CRISPR screen identifies genes that sensitize AML cells to double-negative T-cell therapy.

Acute myeloid leukemia (AML) remains a devastating disease in need of new therapies to improve patient survival. Targeted adoptive T-cell therapies have achieved impressive clinical outcomes in some B-cell leukemias and lymphomas but not in AML. Double-negative T cells (DNTs) effectively kill blast cells from the majority of AML patients and are now being tested in clinical trials. However, AML blasts obtained from ∼30% of patients show resistance to DNT-mediated cytotoxicity; the markers or mechanisms underlying this resistance have not been elucidated. Here, we used a targeted clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) screen to identify genes that cause susceptibility of AML cells to DNT therapy. Inactivation of the Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitinating complex components sensitized AML cells to DNT-mediated cytotoxicity. In contrast, CD64 inactivation resulted in resistance to DNT-mediated cytotoxicity. Importantly, the level of CD64 expression correlated strongly with the sensitivity of AML cells to DNT treatment. Furthermore, the ectopic expression of CD64 overcame AML resistance to DNTs in vitro and in vivo. Altogether, our data demonstrate the utility of CRISPR/Cas9 screens to uncover mechanisms underlying the sensitivity to DNT therapy and suggest CD64 as a predictive marker for response in AML patients.

Venetoclax enhances T cell-mediated antileukemic activity by increasing ROS production.

Venetoclax, a Bcl-2 inhibitor, in combination with the hypomethylating agent azacytidine, achieves complete remission with or without count recovery in ∼70% of treatment-naive elderly patients unfit for conventional intensive chemotherapy. However, the mechanism of action of this drug combination is not fully understood. We discovered that venetoclax directly activated T cells to increase their cytotoxicity against acute myeloid leukemia (AML) in vitro and in vivo. Venetoclax enhanced T-cell effector function by increasing reactive oxygen species generation through inhibition of respiratory chain supercomplexes formation. In addition, azacytidine induced a viral mimicry response in AML cells by activating the STING/cGAS pathway, thereby rendering the AML cells more susceptible to T cell-mediated cytotoxicity. Similar findings were seen in patients treated with venetoclax, as this treatment increased reactive oxygen species generation and activated T cells. Collectively, this study presents a new immune-mediated mechanism of action for venetoclax and azacytidine in the treatment of AML and highlights a potential combination of venetoclax and adoptive cell therapy for patients with AML.

State-of-Art of Cellular Therapy for Acute Leukemia.

With recent clinical breakthroughs, immunotherapy has become the fourth pillar of cancer treatment. Particularly, immune cell-based therapies have been envisioned as a promising treatment option with curative potential for leukemia patients. Hence, an increasing number of preclinical and clinical studies focus on various approaches of immune cell-based therapy for treatment of acute leukemia (AL). However, the use of different immune cell lineages and subsets against different types of leukemia and patient disease statuses challenge the interpretation of the clinical applicability and outcome of immune cell-based therapies. This review aims to provide an overview on recent approaches using various immune cell-based therapies against acute B-, T-, and myeloid leukemias. Further, the apparent limitations observed and potential approaches to overcome these limitations are discussed.

Investigation on urinary and serum alpha klotho in dogs with chronic kidney disease.

BACKGROUND: As a co-receptor for fibroblast growth factor 23, klotho plays a pivotal role in phosphate metabolism. The kidney is known to be the main source of soluble alpha-klotho and the principal regulator of its concentration. Previous studies in human participants showed that the concentration of soluble alpha-klotho in serum and urine decreased in chronic kidney disease (CKD) patients. However, no previous study has assessed soluble alpha-klotho levels in dogs. This study aimed to measure serum and urinary alpha-klotho levels in CKD dogs and identify their associations with International Renal Interest Society (IRIS) CKD stages and other parameters known to be associated with CKD. RESULTS: Serum and urinary alpha klotho concentrations were measured by a commercially available canine-specific sandwich enzyme-linked immunosorbent assay kit and compared between groups by a nonparametric Kruskal-Wallis test. Spearman's correlation coefficient was used to evaluate the relationships between variables. A stepwise multiple regression analysis was performed to estimate the effects of independent predictors on klotho concentrations. The urine klotho-to-creatinine ratio (UrKl/Cr) was significantly lower in stage 3 dogs than the control group and was significantly lower in dogs with stage 3 and 4 CKD than in those with stage 1 and 2 disease. UrKl/Cr was negatively correlated with serum symmetric dimethylarginine (sSDMA), blood urea nitrogen (BUN), creatinine, and phosphorus concentration. Serum alpha-klotho concentration in dogs with stages 2 and 3 CKD was significantly lower than those in the control group. There was no significant correlation between serum alpha-klotho and BUN, creatinine, and phosphorus concentrations. No statistically significant differences were observed in UrKl/Cr and serum alpha-klotho concentration between groups based on sex, age, urine protein-to-creatinine ratio (UPC), or blood pressure. CONCLUSIONS: UrKl/Cr decreased in dogs with advanced CKD, and it was negatively correlated with sSDMA, BUN, creatinine, and phosphorus concentrations. Thus, klotho is associated with CKD and its clinical consequences, including CKD-mineral bone disorder, in dogs. Although serum klotho concentration was negatively correlated with sSDMA levels, it was not apparently related to IRIS CKD stage or other parameters known to be associated with CKD.

Retinal microstructures are altered in patients with idiopathic infantile nystagmus.

PURPOSE: To compare segmented retinal layer thicknesses between patients with idiopathic infantile nystagmus (IIN) and controls. METHODS: This retrospective case-control study included 66 patients with IIN and 66 age-matched controls. The retinal layers were examined using spectral domain optical coherence tomography with autosegmentation. Central foveal thickness (CFT), outer nuclear layer (ONL), and outer segment length (OSL) thickness were measured at the fovea center. Mean values for retinal nerve fiber layer, ganglion cell inner plexiform layer (GCIPL), inner nuclear layer, outer plexiform-outer nuclear layer (OPNL) thicknesses were calculated at two measurement points (nasal and temporal hump points at the macula area). RESULTS: There were no significant between-group differences in age, gender, or refraction error. The CFT was thicker in the IIN group compared with the control group (225.0 µm vs. 217.8 µm, P = 0.017) and OSL was shorter in IIN than in controls (40.0 µm vs. 43.7 µm., P < 0.001). The ONL thickness at the central fovea was not statistically different between the two groups. At the nasal and temporal position where the ganglion cell density was thickest, the GCIPL thickness was thinner in the IIN group compared to the controls (99.5 µm vs. 102.8 µm, P = 0.010). The GCIPL thickness was negatively correlated with logMAR visual acuity (Spearman's rho = -0.502, P < 0.001). CONCLUSIONS: The foveal pit was shallower, OSL was shorter, and the GCIPL thicknesses at macular humps were decreased in the patients with IIN compared with that of controls. The faulty development of the macula may be related to unknown pathophysiologic mechanism during fovea maturation in IIN or continuous eye movement itself interrupt fovea development.

Strabismus surgery and long-term visual outcomes in patients with preadolescent onset ocular myasthenia gravis.

PURPOSE: To investigate long-term strabismus surgical outcomes and visual prognosis in preadolescent ocular myasthenia gravis (OMG). METHODS: The medical records of all patients with preadolescent onset OMG who underwent strabismus surgery were reviewed. Thirteen patients met the study inclusion criteria. The main outcomes, including ocular alignment, number of surgeries, and visual acuity at final visit were evaluated. Outcomes were considered successful if there were ≤10 prism diopters (PD) residual horizontal and ≤4 PD residual vertical deviations at final recorded visit. RESULTS: Among 13 patients, diplopia presented in 11 patients (77.8%). Mean age at disease onset was 5.8 ± 2.7 years (range 1 ∼ 11), mean age at surgery was 20.5 ± 11.3 years (range, 2.5 to 36.6 years), and the time from disease onset to first operation was 14.8 ± 9.6 years (range, 1.5 to 29.6 years). The average length of postoperative follow-up was 4.7 ± 6.6 years (range, 0.5 to 18.9 years). Ocular deviation changed more than 15 PD during stable disease in six patients (46.2%). No patients underwent more than two surgeries. Successful results were achieved in nine patients (69.2%) at final recorded visit. CONCLUSIONS: In our series, nine patients (69.2%) with OMG could obtain good binocular alignment at final visit. Therefore, strabismus surgery can be considered in patients with preadolescent onset OMG who have constant angle of deviation despite medical treatment.

Longitudinal analysis of retinal nerve fiber layer and ganglion cell-inner plexiform layer thickness in ethambutol-induced optic neuropathy.

PURPOSE: The aim of the study was to evaluate longitudinal analysis of peripapillary retinal nerve fiber layer (RNFL) and perifoveal ganglion cell-inner plexiform layer (GCIPL) thickness in patients being treated with ethambutol (EMB). METHODS: This prospective longitudinal cohort study enrolled 37 patients who were treated with EMB for pulmonary tuberculosis. Best-corrected visual acuity, color vision test, automated perimetry, fundus photography, and RNFL and GCIPL thickness were measured at baseline and at 4 and 6 months after the start of EMB treatment, using Cirrus optical coherence tomography. RESULTS: Among 37 patients, EMB-induced optic neuropathy occurred in one patient (2.7 %). In this patient, thickening of the RFNL and thinning of the GCIPL were noted at the onset of symptoms. After discontinuation of EMB, RNFL and GCIPL thickness progressively normalized. Changes in RNFL and GCIPL thickness were not statistically significant in the 36 patients who did not exhibit EMB-induced optic neuropathy-related symptoms during follow-up (all P values > 0.05). CONCLUSIONS: Thickening of the peripapillary RNFL and thinning of the perifoveal GCIPL is an effective quantitative and early marker for diagnosis of EMB-induced optic neuropathy.

Ophthalmic findings in children with nonsyndromic craniosynostosis treated by expansion cranioplasty.

The ocular and systemic abnormalities of nonsyndromic craniosynostosis are often considered to be less severe than those of syndromic craniosynostosis and are less well described. The purpose of this article was to describe the frequency and nature of ophthalmic abnormalities in children treated for nonsyndromic craniosynostosis by expansion cranioplasty. A retrospective review identified 88 consecutive children with nonsyndromic craniosynostosis who underwent expansion cranioplasty with distraction osteogenesis. Assessment of presence and type of strabismus, refractive error, and amblyopia before and 6 months after surgery was recorded. Children with a mean age of 24.4 months were treated for nonsyndromic craniosynostosis (27 with coronal and 61 with sagittal and/or lambdoid). One-fourth of the patients had a fixation preference. Significant refractive errors were found in 45 (51%) of the 88 patients: hyperopia in 27%, myopia in 5%, and astigmatism in 35%. Anisometropia was present in 20%. Of the 85 patients who completed orthoptic examination, 48 (56%) had strabismus: exodeviation in 26%, esodeviation in 14%, and vertical deviation in 5%. Fourteen patients (16%) had abnormal head posture. Significant refractive error and strabismus were more likely to occur in cases with coronal synostosis. The procedures used for cranial vault expansion improved the abnormal head posture but did not affect the refractive error or ocular misalignment. Of children with nonsyndromic craniosynostosis who need neurosurgical correction, more than half were found to have significant refractive error and strabismus. Our findings support the importance of ophthalmic evaluation in these children.

Double-negative T cells utilize a TNFα-JAK1-ICAM-1 cytotoxic axis against acute myeloid leukemia.

ABSTRACT: Allogeneic double-negative T cells (DNTs) are a rare T-cell subset that effectively target acute myeloid leukemia (AML) without inducing graft-versus-host disease in an allogeneic setting. A phase 1 clinical trial demonstrated the feasibility, safety, and potential efficacy of allogeneic DNT therapy among patients with relapsed AML. However, the molecular mechanisms of DNT-mediated cytotoxicity against AML remain elusive. Thus, we used a flow cytometry-based high throughput screening to compare the surface molecule expression profile on DNTs during their interaction with DNT-susceptible or -resistant AML cells and identified a tumor necrosis factor α (TNFα)-dependent cytotoxic pathway in DNT-AML interaction. TNFα secreted by DNTs, upon encountering susceptible AML targets, sensitized AML cells to DNT-mediated killing, including those otherwise resistant to DNTs. Mechanistically, TNFα upregulated ICAM-1 on AML cells through a noncanonical JAK1-dependent pathway. DNTs then engaged with AML cells more effectively through an ICAM-1 receptor, lymphocyte function-associated antigen 1, leading to enhanced killing. These results reveal a TNFα-JAK1-ICAM-1 axis in DNT-mediated cytotoxicity against AML to improve therapeutic efficacy.

Electroretinography in congenital nystagmus patients with a normal fundus examination.

PURPOSE: To identify the ophthalmic causes of congenital nystagmus with normal eye examination by electroretinography (ERG). STUDY DESIGN: Retrospective observational study. METHODS: We reviewed the medical records of patients younger than 6 months of age who presented between June 2008 and November 2011 with nystagmus and no other neurological signs following an otherwise normal eye examination. A complete ophthalmic examination and ERG (Nicolet Bravo system; Nicolet Biomedial & RETIscan; Roland Instruments), fundus photography, and Ishihara color test were performed to identify any ophthalmic causes of congenital nystagmus. RESULTS: Thirty-three patients met the criteria. Rod dysfunction was diagnosed in 4 patients (12.1%), cone dysfunction in 2 patients (6.1%), and cone-rod dysfunction in 1 patient (3.0%). The results of ERG were negative in 2 patients (6.1%). Idiopathic infantile nystagmus was diagnosed in the remaining 24 patients (72.7%) based on their normal ERG examination. CONCLUSIONS: In Korean congenital nystagmus patients with a normal fundus examination, achromatopsia and Leber's congenital amaurosis are uncommon causes. ERG is needed to make a definite diagnosis and provide prognostic information in congenital idiopathic nystagmus patients with a normal fundus examination.

Oseltamivir-resistant influenza viruses isolated in South Korea from 2005 to 2010.

South Korean isolates of oseltamivir-resistant influenza viruses from 2005-2010 were investigated with a total 491 influenza viruses identified from 1702 specimens. Neuraminidase genes from 342 influenza viruses (71 A/H1N1, 74 pandemic A/H1N1 2009, 117 A/H3N2, and 80 B) were analyzed by RT-PCR with molecular markers for oseltamivir resistance. The H274Y mutation in the NA protein was identified in 100 % (n=40) of A/H1N1 viruses circulating in 2008-2009. Influenza A/H1N1 viruses harboring the H274Y substitution exhibited, on average, a 626-fold reduction in oseltamivir susceptibility and clustered with the A/Norway/1736/2007 strain. Close and timely monitoring for resistance to clinically available influenza antivirals should be consistently performed.

Photophobia measurement in intermittent exotropia using the contrast sensitivity test.

BACKGROUND: To assess the presence and extent of photophobia in children with intermittent exotropia (X[T]) using the contrast sensitivity test. METHODS: Fifty-eight children with X(T) and 34 normal controls were studied with the functional acuity contrast test. Each participant viewed the stimuli of contrast monocularly and binocularly under photopic and mesopic conditions, performed with and without glare. Photophobia was defined as a reduction of contrast sensitivity caused by glare light. We compared the photophobia of children with X(T) to that of normal controls, and to the photophobia 3 months after muscle surgery. RESULTS: With stimuli of glare, the contrast sensitivity of children with X(T) was suppressed at intermediate spatial frequencies under mesopic condition (p = 0.006 for 6 cycles per degree [cpd], p = 0.027 for 12 cpd), whereas that of normal controls showed no difference. It occurred when X(T) patients viewed targets binocularly, and significantly improved after strabismus surgery (p = 0.003 at 6 cpd). The measured photophobia of X(T) was strongly correlated to the photophobia symptoms reported by parents (p = 0.002). CONCLUSIONS: The mesopic contrast sensitivity with glare can represent the photophobia of children with X(T). Contrast sensitivity may be a useful measure for monitoring symptoms related to X(T).

Role of heat shock protein 47 in transdifferentiation of human tenon's fibroblasts to myofibroblasts.

BACKGROUND: Heat shock protein 47 (Hsp47) is a well-known molecular chaperone in collagen synthesis and maturation. The aim of this study is to investigate its putative role in the transdifferentiation of Tenon's fibroblasts to myofibroblasts. METHODS: Primary cultured human Tenon's fibroblasts were exposed to transforming growth factor-ß1 (TGF-ß1) for up to 48 hours. The mRNA levels of Hsp47 and α smooth muscle actin (αSMA) were determined by quantitative real time RT-PCR. After delivery of small interfering RNA (siRNA) molecules targeting Hsp47 into the cells, the expression of Hsp47 and αSMA proteins was determined by western immunoblotting. RESULTS: TGF-ß1 increased the mRNA expressions of both Hsp47 and αSMA in human Tenon's fibroblasts, as determined by quantitative real time RT-PCR. However, it induced the protein expression of only αSMA but not Hsp47, as determined by western immunoblots. When siRNAs specific for Hsp47 were introduced into those cells, the TGF-ß1-induced expression of αSMA was significantly attenuated on western immunoblots; after 48 hours of exposure to TGF-ß1, the relative densities of immunobands were 11.58 for the TGF-ß1 only group and 2.75 for the siRNA treatment group, compared with the no treatment control group (p < 0.001). CONCLUSIONS: Our data suggest that Hsp47 may be related to the TGF-ß1-induced transdifferentiation of human Tenon's fibroblasts to myofibroblasts.

Adoptive Cell Therapy for T-Cell Malignancies.

T-cell malignancies are often aggressive and associated with poor prognoses. Adoptive cell therapy has recently shown promise as a new line of therapy for patients with hematological malignancies. However, there are currently challenges in applying adoptive cell therapy to T-cell malignancies. Various approaches have been examined in preclinical and clinical studies to overcome these obstacles. This review aims to provide an overview of the recent progress on adoptive cell therapy for T-cell malignancies. The benefits and drawbacks of different types of adoptive cell therapy are discussed. The potential advantages and current applications of innate immune cell-based adoptive cell therapy for T cell malignancies are emphasized.

Allogeneic double-negative CAR-T cells inhibit tumor growth without off-tumor toxicities.

The development of autologous chimeric antigen receptor T (CAR-T) cell therapies has revolutionized cancer treatment. Nevertheless, the delivery of CAR-T cell therapy faces challenges, including high costs, lengthy production times, and manufacturing failures. To overcome this, attempts have been made to develop allogeneic CAR-T cells using donor-derived conventional CD4+ or CD8+ T cells (Tconvs), but severe graft-versus-host disease (GvHD) and host immune rejection have made this challenging. CD3+CD4-CD8- double-negative T cells (DNTs) are a rare subset of mature T cells shown to fulfill the requirements of an off-the-shelf cellular therapy, including scalability, cryopreservability, donor-independent anticancer function, resistance to rejection, and no observed off-tumor toxicity including GvHD. To overcome the challenges faced with CAR-Tconvs, we evaluated the feasibility, safety, and efficacy of using healthy donor-derived allogeneic DNTs as a CAR-T cell therapy platform. We successfully transduced DNTs with a second-generation anti-CD19-CAR (CAR19) without hampering their endogenous characteristics or off-the-shelf properties. CAR19-DNTs induced antigen-specific cytotoxicity against B cell acute lymphoblastic leukemia (B-ALL). In addition, CAR19-DNTs showed effective infiltration and tumor control against lung cancer genetically modified to express CD19 in xenograft models. CAR19-DNT efficacy was comparable with that of CAR19-Tconvs. However, unlike CAR19-Tconvs, CAR19-DNTs did not cause alloreactivity or xenogeneic GvHD-related mortality in xenograft models. These studies demonstrate the potential of using allogeneic DNTs as a platform for CAR technology to provide a safe, effective, and patient-accessible CAR-T cell treatment option.

Evaluation of the Randox and Fuji Dri-Chem vcCRP-P assays of canine C-reactive protein.

In veterinary medicine, measurement of canine C-reactive protein (cCRP) is used widely to detect inflammatory diseases. We evaluated the precision of Randox and Fuji assays for cCRP, as well as accuracy, correlation, and agreement compared to a reference ELISA. Blood samples from 71 client-owned dogs (20 healthy, 51 diseased) were analyzed with the 3 assays. Inter-assay CVs were ~3.5% with both the Randox and Fuji assays. The mean biases were -1.90% for the Randox and -5.93% for the Fuji test; the targeted biases were ~8.5% for both assays. The CV, bias, and observed total error were acceptable for the 2 assays compared to ASVCP recommendations based on biological variation studies. The Spearman correlation coefficient for cCRP concentration compared with the reference ELISA was 0.83 for the Randox test and 0.92 for the Fuji test. Both assays measured cCRP precisely at intermediate and increased concentrations. Correlation with the reference ELISA was good, and both assays could be used to evaluate cCRP concentrations in veterinary practice. However, the assays did not reach analytical agreement; hence the results obtained by these assays are not interchangeable, and serial monitoring of cCRP requires the use of the same assay.

Central nervous system relapses in 3 dogs with B-cell lymphoma.

This report describes cases of central nervous system (CNS) relapse that occurred during chemotherapy in 3 dogs with lymphoma. Diagnosis was made by a combination of clinical signs and cytology of cerebrospinal fluid. The suspected risk factors, clinical features common to the 3 dogs, and treatment options are discussed.

Mitochondrial and Metabolic Pathways Regulate Nuclear Gene Expression to Control Differentiation, Stem Cell Function, and Immune Response in Leukemia.

Mitochondria are involved in many biological processes including cellular homeostasis, energy generation, and apoptosis. Moreover, mitochondrial and metabolic pathways are interconnected with gene expression to regulate cellular functions such as cell growth, survival, differentiation, and immune recognition. Metabolites and mitochondrial enzymes regulate chromatin-modifying enzymes, chromatin remodeling, and transcription regulators. Deregulation of mitochondrial pathways and metabolism leads to alterations in gene expression that promote cancer development, progression, and evasion of the immune system. This review highlights how mitochondrial and metabolic pathways function as a central mediator to control gene expression, specifically on stem cell functions, differentiation, and immune response in leukemia. SIGNIFICANCE: Emerging evidence demonstrates that mitochondrial and metabolic pathways influence gene expression to promote tumor development, progression, and immune evasion. These data highlight new areas of cancer biology and potential new therapeutic strategies.

Enhancing Therapeutic Efficacy of Double Negative T Cells against Acute Myeloid Leukemia Using Idelalisib.

The double negative T cell (DNT) is a unique subset of T cells with potent anti-leukemic potential. Previously, DNT therapy has been shown to effectively target AML cells in patient-derived xenograft (PDX) models. Further, a recently completed phase I/IIa clinical study demonstrated the safety, feasibility, and potential efficacy in AML patients that relapsed after allogeneic hematopoietic stem cell transplantation. However, the persistence and durability of DNT-mediated anti-leukemic response is less well understood. In this study, we characterized the in vivo persistence of DNTs in PDX models. Further, we improved the efficacy and durability of DNT-mediated activity with phosphoinositide 3-kinase delta (PI3Kδ) inhibition. Mechanistically, DNTs treated with the PI3Kδ inhibitor, Idelalisib (Ide), exhibited early memory phenotype with superior viability and proliferative capacity but less cell exhaustion. Collectively, the findings from this study support the use of Ide-treated DNTs to improve its therapeutic outcome.