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CD38-targeting immunotherapy is approved in combination with lenalidomide and dexamethasone in patients with newly diagnosed multiple myeloma (NDMM) that are transplant ineligible (TI) and is considered the best standard of care (SOC). To improve current SOC, we evaluated the added value of weekly bortezomib (V) to isatuximab plus lenalidomide and dexamethasone (IsaRd versus Isa-VRd). This Intergroupe Francophone of Myeloma phase 3 study randomized 270 patients with NDMM that were TI, aged 65–79 years, to IsaRd versus Isa-VRd arms. The primary endpoint was a minimal residual disease (MRD) negativity rate at 10−5by next-generation sequencing at 18 months from randomization. Key secondary endpoints included response rates, MRD assessment rates, survival and safety. The 18-month MRD negativity rates at 10−5were reported in 35 patients (26%, 95% confidence interval (CI) 19–34) in IsaRd versus 71 (53%, 95% CI 44–61) in Isa-VRd (odds ratio for MRD negativity 3.16, 95% CI 1.89–5.28, P< 0.0001). The MRD benefit was consistent across subgroups at 10−5and 10−6, and was already observed at month 12. The proportion of patients with complete response or better at 18 months was higher with Isa-VRd (58% versus 33%; P< 0.0001), as was the proportion of MRD negativity and complete response or better (37% versus 17%; P= 0.0003). At a median follow-up of 23.5 months, no difference was observed for survival times (immature data). The addition of weekly bortezomib did not significantly affect the relative dose intensity of IsaRd. Isa-VRd significantly increased MRD endpoints, including the 18-month negativity rate at 10−5, the primary endpoint, compared with IsaRd. This study proposes Isa-VRd as a new SOC for patients with NDMM that are TI. ClinicalTrials.gov identifier: NCT04751877.

We report a Pd-catalyzed ring-opening/arylation/cyclization reaction sequence between 2-aminothiazoles and aryl (pseudo)halides that provides modular access to isocytosine analogues. The scope of the reaction is broad with respect to both coupling partners and a robustness test demonstrated the functional group tolerance of the methodology. Visual kinetic analysis revealed that the product may inhibit catalyst turnover for some substrates.

DDX41 germline mutations (DDX41MutGL) are the most common genetic predisposition to myelodysplastic syndrome and acute myeloid leukemia (AML). Recent reports suggest that DDX41MutGL myeloid malignancies could be considered as a distinct entity, even if their specific presentation and outcome remain to be defined. We describe here the clinical and biological features of 191 patients with DDX41MutGL AML. Baseline characteristics and outcome of 86 of these patients, treated with intensive chemotherapy in 5 prospective Acute Leukemia French Association/French Innovative Leukemia Organization trials, were compared with those of 1604 patients with DDX41 wild-type (DDX41WT) AML, representing a prevalence of 5%. Patients with DDX41MutGL AML were mostly male (75%), in their seventh decade, and with low leukocyte count (median, 2 × 109/L), low bone marrow blast infiltration (median, 33%), normal cytogenetics (75%), and few additional somatic mutations (median, 2). A second somatic DDX41 mutation (DDX41MutSom) was found in 82% of patients, and clonal architecture inference suggested that it could be the main driver for AML progression. DDX41MutGL patients displayed higher complete remission rates (94% vs 69%; P < .0001) and longer restricted mean overall survival censored at hematopoietic stem cell transplantation (HSCT) than 2017 European LeukemiaNet intermediate/adverse (Int/Adv) DDX41WT patients (5-year difference in restricted mean survival times, 13.6 months; P < .001). Relapse rates censored at HSCT were lower at 1 year in DDX41MutGL patients (15% vs 44%) but later increased to be similar to Int/Adv DDX41WT patients at 3 years (82% vs 75%). HSCT in first complete remission was associated with prolonged relapse-free survival (hazard ratio, 0.43; 95% confidence interval, 0.21-0.88; P = .02) but not with longer overall survival (hazard ratio, 0.77; 95% confidence interval, 0.35-1.68; P = .5).

ABSTRACTTarnishing is one of the most crucial issues in the conservation of silver alloy objects. This phenomenon is relevant in the field of cultural heritage artifacts: although the blackish patina protects to a degree against ulterior corrosion effects, it significantly affects the aesthetical features of the artwork. Several different approaches are utilized for cleaning tarnished silver–copper alloy surfaces, including mechanical, chemical, electrochemical methods. The use of lasers is also reported. In this paper a dry approach based on the use of erasers is proposed and compared with traditional methods. The different methods were applied on silver–copper alloy mockups which had been artificially tarnished. They were compared based on the degree of sulfide removal and damage to the silver surface. Diagnostic investigations showed that the use of erasers for silver cleaning resulted in fewer surface modifications compared to traditional mechanical methods with an effective tarnish removal. The proposed method could be broadly considered a low-cost, adjustable, and fast one-step cleaning procedure. Finally, two case studies of application of the technique are reported: cleaning of three Islamic artifacts decorated with silver alloy inlay and of the silver letters on the North Door of the Baptistery of Florence.

A screening method for the rapid identification of catalytic conditions for Pd-catalyzed C–N cross-coupling reactions is reported. The strategy evaluates mixtures of precatalysts, ligands, and bases to identify productive conditions that are subsequently optimized through two deconvolution steps, which uncover the active catalyst and identify the optimal solvent and base for the catalytic system. The efficacy of this approach was demonstrated through application to a previously reported reaction, whereby both the literature conditions and additional solutions were retrieved. The same approach to Ni-catalyzed C–N cross-coupling was investigated in parallel but was found to be less successful due to limited activity of the evaluated reagent combinations. Finally, the utility of this method was showcased by identifying effective conditions for the Pd-catalyzed cross-coupling of complex molecules, which not only revealed nonobvious solutions for the processes under evaluation but also resulted in the discovery of new chemical reactions.

The real-world efficacy and safety of gilteritinib was assessed in an ambispective study that included 167 R/R FLT3-mutated AML patients. Among them, 140 received gilteritinib as single agent (cohort B), including 67 previously treated by intensive chemotherapy and midostaurin (cohort C). The main differences in patient characteristics in this study compared to the ADMIRAL trial were ECOG ≥ 2 (83.6% vs. 16.6%), FLT3-TKD mutation (21.0% vs.8.5%), primary induction failure (15.0% vs.40.0%) and line of treatment (beyond 2nd in 37.1% vs.0.0%). The rates of composite complete remission, excluding those that occurred after hematopoietic stem cell transplantation (HSCT), were similar at respectively 25.4% and 27.5% in cohorts B and C. Median overall survival (OS) for these two groups was also similar at respectively 6.4 and 7.8 months. Multivariate analyses for prognostic factors associated with OS identified female gender (HR 1.61), adverse cytogenetic risk (HR 2.52), and allogenic HSCT after gilteritinib (HR 0.13). Although these patients were more heavily pretreated, these real-world data reproduce the results of ADMIRAL and provide new insights into the course of patients previously treated by intensive chemotherapy and midostaurin and beyond the 2nd line of treatment who can benefit from treatment in an outpatient setting.

The mouse line carrying the Tg(Tyr-NRAS*Q61K)1Bee transgene is widely used to model in vivo NRAS-driven melanomagenesis. Although the pathological features of this model are well described, classification and interpretation of the resulting proliferative lesions—including their origin, evolution, grading, and pathobiological significance—are still unclear and not supported by molecular and biological evidence. Focusing on their classification and grading, this work combines histopathology and expression analysis (using both immunohistochemistry [IHC] and quantitative PCR) of selected biomarkers to study the full spectrum of cutaneous and lymph nodal melanocytic proliferations in the Tg(Tyr-NRAS*Q61K)1Bee mouse. The analysis of cutaneous and lymph nodal melanocytic proliferations has demonstrated that a linear correlation exists between tumor grade and Ki-67, microphthalmia-associated transcription factor (MITF), gp100, and nestin IHC, with a significantly increased expression in high-grade lesions compared with low-grade lesions. The accuracy of the assessment of MITF IHC in melanomas was also confirmed by quantitative PCR analysis. In conclusion, we believe the incorporation of MITF, Ki-67, gp100, and nestin analysis into the histopathological classification/grading scheme of melanocytic proliferations described for this model will help to assess with accuracy the nature and evolution of the phenotype, monitor disease progression, and predict response to experimental treatment or other preclinical manipulations.

Haemophilia A and B are X-linked hemorrhagic disorders caused by gene variants in the F8and F9genes. Due to recessive inheritance, males are affected, while female carriers are usually asymptomatic with a wide range of factor VIII (FVIII) or IX (FIX) levels. Bleeding tendency in female carriers is extremely variable and may be associated with low clotting factor levels. This could be explained by F8or F9genetic variations, numerical or structural X chromosomal anomalies, or epigenetic variations such as irregular X chromosome inactivation (XCI). The aim of the study was to determine whether low FVIII or FIX coagulant activity in haemophilia carriers could be related to XCI and bleeding symptoms. HUMARA assay was performed on 73 symptomatic carriers with low clotting activity =50 IU/dL. Bleeding Assessment Tool (BAT) from the International Society on Thrombosis and Haemostasis (ISTH) was used to describe symptoms in the cohort of carriers. In 97% of haemophilia carriers, a specific gene variant in heterozygous state was found, which alone could not justify their low FVIII or FIX levels (=50 IU/dL). A statistical association between XCI pattern and FVIII and FIX levels was observed. Moreover, female carriers with low coagulant activity (=20 IU/dL) and high degree of XCI (?=?80:20) had a higher ISTH-BAT score than the carriers with the opposite conditions (>20 IU/dL and <80:20). In our cohort of haemophilia carriers, XCI was significantly skewed, which may contribute to the low expression of clotting factor levels and bleeding symptoms.

A general method for the quick identification of effective catalytic systems for copper-catalyzed C–N cross-couplings is described. This is based on evaluating mixtures of copper sources, ancillary ligands, and bases in different solvents followed by two deconvolution procedures, which aim at identifying the most proper reagent combination in only three distinct steps. Despite being a high-throughput approach in nature, the proposed method utilizes only frugal technological platforms such as 24-well microplates while offering a screening efficiency—the number of executed experiments vsthe total number of possible experiments—higher than 95%. To facilitate visualization and mining of the high-throughput experimentation (HTE) data, Visual Basic scripts have been developed, which allow streamlining the extraction of raw HPLC data into TIBCO Spotfire for the graphical display in the form of pie charts. The unique capabilities of this “pool and split” approach have been demonstrated by applying it to literature known cross-coupling reactions. In every case, the described experimental setup was validated by retrieving the original literature conditions in addition to exposing several additional solutions with a minimum number of parallel experiments. Finally, examples are provided for the successful application of this HTE screening workflow to internal projects.

The STI571 prospective randomised trial (SPIRIT) French trial is a four-arm study comparing imatinib (IM) 400 mg versus IM 600 mg, IM 400 mg + cytarabine (AraC), and IM 400 mg + pegylated interferon alpha2a (PegIFN-α2a) for the front-line treatment of chronic-phase chronic myeloid leukaemia (CML). Long-term analyses included overall and progression-free survival, molecular responses to treatment, and severe adverse events. Starting in 2003, the trial included 787 evaluable patients. The median overall follow-up of the patients was 13.5 years (range 3 months to 16.7 years). Based on intention-to-treat analyses, at 15 years, overall and progression-free survival were similar across arms: 85%, 83%, 80%, and 82% and 84%, 87%, 79%, and 79% for the IM 400 mg (N= 223), IM 600 mg (N= 171), IM 400 mg + AraC (N= 172), and IM 400 mg + PegIFN-α2a (N= 221) arms, respectively. The rate of major molecular response at 12 months and deep molecular response (MR4) over time were significantly higher with the combination IM 400 mg + PegIFN-α2a than with IM 400 mg: p= 0.0001 and p= 0.0035, respectively. Progression to advanced phases and secondary malignancies were the most frequent causes of death. Toxicity was the main reason for stopping AraC or PegIFN-α2a treatment.

Preventing factor VIII (FVIII) inhibitors following replacement therapies with FVIII products in patients with hemophilia A remains an unmet medical need. Better understanding of the early events of evolving FVIII inhibitors is essential for risk identification and the design of novel strategies to prevent inhibitor development. The Hemophilia Inhibitor Previously Untreated Patients (PUPs) Study (HIPS; www.clinicaltrials.gov #NCT01652027) is the first prospective cohort study to evaluate comprehensive changes in the immune system during the first 50 exposure days (EDs) to FVIII in patients with severe hemophilia A. HIPS participants were enrolled prior to their first exposure to FVIII or blood products (“true PUPs”) and were evaluated for different immunological and clinical parameters at specified time points during their first 50 EDs to a single source of recombinant FVIII. Longitudinal antibody data resulting from this study indicate that there are 4 subgroups of patients expressing distinct signatures of FVIII-binding antibodies. Subgroup 1 did not develop any detectable FVIII-binding immunoglobulin G (IgG) antibodies. Subgroup 2 developed nonneutralizing, FVIII-binding IgG1 antibodies, but other FVIII-binding IgG subclasses were not observed. Subgroup 3 developed transient FVIII inhibitors associated with FVIII-binding IgG1 antibodies, similar to subgroup 2. Subgroup 4 developed persistent FVIII inhibitors associated with an initial development of high-affinity, FVIII-binding IgG1 antibodies, followed by IgG3 and IgG4 antibodies. Appearance of FVIII-binding IgG3 was always associated with persistent FVIII inhibitors and the subsequent development of FVIII-binding IgG4. Some of the antibody signatures identified in HIPS could serve as candidates for early biomarkers of FVIII inhibitor development.

Preventing factor VIII (FVIII) inhibitors following replacement therapies with FVIII products in patients with hemophilia A remains an unmet medical need. Better understanding of the early events of evolving FVIII inhibitors is essential for risk identification and the design of novel strategies to prevent inhibitor development. The Hemophilia Inhibitor Previously Untreated Patients (PUPs) Study (HIPS; www.clinicaltrials.gov#NCT01652027) is the first prospective cohort study to evaluate comprehensive changes in the immune system during the first 50 exposure days (EDs) to FVIII in patients with severe hemophilia A. HIPS participants were enrolled prior to their first exposure to FVIII or blood products (“true PUPs”) and were evaluated for different immunological and clinical parameters at specified time points during their first 50 EDs to a single source of recombinant FVIII. Longitudinal antibody data resulting from this study indicate that there are 4 subgroups of patients expressing distinct signatures of FVIII-binding antibodies. Subgroup 1 did not develop any detectable FVIII-binding immunoglobulin G (IgG) antibodies. Subgroup 2 developed nonneutralizing, FVIII-binding IgG1 antibodies, but other FVIII-binding IgG subclasses were not observed. Subgroup 3 developed transient FVIII inhibitors associated with FVIII-binding IgG1 antibodies, similar to subgroup 2. Subgroup 4 developed persistent FVIII inhibitors associated with an initial development of high-affinity, FVIII-binding IgG1 antibodies, followed by IgG3 and IgG4 antibodies. Appearance of FVIII-binding IgG3 was always associated with persistent FVIII inhibitors and the subsequent development of FVIII-binding IgG4. Some of the antibody signatures identified in HIPS could serve as candidates for early biomarkers of FVIII inhibitor development.

Receptor interacting protein kinase 1 (RIP1) is a critical effector of inflammatory responses and cell death activation. Cell death pathways regulated by RIP1 include caspase‐dependent apoptosis and caspase‐independent necroptosis. The kinase activity of RIP1 has been associated with a number of inflammatory, neurodegenerative, and oncogenic diseases. In this study, we use the RIP1 kinase inhibitor GNE684 to demonstrate that RIP1 inhibition can effectively block skin inflammation and immune cell infiltrates in livers of Sharpinmutant (Cpdm; chronic proliferative dermatitis) mice in an interventional setting, after disease onset. On the other hand, genetic inactivation of RIP1 (RIP1 KD) or ablation of RIP3 (RIP3 KO) or MLKL (MLKL KO) did not affect testicular pathology of aging male mice. Likewise, infection with vaccinia virus or with mouse gammaherpesvirus MHV68 resulted in similar viral clearance in wild‐type, RIP1 KD, and RIP3 KO mice. In summary, this study highlights the benefits of inhibiting RIP1 in skin inflammation, as opposed to its lack of relevance for testicular longevity and the response to certain viral infections. Inhibiting RIP1 has benefits to skin inflammation but not certain viral infections.

An international, multicenter extension study evaluated recombinant fusion protein linking recombinant coagulation factor IX (FIX) with recombinant human albumin (rIX‐FP) in hemophilia B (FIX ≤ 2%) patients previously enrolled in a phase III study or who initiated rIX‐FP prophylaxis following surgery.

Patients with severe hemophilia A display varied bleeding phenotypes despite similar factor VIII (FVIII) activity levels.

AbstractAims:Prophylaxis with standard-acting recombinant factor IX (rFIX) in hemophilia B patients requires frequent injections. Extended half-life (EHL) products allow for prolonged dosing intervals, and so reduce this treatment burden. Three technologies are employed to extend the half-life of FIX; glycopegylation, Fc-fusion, and albumin fusion. rIX-FP is a novel albumin fusion protein, which allows for a prolonged dosing interval of up to 14 days. A systematic review and indirect statistical comparison was performed to evaluate the efficacy of both EHL and standard-acting rFIX products compared with rIX-FP in Phase III trials for prophylaxis in adult hemophilia B patients.Materials and methods:A systematic search was conducted in both EMBASE and PubMed to identify Phase III trials of prophylactic rFIX treatment in previously treated hemophilia B patients aged ≥12 years (FIX ≤2%). Annualized bleeding rate (ABR), spontaneous ABR (AsBR), and joint ABR (AjBR) data were extracted from each study. A z-test was performed using the mean of each parameter, and the mean difference in outcome between studies was calculated.Results:Seven articles investigating six rFIX products were identified. Median ABR, AsBR, and AjBR ranged from 0–3.0, 0–1.0, and 0–1.1 (means = 0.8–4.26, 0.13–2.6, and 0.34–2.85), respectively. rIX-FP achieved the lowest median and mean values in all three parameters. Z-tests showed that mean ABR was significantly lower for rIX-FP 7-day prophylaxis compared with the majority of standard-acting and other EHL rFIX products.Limitations:The low number of appropriate trials available for comparison limits the quantity of data available for comparison, and restricts the use of methods of adjustment for variance in study design or patient characteristics. However, these limitations are shared with similar analyses published in this field.Conclusion:This indirect comparison of Phase III trials indicates that rIX-FP efficacy compares favorably vs other rFIX products for prophylaxis in hemophilia B.

DDX41germline mutations (DDX41MutGL) are the most common genetic predisposition to myelodysplastic syndrome and acute myeloid leukemia (AML). Recent reports suggest that DDX41MutGLmyeloid malignancies could be considered as a distinct entity, even if their specific presentation and outcome remain to be defined. We described here the clinical and biological features of 191 patients with DDX41MutGLAML. Baseline characteristics and outcome of 86 of them, treated with intensive chemotherapy in 5 prospective ALFA/FILO trials were compared with those of 1604 DDX41wild-type (DDX41WT) AML patients, representing a prevalence of 5%. DDX41MutGLAML patients were mostly males (75%) in their seventh decade, with low leukocyte count (median, 2x109/L), low bone marrow blast infiltration (median, 33%), normal cytogenetics (75%) and few additional somatic mutations (median, 2). A second somatic DDX41mutation (DDX41MutSom) was found in 82% of patients and clonal architecture inference suggested that it could be the main driver for AML progression. DDX41MutGLpatients displayed higher complete remission (CR) rates (94% vs.69%, p<0.0001) and longer restricted mean overall survival (OS) censored at hematopoietic stem cell transplantation (HSCT) than ELN-2017 intermediate/adverse (Int/Adv) DDX41WTpatients (5-year ΔRMST of 13.6 months, p < 0.001). Relapse rates censored at HSCT were lower at 1 year in DDX41MutGLpatients (15% vs.44%) but later increased to join that of Int/Adv DDX41WTpatients at 3 years (82% vs 75%). HSCT in first CR was associated with prolonged relapse-free survival (RFS; HR, 0.43 [95%CI, 0.21-0.88]; p = 0.02) but not with longer OS (HR=0.77 [95%CI, 0.35-1.68], p=0.5).

•This report provides a post hoc analysis of males with severe hemophilia A who developed inhibitors in the PUPs A-LONG trial.•High-risk F8variants and factor dosing were associated with high-titer inhibitors (HTI) and low-titer to HTI progression, respectively.

Screening catalysts is an essential step to identify more sustainable and efficient routes for the desired chemical transformations. While several concepts for streamlining high-throughput screenings are already present in chemocatalysis, biocatalysis requires the more challenging dosing of very small amounts of hygroscopic and electrostatic enzyme powders in large numbers to cover the catalyst space of interest. Within this work, the EnzyBeads technology, where enzymes are transiently bound to an inert, free-flowing support to allow dosing of tiny quantities of enzymes, was further developed and coupled with a parallel volumetric dosing approach in a 96-well format. A simplified coating procedure to coat glass beads, an inert carrier that was previously not usable due to inefficient adsorption, with the enzymes of interest was developed, which circumvents the use of resonance acoustic mixing and of the electrostatic polystyrene beads currently required in the status quo procedure. Using a custom-built 3D printable solid dispenser, named the 12X-Dosing Rack, the obtained enzyme-coated glass beads (G-EnzyBeads) can be dispensed in microtiter plates in a quick and reliable fashion to greatly facilitate biocatalysis screenings.

Introduction

Introduction:Gemtuzumab ozogamicin (GO) is an antibody-drug conjugate targeting CD33 with a calicheamicin derivative payload. GO is approved in France in combination with daunorubicin and cytarabine for treatment-naïve patients aged ≥15 years with de novo CD33-positive acute myeloid leukemia (AML). Before the European Medicines Agency granted marketing authorization in 2018, GO was available in France with an authorization for temporary use (ATU) for specific patients since 2010, and between 2018 and 2019 as part of a “post-ATU” cohort until its reimbursement. This study aimed to describe the real-world use, effectiveness, and safety of GO in treatment-naïve patients with CD33-positive AML in France, as requested by the French Transparency Commission.

Background.The current standard of care for non-transplant eligible (NTE) newly diagnosed multiple myeloma (NDMM) is the combination of anti-CD38 immunotherapy daratumumab to lenalidomide-dexamethasone (DRd) on the basis of the Maia study (Facon et al. NEJM). This regimen has considerably improved the survival objectives for the study and the patients, PFS and OS. Interestingly, the MRD sustained 10-5 negative rate of DRd in MAIA is lower than 15%, alluding to the hypothesis that DRd may be immunogenic more than profoundly debulking in its main MOAs.

Introduction

In 1989 ILAR published a list and description of immunodeficient rodents used in research. Since then, advances in understanding of molecular mechanisms; recognition of genetic, epigenetic microbial, and other influences on immunity; and capabilities in manipulating genomes and microbiomes have increased options and opportunities for selecting mice and designing studies to answer important mechanistic and therapeutic questions. Despite numerous scientific breakthroughs that have benefitted from research in mice, there is debate about the relevance and predictive or translational value of research in mice. Reproducibility of results obtained from mice and other research models also is a well-publicized concern. This review summarizes resources to inform the selection and use of immune relevant mouse strains and stocks, aiming to improve the utility, validity, and reproducibility of research in mice. Immune sufficient genetic variations, immune relevant spontaneous mutations, immunodeficient and autoimmune phenotypes, and selected induced conditions are emphasized.

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Haemophilia A and haemophilia B are congenital X-linked bleeding disorders caused by deficiency of coagulation factor VIII (FVIII) and IX (FIX), respectively. The preferred treatment option for patients with haemophilia is replacement therapy. For patients with severe disease, prophylactic replacement of coagulation factor is the treatment of choice; this has been shown to reduce arthropathy significantly, reduce the frequency of bleeds and improve patients’ quality of life. Prophylaxis with standard recombinant factor requires regular intravenous infusion at least two (FIX) to three (FVIII) times a week. Recombinant FVIII and FIX products with an extended half-life are in development, or have been recently licensed. With reported mean half-life extensions of 1.5–1.8 times that of standard products for FVIII and 3–5 times that of standard products for FIX, these products have the potential to address many of the unmet needs of patients currently treated with standard factor concentrates. For example, they may encourage patients to switch from on-demand treatment to prophylaxis and improve the quality of life of patients receiving prophylaxis. Indeed, extended half-life products have the potential to reduce the burden of frequent intravenous injections, reducing the need for central venous lines in children, promote adherence, improve outcomes, potentially allow for more active lifestyles and, depending on the dosing regimen, increase factor trough levels. Members of the Zürich Haemophilia Forum convened for their 19th meeting to discuss the practicalities of incorporating new treatments into the management of people with haemophilia. This review of extended half-life products considers their introduction in haemophilia treatment, including the appropriate dose and schedule of infusions, laboratory monitoring, patient selection, safety considerations, and the economic aspects of care.

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