bearing a deletion that also disrupted neighboring MYOZ2. Age of onset ranged from early infancy to adolescence. Cholestasis tended to be biochemically mild and intermittent, and responsive to medication. Liver fibrosis was, however, present in all 4 patients who were biopsied, and splenomegaly was apparent in 5 of 7 at last ultrasound. CONCLUSIONS: Two groups recently identified patients with liver disease and mutation in USP53. We have now identified biallelic mutation in USP53 in 7 further patients with cholestasis, from 5 families. Most individuals had evidence of chronic liver disease, and long-term follow-up is recommended.

Occupational Localized Cutaneous Argyria With Pseudo-Ochronosis in a Jeweler.

ABSTRACT: A case of localized argyria in a 36-year-old female jeweler is described who presented with 2 discrete and asymptomatic bluish-black pigmented macules on the pulp of her left middle finger. A skin biopsy from both lesions demonstrated deposition of brown/black pigmented granules along the basement membrane zone of eccrine glands, blood vessels, nerves, and the dermo-epidermal junction fully in keeping with silver deposition. In addition, there was yellow-brown deposition seen within the interstitial dermis mimicking an early form of ochronosis, so called "pseudo-ochronosis." This latter feature is rarely described in cases of argyria. Transmission electron microscopy and energy dispersive x-ray spectroscopy confirmed the presence of electron dense particles up to 150 nm in diameter and the presence of silver, respectively. On further questioning, the patient had a history of localized and chronic exposure to silver, which specifically involved holding and manipulating silver wires and rings over the left middle finger. This case highlights an unusual and rare presentation of localized argyria in a jeweler. In addition, our case showed preferential silver deposition on dermal elastic fibers which has not been previously described in the literature.

Rough endoplasmic reticulum expansion: a consistent finding in a patient cohort with vascular Ehlers-Danlos Syndrome and Osteogenesis Imperfecta.

Vascular Ehlers-Danlos Syndrome (vEDS) and Osteogenesis Imperfecta (OI) are two forms of connective tissue disorders. Previously, transmission electron microscopy of skin biopsies was routinely performed on all patients who were clinically suspected to have vEDS. At present, molecular genetics using genomic DNA extracted from a blood sample is the first line investigation for these patients. However, when variants of uncertain clinical significance are identified on genetic testing and individuals do not have the classical features of OI or vEDS, additional phenotypic information obtained from a skin biopsy can be valuable for contributing to the evidence for re-classifying pathogenicity of variants.We present a cohort of six patients with molecularly confirmed vEDS and one patient with a severe form of OI, who each had expanded (or dilated), protein-filled, rough endoplasmic reticulum identified on transmission electron microscopy. The patients were identified through retrospective screening of medical records, and biopsies were taken between 1999-2016. We discuss the potential role for assessing rough endoplasmic reticulum expansion as a useful tool to allow further phenotyping of these individuals.

Microsporidial keratopathy in two dogs.

A microsporidial keratopathy is described in two dogs. Both dogs presented with a unilateral stromal keratopathy characterized by multifocal coalescing opacities, and the diagnosis was made on histopathologic examination of keratectomy specimens. Transmission electron microscopy (TEM) on formalin-fixed, paraffin-embedded corneal tissue was performed in one dog, and the morphologic features were consistent with Nosema species infection. Both dogs were initially diagnosed and treated by superficial keratectomy. One dog received additional antifungal medication and underwent a penetrating keratoplasty following local recurrence two years later. No other systemic lesions attributable to the microsporidial infection were identified clinically. The clinical and diagnostic pathology findings, treatment, and follow-up are discussed.

KCTD7 deficiency defines a distinct neurodegenerative disorder with a conserved autophagy-lysosome defect.

OBJECTIVE: Several small case series identified KCTD7 mutations in patients with a rare autosomal recessive disorder designated progressive myoclonic epilepsy (EPM3) and neuronal ceroid lipofuscinosis (CLN14). Despite the name KCTD (potassium channel tetramerization domain), KCTD protein family members lack predicted channel domains. We sought to translate insight gained from yeast studies to uncover disease mechanisms associated with deficiencies in KCTD7 of unknown function. METHODS: Novel KCTD7 variants in new and published patients were assessed for disease causality using genetic analyses, cell-based functional assays of patient fibroblasts and knockout yeast, and electron microscopy of patient samples. RESULTS: Patients with KCTD7 mutations can exhibit movement disorders or developmental regression before seizure onset, and are distinguished from similar disorders by an earlier age of onset. Although most published KCTD7 patient variants were excluded from a genome sequence database of normal human variations, most newly identified patient variants are present in this database, potentially challenging disease causality. However, genetic analysis and impaired biochemical interactions with cullin 3 support a causal role for patient KCTD7 variants, suggesting deleterious alleles of KCTD7 and other rare disease variants may be underestimated. Both patient-derived fibroblasts and yeast lacking Whi2 with sequence similarity to KCTD7 have impaired autophagy consistent with brain pathology. INTERPRETATION: Biallelic KCTD7 mutations define a neurodegenerative disorder with lipofuscin and lipid droplet accumulation but without defining features of neuronal ceroid lipofuscinosis or lysosomal storage disorders. KCTD7 deficiency appears to cause an underlying autophagy-lysosome defect conserved in yeast, thereby assigning a biological role for KCTD7. Ann Neurol 2018;84:774-788.

P4HB recurrent missense mutation causing Cole-Carpenter syndrome.

BACKGROUND: Cole-Carpenter syndrome (CCS) is commonly classified as a rare Osteogenesis Imperfecta (OI) disorder. This was following the description of two unrelated patients with very similar phenotypes who were subsequently shown to have a heterozygous missense mutation in P4HB. OBJECTIVES: Here, we report a 3-year old female patient with severe OI who on exome sequencing was found to carry the same missense mutation in P4HB as reported in the original cohort. We discuss the genetic heterogeneity of CCS and underlying mechanism of P4HB in collagen production. METHODS: We undertook detailed clinical, radiological and molecular phenotyping in addition, to analysis of collagen in cultured fibroblasts and electron microscopic examination in the patient reported here. RESULTS: The clinical phenotype appears consistent in patients reported so far but interestingly, there also appears to be a definitive phenotypic clue (crumpling metadiaphyseal fractures of the long tubular bones with metaphyseal sclerosis which are findings that are uncommon in OI) to the underlying genotype (P4HB variant). DISCUSSION: P4HB (Prolyl 4-hydroxylase, betasubunit) encodes for PDI (Protein Disulfide isomerase) and in cells, in its tetrameric form, catalyses formation of 4-hydroxyproline in collagen. The recurrent variant in P4HB, c.1178A>G, p.Tyr393Cys, sits in the C-terminal reactive centre and is said to interfere with disulphide isomerase function of the C-terminal reactive centre. P4HB catalyses the hydroxylation of proline residues within the X-Pro-Gly repeats in the procollagen helical domain. Given the inter-dependence of extracellular matrix (ECM) components in assembly of a functional matrix, our data suggest that it is the organisation and assembly of the functional ECM that is perturbed rather than the secretion of collagen type I per se. CONCLUSIONS: We provide additional evidence of P4HB as a cause of a specific form of OI-CCS and expand on response to treatment with bisphosphonates in this rare disorder.

Geroderma osteodysplasticum: Histological features and the role of panel-based exome sequencing in diagnosis.

Geroderma osteodysplasticum (GO) has clinical and histological features that overlap with other causes of wrinkly skin. Here we present the case of a child diagnosed with GO following exome sequencing of a panel of genes covering the wide differential diagnosis. The histological features of the overlapping conditions are presented, highlighting the utility of panel testing for conditions of this type. This is relevant to many genetic conditions and can influence ongoing management as exemplified by this case.

Mutations in DCDC2 (doublecortin domain containing protein 2) in neonatal sclerosing cholangitis.

BACKGROUND & AIMS: Neonatal sclerosing cholangitis (NSC) is a severe neonatal-onset cholangiopathy commonly leading to liver transplantation (LT) for end-stage liver disease in childhood. Liver biopsy findings histopathologically resemble those in biliary atresia (BA); however, in NSC extrahepatic bile ducts are patent, whilst in BA their lumina are obliterated. NSC is commonly seen in consanguineous kindreds, suggesting autosomal recessive inheritance. METHODS: From 29 NSC patients (24 families) identified, DNA was available in 24 (21 families). Thirteen (7 male) patients (12 families) of consanguineous parentage were selected for whole exome sequencing. Sequence variants were filtered for homozygosity, pathogenicity, minor allele frequency, quality score, and encoded protein expression pattern. RESULTS: Four of 13 patients were homozygous and two were compound heterozygous for mutations in the doublecortin domain containing 2 gene (DCDC2), which encodes DCDC2 protein and is expressed in cholangiocyte cilia. Another 11 patients were sequenced: one (with one sibling pair) was compound heterozygous for DCDC2 mutations. All mutations were protein-truncating. In available liver tissue from patients with DCDC2 mutations, immunostaining for human DCDC2 and the ciliary protein acetylated alpha-tubulin (ACALT) showed no expression (n=6) and transmission electron microscopy found that cholangiocytes lacked primary cilia (n=5). DCDC2 and ACALT were expressed in NSC patients without DCDC2 mutations (n=22). Of the patients carrying DCDC2 mutations, one died awaiting LT; five came to LT, of whom one died 2years later. The other 4 are well. CONCLUSION: Among 24 NSC patients with available DNA, 7 had mutations in DCDC2 (6 of 19 families). NSC patients in substantial proportion harbour mutations in DCDC2. Their disease represents a novel liver-based ciliopathy. LAY SUMMARY: Neonatal sclerosing cholangitis (NSC) is a rare genetic form of liver disease presenting in infancy. Through next generation sequencing we identified mutations in the gene encoding for doublecortin domain containing 2 (DCDC2) protein in a group of NSC children. DCDC2 is a signalling and structural protein found in primary cilia of cholangiocytes. Cholangiocytes are the cells forming the biliary system which is the draining system of the liver.

Source and characterization of hepatic macrophages in acetaminophen-induced acute liver failure in humans.

UNLABELLED: Acetaminophen-induced acute liver failure (AALF) is associated with innate immunity activation, which contributes to the severity of hepatic injury and clinical outcome. A marked increase in hepatic macrophages (h-mφ) is observed in experimental models of AALF, but controversy exists regarding their role, implicating h-mφ in both aggravation and resolution of liver injury. The role of h-mφ in human AALF is virtually unexplored. We sought to investigate the role of chemokine (C-C motif) ligand 2 (CCL2) in the recruitment of circulating monocytes to the inflamed liver and to determine how the h-mφ infiltrate and liver microenvironment may contribute to tissue repair versus inflammation in AALF. We evaluated circulating monocytes, their chemokine (C-C motif) receptor 2 (CCR2) expression, and serum CCL2 levels in patients with AALF. Cell subsets and numbers of circulation-derived (MAC387+) or resident proliferating (CD68/Ki67+) h-mφ in hepatic immune infiltrates were determined by immunohistochemistry. Inflammatory cytokine levels were determined in whole and laser microdissected liver tissue by proteome array. In AALF, circulating monocytes were depleted, with the lowest levels observed in patients with adverse outcomes. CCL2 levels were high in AALF serum and hepatic tissue, and circulating monocyte subsets expressed CCR2, suggesting CCL2-dependent hepatic monocyte recruitment. Significant numbers of both MAC387+ and CD68+ h-mφ were found in AALF compared with control liver tissue with a high proportion expressing the proliferation marker Ki67. Levels of CCL2, CCL3, interleukin (IL)-6, IL-10, and transforming growth factor-ß1 were significantly elevated in AALF liver tissue relative to chronic liver disease controls. CONCLUSION: In AALF, the h-mφ population is expanded in areas of necrosis, both through proliferation of resident cells and CCL2-dependent recruitment of circulating monocytes. The presence of h-mφ within an anti-inflammatory/regenerative microenvironment indicates that they are implicated in resolution of inflammation/tissue repair processes during AALF.

Dermatosparaxis (Ehlers-Danlos type VIIC): prenatal diagnosis following a previous pregnancy with unexpected skull fractures at delivery.

Dermatosparaxis Ehlers-Danlos syndrome (or EDS VIIC), a rare autosomal recessive connective tissue disorder, is characterized by extreme skin fragility, premature rupture of membranes in pregnancy, and spontaneous rupture of internal organs. Here we report a second patient with EDS VIIC presenting with congenital skull fractures and skin lacerations at birth, complications which may occur more frequently than previously thought in this condition. We also discuss the role of prenatal diagnosis in the management of a subsequent normal pregnancy.

Cosegregation of focal segmental glomerulosclerosis in a family with familial partial lipodystrophy due to a mutation in LMNA.

BACKGROUND AND AIM: Focal segmental glomerulosclerosis (FSGS) is a common cause of idiopathic nephrotic syndrome in adults (35%). A number of genetic and familial forms of FSGS have been recognized. Here, we report a large pedigree with a pathogenic mutation in LMNA (R349W) in which four members were found to have biopsy-proven FSGS. The LMNA gene codes for lamins A and C, major components of the nuclear lamina which function in nuclear architecture, integrity and the regulation of gene expression. METHODS: Pedigree screening and mutation analysis of LMNA gene in all family members. Renal biopsies were performed in proteinuric patients. A molecular 3D model of the familial LMNA mutation was constructed. RESULTS: There were a total of 16 affected members from four generations, 12 of whom were found to carry the germline LMNA mutation. All affected adults had clinical features of familial partial lipodystrophy (FPLD) of the non-Dunnigan variety. Four patients within the same generation presented with a variable degree of renal impairment and proteinuria. Renal biopsies from all four revealed FSGS. The familial mutation is a missense change (R349W) in exon 6 of LMNA (c.1045C>T). CONCLUSIONS: We report a genetic link between LMNA and biopsy-proven FSGS in a large pedigree with FPLD. This unexpected association extends the disease spectrum of LMNA to the kidney and suggests that the physiological role of LMNA could be relevant to the maintenance of glomerular structure and function.

Subconjunctival Ocular Argyrosis following Treatment with Ruthenium 106 Brachytherapy for Choroidal Melanoma.

Introduction: Ruthenium-106 (Ru-106) brachytherapy is one of the commonest eye-sparing treatments for choroidal melanoma. These patients require long-term surveillance of the treated tumour remnant to ensure there is no local recurrence. New or progressive pigmented lesions in treated eyes are often regarded as suspicious - especially if there are concerns of extra-scleral extension. Case Presentations: We present two cases of posterior choroidal melanoma treated five and 10 years previously with Ru-106. Both cases developed subconjunctival dark/black lesions on the anterior surface of the eye in the quadrant of the conjunctival peritomy during Ru-106 treatment. Both had similar findings on histopathology: black, non-organic, particulate foreign material of varying confluence deposited on elastin and collagen fibres. Energy dispersive X-ray microanalysis confirmed the material contained silver. Discussion: The Ru-106 applicator consists of a radioactive core of Ru-106 encapsulated within pure silver as a radiation shield. During surgical insertion, stainless steel suture needles and forceps can occasionally scratch the applicator's silver eyelets and scatter microscopic particles of elemental silver into the operative field. These particles were likely deposited within the subconjunctival tissues of these patients during brachytherapy administration, leading to localised ocular argyrosis. Iatrogenic ocular argyrosis should be considered in the differential diagnosis of new pigmented lesions in patients treated with Ru-106 brachytherapy. This study is the first to unequivocally identify the cause of some post-brachytherapy ocular surface pigmentation as caused by silver.

Skin gadolinium following use of MR contrast agents in a rat model of nephrogenic systemic fibrosis.

PURPOSE: To detect the ultrastructural site of gadolinium retention in skin by using an animal model of nephrogenic systemic fibrosis and compare a linear, low-stability gadolinium chelate (formulated gadodiamide) with a macrocylic, high-stability gadolinium chelate (gadoterate meglumine). MATERIALS AND METHODS: Experimental procedures were performed according to rules and regulations laid down by the UK Home Office (Animal Procedures Act of 1986). Male Wistar rats were subjected to 5/6 subtotal nephrectomy (creatinine clearance, 25% normal). Gadolinium-based contrast agents, formulated gadodiamide (n = 9) and gadoterate meglumine (n = 11), were administered intravenously (2.5 mmol/kg for 5 days). After 28 days, skin was analyzed by means of morphometric and immunohistochemical techniques and electron microscopy. Data were compared with the Student t test. Skin gadolinium was located by means of energy-filtered transmission electron microscopy. RESULTS: Formulated gadodiamide produced a 40-fold greater increase in gadolinium in skin than did gadoterate meglumine. An electron-dense filamentous material, detected within extracellular matrix, displayed a "halo" appearance, associated with collagen fibrils and electron-dense intracellular fragments of collagen fibrils within activated fibroblasts. Both electron-dense features demonstrated the presence of gadolinium but were much less apparent following gadoterate meglumine administration, where the presence of gadolinium was not detected. Formulated gadodiamide increased dermal cell count, dermal thickness, and collagen bundle density with enhanced immunostain for CD34, fibroblast-specific protein 1,4-hydroxy-prolyl-hydroxylase, and factor XIIIa. Circular staining for α-smooth muscle actin indicated new blood vessel formation. Skin of rats receiving gadoterate meglumine remained unchanged. CONCLUSION: Gadolinium retention in skin following formulated gadodiamide administration was located to the collagen fibril, in both the extracellular matrix and within activated fibroblasts.

Case report: Thirty-year progression of an EMPF1 encephalopathy due to defective mitochondrial and peroxisomal fission caused by a novel de novo heterozygous DNM1L variant.

Mutations in DNM1L (DRP1), which encode a key player of mitochondrial and peroxisomal fission, have been reported in patients with the variable phenotypic spectrum, ranging from non-syndromic optic atrophy to lethal infantile encephalopathy. Here, we report a case of an adult female patient presenting with a complex neurological phenotype that associates axonal sensory neuropathy, spasticity, optic atrophy, dysarthria, dysphasia, dystonia, and ataxia, worsening with aging. Whole-exome sequencing revealed a heterozygous de novo variant in the GTPase domain of DNM1L [NM_001278464.1: c.176C>A p.(Thr59Asn)] making her the oldest patient suffering from encephalopathy due to defective mitochondrial and peroxisomal fission-1. In silico analysis suggested a protein destabilization effect of the variant Thr59Asn. Unexpectedly, Western blotting disclosed profound decrease of DNM1L expression, probably related to the degradation of DNM1L complexes. A detailed description of mitochondrial and peroxisomal anomalies in transmission electron and 3D fluorescence microscopy studies confirmed the exceptional phenotype of this patient.

Vascular histopathology and connective tissue ultrastructure in spontaneous coronary artery dissection: pathophysiological and clinical implications.

AIMS: Spontaneous coronary artery dissection (SCAD) is a cause of acute coronary syndromes and in rare cases sudden cardiac death (SCD). Connective tissue abnormalities, coronary inflammation, increased coronary vasa vasorum (VV) density, and coronary fibromuscular dysplasia have all been implicated in the pathophysiology of SCAD but have not previously been systematically assessed. We designed a study to investigate the coronary histological and dermal collagen ultrastructural findings in SCAD. METHODS AND RESULTS: Thirty-six autopsy SCAD cases were compared with 359 SCAD survivors. Coronary and myocardial histology and immunohistochemistry were undertaken. Transmission electron microscopy (TEM) of dermal extracellular matrix (ECM) components of n = 31 SCAD survivors and n = 16 healthy volunteers were compared. Autopsy cases were more likely male (19% vs. 5%; P = 0.0004) with greater proximal left coronary involvement (56% vs. 18%; P < 0.0001) compared to SCAD survivors. N = 24 (66%) of cases showed no myocardial infarction on macro- or microscopic examination consistent with arrhythmogenic death. There was significantly (P < 0.001) higher inflammation in cases with delayed-onset death vs. sudden death and significantly more inflammation surrounding the dissected vs. non-dissected vessel segments. N = 17 (47%) cases showed limited intimal fibro-elastic thickening but no features of fibromuscular dysplasia and no endothelial or internal elastic lamina abnormalities. There were no differences in VV density between SCAD and control cases. TEM revealed no general ultrastructural differences in ECM components or markers of fibroblast metabolic activity. CONCLUSIONS: Assessment of SCD requires careful exclusion of SCAD, particularly in cases without myocardial necrosis. Peri-coronary inflammation in SCAD is distinct from vasculitides and likely a reaction to, rather than a cause for SCAD. Coronary fibromuscular dysplasia or increased VV density does not appear pathophysiologically important. Dermal connective tissue changes are not common in SCAD survivors.

SARS-COV2 placentitis and pregnancy outcome: A multicentre experience during the Alpha and early Delta waves of coronavirus pandemic in England.

Background: Pregnant women with SARS-CoV-2 infection experience higher rates of stillbirth and preterm birth. A unique pattern of chronic histiocytic intervillositis (CHI) and/or massive perivillous fibrin deposition (MPFD) has emerged, coined as SARS-CoV-2 placentitis. Methods: The aim of this study was to describe a cohort of placentas diagnosed with SARS-CoV-2 placentitis during October 2020-March 2021. Cases with a histological diagnosis of SARS-CoV-2 placentitis and confirmatory immunohistochemistry were reported. Maternal demographic data, pregnancy outcomes and placental findings were collected. Findings: 59 mothers delivered 61 infants with SARS-CoV-2 placentitis. The gestational age ranged from 19 to 41 weeks with most cases (78.6%) being third trimester. 30 infants (49.1%) were stillborn or late miscarriages. Obese mothers had higher rates of pregnancy loss when compared with those with a BMI <30 [67% (10/15) versus 41% (14/34)]. 47/59 (79.7%) mothers had a positive SARS-CoV-2 PCR test either at the time of labour or in the months before, of which 12 (25.5%) were reported to be asymptomatic. Ten reported only CHI, two cases showed MPFD only and in 48 placentas both CHI and MPFD was described. Interpretation: SARS-CoV2 placentitis is a distinct entity associated with increased risk of pregnancy loss, particularly in the third trimester. Women can be completely asymptomatic and still experience severe placentitis. Unlike 'classical' MPFD, placentas with SARS-CoV-2 are generally normal in size with adequate fetoplacental weight ratios. Further work should establish the significance of the timing of maternal SARS-CoV-2 infection and placentitis, the significance of SARS-CoV2 variants, and rates of vertical transmission associated with this pattern of placental inflammation. Funding: There was not funding associated with this study.

Conjunctival epithelial cells resist productive SARS-CoV-2 infection.

Conjunctival epithelial cells, which express viral-entry receptors angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine type 2 (TMPRSS2), constitute the largest exposed epithelium of the ocular surface tissue and may represent a relevant viral-entry route. To address this question, we generated an organotypic air-liquid-interface model of conjunctival epithelium, composed of basal, suprabasal, and superficial epithelial cells, and fibroblasts, which could be maintained successfully up to day 75 of differentiation. Using single-cell RNA sequencing (RNA-seq), with complementary imaging and virological assays, we observed that while all conjunctival cell types were permissive to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome expression, a productive infection did not ensue. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust autocrine and paracrine NF-κB activity, without activation of antiviral interferon signalling. Collectively, these data enrich our understanding of SARS-CoV-2 infection at the human ocular surface, with potential implications for the design of preventive strategies and conjunctival transplantation.