Distinct antibody responses to SARS-CoV-2 in children and adults across the COVID-19 clinical spectrum.

Clinical manifestations of COVID-19 caused by the new coronavirus SARS-CoV-2 are associated with age1,2. Adults develop respiratory symptoms, which can progress to acute respiratory distress syndrome (ARDS) in the most severe form, while children are largely spared from respiratory illness but can develop a life-threatening multisystem inflammatory syndrome (MIS-C)3-5. Here, we show distinct antibody responses in children and adults after SARS-CoV-2 infection. Adult COVID-19 cohorts had anti-spike (S) IgG, IgM and IgA antibodies, as well as anti-nucleocapsid (N) IgG antibody, while children with and without MIS-C had reduced breadth of anti-SARS-CoV-2-specific antibodies, predominantly generating IgG antibodies specific for the S protein but not the N protein. Moreover, children with and without MIS-C had reduced neutralizing activity as compared to both adult COVID-19 cohorts, indicating a reduced protective serological response. These results suggest a distinct infection course and immune response in children independent of whether they develop MIS-C, with implications for developing age-targeted strategies for testing and protecting the population.

Primary differentiated respiratory epithelial cells respond to apical measles virus infection by shedding multinucleated giant cells.

Measles virus (MeV) is highly infectious by the respiratory route and remains an important cause of childhood mortality. However, the process by which MeV infection is efficiently established in the respiratory tract is controversial with suggestions that respiratory epithelial cells are not susceptible to infection from the apical mucosal surface. Therefore, it has been hypothesized that infection is initiated in lung macrophages or dendritic cells and that epithelial infection is subsequently established through the basolateral surface by infected lymphocytes. To better understand the process of respiratory tract initiation of MeV infection, primary differentiated respiratory epithelial cell cultures were established from rhesus macaque tracheal and nasal tissues. Infection of these cultures with MeV from the apical surface was more efficient than from the basolateral surface with shedding of viable MeV-producing multinucleated giant cell (MGC) syncytia from the surface. Despite presence of MGCs and infectious virus in supernatant fluids after apical infection, infected cells were not detected in the adherent epithelial sheet and transepithelial electrical resistance was maintained. After infection from the basolateral surface, epithelial damage and large clusters of MeV-positive cells were observed. Treatment with fusion inhibitory peptides showed that MeV production after apical infection was not dependent on infection of the basolateral surface. These results are consistent with the hypothesis that MeV infection is initiated by apical infection of respiratory epithelial cells with subsequent infection of lymphoid tissue and systemic spread.

Cutaneous T-cell lymphoma of the eyelid masquerading as dermatitis.

Cutaneous T-cell lymphoma (CTCL) may pose a diagnostic challenge for physicians, as clinical presentation and histologic analysis may mimic benign dermatologic conditions. The authors present a case of recurrent CTCL in which the recurrence was limited to the eyelid and misdiagnosed as a contact dermatitis. To the best of the authors' knowledge, this is the first reported case of recurrent CTCL that has presented solely as dermatitis of the eyelid.

[A Project to Improve the Ability of Hemodialysis Staff to Respond to Fire Emergencies].

BACKGROUND & PROBLEMS: In case of fire in the hemodialysis room, it is necessary to help patients get away from dialysis machines smoothly and safely and evacuate the room rapidly. Our unit is located on a higher floor. An investigation showed that the accuracy rate for fire response awareness among the staffs in our unit was only 57.9%, while the accuracy rate of fire response skill operations was only 57.4%. Moreover, 62.0% of the staffs were not clear about the task grouping and task content of fire response. Confusion in our unit regarding the definition of patient mobility led to staffs classifying patients based on subjective perceptions and standards. Moreover, the unit also lacked an audit system for fire emergency operations and fire-response-related learning materials. PURPOSE: To improve staff knowledge and skills related to fire emergency response in the hemodialysis room to 100%. RESOLUTION: The project team worked out solutions such as adding a self-defense fire-fighting group to the dialysis information system, producing fire emergency response learning materials, establishing a seed personnel system, organizing on-the-job education, organizing fire response simulation drills, and implementing an audit system. RESULTS: The awareness of fire emergency response and the accuracy of skill operation among the staff were both improved to 100%, and there were statistically significant differences between the pre-test and post-test paired t-test results. Furthermore, consistent implementation of these resolution measures maintained the staff`s fire emergency response skills at 100% between June 2019 and May 2020. CONCLUSIONS: Tabletop simulation, practice drills, and skill operation audits are effective tools for improving the ability of staff in the hemodialysis room to respond to fire emergencies. It is recommended that institutions produce tabletop simulation props and combine regular on-site drills to improve the readiness of their staffs to respond to fire emergencies, which will shorten the response time during incidents.

Modeling the measles paradox reveals the importance of cellular immunity in regulating viral clearance.

Measles virus (MV) is a highly contagious member of the Morbillivirus genus that remains a major cause of childhood mortality worldwide. Although infection induces a strong MV-specific immune response that clears viral load and confers lifelong immunity, transient immunosuppression can also occur, leaving the host vulnerable to colonization from secondary pathogens. This apparent contradiction of viral clearance in the face of immunosuppression underlies what is often referred to as the 'measles paradox', and remains poorly understood. To explore the mechanistic basis underlying the measles paradox, and identify key factors driving viral clearance, we return to a previously published dataset of MV infection in rhesus macaques. These data include virological and immunological information that enable us to fit a mathematical model describing how the virus interacts with the host immune system. In particular, our model incorporates target cell depletion through infection of host immune cells-a hallmark of MV pathology that has been neglected from previous models. We find the model captures the data well, and that both target cell depletion and immune activation are required to explain the overall dynamics. Furthermore, by simulating conditions of increased target cell availability and suppressed cellular immunity, we show that the latter causes greater increases in viral load and delays to MV clearance. Overall, this signals a more dominant role for cellular immunity in resolving acute MV infection. Interestingly, we find contrasting dynamics dominated by target cell depletion when viral fitness is increased. This may have wider implications for animal morbilliviruses, such as canine distemper virus (CDV), that cause fatal target cell depletion in their natural hosts. To our knowledge this work represents the first fully calibrated within-host model of MV dynamics and, more broadly, provides a new platform from which to explore the complex mechanisms underlying Morbillivirus infection.

[Project for Promoting the Completeness of Electronic Dialysis Nursing Records].

BACKGROUND & PROBLEMS: Obtaining complete electronic dialysis nursing records, a tool that facilitates communication between medical teams, is critical in terms of maintaining the continuity of nursing procedures and nursing quality. An analysis of our unit indicated that nurses lacked sufficient familiarity with electronic dialysis nursing record systems. Moreover, they received insufficient training in operating these systems and lacked the guidelines necessary to maintain these records properly. Furthermore, these systems tend to be poorly designed, and an inspection system for dialysis nursing records is currently unavailable. These factors led to a rate of record completeness of only 58.2%. PURPOSE: To raise the rate of completeness for electronic nursing records to above 90%. RESOLUTION: An intervention was conducted to accomplish seven tasks. These tasks included: modify the electronic dialysis nursing record system, input preset phrases in order to facilitate record compilation in the system, devise a manual to instruct staff on recordkeeping procedures, organize in-service training on system operations, conduct clinical scenario simulations for nurses to practice operating the system, recruit informatics nurses to teach other nurses about the operations, and implement an inspection system for these electronic records. RESULTS: After implementing the intervention, the rate of completeness for electronic nursing records improved to 96% and the average time required for nurses to complete a nursing record decreased from 21 mins 35 s to 8 mins 15 s. CONCLUSIONS: The developed intervention significantly improved the completeness of electronic nursing records, reduced the time required for recordkeeping, and ensured adequate nursing quality for dialysis patients.

Plasma Cytokines and Chemokines in Zambian Children With Measles: Innate Responses and Association With HIV-1 Coinfection and In-Hospital Mortality.

To identify immune factors present during the acute rash phase of measles and associations with outcome and human immunodeficiency virus type 1 (HIV-1) coinfection, we measured the plasma levels of 22 cytokines and chemokines in Zambian children hospitalized with measles (n = 148) and control children (n = 44). Children with measles had higher levels of innate cytokines tumor necrosis factor (TNF) α, interleukin 1ß (IL-1ß), interleukin 18, and interleukin 6; chemokines CCL2, CCL4, CCL11, CCL22, CXCL8, and CXCL10; and T-cell cytokines interferon γ, and interleukin 2, 10, and 17. Children who died in the hospital had higher levels of TNF-α, IL-1ß, interleukin 12p70; CCL2, CCL4, CCL13, CCL17, CXCL8, CXCL10; and interleukin 2 and interferon γ than children who survived, and lower levels of interleukin 4. Children coinfected with HIV-1 had higher levels of TNF-α and IL-1ß than HIV-uninfected children with measles, and lower levels of interleukin 4 and 5. Therefore, acute measles was characterized by activation of macrophages and T cells producing type 1, but not type 2, cytokines, which was more pronounced in fatal disease.

Cutting edge: CXCR4 is critical for CD8+ memory T cell homeostatic self-renewal but not rechallenge self-renewal.

Central memory (CM) CD8(+) T cells "remember" prior encounters because they maintain themselves through cell division in the absence of ongoing challenge (homeostatic self-renewal), as well as reproduce the CM fate while manufacturing effector cells during secondary Ag encounters (rechallenge self-renewal). We tested the consequence of conditional deletion of the bone marrow homing receptor CXCR4 on antiviral T cell responses. CXCR4-deficient CD8(+) T cells have impaired memory cell maintenance due to defective homeostatic proliferation. Upon rechallenge, however, CXCR4-deficient T cells can re-expand and renew the CM pool while producing secondary effector cells. The critical bone marrow-derived signals essential for CD8(+) T cell homeostatic self-renewal appear to be dispensable to yield self-renewing, functionally asymmetric cell fates during rechallenge.

Protein Arginine Methyltransferase 8: Tetrameric Structure and Protein Substrate Specificity.

Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of various proteins, and their dysregulations often correlate with tumorigenesis or developmental deficiency. Recent studies have focused on the in vivo substrate identification and the enzyme mechanism with peptide substrates. However, how PRMTs recognize substrates at the protein level remains unknown. PRMT8 is one of the least characterized type I PRMTs, and its crystal structure has not been reported. Here, we report the crystal structure of the PRMT8:SAH complex, identify a new non-histone protein substrate NIFK, and uncover a previously unknown regulatory region specifically required for recognizing NIFK. Instead of the canonical dimeric structure for other type I PRMTs, PRMT8 exists as a tetramer in solution. Using X-ray crystallography in combination with small-angle X-ray scattering experiments, the dimer of dimers architecture in which two PRMT8 dimers are held together mainly by ß strand interactions was proposed. Mutation of PRMT8-ß15 impedes the methylation of NIFK but still allows methylation of the histone H2A/H2B dimer or a peptide substrate, suggesting a possible structural basis for recognition of protein substrates. Lastly, we observed two PRMT8 dimer orientations resulting in open (without SAH) and closed (with SAH bound) conformations. The comparison between open and closed conformations may provide useful information for PRMT1/8 inhibitor design.

Prolonged persistence of measles virus RNA is characteristic of primary infection dynamics.

Measles virus (MeV) is the poster child for acute infection followed by lifelong immunity. However, recent work shows the presence of MeV RNA in multiple sites for up to 3 mo after infection in a proportion of infected children. Here, we use experimental infection of rhesus macaques to show that prolonged RNA presence is characteristic of primary infection. We found that viral RNA persisted in the blood, respiratory tract, or lymph nodes four to five times longer than the infectious virus and that the clearance of MeV RNA from blood happened in three phases: rapid decline coincident with clearance of infectious virus, a rebound phase with increases up to 10-fold, and a phase of slow decrease to undetectable levels. To examine the effect of individual host immune factors on MeV load dynamics further, we developed a mathematical model that expressed viral replication and elimination in terms of the strength of MeV-specific T-cell responses, antibody responses, target cell limitations, and immunosuppressive activity of regulatory T cells. Based on the model, we demonstrate that viral dynamics, although initially regulated by T cells, require antibody to eliminate viral RNA. These results have profound consequences for our view of acute viral infections, the development of prolonged immunity, and, potentially, viral evolution.

Vaxfectin adjuvant improves antibody responses of juvenile rhesus macaques to a DNA vaccine encoding the measles virus hemagglutinin and fusion proteins.

DNA vaccines formulated with the cationic lipid-based adjuvant Vaxfectin induce protective immunity in macaques after intradermal (i.d.) or intramuscular (i.m.) delivery of 0.5 to 1 mg of codon-optimized DNA encoding the hemagglutinin (H) and fusion (F) proteins of measles virus (MeV). To characterize the effect of Vaxfectin at lower doses of H+F DNA, rhesus macaques were vaccinated twice with 20 µg of DNA plus Vaxfectin i.d., 100 µg of DNA plus Vaxfectin i.d., 100 µg of DNA plus Vaxfectin i.m. or 100 µg of DNA plus phosphate-buffered saline (PBS) i.m. using a needleless Biojector device. The levels of neutralizing (P = 0.036) and binding (P = 0.0001) antibodies were higher after 20 or 100 µg of DNA plus Vaxfectin than after 100 µg of DNA plus PBS. Gamma interferon (IFN-γ)-producing T cells were induced more rapidly than antibody, but were not improved with Vaxfectin. At 18 months after vaccination, monkeys were challenged with wild-type MeV. None developed rash or viremia, but all showed evidence of infection. Antibody levels increased, and IFN-γ- and interleukin-17-producing T cells, including cells specific for the nucleoprotein absent from the vaccine, were induced. At 3 months after challenge, MeV RNA was detected in the leukocytes of two monkeys. The levels of antibody peaked 2 to 4 weeks after challenge and then declined in vaccinated animals reflecting low numbers of bone marrow-resident plasma cells. Therefore, Vaxfectin was dose sparing and substantially improved the antibody response to the H+F DNA vaccine. This immune response led to protection from disease (rash/viremia) but not from infection. Antibody responses after challenge were more transient in vaccinated animals than in an unvaccinated animal.

Successful respiratory immunization with dry powder live-attenuated measles virus vaccine in rhesus macaques.

Measles remains an important cause of childhood mortality worldwide. Sustained high vaccination coverage is the key to preventing measles deaths. Because measles vaccine is delivered by injection, hurdles to high coverage include the need for trained medical personnel and a cold chain, waste of vaccine in multidose vials and risks associated with needle use and disposal. Respiratory vaccine delivery could lower these barriers and facilitate sustained high coverage. We developed a novel single unit dose, dry powder live-attenuated measles vaccine (MVDP) for respiratory delivery without reconstitution. We tested the immunogenicity and protective efficacy in rhesus macaques of one dose of MVDP delivered either with a mask or directly intranasal with two dry powder inhalers, PuffHaler and BD Solovent. MVDP induced robust measles virus (MeV)-specific humoral and T-cell responses, without adverse effects, which completely protected the macaques from infection with wild-type MeV more than one year later. Respiratory delivery of MVDP was safe and effective and could aid in measles control.

Clinical Utility and Reimbursement of Next-Generation Sequencing-Based Testing for Myeloid Malignancies.

Next-generation sequencing is becoming increasingly important for the diagnosis, risk stratification, and management of patients with established or suspected myeloid malignancies. These tests are being incorporated into clinical practice guidelines and many genetic alterations now constitute disease classification criteria. However, the reimbursement for these tests is uncertain. This study analyzed the clinical impact, ordering practices, prior authorization, and reimbursement outcomes of 505 samples from 477 patients sequenced with a 50-gene myeloid next-generation sequencing panel or a 15-gene myeloproliferative neoplasm subpanel. Overall, 98% (496 of 505) of tests provided clinically useful data. Eighty-nine percent of test results, including negative findings, informed or clarified potential diagnoses, 94% of results informed potential prognoses, and 19% of tests identified a potential therapeutic target. Sequencing results helped risk-stratify patients whose bone marrow biopsy specimens were inconclusive for dysplasia, monitor genetic evolution associated with disease progression, and delineate patients with mutation-defined diagnoses. Despite the clinical value, prior authorization from commercial payors or managed government payors was approved for less than half (45%) of requests. Only 51% of all cases were reimbursed, with lack of medical necessity frequently cited as a reason for denial. This study demonstrates the existence of a substantial gap between clinical utility and payor policies on test reimbursement.

Ultrasonic presentation variation of patients with pathological evidence of endometrial atrophy.

OBJECTIVE: Post-menopausal bleeding is one of the most common reasons for attending the gynecology outpatient clinic. The major proportion of the symptoms is endometrial atrophy (about 60%) despite of the endometrial thickness is over 4 mm. Therefore, the aim of this study is to evaluate the endometrial thickness under sonogram in the women with atrophic endometrium, with or without post-menopausal vaginal bleeding. MATERIALS AND METHODS: This is a retrospective study and we enrolled 237 post-menopausal women with pathological evidence of atrophic endometrium from Jan. 2014 to Dec. 2018 in Mackay Memorial hospital. Patient's characteristics taken into account were age, vaginal bleeding status, the methods of obtaining endometrial tissue, hormonal replacement therapy and breast cancer history under tamoxifen treatment. Endometrial thickness was classified as ≤ 4 mm, >4 mm-10 mm and >10 mm. We calculated the proportion of the characteristic mentioned before. RESULTS: In total, 237 patients were enrolled and 35 patients were excluded; therefore, the remaining 202 patients were analyzed. There were 42 (20.8%), 109 (54%) and 51 (25.2%) patients with endometrial thickness ≤4 mm, >4 mm-10 mm and >10 mm respectively. There was significant difference in the numbers of patients with post-menopausal bleeding (p = 0.002) and breast cancer history under tamoxifen therapy (p < 0.05) among the three groups. CONCLUSION: In the patients with endometrial atrophy, the endometrial thickness may be variable. There were only 20.8% of patients with endometrial thickness less than 4 mm in our study. Before endometrial sampling, comprehensive evaluation of the morphology of endometrium under image study, the patient's symptoms and medical history is important.

Reciprocal transmission of activating and inhibitory signals and cell fate in regenerating T cells.

The ability of activated progenitor T cells to self-renew while producing differentiated effector cell descendants may underlie immunological memory and persistent responses to ongoing infection. The nature of stem-like T cells responding to cancer and during treatment with immunotherapy is not clear. The subcellular organization of dividing progenitor CD8+ T cells from mice challenged with syngeneic tumors is examined here. Three-dimensional microscopy reveals an activating hub composed of polarized CD3, CD28, and phosphatidylinositol 3-kinase (PI3K) activity at the putative immunological synapse with an inhibitory hub composed of polarized PD-1 and CD73 at the opposite pole of mitotic blasts. Progenitor T cells from untreated and inhibitory checkpoint blockade-treated mice yield a differentiated TCF1- daughter cell, which inherits the PI3K activation hub, alongside a discordantly fated, self-renewing TCF1+ sister cell. Dynamic organization of opposite activating and inhibitory signaling poles in mitotic lymphocytes may account for the enigmatic durability of specific immunity.

Inflammation and Mortality in COVID-19 Hospitalized Patients With and Without Type 2 Diabetes.

CONTEXT: COVID-19 mortality is increased in patients with diabetes. A common hypothesis is that the relationship of inflammation with COVID-19 mortality differs by diabetes status. OBJECTIVE: The aim of this study was to determine the relationship of inflammation with mortality in COVID-19 hospitalized patients and to assess if the relationship differs by strata of type 2 diabetes status. METHODS: A case-control (died-survived) study of 538 COVID-19 hospitalized patients, stratified by diabetes status, was conducted at Columbia University Irving Medical Center. We quantified the levels of 8 cytokines and chemokines in serum, including interferon (IFN)-α2, IFN-γ, interleukin (IL)-1α, IL-1ß, IL-6, IL-8/CXCL8, IFNγ-induced protein 10 (IP10)/CXCL10 and tumor necrosis factor α (TNF-α) using immunoassays. Logistic regression models were used to model the relationships of log-transformed inflammatory markers (or their principal components) and mortality. RESULTS: In multiple logistic regression models, higher serum levels of IL-6 (adjusted odds ratio [aOR]:1.74, 95% CI [1.48, 2.06]), IL-8 (aOR: 1.75 [1.41, 2.19]) and IP10 (aOR: 1.36 [1.24, 1.51]), were significantly associated with mortality. This association was also seen in second principal component with loadings reflecting similarities among these 3 markers (aOR: 1.88 [1.54-2.31]). Significant positive association of these same inflammatory markers with mortality was also observed within each strata of diabetes. CONCLUSION: We show that mortality in COVID-19 patients is associated with elevated serum levels of innate inflammatory cytokine IL-6 and inflammatory chemokines IL-8 and IP10. This relationship is consistent across strata of diabetes, suggesting interventions targeting these innate immune pathways could potentially also benefit patients with diabetes.

Pharmacologic Inhibition of NT5C2 Reverses Genetic and Nongenetic Drivers of 6-MP Resistance in Acute Lymphoblastic Leukemia.

Low-intensity maintenance therapy with 6-mercaptopurine (6-MP) limits the occurrence of acute lymphoblastic leukemia (ALL) relapse and is central to the success of multiagent chemotherapy protocols. Activating mutations in the 5'-nucleotidase cytosolic II (NT5C2) gene drive resistance to 6-MP in over 35% of early relapse ALL cases. Here we identify CRCD2 as a first-in-class small-molecule NT5C2 nucleotidase inhibitor broadly active against leukemias bearing highly prevalent relapse-associated mutant forms of NT5C2 in vitro and in vivo. Importantly, CRCD2 treatment also enhanced the cytotoxic activity of 6-MP in NT5C2 wild-type leukemias, leading to the identification of NT5C2 Ser502 phosphorylation as a novel NT5C2-mediated mechanism of 6-MP resistance in this disease. These results uncover an unanticipated role of nongenetic NT5C2 activation as a driver of 6-MP resistance in ALL and demonstrate the potential of NT5C2 inhibitor therapy for enhancing the efficacy of thiopurine maintenance therapy and overcoming resistance at relapse. SIGNIFICANCE: Relapse-associated NT5C2 mutations directly contribute to relapse in ALL by driving resistance to chemotherapy with 6-MP. Pharmacologic inhibition of NT5C2 with CRCD2, a first-in-class nucleotidase inhibitor, enhances the cytotoxic effects of 6-MP and effectively reverses thiopurine resistance mediated by genetic and nongenetic mechanisms of NT5C2 activation in ALL. This article is highlighted in the In This Issue feature, p. 2483.

Oncogenic Vav1-Myo1f induces therapeutically targetable macrophage-rich tumor microenvironment in peripheral T cell lymphoma.

Peripheral T cell lymphoma not otherwise specified (PTCL-NOS) comprises heterogeneous lymphoid malignancies characterized by pleomorphic lymphocytes and variable inflammatory cell-rich tumor microenvironment. Genetic drivers in PTCL-NOS include genomic alterations affecting the VAV1 oncogene; however, their specific role and mechanisms in PTCL-NOS remain incompletely understood. Here we show that expression of Vav1-Myo1f, a recurrent PTCL-associated VAV1 fusion, induces oncogenic transformation of CD4+ T cells. Notably, mouse Vav1-Myo1f lymphomas show T helper type 2 features analogous to high-risk GATA3+ human PTCL. Single-cell transcriptome analysis reveals that Vav1-Myo1f alters T cell differentiation and leads to accumulation of tumor-associated macrophages (TAMs) in the tumor microenvironment, a feature linked with aggressiveness in human PTCL. Importantly, therapeutic targeting of TAMs induces strong anti-lymphoma effects, highlighting the lymphoma cells' dependency on the microenvironment. These results demonstrate an oncogenic role for Vav1-Myo1f in the pathogenesis of PTCL, involving deregulation in T cell polarization, and identify the lymphoma-associated macrophage-tumor microenvironment as a therapeutic target in PTCL.

Incidental intra-adrenal splenule.

Splenules can be found in the adrenals and should be considered in the differential diagnosis of adrenal incidentalomas.

Approaching the Interpretation of Discordances in SARS-CoV-2 Testing.

The coronavirus disease 2019 pandemic has upended life throughout the globe. Appropriate emphasis has been placed on developing effective therapies and vaccines to curb the pandemic. While awaiting such countermeasures, mitigation efforts coupled with robust testing remain essential to controlling spread of the disease. In particular, serological testing plays a critical role in providing important diagnostic, prognostic, and therapeutic information. However, this information is only useful if the results can be accurately interpreted. This pandemic placed clinical testing laboratories and requesting physicians in a precarious position because we are actively learning about the disease and how to interpret serological results. Having developed robust assays to detect antibodies generated against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and serving the hardest-hit areas within the New York City epicenter, we found 3 types of discordances in SARS-CoV-2 test results that challenge interpretation. Using representative clinical vignettes, these interpretation dilemmas are highlighted, along with suggested approaches to resolve such cases.