Hematopoietic stem cell gene editing rescues B-cell development in X-linked agammaglobulinemia.

BACKGROUND: X-linked agammaglobulinemia (XLA) is an inborn error of immunity that renders boys susceptible to life-threatening infections due to loss of mature B cells and circulating immunoglobulins. It is caused by defects in the gene encoding the Bruton tyrosine kinase (BTK) that mediates the maturation of B cells in the bone marrow and their activation in the periphery. This paper reports on a gene editing protocol to achieve "knock-in" of a therapeutic BTK cassette in hematopoietic stem and progenitor cells (HSPCs) as a treatment for XLA. METHODS: To rescue BTK expression, this study employed a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system that creates a DNA double-strand break in an early exon of the BTK locus and an adeno-associated virus 6 virus that carries the donor template for homology-directed repair. The investigators evaluated the efficacy of the gene editing approach in HSPCs from patients with XLA that were cultured in vitro under B-cell differentiation conditions or that were transplanted in immunodeficient mice to study B-cell output in vivo. RESULTS: A (feeder-free) B-cell differentiation protocol was successfully applied to blood-mobilized HSPCs to reproduce in vitro the defects in B-cell maturation observed in patients with XLA. Using this system, the investigators could show the rescue of B-cell maturation by gene editing. Transplantation of edited XLA HSPCs into immunodeficient mice led to restoration of the human B-cell lineage compartment in the bone marrow and immunoglobulin production in the periphery. CONCLUSIONS: Gene editing efficiencies above 30% could be consistently achieved in human HSPCs. Given the potential selective advantage of corrected cells, as suggested by skewed X-linked inactivation in carrier females and by competitive repopulating experiments in mouse models, this work demonstrates the potential of this strategy as a future definitive therapy for XLA.

Extracellular vesicles as a promising source of lipid biomarkers for breast cancer detection in blood plasma.

Extracellular vesicles (EVs), including exosomes and microvesicles, mediate intercellular communication in cancer, from development to metastasis. EV-based liquid biopsy is a promising strategy for cancer diagnosis as EVs can be found in cancer patients' body fluids. In this study, the lipid composition of breast cancer-derived EVs was studied as well as the potential of blood plasma EVs for the identification of lipid biomarkers for breast cancer detection. Initially, an untargeted lipidomic analysis was carried out for a panel of cancerous and non-cancerous mammary epithelial cells and their secreted EVs. We found that breast cancer-derived EVs are enriched in sphingolipids and glycerophospholipids compared to their parental cells. The initial in vitro study showed that EVs and their parental cells can be correctly classified (100% accuracy) between cancerous and non-cancerous, as well as into their respective breast cancer subtypes, based on their lipid composition. Subsequently, an untargeted lipidomic analysis was carried out for blood plasma EVs from women diagnosed with breast cancer (primary or progressive metastatic breast cancer) as well as healthy women. Correspondingly, when blood plasma EVs were analysed, breast cancer patients and healthy women were correctly classified with an overall accuracy of 93.1%, based on the EVs' lipid composition. Similarly, the analysis of patients with primary breast cancer and healthy women showed an overall accuracy of 95% for their correct classification. Furthermore, primary and metastatic breast cancers were correctly classified with an overall accuracy of 89.5%. This reveals that the blood plasma EVs' lipids may be a promising source of biomarkers for detection of breast cancer. Additionally, this study demonstrates the usefulness of untargeted lipidomics in the study of EV lipid composition and EV-associated biomarker discovery studies. This is a proof-of-concept study and a starting point for further analysis on the identification of EV-based biomarkers for breast cancer.

Wide-range soft anisotropic thermistor with a direct wireless radio frequency interface.

Temperature sensors are one of the most fundamental sensors and are found in industrial, environmental, and biomedical applications. The traditional approach of reading the resistive response of Positive Temperature Coefficient thermistors at DC hindered their adoption as wide-range temperature sensors. Here, we present a large-area thermistor, based on a flexible and stretchable short carbon fibre incorporated Polydimethylsiloxane composite, enabled by a radio frequency sensing interface. The radio frequency readout overcomes the decades-old sensing range limit of thermistors. The composite exhibits a resistance sensitivity over 1000 °C-1, while maintaining stability against bending (20,000 cycles) and stretching (1000 cycles). Leveraging its large-area processing, the anisotropic composite is used as a substrate for sub-6 GHz radio frequency components, where the thermistor-based microwave resonators achieve a wide temperature sensing range (30 to 205 °C) compared to reported flexible temperature sensors, and high sensitivity (3.2 MHz/°C) compared to radio frequency temperature sensors. Wireless sensing is demonstrated using a microstrip patch antenna based on a thermistor substrate, and a battery-less radio frequency identification tag. This radio frequency-based sensor readout technique could enable functional materials to be directly integrated in wireless sensing applications.

Multidimensional Response Surface Methodology for the Development of a Gene Editing Protocol for p67phox-Deficient Chronic Granulomatous Disease.

Replacing a faulty gene with a correct copy has become a viable therapeutic option as a result of recent progress in gene editing protocols. Targeted integration of therapeutic genes in hematopoietic stem cells has been achieved for multiple genes using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system and Adeno-Associated Virus (AAV) to carry a donor template. Although this is a promising strategy to correct genetic blood disorders, it is associated with toxicity and loss of function in CD34+ hematopoietic stem and progenitor cells, which has hampered clinical application. Balancing the maximum achievable correction against deleterious effects on the cells is critical. However, multiple factors are known to contribute, and the optimization process is laborious and not always clearly defined. We have developed a flexible multidimensional Response Surface Methodology approach for optimization of gene correction. Using this approach, we could rapidly investigate and select editing conditions for CD34+ cells with the best possible balance between correction and cell/colony-forming unit (CFU) loss in a parsimonious one-shot experiment. This method revealed that using relatively low doses of AAV2/6 and CRISPR/Cas9 ribonucleoprotein complex, we can preserve the fitness of CD34+ cells and, at the same time, achieve high levels of targeted gene insertion. We then used these optimized editing conditions for the correction of p67phox-deficient chronic granulomatous disease (CGD), an autosomal recessive disorder of blood phagocytic cells resulting in severe recurrent bacterial and fungal infections and achieved rescue of p67phox expression and functional correction of CD34+-derived neutrophils from a CGD patient.

Genomic investigations of unexplained acute hepatitis in children.

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children.

Genome Editing With TALEN, CRISPR-Cas9 and CRISPR-Cas12a in Combination With AAV6 Homology Donor Restores T Cell Function for XLP.

X-linked lymphoproliferative disease is a rare inherited immune disorder, caused by mutations or deletions in the SH2D1A gene that encodes an intracellular adapter protein SAP (Slam-associated protein). SAP is essential for mediating several key immune processes and the immune system - T cells in particular - are dysregulated in its absence. Patients present with a spectrum of clinical manifestations, including haemophagocytic lymphohistiocytosis (HLH), dysgammaglobulinemia, lymphoma and autoimmunity. Treatment options are limited, and patients rarely survive to adulthood without an allogeneic haematopoietic stem cell transplant (HSCT). However, this procedure can have poor outcomes in the mismatched donor setting or in the presence of active HLH, leaving an unmet clinical need. Autologous haematopoeitic stem cell or T cell therapy may offer alternative treatment options, removing the need to find a suitable donor for HSCT and any risk of alloreactivity. SAP has a tightly controlled expression profile that a conventional lentiviral gene delivery platform may not be able to fully replicate. A gene editing approach could preserve more of the endogenous regulatory elements that govern SAP expression, potentially providing a more optimum therapy. Here, we assessed the ability of TALEN, CRISPR-Cas9 and CRISPR-Cas12a nucleases to drive targeted insertion of SAP cDNA at the first exon of the SH2D1A locus using an adeno-associated virus serotype 6 (AAV6)-based vector containing the donor template. All nuclease platforms were capable of high efficiency gene editing, which was optimised using a serum-free AAV6 transduction protocol. We show that T cells from XLP patients corrected by gene editing tools have restored physiological levels of SAP gene expression and restore SAP-dependent immune functions, indicating a new therapeutic opportunity for XLP patients.

Delay to elective colorectal cancer surgery and implications for survival: a systematic review and meta-analysis.

AIM: The Covid-19 pandemic has delayed elective colorectal cancer (CRC) surgery. The aim of this study was to see whether or not this may affect overall survival (OS) and disease-free survival (DFS). METHOD: A systematic review was carried out according to PRISMA guidelines (PROSPERO ID: CRD42020189158). Medline, EMBASE and Scopus were interrogated. Patients aged over 18 years with a diagnosis of colon or rectal cancer who received elective surgery as their primary treatment were included. Delay to elective surgery was defined as the period between CRC diagnosis and the day of surgery. Meta-analysis of the outcomes OS and DFS were conducted. Forest plots, funnel plots and tests of heterogeneity were produced. An estimated number needed to harm (NNH) was calculated for statistically significant pooled hazard ratios (HRs). RESULTS: Of 3753 articles identified, seven met the inclusion criteria. Encompassing 314 560 patients, three of the seven studies showed that a delay to elective resection is associated with poorer OS or DFS. OS was assessed at a 1 month delay, the HR for six datasets was 1.13 (95% CI 1.02-1.26, p = 0.020) and at 3 months the pooled HR for three datasets was 1.57 (95% CI 1.16-2.12, p = 0.004). The estimated NNH for a delay at 1 month and 3 months was 35 and 10 respectively. Delay was nonsignificantly negatively associated with DFS on meta-analysis. CONCLUSION: This review recommends that elective surgery for CRC patients is not postponed longer than 4 weeks, as available evidence suggests extended delays from diagnosis are associated with poorer outcomes. Focused research is essential so patient groups can be prioritized based on risk factors in future delays or pandemics.

Molecular imaging of extracellular vesicles in vitro via Raman metabolic labelling.

Extracellular vesicles (EVs) are biologically-derived nanovectors important for intercellular communication and trafficking. As such, EVs show great promise as disease biomarkers and therapeutic drug delivery vehicles. However, despite the rapidly growing interest in EVs, understanding of the biological mechanisms that govern their biogenesis, secretion, and uptake remains poor. Advances in this field have been hampered by both the complex biological origins of EVs, which make them difficult to isolate and identify, and a lack of suitable imaging techniques to properly study their diverse biological roles. Here, we present a new strategy for simultaneous quantitative in vitro imaging and molecular characterisation of EVs in 2D and 3D based on Raman spectroscopy and metabolic labelling. Deuterium, in the form of deuterium oxide (D2O), deuterated choline chloride (d-Chol), or deuterated d-glucose (d-Gluc), is metabolically incorporated into EVs through the growth of parent cells on medium containing one of these compounds. Isolated EVs are thus labelled with deuterium, which acts as a bio-orthogonal Raman-active tag for direct Raman identification of EVs when introduced to unlabelled cell cultures. Metabolic deuterium incorporation demonstrates no apparent adverse effects on EV secretion, marker expression, morphology, or global composition, indicating its capacity for minimally obstructive EV labelling. As such, our metabolic labelling strategy could provide integral insights into EV biocomposition and trafficking. This approach has the potential to enable a deeper understanding of many of the biological mechanisms underpinning EVs, with profound implications for the design of EVs as therapeutic delivery vectors and applications as disease biomarkers.

Gold Nanocluster Extracellular Vesicle Supraparticles: Self-Assembled Nanostructures for Three-Dimensional Uptake Visualization.

Extracellular vesicles (EVs) are secreted by the vast majority of cells and are being intensively studied due to their emerging involvement in a variety of cellular communication processes. However, the study of their cellular uptake and fate has been hampered by difficulty in imaging EVs against the cellular background. Here, we show that EVs combined with hydrophobic gold nanoclusters (AuNCs) can self-assemble into supraparticles, offering an excellent labeling strategy for high-resolution electron microscopic imaging in vitro. We have tracked and visualized the reuptake of breast cancer cell-derived EV AuNC supraparticles into their parent cells, from early endocytosis to lysosomal degradation, using focused ion beam-scanning electron microscopy (FIB-SEM). The presence of gold within the EVs and lysosomes was confirmed via DF-STEM EDX analysis of lift-out sections. The demonstrated formation of AuNC EV supraparticles will facilitate future applications in EV imaging as well as the EV-assisted cellular delivery of AuNCs.

Glycosaminoglycan-based biomaterials for growth factor and cytokine delivery: Making the right choices.

Controlled, localized drug delivery is a long-standing goal of medical research, realization of which could reduce the harmful side-effects of drugs and allow more effective treatment of wounds, cancers, organ damage and other diseases. This is particularly the case for protein "drugs" and other therapeutic biological cargoes, which can be challenging to deliver effectively by conventional systemic administration. However, developing biocompatible materials that can sequester large quantities of protein and release them in a sustained and controlled manner has proven challenging. Glycosaminoglycans (GAGs) represent a promising class of bio-derived materials that possess these key properties and can additionally potentially enhance the biological effects of the delivered protein. They are a diverse group of linear polysaccharides with varied functionalities and suitabilities for different cargoes. However, most investigations so far have focused on a relatively small subset of GAGs - particularly heparin, a readily available, promiscuously-binding GAG. There is emerging evidence that for many applications other GAGs are in fact more suitable for regulated and sustained delivery. In this review, we aim to illuminate the beneficial properties of various GAGs with reference to specific protein cargoes, and to provide guidelines for informed choice of GAGs for therapeutic applications.

Vision loss with pembrolizumab treatment: A report of two cases.

BACKGROUND: Vision loss and other ocular toxicities are rare, but detrimental, side effects of immune checkpoint inhibitors. Herein, we report two patients who developed vision loss while on pembrolizumab treatment. CASES PRESENTATION: Case 1 - A 58-year-old man was started on pembrolizumab for advanced melanoma. He was tolerating the treatment well. After receiving 14 cycles of pembrolizumab, he developed acute bilateral vision loss and occipital headaches. An emergent ophthalmologic evaluation revealed bilateral shallow choroidal effusion with bilateral focal exudative retinal detachment. After excluding other possible etiologies, inflammatory process secondary to pembrolizumab was suspected. Pembrolizumab was stopped, and the patient was started on a course of systemic and topical steroids. His vision improved within days and he recovered completely within two months. Calculated Naranjo Nomogram score was 7 indicating a "probable" correlation; Case 2 - A 57-year-old man with stage IIIC melanoma was started on adjuvant pembrolizumab. After a few weeks of treatments, he reported minor bilateral vision changes that progressively worsened over a period of six months. An ophthalmologic evaluation revealed bilateral posterior uveitis with right optic disc edema. Pembrolizumab-related inflammatory changes were suspected, and he was started on systemic and topical steroids. His symptoms improved within a few weeks and steroids were tapered. He was re-challenged with pembrolizumab and his symptoms quickly re-occurred. Pembrolizumab was stopped indefinitely and the patient again treated with systemic and topical steroids. His symptoms resolved and his vision returned to baseline within two months. The Naranjo Nomogram score was 9 indicating a "definite" correlation. CONCLUSIONS: Vision loss is a serious complication that may occur at any point during treatment with PD-1 inhibitors. Vision loss is very distressing to the patients and their families. It is prudent for practitioners to recognize early vision abnormalities in patients receiving PD-1 antagonists to prevent permanent vision loss.

Bisphosphonate-induced orbital inflammation in a patient on chronic immunosuppressive therapy.

BACKGROUND: To report a case of orbital inflammation after bisphosphonate infusion in a patient who was already receiving immunosuppressive therapy. CASE PRESENTATION: A 56-year-old woman presented to the ophthalmology clinic with acute onset of right eye pain 24 h after receiving her first Zolendronic acid infusion. She has a past medical history of chronic inflammatory demyelinating polyneuropathy, Sjogren's syndrome, and systemic lupus erythematosus that have been controlled with immunosuppressive therapy for three years. Clinical ophthalmic exam and MRI studies were significant for right orbital inflammation. The patient was started on oral prednisone with rapid resolution of symptoms. CONCLUSIONS: This is the first case report of a patient receiving chronic immunosuppressive therapy to develop orbital inflammation after Zoledronic acid infusion. In addition, it demonstrates that corticosteroids can be an effective first line therapy in treating orbital inflammation in similar patients. Physicians should be aware of this rare but serious potential side effect of bisphosphonates, and have bisphosphonate-related orbital inflammation on their differential for proper initiation of treatment.

Deep lateral wall orbital decompression following strabismus surgery in patients with Type II ophthalmic Graves' disease.

PURPOSE: Surgical management of ophthalmic Graves' disease traditionally involves, in order, orbital decompression, followed by strabismus surgery and eyelid surgery. Nunery et al. previously described two distinct sub-types of patients with ophthalmic Graves' disease; Type I patients exhibit no restrictive myopathy (no diplopia) as opposed to Type II patients who do exhibit restrictive myopathy (diplopia) and are far more likely to develop new-onset worsening diplopia following medial wall and floor decompression. Strabismus surgery involving extra-ocular muscle recession has, in turn, been shown to potentially worsen proptosis. Our experience with Type II patients who have already undergone medial wall and floor decompression and strabismus surgery found, when additional decompression is necessary, deep lateral wall decompression (DLWD) appears to have a low rate of post-operative primary-gaze diplopia. METHODS: A case series of four Type II ophthalmic Graves' disease patients, all of whom had already undergone decompression and strabismus surgery, and went on to develop worsening proptosis or optic nerve compression necessitating further decompression thereafter. In all cases, patients were treated with DLWD. Institutional Review Board approval was granted by the University of Kansas. RESULTS: None of the four patients treated with this approach developed recurrent primary-gaze diplopia or required strabismus surgery following DLWD. CONCLUSIONS: While we still prefer to perform medial wall and floor decompression as the initial treatment for ophthalmic Graves' disease, for proptosis following consecutive strabismus surgery, DLWD appears to be effective with a low rate of recurrent primary-gaze diplopia.

A case series of dural venous sinus stenting in idiopathic intracranial hypertension: association of outcomes with optical coherence tomography.

Purpose/Aim: Pseudotumor cerebri or idiopathic intracranial hypertension (IIH) is characterized by increased intracranial pressure of unknown etiology. A subset of patients has shown benefit from endovascular dural venous sinus stenting (DVSS). We sought to identify a population of IIH patients who underwent DVSS to assess outcomes. MATERIALS AND METHODS: A retrospective study was performed to identify IIH patients with dural sinus stenosis treated with DVSS. Outcome measures included dural sinus pressure gradients, peripapillary retinal nerve fiber layer (RNFL) thickness using optical coherence tomography and improvement in symptoms. RESULTS: Seventeen patients underwent DVSS. Average pre- and post-intervention pressure gradients were 23.06 and 1.18 mmHg, respectively (p < 0.0001). Sixteen (94%) noted improvement in headache, fourteen (82%) had visual improvement and all (100%) patients had improved main symptom. Of 11 patients with optical coherence tomography, 8 showed decreased RNFL thickness and 3 remained stable; furthermore, these 11 patients had improved vision with improved papilledema in 8, lack of pre-existing papilledema in 2 and stable, mild edema in 1 patient. CONCLUSIONS: Our series of patients with dural sinus stenosis demonstrated improvement in vision and reduction in RNFL thickness. DVSS appears to be a useful treatment for IIH patients with dural sinus stenosis.

Farris-Tang retractor in optic nerve sheath decompression surgery.

PURPOSE: Our purpose is to introduce the use of the Farris-Tang retractor in optic nerve sheath decompression surgery. METHODS: The procedure of optic nerve sheath fenestration was reviewed at our tertiary care teaching hospital, including the use of the Farris-Tang retractor. RESULTS: Pseudotumor cerebri is a syndrome of increased intracranial pressure without a clear cause. Surgical treatment can be effective in cases in which medical therapy has failed and disc swelling with visual field loss progresses. Optic nerve sheath decompression surgery (ONDS) involves cutting slits or windows in the optic nerve sheath to allow cerebrospinal fluid to escape, reducing the pressure around the optic nerve. We introduce the Farris-Tang retractor, a retractor that allows for excellent visualization of the optic nerve sheath during this surgery, facilitating the fenestration of the sheath and visualization of the subsequent cerebrospinal fluid egress. Utilizing a medial conjunctival approach, the Farris-Tang retractor allows for easy retraction of the medial orbital tissue and reduces the incidence of orbital fat protrusion through Tenon's capsule. CONCLUSION: The Farris-Tang retractor allows safe, easy, and effective access to the optic nerve with good visualization in optic nerve sheath decompression surgery. This, in turn, allows for greater surgical efficiency and positive patient outcomes.

Relationship Between Age, Gender, and Race in Patients Presenting With Myasthenia Gravis With Only Ocular Manifestations.

BACKGROUND: The demographic associations among patients presenting with myasthenia gravis with only ocular manifestations (OMG) is not clear. METHODS: In this 5-center case series, we collected the race, gender, and age at diagnosis of patients diagnosed with myasthenia gravis who had no signs or symptoms of generalized myasthenia gravis (GMG). An a priori sample size calculation determined that 140 patients were required to accept that there was a ≤10-year difference in mean age (equivalence testing: power 90%, α = 0.05). Robust Bayesian analysis and linear regression were applied to evaluate whether age differed by gender or race. RESULTS: Of 433 patients included, 258 (60%) were men. Mean age among men was 57 years (SD = 19) and 52 years (SD = 21) among women. The 95% credible interval (CI) (Bayesian equivalent of confidence interval) was 0.8-8.7 years for mean age, and there was a 99.6% probability that the mean difference in age between sexes was <10 years. Race was documented in 376 (68 [18%] non-Caucasian). Caucasians were 17.3 years older than non-Caucasians at diagnosis (95% CI, 12.2-22.3 y; P < 0.001) controlling for gender. There was no additive interaction of gender and race (P = 0.74). There was a bimodal distribution for women peaking around 30 and 60 years. Men had a left skewed unimodal age distribution peaking at age 70. CONCLUSIONS: The distribution of age at presentation in patients with OMG is different between men and women, similar to GMG. Non-Caucasian patients tend to develop OMG at a younger age.

Cytokine profiles in clinical subtypes of ophthalmic Graves' disease.

PURPOSE: To examine the association of cytokines in the two clinical subtypes of ophthalmic Graves' disease by comparing cytokine expression in the fat and ethmoid tissue of type I and type II patients. METHODS: Patients needing orbital decompression or eyelid surgery were identified and enrolled into a prospective study. Patients were assigned to the type I or type II subclassification, based on the presence of diplopia. Orbital fat, sinus tissue or muscle removed during surgery was evaluated. The mRNA expression profiles of Th1 cytokines (TNF-alpha/beta, IFN-gamma, IL-2) and Th2 cytokines (IL-4, IL-5, IL-6, IL-10) were analyzed using real time PCR. RESULTS: 30 patients were enrolled in the study: 5 type I (80% female), 14 type II (71% female) and 11 controls (73% female). There were 14 decompressions (3 type I and 11 type II), 17 lid procedures (2 type I, 4 type II and 11 controls) and 10 ethmoidectomies (3 type I and 7 type II). The average ages were 45, 56 and 66 in the type I, type II and control groups, respectively. There was more TNF-alpha (p value 0.009) and IL-6 (p value 0.04) in ethmoid sinus cells of type II patients compared to ethmoid sinus cells of type I patients and a trend of higher expression of all cytokines in type II patients. CONCLUSIONS: There is a trend towards greater mRNA expression of both Th1 and Th2 cytokines in both orbital fat and ethmoidal sinus tissue of type II patients compared to type I patients.