Mask R-CNN provides efficient and accurate measurement of chondrocyte viability in the label-free assessment of articular cartilage.

Objective: Chondrocyte viability (CV) can be measured with the label-free method using second harmonic generation (SHG) and two-photon excitation autofluorescence (TPAF) imaging. To automate the image processing for the label-free CV measurement, we previously demonstrated a two-step deep-learning method: Step 1 used a U-Net to segment the lacuna area on SHG images; Step 2 used dual CNN networks to count live cells and the total number of cells in extracted cell clusters from TPAF images. This study aims to develop one-step deep learning methods to improve the efficiency of CV measurement. Method: TPAF/SHG images were acquired simultaneously on cartilage samples from rats and pigs using two-photon microscopes and were merged to form RGB color images with red, green, and blue channels assigned to emission bands of oxidized flavoproteins, reduced forms of nicotinamide adenine dinucleotide, and SHG signals, respectively. Based on the Mask R-CNN, we designed a deep learning network and its denoising version using Wiener deconvolution for CV measurement. Results: Using training and test datasets from rat and porcine cartilage, we have demonstrated that Mask R-CNN-based networks can segment and classify individual cells with a single-step processing flow. The absolute error (difference between the measured and the ground-truth CV) of the CV measurement using the Mask R-CNN with or without Wiener deconvolution denoising reaches 0.01 or 0.08, respectively; the error of the previous CV networks is 0.18, significantly larger than that of the Mask R-CNN methods. Conclusions: Mask R-CNN-based deep-learning networks improve efficiency and accuracy of the label-free CV measurement.

PD-1 blockade in combination with dasatinib potentiates induction of anti-acute lymphocytic leukemia immunity.

Immunotherapy, in the form of hematopoietic stem cell transplantation (HSCT), has been part of the standard of care in the treatment of acute leukemia for over 40 years. Trials evaluating novel immunotherapeutic approaches, such as targeting the programmed death-1 (PD-1) pathway, have unfortunately not yielded comparable results to those seen in solid tumors. Major histocompatibility complex (MHC) proteins are cell surface proteins essential for the adaptive immune system to recognize self versus non-self. MHC typing is used to determine donor compatibility when evaluating patients for HSCT. Recently, loss of MHC class II (MHC II) was shown to be a mechanism of immune escape in patients with acute myeloid leukemia after HSCT. Here we report that treatment with the tyrosine kinase inhibitor, dasatinib, and an anti-PD-1 antibody in preclinical models of Philadelphia chromosome positive B-cell acute lymphoblastic leukemia is highly active. The dasatinib and anti-PD-1 combination reduces tumor burden, is efficacious, and extends survival. Mechanistically, we found that treatment with dasatinib significantly increased MHC II expression on the surface of antigen-presenting cells (APC) in a tumor microenvironment-independent fashion and caused influx of APC cells into the leukemic bone marrow. Finally, the induction of MHC II may potentiate immune memory by impairing leukemic engraftment in mice previously cured with dasatinib, after re-inoculation of leukemia cells. In summary, our data suggests that anti-PD-1 therapy may enhance the killing ability of dasatinib via dasatinib driven APC growth and expansion and upregulation of MHC II expression, leading to antileukemic immune rewiring.

Nuclear export signal mutation of epidermal growth factor receptor enhances malignant phenotypes of cancer cells.

Nuclear epidermal growth factor receptor (EGFR) has been shown to be correlated with drug resistance and a poor prognosis in patients with cancer. Previously, we have identified a tripartite nuclear localization signal (NLS) within EGFR. To comprehensively determine the functions and underlying mechanism of nuclear EGFR and its clinical implications, we aimed to explore the nuclear export signal (NES) sequence of EGFR that is responsible for interacting with the exportins. We combined in silico prediction with site-directed mutagenesis approaches and identified a putative NES motif of EGFR, which is located in amino acid residues 736-749. Mutation at leucine 747 (L747) in the EGFR NES led to increased nuclear accumulation of the protein via a less efficient release of the exportin CRM1. Interestingly, L747 with serine (L747S) and with proline (L747P) mutations were found in both tyrosine kinase inhibitor (TKI)-treated and -naïve patients with lung cancer who had acquired or de novo TKI resistance and a poor outcome. Reconstituted expression of the single NES mutant EGFRL747P or EGFRL747S, but not the dual mutant along with the internalization-defective or NLS mutation, in lung cancer cells promoted malignant phenotypes, including cell migration, invasiveness, TKI resistance, and tumor initiation, supporting an oncogenic role of nuclear EGFR. Intriguingly, cells with germline expression of the NES L747 mutant developed into B cell lymphoma. Mechanistically, nuclear EGFR signaling is required for sustaining nuclear activated STAT3, but not for Erk. These findings suggest that EGFR functions are compartmentalized and that nuclear EGFR signaling plays a crucial role in tumor malignant phenotypes, leading to tumorigenesis in human cancer.

Presumed Multidrug-Resistant Mycoplasma genitalium Urethritis in a Man with HIV Infection.

Sexually transmitted infection (STI) rates in the U.S. have rapidly increased in the past decade. Although most of this rise is due to syphilis, gonorrhea, and chlamydia, less common STIs are also rising, including Mycoplasma genitalium. We present the case of a 40-year-old male with a history of virologically-suppressed human immunodeficiency virus (HIV) infection who presented with recurrent nongonococcal urethritis. Unfortunately, his symptoms were refractory to multiple empiric drug regimens, and he was eventually diagnosed with Mycoplasma genitalium. After consultation with the Centers for Disease Control and Prevention STI branch, minocycline was successfully used to eradicate the infection.

Measuring chondrocyte viability of articular cartilage based on label-free two-photon microscopy and deep learning image analysis.

Objective: Chondrocyte viability (CV) is an important indicator of articular cartilage health. Two-photon excitation autofluorescence (TPAF) and second harmonic generation (SHG) microscopy provide a label-free method for imaging chondrocytes. In this study, we propose an automated assessment of CV using deep learning cell segmentation and counting based on acquired TPAF/SHG images. Design: Label-free TPAF/SHG images of cartilage samples from rats and porcine were acquired using both commercial and home-built two-photon microscopes, respectively. TPAF/SHG images were merged to form RGB color images with red, green, and blue channels assigned to TPAF (two channels) and SHG signals, respectively. To make the training datasets for the deep learning networks, individual chondrocyte areas on the RGB color images were manually circled and live or dead chondrocytes were validated by using Calcein-AM and Ethidium homodimer-1 dye labeling. We first built a chondrocyte viability network (MCV-Net) using the Mask R-CNN architecture, which could provide individual segmented cellular areas with live or dead status. Wiener deconvolution preprocessing was added before the input of MCV-Net to improve the accuracy of the CV analysis, forming the Wiener deconvolution CV network (wMCV-Net). Results: Training (300 images) and test (120 images) datasets were built for rats and porcine cartilage respectively. Wiener deconvolution could improve the Peak Signal-to-Noise Ratio (PSNR) for 30-40%. We demonstrated that both MCV-Net and wMCV-Net significantly improved the accuracy of the CV measurement. Conclusion: A custom desktop TPAF/SHG microscope was used in collaboration with deep learning algorithm wMCV-Net based label-free method to assess the CV and get 95% accuracy with both rats and porcine samples.

Targeting the ALK-CDK9-Tyr19 kinase cascade sensitizes ovarian and breast tumors to PARP inhibition via destabilization of the P-TEFb complex.

Poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated promising clinical activity in multiple cancers. However, resistance to PARP inhibitors remains a substantial clinical challenge. In the present study, we report that anaplastic lymphoma kinase (ALK) directly phosphorylates CDK9 at tyrosine-19 to promote homologous recombination (HR) repair and PARP inhibitor resistance. Phospho-CDK9-Tyr19 increases its kinase activity and nuclear localization to stabilize positive transcriptional elongation factor b and activate polymerase II-dependent transcription of HR-repair genes. Conversely, ALK inhibition increases ubiquitination and degradation of CDK9 by Skp2, an E3 ligase. Notably, combination of US Food and Drug Administration-approved ALK and PARP inhibitors markedly reduce tumor growth and improve survival of mice in PARP inhibitor-/platinum-resistant tumor xenograft models. Using human tumor biospecimens, we further demonstrate that phosphorylated ALK (p-ALK) expression is associated with resistance to PARP inhibitors and positively correlated with p-Tyr19-CDK9 expression. Together, our findings support a biomarker-driven, combinatorial treatment strategy involving ALK and PARP inhibitors to induce synthetic lethality in PARP inhibitor-/platinum-resistant tumors with high p-ALK-p-Tyr19-CDK9 expression.

Two Cases Comparing the Presentations and Outcomes of Heroin-induced Toxic Leukoencephalopathy.

Heroin-induced toxic leukoencephalopathy (TLE) is an uncommon condition that presents with nonspecific and variable neuropsychiatric findings. It may result in satisfactory recovery or death. Traditionally referred to as "chasing the dragon" syndrome and associated with inhalation of pyrolyzed heroin, recent publications have reported forms of the syndrome associated with noninhaled heroin. We report 2 cases of heroin-induced TLE associated with noninhaled routes of administration and a well-documented history of opioid use disorder. The patient in the first case presented with moderate to severe symptoms. Magnetic resonance imaging of the brain revealed increased T2 and fluid-attenuated inversion recovery signals bilaterally throughout subcortical and periventricular white matter. She survived with significant cognitive issues at discharge from which she adequately recovered by 11-month follow up. The patient in the second case presented with severe symptoms. Magnetic resonance imaging of the brain showed diffuse abnormal increased T2 and fluid-attenuated inversion recovery signals in the white matter of the centrum semiovale and corona radiata. The patient died within 3 weeks of presentation. Both cases illustrate the underrecognition of the form of TLE associated with noninhaled heroin and the difficulties involved in confirming recent heroin use that likely delayed the diagnosis. Further, noninhaled heroin-induced TLE can present with specific signs and symptoms that may help clinicians delineate it from the inhaled form. Given the ongoing opioid epidemic, early and accurate recognition of this condition is of paramount importance.

Phosphorylation and Stabilization of PD-L1 by CK2 Suppresses Dendritic Cell Function.

Targeting immune checkpoints such as programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) has transformed cancer treatment, with durable clinical responses across a wide range of tumor types. However, a high percentage of patients fail to respond to anti-PD-1/PD-L1 treatment. A greater understanding of PD-L1 regulation is critical to improving the clinical response rate of PD-1/PD-L1 blockade. Here, we demonstrate that PD-L1 is phosphorylated and stabilized by casein kinase 2 (CK2) in cancer and dendritic cells (DC). Phosphorylation of PD-L1 at Thr285 and Thr290 by CK2 disrupted PD-L1 binding with speckle-type POZ protein, an adaptor protein of the cullin 3 (CUL3) ubiquitin E3 ligase complex, protecting PD-L1 from CUL3-mediated proteasomal degradation. Inhibition of CK2 decreased PD-L1 protein levels by promoting its degradation and resulted in the release of CD80 from DC to reactivate T-cell function. In a syngeneic mouse model, combined treatment with a CK2 inhibitor and an antibody against T-cell immunoglobulin mucin-3 (Tim-3) suppressed tumor growth and prolonged survival. These findings uncover a mechanism by which PD-L1 is regulated and suggest a potential antitumor treatment option to activate DC function by blocking the CK2-PD-L1 pathway and inhibiting Tim-3. SIGNIFICANCE: This work identifies a role for CK2 in immunosuppression by phosphorylation and stabilization of PD-L1, identifying CK2 inhibition as an immunotherapeutic approach for treating cancer.

Ephrin receptor A10 monoclonal antibodies and the derived chimeric antigen receptor T cells exert an antitumor response in mouse models of triple-negative breast cancer.

Expression of the receptor tyrosine kinase ephrin receptor A10 (EphA10), which is undetectable in most normal tissues except for the male testis, has been shown to correlate with tumor progression and poor prognosis in several malignancies, including triple-negative breast cancer (TNBC). Therefore, EphA10 could be a potential therapeutic target, likely with minimal adverse effects. However, no effective clinical drugs against EphA10 are currently available. Here, we report high expression levels of EphA10 in tumor regions of breast, lung, and ovarian cancers as well as in immunosuppressive myeloid cells in the tumor microenvironment. Furthermore, we developed anti-EphA10 monoclonal antibodies (mAbs) that specifically recognize cell surface EphA10, but not other EphA family isoforms, and target tumor regions precisely in vivo with no apparent accumulation in other organs. In syngeneic TNBC mouse models, we found that anti-EphA10 mAb clone #4 enhanced tumor regression, therapeutic response rate, and T cell-mediated antitumor immunity. Notably, the chimeric antigen receptor T cells derived from clone #4 significantly inhibited TNBC cell viability in vitro and tumor growth in vivo. Together, our findings suggest that targeting EphA10 via EphA10 mAbs and EphA10-specific chimeric antigen receptor-T cell therapy may represent a promising strategy for patients with EphA10-positive tumors.

Despite The FDA's Five-Year Plan, Black Patients Remain Inadequately Represented In Clinical Trials For Drugs.

For decades Black patients have been underrepresented in clinical trials of new treatments. In response, in 2015 the Food and Drug Administration (FDA) launched a five-year action plan aimed at improving diversity in and transparency of pivotal clinical trials for newly approved drugs. The plan contained many action steps that were aimed at improving the racial representativeness of clinical trials and enhancing the reporting of new drug side effects and benefits across diverse populations. Yet, relying on the FDA's Drug Trials Snapshots website, we failed to find evidence that the action plan improved representation of Black trial participants. Black patients remained inadequately represented in clinical trials for drugs, with a median of one-third the enrollment that would be required, whether the trials were started before, during, or after the action plan. Fewer than 20 percent of drugs had data regarding treatment benefits or side effects reported for Black patients; neither measure improved during the action plan period.

A Unique Case of Community Acquired Proteus mirabilis Meningitis.

Proteus mirabilis, a gram-negative bacterium commonly known for causing urinary tract infections (UTI) can rarely present with central nervous system (CNS) infections. Proteus mirabilis CNS infections are usually encountered in the neonatal and infantile period and occasionally cause brain abscesses. It is an uncommon cause of adult CNS infection. We report the first case of a community-acquired Proteus mirabilis meningitis (PMM) in a patient with Proteus mirabilis UTI, urolithiasis, and bacteremia. Risk factors for gram-negative bacillary meningitis (GNBM) include extremes of age, cancer history, diabetes mellitus, UTI, and nosocomial exposure, with the latter being a more prominent cause of PMM. Compromise of the anatomical defense against CNS infections whether accidental or neurosurgical is another important cause, and approximately two-thirds of reported cases of PMM have occurred after neurosurgical procedures. PMM patients develop fever, altered consciousness, and have an acute clinical course. Antimicrobials that can be used for treatment include third-generation cephalosporins, ciprofloxacin, imipenem/ cilastatin, aztreonam, and intraventricular aminoglycosides. Despite appropriate antibiotic therapy outcomes are poor with severe neurological deficit and death commonly resulting. Nosocomial infections can be drug-resistant and multiple antibiotics should be started while awaiting culture results. Literature review reveals that treatment with intraventricular aminoglycosides when attempted has shown bacteriological cure indicating this can be an important treatment approach. Due to the acute clinical course and high morbidity and mortality, we recommend starting multiple antibiotics with different mechanisms of action as soon as the disease is suspected. Our patient was initially started on ceftriaxone, vancomycin, acyclovir, and ampicillin for UTI and meningoencephalitis. The antibiotics were later consolidated to cefepime based on blood, urine and, cerebrospinal fluid cultures growing pan-sensitive Proteus mirabilis. Her clinical condition continued to worsen and ciprofloxacin was added. However, due to the progressive decline in her condition, the family elected for inpatient hospice care and intraventricular aminoglycosides were not attempted.

Factors That Affect Patient Attrition in Buprenorphine Treatment for Opioid Use Disorder: A Retrospective Real-World Study Using Electronic Health Records.

PURPOSE: To describe attrition patterns of opioid use disorder (OUD) patients treated with buprenorphine (BUP) and to assess how clinical, sociodemographic, or BUP medication dosing features are associated with attrition. PATIENTS AND METHODS: Electronic health records of adults (16+ year-olds) with OUD treated with BUP from 23 different substance use or mental health care programs across 11 US states were examined for one year following BUP initiation in inpatient (IP), intensive outpatient (IOP), or outpatient (OP) settings. Treatment attrition was declared at >37 days following the last recorded visit. Survival analyses and predictive modelling were used. RESULTS: Retention was consistently 2-3 times higher following BUP initiation in OP (n = 2409) than in IP/IOP (n = 2749) settings after 2 (50% vs 25%), 6 (27% vs 9%) and 12 months (14% vs 4%). Retention was higher for females, whites (vs blacks), and those with less severe OUD, better global function, or not using non-psychotropic medications. Comorbid substance use, other psychiatric disorders, and the number of psychotropic medications were variously related to retention depending on the setting in which BUP was initiated. Predictive modelling revealed that a higher global assessment of functioning and a smaller OUD severity based on the Clinical Global Impression - Severity led to longer retentions, a higher initial BUP dose led to higher retention in a few cases, an OP setting of BUP initiation led to longer retentions, and a lower total number of psychotropic and non-psychotropic medications led to longer retentions. These were the most important parameters in the model, which identified 75.2% of patients who left BUP treatment within three months post-initiation, with a precision of 90.5%. CONCLUSION: Of all the OUD patients who began BUP, 50-75% left treatment within three months, and most could be accurately identified. This could facilitate patient-centered management to better retain OUD patients in BUP treatment.

Treatment of Cannabinoid Hyperemesis With Olanzapine: A Case Series.

The use and availability of cannabis for recreational and medical purposes has become more widespread with increased legalization. Adverse health outcomes of this increased use include cannabinoid hyperemesis syndrome (CHS), which is underrecognized in medical settings. Cessation of substance use is the recommendation of choice for the complete resolution of CHS. However, interventions that provide rapid relief may be necessary in treatment-refractory cases. Little evidence is available to guide care in these cases. Here we report 4 cases of treatment-refractory CHS, all of which remitted after treatment with olanzapine. Olanzapine is known to block multiple neurotransmitter receptors involved in nausea and vomiting in chemotherapy-induced nausea and vomiting. Outcomes of the cases reported here suggest that off-label use of olanzapine may be effective in the symptomatic treatment of refractory CHS and may be the preferred treatment in cases in which comorbid psychotic symptoms or agitation are present.

PKCδ-mediated SGLT1 upregulation confers the acquired resistance of NSCLC to EGFR TKIs.

The tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) have been widely used for non-small cell lung cancer (NSCLC) patients, but the development of acquired resistance remains a therapeutic hurdle. The reduction of glucose uptake has been implicated in the anti-tumor activity of EGFR TKIs. In this study, the upregulation of the active sodium/glucose co-transporter 1 (SGLT1) was found to confer the development of acquired EGFR TKI resistance and was correlated with the poorer clinical outcome of the NSCLC patients who received EGFR TKI treatment. Blockade of SGLT1 overcame this resistance in vitro and in vivo by reducing glucose uptake in NSCLC cells. Mechanistically, SGLT1 protein was stabilized through the interaction with PKCδ-phosphorylated (Thr678) EGFR in the TKI-resistant cells. Our findings revealed that PKCδ/EGFR axis-dependent SGLT1 upregulation was a critical mechanism underlying the acquired resistance to EGFR TKIs. We suggest co-targeting PKCδ/SGLT1 as a potential strategy to improve the therapeutic efficacy of EGFR TKIs in NSCLC patients.

Targeting fibroblast growth factor receptors to combat aggressive ependymoma.

Ependymomas (EPN) are central nervous system tumors comprising both aggressive and more benign molecular subtypes. However, therapy of the high-risk subtypes posterior fossa group A (PF-A) and supratentorial RELA-fusion positive (ST-RELA) is limited to gross total resection and radiotherapy, as effective systemic treatment concepts are still lacking. We have recently described fibroblast growth factor receptors 1 and 3 (FGFR1/FGFR3) as oncogenic drivers of EPN. However, the underlying molecular mechanisms and their potential as therapeutic targets have not yet been investigated in detail. Making use of transcriptomic data across 467 EPN tissues, we found that FGFR1 and FGFR3 were both widely expressed across all molecular groups. FGFR3 mRNA levels were enriched in ST-RELA showing the highest expression among EPN as well as other brain tumors. We further identified high expression levels of fibroblast growth factor 1 and 2 (FGF1, FGF2) across all EPN subtypes while FGF9 was elevated in ST-EPN. Interrogation of our EPN single-cell RNA-sequencing data revealed that FGFR3 was further enriched in cycling and progenitor-like cell populations. Corroboratively, we found FGFR3 to be predominantly expressed in radial glia cells in both mouse embryonal and human brain datasets. Moreover, we detected alternative splicing of the FGFR1/3-IIIc variant, which is known to enhance ligand affinity and FGFR signaling. Dominant-negative interruption of FGFR1/3 activation in PF-A and ST-RELA cell models demonstrated inhibition of key oncogenic pathways leading to reduced cell growth and stem cell characteristics. To explore the feasibility of therapeutically targeting FGFR, we tested a panel of FGFR inhibitors in 12 patient-derived EPN cell models revealing sensitivity in the low-micromolar to nano-molar range. Finally, we gain the first clinical evidence for the activity of the FGFR inhibitor nintedanib in the treatment of a patient with recurrent ST-RELA. Together, these preclinical and clinical data suggest FGFR inhibition as a novel and feasible approach to combat aggressive EPN.