HDAC Inhibitors recapitulate Human Disease-Associated Microglia Signatures in vitro.

Disease-associated microglia (DAM), initially described in mouse models of neurodegenerative diseases, have been classified into two related states; starting from a TREM2-independent DAM1 state to a TREM2 dependent state termed DAM2, with each state being characterized by the expression of specific marker genes1. Recently, single-cell (sc)RNA-Seq studies have reported the existence of DAMs in humans2-6; however, whether DAMs play beneficial or detrimental roles in the context of neurodegeneration is still under debate7,8. Here, we present a pharmacological approach to mimic human DAM in vitro by exposing different human microglia models to selected histone deacetylase (HDAC) inhibitors. We also provide an initial functional characterization of our model system, showing a specific increase of amyloid beta phagocytosis along with a reduction of MCP-1 secretion. Additionally, we report an increase in MITF expression, a transcription factor previously described to drive expression towards the DAM phenotype. We further identify CADM1, LIPA and SCIN as DAM-marker genes shared across various proposed DAM signatures and in our model systems. Overall, our strategy for targeted microglial polarization bears great potential to further explore human DAM function and biology.

A modular chemigenetic calcium indicator for multiplexed in vivo functional imaging.

Genetically encoded fluorescent calcium indicators allow cellular-resolution recording of physiology. However, bright, genetically targetable indicators that can be multiplexed with existing tools in vivo are needed for simultaneous imaging of multiple signals. Here we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several that efficiently label the brain in animals. When bound to a near-infrared dye-ligand, WHaloCaMP shows a 7× increase in fluorescence intensity and a 2.1-ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a to image Ca2+ responses in vivo in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae and to quantify Ca2+ concentration using fluorescence lifetime imaging microscopy (FLIM).

Pulmonary artery stenting in cancer patients: A single-center experience.

Endovascular stenting of the pulmonary artery treats arterial stenosis from pulmonary hypertension, congenital heart defects, or post-transplant stricture. Patients with malignant extrinsic pulmonary arterial compression, secondary to large mediastinal or pulmonary masses, often present with dyspnea, hypoxemia, and right ventricular failure. Conventional therapies like surgery, chemotherapy, and radiation are often slow and fail to promptly resolve acute symptoms. Balloon angioplasty and stenting have been explored as a rapid treatment to alleviate symptoms of external pulmonary artery compression. Despite its potential, the adoption of this procedure is limited due to risks like stent misplacement, migration, cardiac arrhythmias, and arterial rupture. This paper presents 3 cases of pulmonary angiography and stenting performed for malignant extrinsic pulmonary artery compression. These cases aim to demonstrate the feasibility of pulmonary artery stenting, encouraging its consideration as a palliative option for symptomatic patients with this condition.

Improving Time to Antibiotics in Patients With Long Bone Open Fractures Presenting to the Pediatric Emergency Department.

OBJECTIVES: To evaluate a new triage workflow aimed at improving time to intravenous antibiotics in open fractures to under less than 60 minutes of arrival to the Pediatric Emergency Department. DESIGN: A prospective, multidisciplinary, quality improvement project. SETTING: A tertiary care, Level 1 pediatric trauma hospital in New York. PATIENT SELECTION CRITERIA: Patients ages 17 years and younger with long bone open fractures between June 1, 2020, and May 31, 2021, excluding those transferred from an outside hospital, with nonlong bone fractures and nonfractured, injured extremities. OUTCOME MEASURES AND COMPARISONS: The new workflow involved splint removal and skin assessment during triage to identify open fractures. The aim of serial Plan-Do-Study-Act cycles was to refine this workflow and reduce antibiotic administration time. Primary outcome: percentage of patients with open fracture receiving intravenous antibiotics within 60 minutes. Secondary outcome: assessment of triage documentation regarding splint presence and removal. An exact Wilcoxon two-sample test compared time from patient arrival (quick registration) with antibiotic administration before, during, and after workflow implementation on June 1, 2020. RESULTS: A total of 51 patients (33 male) ages 17 years and younger, with open fractures, were reviewed: 25 during the preintervention phase January 1, 2018 to May 31, 2020; 14 during the intervention phase June 1, 2020 to May 31, 2021; and 12 during the postintervention phase June 1, 2021 to November 30, 2021. Continuous improvement efforts through Plan-Do-Study-Act cycles focusing on education, reinforcement, recognition, and barrier identification increased the percentage of patients receiving antibiotics within 60 minutes from 36% to 87.5%. The median time and interquartile range (interquartile range: 25th percentile-75th percentile) from quick registration to administration was 86 minutes (interquartile range: 51-147) before June 1, 2020, and 34 minutes (interquartile range: 16-42) thereafter. CONCLUSION: The implemented triage workflow led to improved time to antibiotics to within 60 minutes for patients with long bone open fractures in the pediatric emergency department. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Contraindications to tissue plasminogen activator thrombolysis for acute lower extremity ischemia.

OBJECTIVE: Previous randomized prospective trials have demonstrated the effectiveness of transcatheter tissue plasminogen activator (tPA) thrombolysis in treating acute limb ischemia (ALI) compared to conventional surgery. These pivotal trials have also highlighted contraindications for these procedures. Given recent advancements in techniques and technology, our aim is to reassess the relevance of these contraindications in contemporary practice. METHODS: A retrospective chart analysis was performed utilizing the inpatient medical records of consecutive individuals who underwent tPA treatment for acute limb ischemia (ALI) from September 2016 to April 2022. Inclusion criteria encompassed patients aged 18 and above displaying clinical symptoms and imaging evidence of ALI within 14 days. All patients received tPA with suction thrombectomy following the fast-track thrombolysis protocol. In cases where a persistent thrombus or stenosis was detected, catheter-directed thrombolysis was considered overnight, and patients underwent angiography and reassessment in the operating room subsequently. RESULTS: Patients were classified into two groups based on the STILE trial's established contraindications for endovascular treatment in acute limb ischemia (ALI). If a patient had any of these contraindications, they were placed in the contraindicated group. This resulted in 24 patients (32%) in the contraindicated group and 52 patients (68%) in the non-contraindicated group. No statistically significant demographic variations were observed between these groups. Contraindications in our study included uncontrolled hypertension (12/24, 50%), recent invasive procedures (7/27, 29%), history of cerebrovascular accident (CVA) within 6 months (3/24, 12%), and intracranial malformation/neoplasms (2/24, 8%). Three patients within the non-contraindicated group experienced bleeding complications: two with puncture site bleeds and one with nasal bleeding. In contrast, one patient in the contraindicated group had transient postoperative hematuria. There were no significant differences in bleeding complications observed between the two groups (p = .771). Additionally, no amputations were observed within our population. CONCLUSIONS: In light of our study results and advancements in endovascular therapies, we can now safely and efficiently treat patients who were previously considered contraindicated for such treatments. It is essential to individualize treatments and carefully balance the risks and benefits of endovascular versus open surgical revascularization for these patients. Additionally, we believe that the nearly 30-year-old guidelines for endovascular therapies need to be revisited and updated to align with modern technology.

Reduced SH3RF3 may protect against Alzheimer's disease by lowering microglial pro-inflammatory responses via modulation of JNK and NFkB signaling.

Understanding how high-risk individuals are protected from Alzheimer's disease (AD) may illuminate potential therapeutic targets. A previously identified non-coding SNP in SH3RF3/POSH2 significantly delayed disease onset in a Caribbean Hispanic cohort carrying the PSEN1 G206A mutation sufficient to cause early-onset AD and microglial expression of SH3RF3 has been reported to be a key driver of late-onset AD. SH3RF3 acts as a JNK pathway scaffold and can activate NFκB signaling. While effects of SH3RF3 knockdown in human neurons were subtle, including decreased phospho-tau S422, knockdown in human microglia significantly reduced inflammatory cytokines in response to either a viral mimic or oligomeric Aß42. This was associated with reduced activation of JNK and NFκB pathways in response to these stimuli. Pharmacological inhibition of JNK or NFκB signaling phenocopied SH3RF3 knockdown. We also found PSEN1 G206A microglia have reduced inflammatory responses to oAß42. Thus, further reduction of microglial inflammatory responses in PSEN1 mutant carriers by protective SNPs in SH3RF3 might reduce the link between amyloid and neuroinflammation to subsequently delay the onset of AD.

Sodium-Glucose Transport Protein 2 (SGLT2) Inhibitors and the Risk of Pancreatitis: A Case Report.

Acute pancreatitis is a condition seldom encountered with the use of sodium-glucose cotransporter 2 (SGLT2) inhibitors. They are beneficial in the treatment of various conditions and offer great promise. Despite this, they are associated with several adverse effects, necessitating vigilance and further research. This case study reports a 69-year-old male with multiple comorbidities who presented with epigastric pain radiating to the back. Laboratory tests revealed elevated AST, ALT, GGT and lipase. The patient was diagnosed with acute pancreatitis secondary to the SGLT2 inhibitor therapy regimen. Cessation of dapagliflozin resulted in a complete resolution of symptoms. There is credible evidence to suggest the presence of an association between SGLT2 inhibitors and acute pancreatitis, although extensive research is warranted to consolidate this association.

Risk Factors for Upper Extremity Impairment after Mastectomy: A Single Institution Retrospective Review.

Background: Patients with breast cancer treated with mastectomy are more likely to develop upper extremity dysfunction compared with those treated with breast-conserving therapy. This study aimed to identify cancer and treatment characteristics that may be risk factors for development of upper extremity dysfunction in patients treated with mastectomy. Methods: The authors performed a retrospective chart review of patients at the University of Chicago who were treated with a unilateral or bilateral mastectomy from 2010 to 2020 and developed upper extremity dysfunction based on International Classification of Disease-10 codes. Patients were analyzed by side of body (left or right). Patient demographics and treatment characteristics were extracted from the electronic medical record. Results: In total, 259 patients met criteria and were included in our study. A total of 396 upper extremities were recorded as experiencing dysfunction and were analyzed. Mean age was 60 years (range = 28-96), and mean body mass index was 28.4 (SD = 7.5). An estimated 54% of patients underwent breast reconstruction. After multivariable analysis, chronic upper extremity pain was found to be associated with ipsilateral radiotherapy (P < 0.001) and ipsilateral in situ cancer (0.041). Limited range of motion was found to be associated with ipsilateral invasive cancer (P = 0.01), any ipsilateral mastectomy surgery (P < 0.001), and ipsilateral radiotherapy (P = 0.03). Musculoskeletal dysfunction was found to be associated with no ipsilateral modified radical mastectomy (P = 0.033). No oncological or treatment characteristics were found to be associated with decreased strength or adhesive capsulitis. Furthermore, breast reconstruction (implant or autologous tissue based) was not associated with upper extremity dysfunction. Conclusion: Breast cancer characteristics and treatment modalities may predispose patients treated with mastectomy to developing types of upper extremity dysfunction.

Generation of hiPSC-derived brain microvascular endothelial cells using a combination of directed differentiation and transcriptional reprogramming strategies.

The blood-brain barrier (BBB), formed by specialized brain microvascular endothelial cells (BMECs), regulates brain function in health and disease. In vitro modeling of the human BBB is limited by the lack of robust hiPSC protocols to generate BMECs. Here, we report generation, transcriptomic and functional characterization of reprogrammed BMECs (rBMECs) by combining hiPSC differentiation into BBB-primed endothelial cells and reprogramming with two BBB transcription factors FOXF2 and ZIC3. rBMECs express a subset of the BBB gene repertoire including tight junctions and transporters, exhibit stronger paracellular barrier properties, lower caveolar-mediated transcytosis, and similar p-Glycoprotein activity compared to primary HBMECs. They can acquire an inflammatory phenotype when treated with oligomeric Aß42. rBMECs integrate with hiPSC-derived pericytes and astrocytes to form a 3D neurovascular system using the MIMETAS microfluidics platform. This novel 3D system resembles the in vivo BBB at structural and functional levels to enable investigation of pathogenic mechanisms of neurological diseases.

Fusobacterium nucleatum Bacteremia Presenting as Isolated Superior Mesenteric Vein Thrombophlebitis.

Fusobacterium nucleatum (F. nucleatum) is a commensal Gram-negative anaerobic bacterium that lives in the oral cavity and gastrointestinal tract of humans. While it is a regular resident of the human oral cavity, F. nucleatum has been implicated in various infections and inflammatory conditions. This case report highlights an unusual association between F. nucleatum and isolated superior mesenteric vein (SMV) thrombosis.

Imaging the extracellular matrix in live tissues and organisms with a glycan-binding fluorophore.

All multicellular systems produce and dynamically regulate extracellular matrices (ECM) that play important roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, nonperturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.

Intravenous Immunoglobulin-Induced Aseptic Meningitis in a Dermatomyositis Patient.

Aseptic meningitis is a rare but serious complication of treatment with intravenous immunoglobulin (IVIG) and often mimics meningitis of infectious etiology which poses a challenge for timely diagnosis. Although there are published recommendations on the management of IVIG-induced complications, there are no clear guidelines on the continuation of IVIG use after resolution of aseptic meningitis. We present a case of IVIG-induced aseptic meningitis in a patient with a history of refractory dermatomyositis who had been treated with immunosuppressive therapy and IVIG infusions for over a year. The patient developed intense head and neck pain with associated photophobia 24 hours after the most recent IVIG infusion. The patient was managed with supportive care consisting of intravenous fluids and analgesics. The patient's aseptic meningitis resolved without neurological complications. Ultimately, the patient was restarted on IVIG due to the recurrence of weakness from dermatomyositis. The patient tolerated re-initiation of IVIG without recurrence of IVIG-induced complications. This case highlights the importance of considering IVIG-induced aseptic meningitis as a differential diagnosis in evaluating patients with non-infectious meningitis even after regular IVIG infusions. This case also demonstrates that it is safe to reinitiate IVIG after the resolution of IVIG-induced aseptic meningitis.

Carotid intima media thickness in multiple sclerosis: A CLSA study.

BACKGROUND: People with multiple sclerosis (MS) have an increased incidence of atherosclerotic disease, including ischemic heart disease and stroke, compared to people without MS even after accounting for risk factors such as hypertension, dyslipidemia, diabetes and smoking. We compared carotid intima media thickness (CIMT), a surrogate of atherosclerosis, in people with MS and in two groups of people without MS (rheumatoid arthritis [RA]; all other participants). METHODS: We used data from participants in the Canadian Longitudinal Study on Aging (CLSA) who did not have known vascular disease (ischemic heart disease, stroke, transient ischemic attack, peripheral vascular disease) and who underwent carotid ultrasound for assessment of CIMT. We selected participants with MS, RA and controls who did not have MS or RA. Using age and gender-stratified norms for average CIMT in the CLSA, we identified participants in each cohort with a CIMT ≥75th percentile (subclinical atherosclerosis). We also calculated ten-year level of cardiovascular risk using the Framingham Risk Score (FRS). We tested the association between cohort membership (MS, RA, controls) and atherosclerosis using logistic regression, adjusted for FRS, abdominal obesity, excess alcohol intake, education and elevated symptoms of depression. We adjusted all analyses for the stratified sampling design. RESULTS: We included 78 participants with MS, 364 participants with RA and 13,891 controls. Overall, the average (SE) CIMT was 0.699 (0.002), and this did not differ between cohorts. Logistic regression analyses revealed that cohort membership was not associated with atherosclerosis based on the average CIMT in unadjusted or adjusted models. However, a 1-point higher FRS was associated with 1.032 (95 %CI: 1.021, 1.043) increased odds of atherosclerosis. CONCLUSION: Average CIMT does not differ between people with MS, people with RA and people without these diseases. Subclinical atherosclerosis as defined by a CIMT ≥75 % is not observed in people with MS at an increased rate beyond what FRS would predict. Further evaluation is needed to determine what mechanisms underlie the increased rates of cardiovascular disease and stroke in MS.

Treatment with nanosomal paclitaxel lipid suspension versus conventional paclitaxel in metastatic breast cancer patients - a multicenter, randomized, comparative, phase II/III clinical study.

Background: A novel nanosomal paclitaxel lipid suspension (NPLS), free from Cremophor EL (CrEL) and ethanol, was developed to address the solvent-related toxicities associated with conventional paclitaxel formulation. Objective: To evaluate the efficacy and safety of NPLS versus CrEL-based paclitaxel (conventional paclitaxel) in patients with metastatic breast cancer (MBC). Design: A prospective, open-label, randomized, multiple-dose, parallel, phase II/III study. Methods: Adult (18-65 years) female patients with MBC who had previously failed at least one line of chemotherapy were randomized (2:2:1) to NPLS 175 mg/m2 every 3 weeks (Q3W, n = 48, arm A), NPLS 80 mg/m2 every week (QW, n = 45, arm B) without premedication or conventional paclitaxel (Taxol®, manufactured by Bristol-Myers Squibb, Princeton, NJ, USA) 175 mg/m2 Q3W (n = 27, arm C) with premedication. In the extension study, an additional 54 patients were randomized (2:1) to arm A (n = 37) or arm C (n = 17). Results: Pooled data from the primary study and its extension phase included 174 patients. The primary endpoint was the overall response rate (ORR). As per intent-to-treat analysis, ORR was significantly better in the NPLS QW arm as compared to conventional paclitaxel [44.4% (20/45) versus 22.7% (10/44), (p = 0.04)]. An improvement in ORR with NPLS Q3W versus conventional paclitaxel arm [29.4% (25/85) versus 22.7% (10/44)] (p = 0.53) was observed. Disease control rates observed were improved with NPLS Q3W versus conventional paclitaxel Q3W (77.7% versus 72.7%, p = 0.66) and with NPLS QW versus conventional paclitaxel Q3W (84.4% versus 72.7%, p = 0.20), although not significant. A lower incidence of grade III/IV peripheral sensory neuropathy, vomiting, and dyspnea was reported with NPLS Q3W versus conventional paclitaxel Q3W arms. Conclusion: NPLS demonstrated an improved tumor response rate and a favorable safety profile versus conventional paclitaxel. NPLS 80 mg/m2 QW demonstrated a significantly better response versus conventional paclitaxel 175 mg/m2 Q3W. Trial registration: Clinical Trial Registry-India (CTRI), CTRI/2010/091/001344 Registered on: 18 October 2010 (https://ctri.nic.in/Clinicaltrials/pmaindet2.php?EncHid=MjEzNQ==&Enc=&userName=CTRI/2010/091/001344), CTRI/2015/07/006062 Registered on: 31 July 2015 (https://ctri.nic.in/Clinicaltrials/pmaindet2.php?EncHid=MTE2Mjc=&Enc=&userName=CTRI/2015/07/006062).

Role of nanosomal paclitaxel lipid suspension (NPLS) in the treatment of patients with metastatic breast cancer (MBC) Why was the study done? Paclitaxel is a commonly used drug for the treatment of breast cancer. Conventional formulation of paclitaxel is known to cause side effects like injection site reactions. A newer formulation named NPLS was developed to overcome the limitations of the conventional paclitaxel. The current study was done to compare the safety and effectiveness of NPLS and conventional paclitaxel in patients with advanced breast cancer. What did the researchers do? The research team conducted a large study in multiple hospitals across India, involving women with advanced breast cancer who had experienced treatment failure with previous chemotherapy. A total of 174 patients were randomly assigned to receive either of the three treatment schedules: (1) NPLS every 3 weeks, (2) NPLS every week, (3) conventional paclitaxel every 3 weeks. What did the researchers find? The results showed that NPLS, in a weekly schedule, led to better tumor response rates compared to conventional paclitaxel given every 3 weeks. Additionally, NPLS demonstrated a favorable safety profile, as compared to conventional paclitaxel. What do the findings mean? These findings suggest that NPLS could be a promising alternative for women with advanced breast cancer. NPLS improved the response to treatment, with a better safety profile compared to conventional paclitaxel.

ALSUntangled #75: Portable neuromodulation stimulator therapy.

Spurred by patient interest, ALSUntangled herein examines the potential of the Portable Neuromodulation Stimulator (PoNS™) in treating amyotrophic lateral sclerosis (ALS). The PoNS™ device, FDA-approved for the treatment of gait deficits in adult patients with multiple sclerosis, utilizes translingual neurostimulation to stimulate trigeminal and facial nerves via the tongue, aiming to induce neuroplastic changes. While there are early, promising data for PoNS treatment to improve gait and balance in multiple sclerosis, stroke, and traumatic brain injury, no pre-clinical or clinical studies have been performed in ALS. Although reasonably safe, high costs and prescription requirements will limit PoNS accessibility. At this time, due to the lack of ALS-relevant data, we cannot endorse the use of PoNS as an ALS treatment.

Subclinical atherosclerosis in multiple sclerosis.

Background: People with multiple sclerosis (MS) have an increased risk of ischemic heart disease as compared to people without MS after accounting for traditional vascular risk factors. Objective: We assessed whether subclinical atherosclerosis, an inflammatory disease of arteries, occurs in persons with MS who do not have traditional vascular risk factors, and whether the Framingham Score (FRS) predicted carotid intima media thickness (CIMT) similarly in people with and without MS. Methods: We recruited participants with and without MS who did not have vascular disease. Participants completed questionnaires, physical assessments, underwent an ultrasound (CIMT), and provided samples for HbA1c and lipid measurements. We defined subclinical atherosclerosis as an average CIMT ≥75th percentile, and tested the association between MS/not-MS, FRS, and atherosclerosis using logistic regression. Results: We recruited 106 participants with MS 101 without MS. The average (SD) CIMT did not differ between the MS (0.60 [0.11]) and non-MS (0.61 [0.12]) cohorts (p = 0.69), nor did the proportion with atherosclerosis (MS: 11.3% vs. non-MS 13.4%, p = 0.58). On regression analysis a 1-point increase in the FRS was associated with 11% increased odds of having atherosclerosis (95%CI: 1.04, 1.19) but MS was not. Conclusion: MS was not associated with subclinical atherosclerosis.

A pharmacological toolkit for human microglia identifies Topoisomerase I inhibitors as immunomodulators for Alzheimer's disease.

While efforts to identify microglial subtypes have recently accelerated, the relation of transcriptomically defined states to function has been largely limited to in silico annotations. Here, we characterize a set of pharmacological compounds that have been proposed to polarize human microglia towards two distinct states - one enriched for AD and MS genes and another characterized by increased expression of antigen presentation genes. Using different model systems including HMC3 cells, iPSC-derived microglia and cerebral organoids, we characterize the effect of these compounds in mimicking human microglial subtypes in vitro. We show that the Topoisomerase I inhibitor Camptothecin induces a CD74high/MHChigh microglial subtype which is specialized in amyloid beta phagocytosis. Camptothecin suppressed amyloid toxicity and restored microglia back to their homeostatic state in a zebrafish amyloid model. Our work provides avenues to recapitulate human microglial subtypes in vitro, enabling functional characterization and providing a foundation for modulating human microglia in vivo.

Role of Lateral Extra-articular Tenodesis in Restraining Internal Tibial Rotation: In Vitro Biomechanical Assessment of Lateral Tissue Engagement.

BACKGROUND: The way in which force increases in the anterolateral tissues and the lateral extra-articular tenodesis (LET) tissue to resist internal rotation (IR) of the tibia after anterior cruciate ligament (ACL) reconstruction in isolation and after LET augmentation, respectively, is not well understood. PURPOSE: (1) To compare in a cadaveric model how force increases (ie, engages) in the anterolateral tissues with IR of the tibia after isolated ACL reconstruction and in the LET tissue after augmentation of the ACL reconstruction with LET and (2) to determine whether IR of the tibia is related to engagement of the LET tissue. STUDY DESIGN: Controlled laboratory study. METHODS: IR moments were applied to 9 human cadaveric knees at 0°, 30°, 60°, and 90° of flexion using a robotic manipulator. Each knee was tested in 2 states: (1) after isolated ACL reconstruction with intact anterolateral tissues and (2) after LET was performed using a modified Lemaire technique with the LET tissue fixed at 60° of flexion under 44 N of tension. Resultant forces carried by the anterolateral tissues and the LET tissue were determined via superposition. The way force increased in these tissues was characterized via parameters of tissue engagement, namely in situ slack, in situ stiffness, and tissue force at peak applied IR moment, and then compared (α < .05). IR was related to parameters of engagement of the LET tissue via simple linear regression (α < .05). RESULTS: The LET tissue exhibited less in situ slack than the anterolateral tissues at 30°, 60°, and 90° of flexion (P≤ .04) and greater in situ stiffness at 30° and 90° of flexion (P≤ .043). The LET tissue carried greater force at the peak applied IR moment at 0° and 30° of flexion (P≤ .01). IR was related to the in situ slack of the LET tissue (R2≥ 0.88; P≤ .0003). CONCLUSION: LET increased restraint to IR of the tibia compared with the anterolateral tissue, particularly at 30°, 60°, and 90° of flexion. IR of the tibia was positively associated with in situ slack of the LET tissue. CLINICAL RELEVANCE: Fixing the LET at 60° of flexion still provided IR restraint in the more functionally relevant flexion angle of 30°. Surgeons should pay close attention to the angle of internal and/or external tibial rotation when fixing the LET tissue intraoperatively because this surgical parameter is related to in situ slack of the LET tissue and, therefore, the amount of IR of the tibia.