The effects of pathogenic and likely pathogenic variants for inherited hemostasis disorders in 140 214 UK Biobank participants.

Rare genetic diseases affect millions, and identifying causal DNA variants is essential for patient care. Therefore, it is imperative to estimate the effect of each independent variant and improve their pathogenicity classification. Our study of 140 214 unrelated UK Biobank (UKB) participants found that each of them carries a median of 7 variants previously reported as pathogenic or likely pathogenic. We focused on 967 diagnostic-grade gene (DGG) variants for rare bleeding, thrombotic, and platelet disorders (BTPDs) observed in 12 367 UKB participants. By association analysis, for a subset of these variants, we estimated effect sizes for platelet count and volume, and odds ratios for bleeding and thrombosis. Variants causal of some autosomal recessive platelet disorders revealed phenotypic consequences in carriers. Loss-of-function variants in MPL, which cause chronic amegakaryocytic thrombocytopenia if biallelic, were unexpectedly associated with increased platelet counts in carriers. We also demonstrated that common variants identified by genome-wide association studies (GWAS) for platelet count or thrombosis risk may influence the penetrance of rare variants in BTPD DGGs on their associated hemostasis disorders. Network-propagation analysis applied to an interactome of 18 410 nodes and 571 917 edges showed that GWAS variants with large effect sizes are enriched in DGGs and their first-order interactors. Finally, we illustrate the modifying effect of polygenic scores for platelet count and thrombosis risk on disease severity in participants carrying rare variants in TUBB1 or PROC and PROS1, respectively. Our findings demonstrate the power of association analyses using large population datasets in improving pathogenicity classifications of rare variants.

A genome-wide association study of neutrophil count in individuals associated to an African continental ancestry group facilitates studies of malaria pathogenesis.

BACKGROUND: 'Benign ethnic neutropenia' (BEN) is a heritable condition characterized by lower neutrophil counts, predominantly observed in individuals of African ancestry, and the genetic basis of BEN remains a subject of extensive research. In this study, we aimed to dissect the genetic architecture underlying neutrophil count variation through a linear-mixed model genome-wide association study (GWAS) in a population of African ancestry (N = 5976). Malaria caused by P. falciparum imposes a tremendous public health burden on people living in sub-Saharan Africa. Individuals living in malaria endemic regions often have a reduced circulating neutrophil count due to BEN, raising the possibility that reduced neutrophil counts modulate severity of malaria in susceptible populations. As a follow-up, we tested this hypothesis by conducting a Mendelian randomization (MR) analysis of neutrophil counts on severe malaria (MalariaGEN, N = 17,056). RESULTS: We carried out a GWAS of neutrophil count in individuals associated to an African continental ancestry group within UK Biobank, identifying 73 loci (r2 = 0.1) and 10 index SNPs (GCTA-COJO loci) associated with neutrophil count, including previously unknown rare loci regulating neutrophil count in a non-European population. BOLT-LMM was reliable when conducted in a non-European population, and additional covariates added to the model did not largely alter the results of the top loci or index SNPs. The two-sample bi-directional MR analysis between neutrophil count and severe malaria showed the greatest evidence for an effect between neutrophil count and severe anaemia, although the confidence intervals crossed the null. CONCLUSION: Our GWAS of neutrophil count revealed unique loci present in individuals of African ancestry. We note that a small sample-size reduced our power to identify variants with low allele frequencies and/or low effect sizes in our GWAS. Our work highlights the need for conducting large-scale biobank studies in Africa and for further exploring the link between neutrophils and severe malaria.

Neutrophils cultured ex vivo from CD34+ stem cells are immature and genetically tractable.

BACKGROUND: Neutrophils are granulocytes with essential antimicrobial effector functions and short lifespans. During infection or sterile inflammation, emergency granulopoiesis leads to release of immature neutrophils from the bone marrow, serving to boost circulating neutrophil counts. Steady state and emergency granulopoiesis are incompletely understood, partly due to a lack of genetically amenable models of neutrophil development. METHODS: We optimised a method for ex vivo production of human neutrophils from CD34+ haematopoietic progenitors. Using flow cytometry, we phenotypically compared cultured neutrophils with native neutrophils from donors experiencing emergency granulopoiesis, and steady state neutrophils from non-challenged donors. We carry out functional and proteomic characterisation of cultured neutrophils and establish genome editing of progenitors. RESULTS: We obtain high yields of ex vivo cultured neutrophils, which phenotypically resemble immature neutrophils released into the circulation during emergency granulopoiesis. Cultured neutrophils have similar rates of ROS production and bacterial killing but altered degranulation, cytokine release and antifungal activity compared to mature neutrophils isolated from peripheral blood. These differences are likely due to incomplete synthesis of granule proteins, as demonstrated by proteomic analysis. CONCLUSION: Ex vivo cultured neutrophils are genetically tractable via genome editing of precursors and provide a powerful model system for investigating the properties and behaviour of immature neutrophils.

Outcomes in grade 3B follicular lymphoma: an international study led by the Australasian Lymphoma Alliance.

Grade (G) 3B follicular lymphoma (FL) is a rare FL subtype which exists on a histological continuum between 'lowgrade' (Grade 1, 2 and 3A FL) and diffuse large B-cell lymphoma (DLBCL) appearing to share features with each. Clinical characteristics and outcomes are poorly understood due to lack of adequate representation in prospective trials and large-scale analyses. We analyzed 157 G3BFL cases from 18 international centers, and two comparator groups; G3AFL (n=302) and DLBCL (n=548). Composite histology with DLBCL or low-grade FL occurred in approximately half of the G3BFL cases. With a median of 5 years follow-up, the overall survival and progression-free survival of G3BFL patients was better than that of DLBCL patients (P<0.001 and P<0.001, respectively); however, G3BFL patients were younger (P<0.001) with better performance status (P<0.001), less extranodal disease (P<0.001) and more frequently had normal lactate dehydrogenase (P<0.001) at baseline. The overall and progression-free survival of patients with G3BFL and G3AFL were similar (P=0.83 and P=0.80, respectively). After frontline immunochemotherapy, 24% of G3BFL relapsed; relapse rates were 63% in the DLBCL cohort and 19% in the low-grade FL cohort. Eight percent of relapses occurred beyond 5 years. In this G3BFL cohort, the revised International Prognostic Index successfully delineated risk groups, but the Follicular Lymphoma International Prognostic Index did not. We conclude that patients with immunochemotherapy-treated G3BFL have similar survival outcomes to those with G3AFL, yet a favorable baseline profile and distinctly superior prognosis compared to patients with DLBCL.

MRI ischemic and hemorrhagic lesions in arterial and venous territories characterize central nervous system intravascular lymphoma in dogs.

Intravascular lymphoma (IVL) is characterized by the proliferation of large malignant lymphocytes within the lumen of blood vessels. This retrospective, multi-center, case series study aimed to describe the MRI features of confirmed central nervous system IVL in dogs and compare them with histopathological findings. Medical record databases from seven veterinary centers were searched for cases of histologically confirmed IVL. Dogs were included if an MRI was performed. The MRI studies and histopathology samples were reviewed to compare the MRI changes with the histopathological findings. Twelve dogs met the inclusion criteria (12 brains and three spinal cords). Imaging of the brains revealed multifocal T2-weighted/FLAIR hyperintense and T1-weighted iso-hypointense lesions, with variable contrast enhancement; areas of abnormal diffusion both in arterial and venous territories in diffusion-weighted imaging; and meningeal enhancement. On gradient echo images (GRE), the changes comprised tubular susceptibility artifacts, consistent with the "susceptibility vessel sign", and additional variably sized/shaped intraparenchymal susceptibility artifacts. Spinal cord lesions presented as fusiform T2-weighted hyperintensities with scattered susceptibility artifacts on GRE and variable parenchymal and meningeal contrast enhancement. On histopathology, subarachnoid hemorrhages and neuroparenchymal areas of edema and necrosis, with or without hemorrhage, indicating ischemic and hemorrhagic infarctions, were found. These lesions were concurrent with severely dilated meningeal and parenchymal arteries and veins plugged by neoplastic lymphocytes and fibrin. Due to the unique angiocentric distribution of IVL, ischemic and hemorrhagic infarcts of variable chronicity affecting both the arterial and venous territories associated with thrombi formation can be detected on MRI.

Clinical Nurses' View of Staffing During the Pandemic.

The COVID-19 pandemic presented staffing challenges in providing care during the surge of critically ill patients. This qualitative descriptive study was conducted to obtain an understanding of clinical nurses' perspective of staffing in units during the first wave of the pandemic. Eighteen focus groups were conducted with registered nurses who worked on intensive care, telemetry, or medical-surgical units at 9 acute care hospitals. The focus group transcripts were thematically analyzed to identify codes and themes. The overarching theme was staffing, a bit of a mess, which sums up the general perception of nurses during the initial phase of the pandemic. The following additional themes underscore the overarching theme: challenging physical work environment; supplementing the frontline: buddies, helpers, runners, agency, and travel nurses; nurses do everything; getting through as a team; and emotional toll. Nurse leaders can utilize these findings to guide staffing decisions today and in the future, such as ensuring nurses are oriented to their deployed unit, keeping team members together when reassigned, and striving for consistency with staffing. Learning from the experience of clinical nurses who worked during this unprecedented time will assist in improving nurse and patient outcomes.

Determining the efficacy of the bovine amniotic membrane homogenate during the healing process in rabbits' ex vivo corneas.

OBJECTIVE: To evaluate the efficacy of bovine amniotic membrane homogenate (BAMH) on wounded ex vivo rabbit corneas. PROCEDURE: Eighteen corneas obtained from normal rabbit eyes were wounded equally using a 6 mm trephine and cultured into an air-liquid interface model. Corneas were treated with phosphate-buffered saline (PBS) (n = 6, control group), 0.2% ethylenediaminetetraacetic acid (EDTA; n = 6), or BAMH (n = 6). All treatments were applied topically 6 times/day. Each cornea was macrophotographed daily with and without fluorescein stain to assess epithelialization and haziness. After 7 days, corneal transparency was evaluated, and the tissues prepared for histologic analysis of viability, and total and epithelial thickness. RESULTS: The mean epithelialization time was 6.2 ± 0.82 days for the control group, 6.2 ± 0.75 days for the EDTA-treated group, and 5.1 ± 0.40 days for the BAMH-treated group, demonstrating a significant difference between the BAMH and the other groups. The corneas that received EDTA had better transparency compared with the other groups. Histologically, all corneas had adequate morphology and architecture after healing. Analysis of corneal and epithelial thickness revealed no significant difference among groups. CONCLUSION: Bovine amniotic membrane homogenate is an effective and promising treatment for stromal and epithelial ulcers.

Determining MMP-2 and MMP-9 reductive activities of bovine and equine amniotic membranes homogenates using fluorescence resonance energy transfer.

INTRODUCTION: Matrix metalloproteinases (MMPs)-2 and -9 are present in corneal ulcers, and an imbalance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) leads to further corneal degradation. Amniotic membrane homogenate (AMH) has proteolytic properties beneficial for corneal healing, but it is unknown whether AMH possesses TIMPs or effectively inhibits MMP-2 and MMP-9 activity. OBJECTIVE: To determine if bovine and equine AMH reduce in vitro MMP-2 and MMP-9 activities associated with the presence of TIMPs. PROCEDURES: Undiluted and diluted twofold series (0-fold to 16-fold dilutions) of equine amniotic membrane homogenates (EAMH, n = 8) and bovine amniotic membrane homogenates (BAMH, n = 8) were subjected to fluorescence resonance energy transfer, and the fluorescence emitted was recorded over time. Average fluorescence was calculated versus recombinant concentration. Enzyme-linked immunosorbent assays for TIMPs 1-4 were applied to quantify TIMPs in the samples. RESULTS: AMH from both species were able to inhibit MMP-2 and MMP-9 activities in vitro, and the inhibition efficacy decreased gradually with dilution. BAMH was significantly more effective than EAMH at inhibiting MMP-2 and MMP-9 in vitro. TIMPs -2 and -3 were present in EAMH and BAMH. TIMP-1 was detected only in BAMH, and TIMP-4 was not detected in any samples. CONCLUSION: Both EAMH and BAMH directly inhibited MMP-2 and MMP-9 in vitro without dilution, and BAMH showed better inhibition of MMP-2 and MMP-9 before and after dilution compared to EAMH.

Osmolarity of basal and reflex tears of normal dogs.

OBJECTIVE: To compare basal and reflex tear osmolarity in healthy dogs and to evaluate for correlation among tear film osmolarity, tear production, and tear fern pattern. ANIMALS STUDIED: The population consisted of 22 healthy adult dogs. PROCEDURES: Reflex tear osmolarity was measured in both eyes using the I-PEN®  VET osmometer 30 minutes following the Schirmer tear test (STT-1) measurement. Subsequently, two minutes following topical anesthetic application, the lacrimal lake and conjunctival fornices were dried, and 3 minutes later, basal tear osmolarity was measured. Tears were extracted from the dye-free STT-1 strip by centrifugation, placed on a glass slide, and the ferning pattern was determined by light microscopy. Comparisons between basal and reflex tears were performed with one-way ANOVA. Correlations between tear osmolarity, STT-1, and tear ferning were verified by Pearson's correlation coefficient. RESULTS: No statistical difference was found between right and left eyes for STT-1, tear fern pattern, and osmolarity of reflex or basal tears (P < .05). There was no statistical difference between osmolarity of basal and reflex tears among individuals (P < .05). No correlation was detected between tear fern pattern and osmolarity of reflex tears. A weak positive correlation was detected between STT-1 values and osmolarity of reflex, but not basal tears. CONCLUSIONS: The osmolarity of basal tears did not differ from that of reflex tears. A positive weak correlation exists between tear production and osmolarity of reflex tears. No correlation was detected between tear osmolarity measured by handheld osmometer and tear ferning.

Compassion fatigue and the ART model.

PURPOSE: To understand perceptions of compassion fatigue (CF), including compassion satisfaction, burnout, and work-related secondary traumatic stress in a random sampling of nurses from the authors' healthcare facility. METHODS: The Professional Quality-of-Life (ProQOL) nurse survey was used to measure the positive and negative aspects of caring and to identify elements of CF. Quantitative data were analyzed using descriptive statistics and statistical software, as well as a thematic analysis to identify themes in the interview transcripts. RESULTS: The level of compassion satisfaction was considered "average" by 76% of participants, suggesting that they were likely to derive pleasure from their work. Conversely, burnout and secondary traumatic stress were reported as average-to-low in more than 90% of the nurses. ProQOL scores reflected positive participant feelings about their ability to be effective in their work and indicated that they do not consider their work environment frightening. CONCLUSION: Providing nurses the tools, education, and support to implement self-care practices on the unit can help reduce CF.

Three-dimensional T1-weighted gradient echo is a suitable alternative to two-dimensional T1-weighted spin echo for imaging the canine brain.

Volumetric imaging (VOL), a three-dimensional magnetic resonance imaging (MRI) technique, has been described in the literature for evaluation of the human brain. It offers several advantages over conventional two-dimensional (2D) spin echo (SE), allowing rapid, whole-brain, isotropic imaging with submillimeter voxels. This retrospective, observational study compares the use of 2D T1-weighted SE (T1W SE), with T1W VOL, for the evaluation of dogs with clinical signs of intracranial disease. Brain MRI images from 160 dogs who had T1W SE and T1W VOL sequences acquired pre- and postcontrast, were reviewed for presence and characteristics of intracranial lesions. Twenty-nine of 160 patients were found to have intracranial lesions, all visible on both sequences. Significantly better grey-white matter (GWM) differentiation was identified with T1W VOL (P < .001), with fair agreement between the two sequences (weighted κ = 0.35). Excluding a mild reduction in lesion intensity in three dogs precontrast on the T1W VOL images compared to T1W SE, and meningeal enhancement noted on the T1W VOL images in one dog, not identified on T1W SE, there was otherwise complete agreement between the two sequences. The T1W VOL sequence provided equivalent lesion evaluation and significantly improved GWM differentiation. Images acquired were of comparable diagnostic quality to those produced using a conventional T1W SE technique, for assessment of lesion appearance, number, location, mass effect, and postcontrast enhancement. T1W VOL, therefore, provides a suitable alternative T1W sequence for canine brain evaluation and can facilitate a reduction in total image acquisition time.

Primary small intestinal lymphangiosarcoma in a dog presenting with a segmental partial mesenteric volvulus.

A 3-year-old Great Dane presented with a history of chronic vomiting and diarrhoea. Abdominal computed tomography followed by exploratory laparotomy revealed a perforated, segmental partial mesenteric volvulus, affecting an abnormal section of distal jejunum, which was resected. Histopathology and immunohistochemistry results were consistent with jejunal lymphangiosarcoma. This case represents the first report of primary small intestinal lymphangiosarcoma in dogs and the importance of immunohistochemistry for definitive diagnosis.

Accuracy of radiographic diagnosis of pneumoperitoneum secondary to gastrointestinal perforation in dogs and cats.

BACKGROUND: Radiography is commonly included in the diagnostic investigation of animals with suspected gastrointestinal perforation and resultant pneumoperitoneum. This study aimed to (1) identify the accuracy of radiographic diagnosis of pneumoperitoneum and (2) determine if observer experience affected accuracy. METHODS: This was a retrospective case-controlled study evaluating abdominal radiographs of dogs and cats with surgical confirmation of gastrointestinal perforation or peritonitis without pneumoperitoneum. Radiographs were reviewed by a radiologist, an imaging resident, a general practitioner and a veterinary student. RESULTS: Radiographs from 60 dogs and eight cats were evaluated; 34 animals had confirmed perforation. Accuracy was fair to excellent for all observers: student 61.8%, general practitioner 70.6%, resident 85.3% and radiologist 83.8%. There was no significant difference in sensitivity between observers (all 70.6%-85.3%); however, there was a significant difference in specificity between both the resident (91.2%) and radiologist (91.2%) compared to the student (52.9%) (p < 0.001) and the general practitioner (55.9%) (p = 0.002). Overall, there was slight to substantial agreement between observers (κ = 0.28-0.73), with the highest being between the radiologist and resident. CONCLUSION: Abdominal radiography, when interpreted by veterinarians with differing experience, has variable accuracy for diagnosis of pneumoperitoneum for animals in this study. Further studies are needed to determine if these results are applicable for other populations of animals with pneumoperitoneum.

Proteome Composition of Bovine Amniotic Membrane and Its Potential Role in Corneal Healing.

Purpose: To investigate the protein profile of bovine amniotic membranes (bAM) and to determine putative associations between protein composition in bAM and known corneal healing pathways. Methods: The bAM were acquired from normal full-term births (n = 10), processed, and stored at -80°C for two days. Subsequently, the frozen membranes were thawed at room temperature and prepared for proteomic exploration using high-resolution liquid chromatography-mass spectrometry, followed by bioinformatics analysis. Recently identified corneal healing pathways were contrasted with protein profiles and pathways present in bAM. Results: The analyses identified 2105 proteins, with an interactive network of 1271 nodes (proteins) and 8757 edges (interactions). The proteins with higher betweenness centrality measurements include microfibril-associated protein 4, HSD3B1, CAPNS1, ATP1B3, CAV1, ANXA2, YARS, and GAPDH. The top four pathways in Kyoto Encyclopedia of Genes and Genomes were ribosome, metabolic pathway, spliceosome, and oxidative phosphorylation. The bAM and cornea shared abundant proteins, genome ontology, and signaling pathways. Conclusions: The high-throughput proteomic profile of the bAM demonstrated that numerous proteins present in the cornea are also present in this fetal membrane. Our findings collectively demonstrate the similarity between bAM and the cornea's protein composition, supporting our hypothesis that bAM can be used to treat corneal diseases.

DDB2 Genetic Risk Factor for Ocular Squamous Cell Carcinoma Identified in Three Additional Horse Breeds.

Squamous cell carcinoma (SCC) is the most common cancer affecting the equine eye. A missense variant within the gene damage-specific DNA binding protein 2 (DDB2 c.1013C>T, p.Thr338Met) was previously identified as a causal recessive genetic risk factor for the development of ocular SCC within Haflingers, Belgian Draft horses, and Rocky Mountain Horses, but not in the Appaloosa or Arabian breeds. This study aimed to evaluate three cases of ocular SCC in additional breeds and determine if DNA testing for the DDB2 variant in warmblood horses and Connemara ponies is warranted. Histopathology confirmed ocular SCC in all three cases and DNA testing confirmed each horse was homozygous for the DDB2 risk factor. The DDB2 risk allele frequency was estimated to be 0.0043 for Holsteiners (N = 115), 0.014 for Belgian Warmbloods (N = 71), and 0.22 for Connemara Ponies (N = 86). Taken together these data support using DNA testing for DDB2 in Connemara Ponies to assist in mate selection and clinical management. Given the low observed allele frequencies in both the Holsteiner and Belgian Warmblood breeds and that the case under investigation was a warmblood cross-bred, evaluating additional SCC affected warmbloods is warranted to fully determine the importance of DDB2 genotyping as a risk factor in warmblood breeds.

Ocular Surface Disease in Reptiles.

In this article, ocular examination techniques are reviewed in the context of the reptilian patient. Subsequently, ocular surface anatomy and diseases are discussed. Sections for 4 of the 5 Reptilia orders are included: lizards, chelonians, crocodilians, and snakes. Literature pertaining to both wild and captive animals is included.

A Case of Lenvatinib-Induced Focal Segmental Glomerulosclerosis (FSGS) in Metastatic Medullary Thyroid Cancer.

We describe a case of lenvatinib (E7080) associated focal segmental glomerulosclerosis (FSGS) encountered during the treatment of metastatic medullary thyroid cancer. Proteinuria is a relatively common side effect of VEGF-targeted treatments and can occasionally result in treatment discontinuation. Here, we describe a patient who developed secondary FSGS necessitating discontinuation of treatment at first but who was subsequently rechallenged with anti-VEGF-targeted treatment without recurrence of proteinuria. Lenvatinib was a novel experimental agent at the time the treatment took place; however, its recent licensing for the treatment of thyroid malignancies in the UK makes reporting of these adverse effects all the more important now.