Abstract Human anucleate platelets cannot be directly modified using traditional genetic approaches. Instead, studies of platelet gene function depend on alternative models. Megakaryocytes (the nucleated precursor to platelets) are the nearest cell to platelets in origin, structure, and function. However, achieving consistent genetic modifications in primary megakaryocytes has been challenging, and the functional effects of induced gene deletions on human megakaryocytes for even well-characterized platelet genes (eg, ITGA2B) are unknown. Here we present a rapid and systematic approach to screen genes for platelet functions in CD34+ cell-derived megakaryocytes called CRIMSON (CRISPR-edited megakaryocytes for rapid screening of platelet gene functions). By using CRISPR/Cas9, we achieved efficient nonviral gene editing of a panel of platelet genes in megakaryocytes without compromising megakaryopoiesis. Gene editing induced loss of protein in up to 95% of cells for platelet function genes GP6, RASGRP2, and ITGA2B; for the immune receptor component B2M; and for COMMD7, which was previously associated with cardiovascular disease and platelet function. Gene deletions affected several select responses to platelet agonists in megakaryocytes in a manner largely consistent with those expected for platelets. Deletion of B2M did not significantly affect platelet-like responses, whereas deletion of ITGA2B abolished agonist-induced integrin activation and spreading on fibrinogen without affecting the translocation of P-selectin. Deletion of GP6 abrogated responses to collagen receptor agonists but not thrombin. Deletion of RASGRP2 impaired functional responses to adenosine 5′-diphosphate (ADP), thrombin, and collagen receptor agonists. Deletion of COMMD7 significantly impaired multiple responses to platelet agonists. Together, our data recommend CRIMSON for rapid evaluation of platelet gene phenotype associations.
The purpose of the study was to evaluate, as a pilot trial, safety and tolerability of CAM-101 10% and 30% topical ophthalmic fibrinogen-depleted human platelet lysate (FD hPL) solution in patients with dry eye disease (DED) secondary to graft-versus-host disease (GvHD) after 6 weeks of treatment.A phase I/II, pilot, prospective, multicenter, randomized, double-masked clinical trial.Patients with DED secondary to GvHD.Sixty-four adult patients were stratified by "symptom severity" (Ocular Surface Disease Index [OSDI], ocular discomfort Visual Analog Scale (VAS), ocular symptom frequency, and use of artificial tears) and then randomized 1:1:1 to CAM-101 (FD hPL) at 10% or 30% concentration or an electrolyte (Plasma-Lyte A) vehicle control, 1 drop in both eyes, 4 times daily, for 42 days. After 42 days, control patients were offered 42 days of open-label treatment with 30% FD hPL.Primary outcome safety measures were ocular and systemic adverse events and the number of patients in each group with clinically significant change from normal to abnormal in any ocular findings. Secondary outcomes were changes from baseline to day 42 in ocular discomfort, OSDI, fluorescein corneal staining, and lissamine green conjunctival staining relative to the vehicle control. The ocular symptom frequency was assessed on a 100-point VAS.FD hPL 10% and 30% were safe and well tolerated. Relative to the vehicle control, significant decreases from baseline to day 42 were seen in the FD hPL 30% group with regard to ocular discomfort (mean decrease = -18.04; P = 0.018), frequency of burning/stinging (-20.23; P = 0.022), eye discomfort (-32.97; P < 0.001), eye dryness (-21.61; P = 0.020), pain (-15.12; P = 0.044), photophobia (-24.33; P = 0.0125), and grittiness (-20.08; P = 0.0185). Decreases were also seen for itching and foreign body sensation, though not statistically significant. Improvements were seen in tear breakup time (mean increase = 1.30 seconds; P = 0.082) and the investigator's global evaluation 4-point scale (mean decrease = -0.86; P = 0.026). Corneal fluorescein staining was not improved. The OSDI had a mean decrease of -8.88 compared to the vehicle, although not statistically significant.Fibrinogen-depleted human platelet lysate appears to be well tolerated, with no significant toxicity at concentrations of 10% and 30%. These initial data suggest some efficacy, especially for subjective outcome measures relative to baseline assessments and treatment with the vehicle, but larger studies are needed to confirm these effects.
In response to increasing rates of self-reported latex allergies, changes have been made to prevent anaphylaxis in the operating room, including the use of latex-free gloves. However, the impact of these changes on the risk of prosthetic joint infection (PJI) after arthroplasty is unclear. This study evaluated whether documented latex allergy is an independent risk factor for PJI and aseptic revision surgery after total hip arthroplasty (THA) and total knee arthroplasty (TKA). A retrospective matched cohort study was conducted with an administrative claims database. A total of 17,501 patients who underwent TKA and had documented latex allergy were matched 1:4 with 70,004 control subjects, and 8221 patients who underwent THA and had documented latex allergy were matched 1:4 with 32,884 control subjects. Multivariable logistic regression showed that patients who had TKA and had a latex allergy showed significantly higher risk of PJI at both 90 days (odds ratio [OR], 1.26) and 1 year (OR, 1.22) and significantly higher risk of aseptic revision TKA at 1 year (OR, 1.21) after surgery compared with control subjects. Patients who had THA and had a latex allergy had significantly higher risk of PJI at 1 year (OR, 1.19) compared with control subjects. Rates of aseptic revision THA were higher in the latex allergy cohort but statistically comparable (P>.05). Latex allergy was associated with significantly increased risk of PJI and aseptic revision after TKA and significantly increased risk of PJI after THA. More work is needed to determine whether these risks can be mitigated or if latex allergy is an inherent, nonmodifiable risk factor requiring modification to typical arthroplasty pathways. [Orthopedics. 2022;45(4):244-250.].
