TGF-ß1 mediates hypoxia-preconditioned olfactory mucosa mesenchymal stem cells improved neural functional recovery in Parkinson's disease models and patients.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder characterized by the degeneration of dopaminergic neurons in the substantia nigra (SN). Activation of the neuroinflammatory response has a pivotal role in PD. Mesenchymal stem cells (MSCs) have emerged as a promising therapeutic approach for various nerve injuries, but there are limited reports on their use in PD and the underlying mechanisms remain unclear. METHODS: We investigated the effects of clinical-grade hypoxia-preconditioned olfactory mucosa (hOM)-MSCs on neural functional recovery in both PD models and patients, as well as the preventive effects on mouse models of PD. To assess improvement in neuroinflammatory response and neural functional recovery induced by hOM-MSCs exposure, we employed single-cell RNA sequencing (scRNA-seq), assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) combined with full-length transcriptome isoform-sequencing (ISO-seq), and functional assay. Furthermore, we present the findings from an initial cohort of patients enrolled in a phase I first-in-human clinical trial evaluating the safety and efficacy of intraspinal transplantation of hOM-MSC transplantation into severe PD patients. RESULTS: A functional assay identified that transforming growth factor-ß1 (TGF-ß1), secreted from hOM-MSCs, played a critical role in modulating mitochondrial function recovery in dopaminergic neurons. This effect was achieved through improving microglia immune regulation and autophagy homeostasis in the SN, which are closely associated with neuroinflammatory responses. Mechanistically, exposure to hOM-MSCs led to an improvement in neuroinflammation and neural function recovery partially mediated by TGF-ß1 via activation of the anaplastic lymphoma kinase/phosphatidylinositol-3-kinase/protein kinase B (ALK/PI3K/Akt) signaling pathway in microglia located in the SN of PD patients. Furthermore, intraspinal transplantation of hOM-MSCs improved the recovery of neurologic function and regulated the neuroinflammatory response without any adverse reactions observed in patients with PD. CONCLUSIONS: These findings provide compelling evidence for the involvement of TGF-ß1 in mediating the beneficial effects of hOM-MSCs on neural functional recovery in PD. Treatment and prevention of hOM-MSCs could be a promising and effective neuroprotective strategy for PD. Additionally, TGF-ß1 may be used alone or combined with hOM-MSCs therapy for treating PD.

A Cullin 5-based complex serves as an essential modulator of ORF9b stability in SARS-CoV-2 replication.

The ORF9b protein, derived from the nucleocapsid's open-reading frame in both SARS-CoV and SARS-CoV-2, serves as an accessory protein crucial for viral immune evasion by inhibiting the innate immune response. Despite its significance, the precise regulatory mechanisms underlying its function remain elusive. In the present study, we unveil that the ORF9b protein of SARS-CoV-2, including emerging mutant strains like Delta and Omicron, can undergo ubiquitination at the K67 site and subsequent degradation via the proteasome pathway, despite certain mutations present among these strains. Moreover, our investigation further uncovers the pivotal role of the translocase of the outer mitochondrial membrane 70 (TOM70) as a substrate receptor, bridging ORF9b with heat shock protein 90 alpha (HSP90α) and Cullin 5 (CUL5) to form a complex. Within this complex, CUL5 triggers the ubiquitination and degradation of ORF9b, acting as a host antiviral factor, while HSP90α functions to stabilize it. Notably, treatment with HSP90 inhibitors such as GA or 17-AAG accelerates the degradation of ORF9b, leading to a pronounced inhibition of SARS-CoV-2 replication. Single-cell sequencing data revealed an up-regulation of HSP90α in lung epithelial cells from COVID-19 patients, suggesting a potential mechanism by which SARS-CoV-2 may exploit HSP90α to evade the host immunity. Our study identifies the CUL5-TOM70-HSP90α complex as a critical regulator of ORF9b protein stability, shedding light on the intricate host-virus immune response dynamics and offering promising avenues for drug development against SARS-CoV-2 in clinical settings.

Discovery of a signaling feedback circuit that defines interferon responses in myeloproliferative neoplasms.

Interferons (IFNs) are key initiators and effectors of the immune response against malignant cells and also directly inhibit tumor growth. IFNα is highly effective in the treatment of myeloproliferative neoplasms (MPNs), but the mechanisms of action are unclear and it remains unknown why some patients respond to IFNα and others do not. Here, we identify and characterize a pathway involving PKCδ-dependent phosphorylation of ULK1 on serine residues 341 and 495, required for subsequent activation of p38 MAPK. We show that this pathway is essential for IFN-suppressive effects on primary malignant erythroid precursors from MPN patients, and that increased levels of ULK1 and p38 MAPK correlate with clinical response to IFNα therapy in these patients. We also demonstrate that IFNα treatment induces cleavage/activation of the ULK1-interacting ROCK1/2 proteins in vitro and in vivo, triggering a negative feedback loop that suppresses IFN responses. Overexpression of ROCK1/2 is seen in MPN patients and their genetic or pharmacological inhibition enhances IFN-anti-neoplastic responses in malignant erythroid precursors from MPN patients. These findings suggest the clinical potential of pharmacological inhibition of ROCK1/2 in combination with IFN-therapy for the treatment of MPNs.

Association of HLA class I homozygosity with unfavorable clinical outcomes in patients with non-small cell lung cancer treated with chemo-immunotherapy or immunotherapy as first-line therapy.

BACKGROUND: Homozygosity at HLA-I locus has been reported to be an unfavorable predictive biomarker of second-line or beyond immunotherapy in patients with different types of cancer. The linkage between HLA-I zygosity and survival in NSCLC patients treated with first-line immunotherapy with or without chemotherapy has not been reported. METHODS: Next generation sequencing with HLA genotyping was performed on patients with advanced NSCLC treated with immune checkpoint inhibitors with or without chemotherapy as first-line (N = 29). Progression free survival was compared between HLA-I homozygous (defined as homozygosity in at least one locus A, B, or C) and heterozygous patients. Kaplan-Meier curves were built, and log-rank test was used. RESULTS: Among 29 enrollees, 25 patients (86.2%) were HLA-I heterozygous and four patients (13.8%) were HLA-I homozygous. Treatment response was not available in five patients with HLA-I heterozygosity. Among 20 patients with HLA-I heterozygosity, five patients (20.0%) had partial response, 10 patients (50.0%) had stable disease, two patients (8.0%) had non-complete response/non-progressive disease, and three patients (12.0%) had progressive disease. Among four patients with HLA-I heterozygosity, one patient (25.0%) had partial response, one patient (25.0%) had stable disease, and two patients (50.0%) had progressive disease. The median progression free survival was not reached in heterozygous group and was 2.97 months in homozygous group (Log-rank p = 0.68). CONCLUSIONS: We observed a trend toward an inverse association between HLA-I homozygosity and survival outcomes in patients with NSCLC treated with first-line therapy in conjunction with immunotherapy. Further prospective studies to validate aforementioned relationship are warranted.

Avenues Toward microRNA Detection In Vitro: A Review of Technical Advances and Challenges.

Over the decades, the biological role of microRNAs (miRNAs) in the post-transcriptional regulation of gene expression has been discovered in many cancer types, thus initiating the tremendous expectation of their application as biomarkers in the diagnosis, prognosis, and treatment of cancer. Hence, the development of efficient miRNA detection methods in vitro is in high demand. Extensive efforts have been made based on the intrinsic properties of miRNAs, such as low expression levels, high sequence homology, and short length, to develop novel in vitro miRNA detection methods with high accuracy, low cost, practicality, and multiplexity at point-of-care settings. In this review, we mainly summarized the newly developed in vitro miRNA detection methods classified by three key elements, including biological recognition elements, additional micro-/nano-materials and signal transduction/readout elements, their current challenges and further applications are also discussed.

Proliferation saturation index in an adaptive Bayesian approach to predict patient-specific radiotherapy responses.

Purpose: Radiotherapy prescription dose and dose fractionation protocols vary little between individual patients having the same tumor grade and stage. To personalize radiotherapy a predictive model is needed to simulate radiation response. Previous modeling attempts with multiple variables and parameters have been shown to yield excellent data fits at the cost of non-identifiability and clinically unrealistic results. Materials and methods: We develop a mathematical model based on a proliferation saturation index (PSI) that is a measurement of pre-treatment tumor volume-to-carrying capacity ratio that modulates intrinsic tumor growth and radiation response rates. In an adaptive Bayesian approach, we utilize an increasing number of data points for individual patients to predict patient-specific responses to subsequent radiation doses. Results: Model analysis shows that using PSI as the only patient-specific parameter, model simulations can fit longitudinal clinical data with high accuracy (R2=0.84). By analyzing tumor response to radiation using daily CT scans early in the treatment, response to the remaining treatment fractions can be predicted after two weeks with high accuracy (c-index = 0.89). Conclusion: The PSI model may be suited to forecast treatment response for individual patients and offers actionable decision points for mid-treatment protocol adaptation. The presented work provides an actionable image-derived biomarker prior to and during therapy to personalize and adapt radiotherapy.

Culture-Negative Periprosthetic Joint Infection: An Update on What to Expect.

BACKGROUND: Culture-negative periprosthetic joint infection (PJI) is a challenging condition to treat. The most appropriate management of culture-negative PJI is not known, and there is immense variability in the treatment outcome of this condition. The purpose of this study was to elucidate the characteristics, outcomes, and risk factors for failure of treatment of culture-negative PJI. METHODS: A retrospective review of 219 patients (138 hips and 81 knees) who had undergone surgery for the treatment of culture-negative PJI was performed utilizing a prospectively collected institutional PJI database. PJIs for which the results of culture were unavailable were excluded. An electronic query and manual review of the medical records were completed to obtain patient demographics, treatment, microbiology data, comorbidities, and other surgical characteristics. Treatment failure was assessed using the Delphi consensus criteria. RESULTS: The prevalence of suspected culture-negative PJI was 22.0% (219 of 996), and the prevalence of culture-negative PJI as defined by the Musculoskeletal Infection Society (MSIS) was 6.4% (44 of 688). Overall, the rate of treatment success was 69.2% (110 of 159) in patients with >1 year of follow-up. Of the 49 culture-negative PJIs for which treatment failed, 26 (53.1%) subsequently had positive cultures; of those 26, 10 (38.5%) were positive for methicillin-sensitive Staphylococcus aureus. The rate of treatment success was greater (p = 0.019) for patients who had 2-stage exchange than for those who underwent irrigation and debridement. CONCLUSIONS: The present study demonstrates that culture-negative PJI is a relatively frequent finding with unacceptable rates of treatment failure. Every effort should be made to isolate the infecting organism prior to surgical intervention, including extending the incubation period for cultures, withholding antibiotics prior to obtaining culture specimens, and possibly using newly introduced molecular techniques. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Highly Effective and Low-Cost MicroRNA Detection with CRISPR-Cas9.

MicroRNAs have been reported as related to multiple diseases and have potential applications in diagnosis and therapeutics. However, detection of miRNAs remains improvable, given their complexity, high cost, and low sensitivity as of currently. In this study, we attempt to build a novel platform that detects miRNAs at low cost and high efficacy. This detection system contains isothermal amplification, detecting and reporting process based on rolling circle amplification, CRISPR-Cas9, and split-horseradish peroxidase techniques. It is able to detect trace amount of miRNAs from samples with mere single-base specificity. Moreover, we demonstrated that such scheme can effectively detect target miRNAs in clinical serum samples and significantly distinguish patients of non-small cell lung cancer from healthy volunteers by detecting the previously reported biomarker: circulating let-7a. As the first to use CRISPR-Cas9 in miRNA detection, this method is a promising approach capable of being applied in screening, diagnosing, and prognosticating of multiple diseases.

Diagnosing Periprosthetic Joint Infection: And the Winner Is?

BACKGROUND: Diagnosis of periprosthetic joint infection (PJI) remains a challenge despite a wide variety of available diagnostic tests. The question that arises is which of these tests has a better performance for diagnosing PJI. Diagnostic odds ratio (DOR) has been described as the best indicator for test performance. METHODS: To compare the performance of the standard diagnostic tests, the DOR of serum erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), synovial fluid (SF) white blood cell (WBC) count, SF polymorphonuclear (PMN%), and leukocyte esterase (LE) were calculated. RESULTS: We obtained 4662 ESRs, 4392 CRPs, 836 SF WBC, 804 SF PMN%, and 659 LE results. LE had the highest DOR for diagnosing PJI: 30.06 (95% confidence interval [CI]: 17.8-50.7). The rest of the DORs in the descending order were SF WBC: 29.4 (95% CI: 20.2-42.8), CRP: 25.6 (95% CI: 19.5-33.7), SF PMN%: 25.5 (95% CI: 17.5-37.0), and ESR: 14.6 (95% CI: 11.5-18.6). CONCLUSION: Based on our findings, it appears that among the minor diagnostic criteria, LE has the best performance.

Vancomycin Prophylaxis for Total Joint Arthroplasty: Incorrectly Dosed and Has a Higher Rate of Periprosthetic Infection Than Cefazolin.

BACKGROUND: In total joint arthroplasty (TJA), vancomycin is used as perioperative antibiotic prophylaxis in patients with penicillin allergy or in patients colonized with methicillin-resistant Staphylococcus aureus (MRSA). Although vancomycin dosing should be weight-based (15 mg/kg), not all surgeons are aware of this; a fixed 1-g dose is instead frequently administered. QUESTIONS/PURPOSES: (1) Is there a difference in the risk of periprosthetic joint infection (PJI) in patients receiving vancomycin or cefazolin prophylaxis after primary TJA? (2) What proportion of patients is adequately dosed with vancomycin? (3) Compared with actual fixed dosing, does weight-based dosing result in a greater proportion of patients staying above the recommended 15-mg/L level at the beginning and end of surgery? (4) Are patients overdosed with vancomycin at greater risk of developing nephrotoxicity and acute kidney injury? METHODS: A single-institution, retrospective study was performed on 1828 patients undergoing primary TJAs who received vancomycin prophylaxis between 2008 and 2014. During the same period, 5810 patients underwent primary TJA and received cefazolin monotherapy. A chart review was performed to obtain patient characteristics, antibiotic dose and timing of administration, and microbiology data. Adequate vancomycin dosing was defined as 15 mg/kg and within the 125-mg range. Vancomycin levels were calculated at the beginning and end of surgery using pharmacokinetic equations. Levels of 15 mg/L were considered adequate. Logistic regression, chi square tests, and analysis of variance were performed. RESULTS: Among primary TJAs, patients receiving vancomycin had a higher rate of PJI (32 of 1828 [2%]) compared with patients receiving cefazolin prophylaxis (62 of 5810 [1%]; adjusted odds ratio, 1.587 [1.004-2.508]; p = 0.048). Ten percent of PJIs in the vancomycin underdosed group (two of 20) was caused by MRSA, and no patients with adequate dosing or overdosing of vancomycin developed PJI with MRSA. Of all procedures in which vancomycin monotherapy was used, 28% (518 of 1828) was adequately dosed according to weight-based dosage recommendations. Furthermore, 94% (1726 of 1828) of patients received a fixed 1-g dose of vancomycin, of whom 64% (1105 of 1726) were underdosed. All patients had vancomycin infusion initiated within 2 hours before incision. A weight-based protocol would have resulted in fewer patients having unacceptably low vancomycin levels (< 15 mg/L) compared with those with actual fixed dosing, both for the beginning of surgery at the time of incision (zero of 1828 [0%] versus 471 of 1828 [26%]; odds ratio, 0.001 [0.000-0.013]; p < 0.001) and at the end of surgery (33 of 1828 [2%] versus 746 of 1828 [41%]; odds ratio, 0.027 [0.019-0.038]; p < 0.001). Between the vancomycin dosage groups, there were no differences in the rate of nephrotoxicity (underdosed: 12 of 1130 [1%], adequately dosed: five of 518 [1%], overdosed: four of 180 [2%], p = 0.363) and acute kidney injury (underdosed: 28 of 1130 [2%], adequately dosed: 10 of 518 [2%], overdosed: six of 180 [3%], p = 0.561). CONCLUSIONS: The majority of patients given vancomycin prophylaxis are underdosed according to the weight-based dosage recommendations, and MRSA did not occur in patients who were adequately dosed with vancomycin. Surgeons should thus ensure that their patients are adequately dosed with vancomycin using the recommendation of 15 mg/kg and that the dose of vancomycin is administered in a timely fashion. Furthermore, and based on the findings of this study, we have moved toward limiting the utilization of vancomycin prophylaxis for patients undergoing elective arthroplasty at our institution. LEVEL OF EVIDENCE: Level III, therapeutic study.

Polymicrobial Periprosthetic Joint Infections: Outcome of Treatment and Identification of Risk Factors.

BACKGROUND: The treatment outcomes of periprosthetic joint infection are frequently dependent on characteristics of the causative organism. The objective of this comparative study was to investigate the prevalence of and risk factors for development of polymicrobial periprosthetic joint infection, and the outcome of surgical treatment of these patients. METHODS: All patients with polymicrobial, monomicrobial, or culture-negative periprosthetic joint infection treated from 2000 to 2014 were identified at a single institution. Ninety-five patients with a polymicrobial periprosthetic joint infection had a minimum follow-up of 12 months. We matched patients with a polymicrobial periprosthetic joint infection with the other cohorts using propensity score matching for several important parameters. Treatment success was defined according to the Delphi criteria; Kaplan-Meier survivorship curves were generated to demonstrate this. A multiple logistic regression analysis was performed to determine risk factors for a polymicrobial periprosthetic joint infection. RESULTS: Overall, 10.3% (108 of 1,045) of the periprosthetic joint infections treated at our institution were polymicrobial in nature. Patients with a polymicrobial periprosthetic joint infection had a higher failure rate at 50.5% (48 of 95) compared with the monomicrobial periprosthetic joint infection cohort at 31.5% (63 of 200) and the culture-negative periprosthetic joint infection cohort at 30.2% (48 of 159) (p = 0.003). The survivorship of the polymicrobial periprosthetic joint infection group was 52.2% at the 2-year follow-up, 49.3% at the 5-year follow-up, and 46.8% at the 10-year follow-up. Patients with polymicrobial periprosthetic joint infection had a higher rate of amputation (odds ratio [OR], 3.80 [95% confidence interval (CI), 1.34 to 10.80]), arthrodesis (OR, 11.06 [95% CI, 1.27 to 96.00]), and periprosthetic joint infection-related mortality (OR, 7.88 [95% CI, 1.60 to 38.67]) compared with patients with monomicrobial periprosthetic joint infection. Isolation of gram-negative organisms (p < 0.01), enterococci (p < 0.01), Escherichia coli (p < 0.01), and atypical organisms (p < 0.01) was associated with polymicrobial periprosthetic joint infection. Only the presence of a sinus tract (OR, 2.20 [95% CI, 1.39 to 3.47]; p = 0.001) was a significant risk factor for polymicrobial periprosthetic joint infection on multivariate analysis. CONCLUSIONS: This study reveals that polymicrobial periprosthetic joint infection, occurring at a relatively low rate, is associated with poor outcomes when compared with monomicrobial and culture-negative periprosthetic joint infection. Patients with polymicrobial infections were more likely to require a salvage procedure or to have periprosthetic joint infection-related mortality. Polymicrobial periprosthetic joint infection was associated with soft-tissue defects such as a sinus tract and certain types of organisms, which should be considered when administering antibiotics to these patients. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Chronic Kidney Disease Linearly Predicts Outcomes After Elective Total Joint Arthroplasty.

BACKGROUND: Kidney disease is associated with increased complications in total joint arthroplasty (TJA). The purpose of this study was to determine the association of kidney disease severity as measured by the chronic kidney disease (CKD) staging system with complications after TJA. METHODS: A retrospective review of 12,308 primary TJAs (6361 hips and 5947 knees) from 2008 to 2013 was performed. The following preoperative variables were obtained from medical records: chemistry 7 panel, Elixhauser comorbidities, and demographic factors. CKD stages were defined based on estimated glomerular filtration rate (eGFR) in mL/min/1.73m(2): (1) 90+, (2) 60-89, (3A) 45-59, (3B) 30-44, (4) 15-29, and (5) <15. Multivariate analysis was performed to assess the independent influence of CKD stage on the aforementioned end points. RESULTS: Patients with CKD stage greater than 2 demonstrated an increased odds of receiving transfusions (P = .001), length of stay >3 days (P = .010), acute kidney injury (P < .001), septic revisions (P = .002), and in-hospital complications (P < .001) compared with all patients with eGFR ≥60 when controlling for potential confounders. Only CKD stage 3A was significantly associated with septic revisions (90 days, P = .004; 2 years P = .002). In addition, the relationship between eGFR and the previously mentioned complications increased linearly rather than demonstrating a clear threshold at which the risk increased substantially. CONCLUSION: Severe CKD is associated with increased transfusion, length of stay, and in-hospital complications; and complications increased linearly with disease severity. Surgeons should be cognizant of this increase when evaluating TJA patients with renal disease.