Identifying the core genome of the nucleus-forming bacteriophage family and characterization of Erwinia phage RAY.

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were still to be determined. Here, we show that phages encoding the major phage nucleus protein chimallin share 72 conserved genes encoded within seven gene blocks. Of these, 21 core genes are unique to nucleus-forming phage, and all but one of these genes encode proteins of unknown function. We propose that these phages comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryoelectron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication are conserved among diverse chimalliviruses and reveal variations on this replication mechanism. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.

Identifying the core genome of the nucleus-forming bacteriophage family and characterization of Erwinia phage RAY.

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were unknown. By studying phages that encode the major phage nucleus protein chimallin, including previously sequenced yet uncharacterized phages, we discovered that chimallin-encoding phages share a set of 72 highly conserved genes encoded within seven distinct gene blocks. Of these, 21 core genes are unique to this group, and all but one of these unique genes encode proteins of unknown function. We propose that phages with this core genome comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryo-electron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication encoded in the core genome are conserved among diverse chimalliviruses, and reveal that non-core components can confer intriguing variations on this replication mechanism. For instance, unlike previously studied nucleus-forming phages, RAY doesn't degrade the host genome, and its PhuZ homolog appears to form a five-stranded filament with a lumen. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.

Increased Comorbidity Burden Among Hip Fracture Patients During the COVID-19 Pandemic in New York City.

BACKGROUND: The coronavirus disease 19 (COVID-19) pandemic had a devastating effect on New York City in the spring of 2020. Several global reports suggested worse early outcomes among COVID-positive patients with hip fractures. However, there is limited data comparing baseline comorbidities among patients treated during the pandemic relative to those treated in non-pandemic conditions. MATERIALS AND METHODS: A multicenter retrospective cohort study was performed at two Level 1 Trauma centers and one orthopedic specialty hospital to assess demographics, comorbidities, and outcomes among 67 hip fracture patients treated (OTA/AO 31, 32.1) during the peak of the COVID-19 pandemic in New York City (March 20, 2020 to April 24, 2020), including 9 who were diagnosed with COVID-19. These patients were compared to a cohort of 76 hip fracture patients treated 1 year prior (March 20, 2019 to April 24, 2019). Baseline demographics, comorbidities, treatment characteristics, and respiratory symptomatology were evaluated. The primary outcome was inpatient mortality. RESULTS: Relative to patients treated in 2019, patients with hip fractures during the pandemic had worse Charlson Comorbidity Indices (median 5.0 vs 6.0, P = .03) and American Society of Anesthesiologists (ASA) scores (mean 2.4 vs 2.7, P = .04). Patients during the COVID-19 pandemic were more likely to have decreased ambulatory status (P<.01) and a smoking history (P = .04). Patients in 2020 had longer inpatient stays (median 5 vs 7 days, P = .01), and were more likely to be discharged home (61% vs 9%, P<.01). Inpatient mortality was significantly increased during the COVID-19 pandemic (12% vs 0%, P = .002). CONCLUSIONS: Patients with hip fractures during the COVID-19 pandemic had worse comorbidity profiles and decreased functional status compared to patients treated the year prior. This information may be relevant in negotiations regarding reimbursement for cost of care of hip fracture patients with COVID-19, as these patients may require more expensive care.

The association between spondylolisthesis and decreased muscle health throughout the lumbar spine for patients with operative lumbar spinal stenosis.

PURPOSE: There are data underlining the relationship between muscle health and spine related pathology, but little data regarding changes in paralumbar muscle associated with lumbar spondylolisthesis. We aimed to define changes in paralumbar muscle health associated with spondylolisthesis. METHODS: A retrospective review was performed on consecutive patients with lumbar spine pathology requiring an operation. A pre-operative lumbar MRI was analysed for muscle health measurements including lumbar indentation value (LIV), paralumbar cross-sectional area divided by body mass index (PL-CSA/BMI), and Goutallier classification of fatty atrophy. All measurements were taken from an axial slice of a T2-weighted image at lumbar disc spaces. Baseline health-related quality of life scores (HRQOLs), narcotic use and areas of stenosis were tracked. We performed Chi-square analyses and student's t test to determine statistically significant differences between cohorts. RESULTS: There were 307 patients (average age 56.1 ± 16.7 years, 141 females) included within our analysis. 112 patients had spondylolisthesis. There were no differences in baseline HRQOLs between the spondylolisthesis cohort (SC) and non-spondylolisthesis cohort (non-SC). There were significantly worse PL-CSA/BMI at L2-L3 (p = 0.03), L3-L4 (p = 0.04) and L4-L5 (p = 0.02) for the SC. Goutallier classification of paralumbar muscle was worse for SC at L1-L2 (p = 0.04) and at L4-L5 (p < 0.001). Increased grade of spondylolisthesis was associated with worse PL-CSA at L1-L2 (p = 0.02), L2-L3 (p = 0.03) and L3-L4 (p = 0.05). Similarly, there were worse Goutallier classification scores associated with higher-grade spondylolisthesis at all levels (p < 0.05). CONCLUSION: There are significant detrimental changes to paralumbar muscle health throughout the lumbar spine associated with spondylolisthesis.

A Novel Magnetic Resonance Imaging-based Lumbar Muscle Grade to Predict Health-related Quality of Life Scores Among Patients Requiring Surgery.

STUDY DESIGN: Retrospective cross-sectional cohort. OBJECTIVE: The aim of this sudy was to determine whether muscle health measurements are associated with health-related quality of life scores (HRQOLs) for patients with lumbar spine pathology. SUMMARY OF BACKGROUND DATA: Poor muscle health has been implicated as a source of pain/dysfunction for patients with lumbar spine pathology. Our aim was to quantify the relationship using muscle health measurements and HRQOLs. METHODS: Three hundred and eight patients were included (mean age 57.7 ±â€Šstandard deviation 18.2 years' old). We randomly selected patients into a derivation cohort (200) and validation cohort (108) to create our muscle health grade. We measured muscle health by the lumbar indentation value (LIV), goutallier classification (GC), and ratio of paralumbar muscle cross-sectional area over body mass index (PL-CSA/BMI). A muscle health grade was derived based on whether a measurement showed a statistically significant impact on visual analog scale back and leg pain (VAS-leg and VAS-leg), Oswestry Disability Index (ODI), short-form 12 physical health score (SF-12 PHS), short-form 12 mental health score (SF-12 MHS) and Patient-reported Outcomes Measurement Information System (PROMIS). A variety of statistical tools were used to determine whether there was a relationship between a measurement and HRQOLs. RESULTS: In the derivation cohort, a muscle health grade was created based on the GC and PL-CSA/BMI ratio. For patients with a GC ≤2, one point was given. For patients with a PL-CSA/BMI ≥130, one point was given. Patients with 2 points were graded as "A" and 0 or 1 point were graded "B." Within the validation cohort of patients, there was a statistically significant higher PROMIS (mean 34.5 ±â€Šstandard deviation 12.6 vs. 27.6 ±â€Š14.0, P = 0.002), ODI (38.8 ±â€Š18.3 vs. 45.8 ±â€Š18.1, P = 0.05) and SF-12 PHS (34.7 ±â€Š11.3 vs. 29.1 ±â€Š6.3, P = 0.002) for patients with a good muscle health grade of "A." CONCLUSION: This study offers an objective measurement of muscle health that correlates with HRQOLs for patients with lumbar spine pathology.Level of Evidence: 3.

Clinical Experience with COVID-19 at a Specialty Orthopedic Hospital Converted to a Pandemic Overflow Field Hospital.

BACKGROUND: COVID-19, the illness caused by the novel coronavirus, SARS-CoV-2, has sickened millions and killed hundreds of thousands as of June 2020. New York City was affected gravely. Our hospital, a specialty orthopedic hospital unaccustomed to large volumes of patients with life-threatening respiratory infections, underwent rapid adaptation to care for COVID-19 patients in response to emergency surge conditions at neighboring hospitals. PURPOSES: We sought to determine the attributes, pharmacologic and other treatments, and clinical course in the cohort of patients with COVID-19 who were admitted to our hospital at the height of the pandemic in April 2020 in New York City. METHODS: We conducted a retrospective observational cohort study of all patients admitted between April 1 and April 21, 2020, who had a diagnosis of COVID-19. Data were gathered from the electronic health record and by manual chart abstraction. RESULTS: Of the 148 patients admitted with COVID-19 (mean age, 62 years), ten patients died. There were no deaths among non-critically ill patients transferred from other hospitals, while 26% of those with critical illness died. A subset of COVID-19 patients was admitted for orthopedic and medical conditions other than COVID-19, and some of these patients required intensive care and ventilatory support. CONCLUSION: Professional and organizational flexibility during pandemic conditions allowed a specialty orthopedic hospital to provide excellent care in a global public health emergency.

Hip Fracture Outcomes During the COVID-19 Pandemic: Early Results From New York.

OBJECTIVE: To evaluate inpatient outcomes among patients with hip fracture treated during the COVID-19 pandemic in New York City. DESIGN: Multicenter retrospective cohort study. SETTING: One Level 1 trauma center and one orthopaedic specialty hospital in New York City. PATIENTS/PARTICIPANTS: Fifty-nine consecutive patients (average age 85 years, range: 65-100 years) treated for a hip fracture (OTA/AO 31, 32.1) over a 5-week period, March 20, 2020, to April 24, 2020, during the height of the COVID-19 crisis. MAIN OUTCOME MEASUREMENTS: COVID-19 infection status was used to stratify patients. The primary outcome was inpatient mortality. Secondary outcomes were admission to the intensive care unit, unexpected intubation, pneumonia, deep vein thrombosis, pulmonary embolus, myocardial infarction, cerebrovascular accident, urinary tract infection, and transfusion. Baseline demographics, comorbidities, treatment characteristics, and COVID-related symptomatology were also evaluated. RESULTS: Ten patients (15%) tested positive for COVID-19 (COVID+) (n = 9; 7 preoperatively and 2 postoperatively) or were presumed positive (n = 1), 40 (68%) patients tested negative, and 9 (15%) patients were not tested in the primary hospitalization. American Society of Anesthesiologists' scores were higher in the COVID+ group (d = -0.83; P = 0.04); however, the Charlson Comorbidity Index was similar between the study groups (d = -0.17; P = 0.63). Inpatient mortality was significantly increased in the COVID+ cohort (56% vs. 4%; odds ratio 30.0, 95% confidence interval 4.3-207; P = 0.001). Including the one presumed positive case in the COVID+ cohort increased this difference (60% vs. 2%; odds ratio 72.0, 95% confidence interval 7.9-754; P < 0.001). CONCLUSIONS: Hip fracture patients with concomitant COVID-19 infection had worse American Society of Anesthesiologists' scores but similar baseline comorbidities with significantly higher rates of inpatient mortality compared with those without concomitant COVID-19 infection. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

How does preoperative opioid use impact postoperative health-related quality of life scores for patients undergoing lumbar microdiscectomy?

BACKGROUND CONTEXT: Narcotic use amongst patients suffering from lumbar radiculopathy is common, but the clinical benefit of narcotics for lumbar radiculopathy is likely minimal. It is unknown what the impact of preoperative use of narcotics has on outcomes related to lumbar microdiscectomy. PURPOSE: Determine the impact that preoperative opioid use has on postoperative outcomes after lumbar microdisectomy. STUDY DESIGN: Retrospective analysis of a prospectively collected database. PATIENT SAMPLE: One hundred and twenty-six patients undergoing a microdiscectomy for a lumbar disc herniation. OUTCOME MEASURES: Patient-reported outcomes measurement information system mental health scores (PROMIS MHS), patient-reported outcomes measurement information system physical health scores (PROMIS PHS) and oswestry disability index (ODI). METHODS: We analyzed a prospectively collected database of patients undergoing a lumbar microdiscectomy for preoperative opioid use. We measured the severity of lumbar pathology on MRI based on degree of facet/disc degeneration and cross-sectional area of the dural tube at the disc herniation. We tracked PROMIS MHS, PROMIS PHS and ODI for patients both preoperatively and postoperatively. A Mann-Whitney test was used to compare HRQOL scores and time to MCID for the opioid using cohort (OC) and the nonopioid using cohort (non-OC). We performed a linear regression analysis to determine correlation between preoperative opioid use and postoperative HRQOLs. RESULTS: There were 44 of 126 microdiscectomy patients in the OC (32.5%). There was no difference in the dural cross-sectional area (p=.91), degree of facet degeneration (p=.38), or disc degeneration (p=.5) between OC and non-OC. There were no differences in PROMIS PHS, PROMIS MHS or ODI between the OC and non-OC at the preoperative visit and all postoperative time points. There were no differences in time to reach MCID between the OC and non-OC for ODI (p=.9), PROMIS PHS (p=.64) or PROMIS MHS (p=.90). At three months out from surgery there was a statistically significant correlation between pre-op opioid use and ODI (p=.02), PROMIS MHS (p=.02) and PROMIS PHS (p=.049). CONCLUSIONS: Our results demonstrate that patients that use opioids prior to lumbar microdiscectomy have equivalent postoperative outcomes as those that do not use opioids. Use of higher doses of opioids is associated with worse short-term outcomes.

The effect of anti-CTLA4 treatment on peripheral and intra-tumoral T cells in patients with hepatocellular carcinoma.

BACKGROUND: Checkpoint inhibitors have recently been approved for the treatment of patients with hepatocellular carcinoma (HCC). However, biomarkers, which will help identify patients responding to therapy, are missing. We recently tested the combination of anti-CTLA4 treatment (tremelimumab) with loco-regional therapy in patients with HCC and reported a partial response rate of 26%. METHODS: Here, we report updated survival analyses and results from our immune monitoring studies on peripheral blood mononuclear cells (PBMCs) and tumors from these patients. RESULTS: Tremelimumab therapy increased CD4+-HLA-DR+, CD4+PD-1+, CD8+HLA-DR+, CD8+PD-1+, CD4+ICOS+ and CD8+ICOS+ T cells in the peripheral blood of the treated patients. Patients with higher CD4+PD1+ cell frequency at baseline were more likely to respond to tremelimumab therapy. PD-1 expression was increased on alpha fetal protein (AFP) and survivin-specific CD8 T cells upon tremelimumab treatment. An increase of tumor infiltrating CD3+ T cells were also seen in these patients. Immunosequencing of longitudinal PBMC showed that one cycle of tremelimumab significantly decreased peripheral clonality, while no additional effects were seen after loco-regional therapy. CONCLUSION: In summary, we observed a clear activation of T cell responses in HCC patients treated with tremelimumab and identified potential biomarkers which will help identify patients responding to immunotherapy with anti-CTLA4.

Targeting the crosstalk between cytokine-induced killer cells and myeloid-derived suppressor cells in hepatocellular carcinoma.

BACKGROUND & AIMS: Cytokine-induced killer (CIK) cell-based immunotherapy is effective as an adjuvant therapy in early stage hepatocellular carcinoma (HCC) but lacks efficacy in advanced HCC. We aimed to investigate immune suppressor mechanisms in HCC, focusing on the role of myeloid-derived suppressor cells (MDSCs) in response to CIK therapy. METHODS: MDSCs were quantified by flow cytometry and quantitative real-time PCR. Cytokines were detected by cytokine array. A lactate dehydrogenase cytotoxicity assay was performed in the presence or absence of MDSCs to study CIK function against HCC cells in vitro. An FDA-approved PDE5 inhibitor, tadalafil, was used to target MDSCs in vitro and in vivo. Two different murine HCC cell lines were tested in subcutaneous and orthotopic tumor models in C57BL/6 and BALB/c mice. The antitumor effects of human CIKs and MDSCs were also tested in vitro. RESULTS: Adoptive cell transfer of CIKs into tumor-bearing mice induced inflammatory mediators (e.g., CX3CL1, IL-13) in the tumor microenvironment and an increase of tumor-infiltrating MDSCs, leading to impaired antitumor activity in 2 different HCC models. MDSCs efficiently suppressed the cytotoxic activity of CIKs in vitro. In contrast, treatment with a PDE5 inhibitor reversed the MDSC suppressor function via ARG1 and iNOS blockade and systemic treatment with a PDE5 inhibitor prevented MDSC accumulation in the tumor microenvironment upon CIK cell therapy and increased its antitumor efficacy. Similar results were observed when human CIKs were tested in vitro in the presence of CD14+HLA-DR-/low MDSCs. Treatment of MDSCs with a PDE5 inhibitor suppressed MDSC suppressor function and enhanced CIK activity against human HCC cell lines in vitro. CONCLUSION: Our results suggest that targeting MDSCs is an efficient strategy to enhance the antitumor efficacy of CIKs for the treatment of patients with HCC. LAY SUMMARY: Cytokine-induced killer cells are a mixture of immune cells given to eliminate cancer cells. However, not all patients respond to this treatment. Herein, we show in 2 different liver cancer models that myeloid-derived suppressor cells are increased in response to cytokine-induced killer cell therapy. Targeting these myeloid-derived suppressor cells may provide an additional therapeutic benefit alongside cytokine-induced killer cell therapy.

Tremelimumab in Combination With Microwave Ablation in Patients With Refractory Biliary Tract Cancer.

Treatment options for patients with advanced biliary tract cancer are limited. Dysregulation of the immune system plays an important role in the pathogenesis of biliary tract cancer (BTC). This study aimed to investigate whether tremelimumab, an anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4) inhibitor, could be combined safely with microwave ablation to enhance the effect of anti-CTLA4 treatment in patients with advanced BTC. Patients were enrolled to receive monthly tremelimumab (10 mg/kg, intravenously) for six doses, followed by infusions every 3 months until off-treatment criteria were met. Thirty-six days after the first tremelimumab dose, patients underwent subtotal microwave ablation. Interval imaging studies were performed every 8 weeks. Adverse events (AEs) were noted and managed. Tumor and peripheral blood samples were collected to perform immune monitoring and whole-exome sequencing (WES). Twenty patients with refractory BTC were enrolled (median age, 56.5 years). No dose-limiting toxicities were encountered. The common treatment-related AEs included lymphopenia, diarrhea, and elevated transaminases. Among 16 patients evaluable for efficacy analysis, 2 (12.5%) patients achieved a confirmed partial response (lasting for 8.0 and 18.1 months, respectively) and 5 patients (31.3%) achieved stable disease. Median progression free survival (PFS) and overall survival (OS) were 3.4 months (95% confidence interval [CI], 2.5-5.2) and 6.0 months (95% CI, 3.8-8.8), respectively. Peripheral blood immune cell subset profiling showed increased circulating activated human leukocyte antigen, DR isotype ([HLA-DR] positive) CD8+ T cells. T-cell receptor (TCR)ß screening showed tremelimumab expanded TCR repertoire, but not reaching statistical significance (P = 0.057). Conclusion: Tremelimumab in combination with tumor ablation is a potential treatment strategy for patients with advanced BTC. Increased circulating activated CD8+ T cells and TCR repertoire expansion induced by tremelimumab may contribute to treatment benefit.

Indoleamine 2,3-dioxygenase provides adaptive resistance to immune checkpoint inhibitors in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. Immune checkpoint blockade with anti-CTLA-4 and anti-PD-1 antibodies has shown promising results in the treatment of patients with advanced HCC. The anti-PD-1 antibody, nivolumab, is now approved for patients who have had progressive disease on the current standard of care. However, a subset of patients with advanced HCC treated with immune checkpoint inhibitors failed to respond to therapy. Here, we provide evidence of adaptive resistance to immune checkpoint inhibitors through upregulation of indoleamine 2,3-dioxygenase (IDO) in HCC. Anti-CTLA-4 treatment promoted an induction of IDO1 in resistant HCC tumors but not in tumors sensitive to immune checkpoint blockade. Using both subcutaneous and hepatic orthotopic models, we found that the addition of an IDO inhibitor increases the efficacy of treatment in HCC resistant tumors with high IDO induction. Furthermore, in vivo neutralizing studies demonstrated that the IDO induction by immune checkpoint blockade was dependent on IFN-γ. Similar findings were observed with anti-PD-1 therapy. These results provide evidence that IDO may play a role in adaptive resistance to immune checkpoint inhibitors in patients with HCC. Therefore, inhibiting IDO in combination with immune checkpoint inhibitors may add therapeutic benefit in tumors which overexpress IDO and should be considered for clinical evaluation in HCC.

Gut microbiome-mediated bile acid metabolism regulates liver cancer via NKT cells.

Primary liver tumors and liver metastasis currently represent the leading cause of cancer-related death. Commensal bacteria are important regulators of antitumor immunity, and although the liver is exposed to gut bacteria, their role in antitumor surveillance of liver tumors is poorly understood. We found that altering commensal gut bacteria in mice induced a liver-selective antitumor effect, with an increase of hepatic CXCR6+ natural killer T (NKT) cells and heightened interferon-γ production upon antigen stimulation. In vivo functional studies showed that NKT cells mediated liver-selective tumor inhibition. NKT cell accumulation was regulated by CXCL16 expression of liver sinusoidal endothelial cells, which was controlled by gut microbiome-mediated primary-to-secondary bile acid conversion. Our study suggests a link between gut bacteria-controlled bile acid metabolism and liver antitumor immunosurveillance.