A first-in-human study of the anti-LAG-3 antibody favezelimab plus pembrolizumab in previously treated, advanced microsatellite stable colorectal cancer.

BACKGROUND: Treatment options are limited for participants with microsatellite stable (MSS) metastatic colorectal cancer (mCRC) that progressed after two or more prior therapies. Studies have shown that blockade of both lymphocyte-activation gene 3 (LAG-3) and programmed cell death protein 1 (PD-1) can improve antitumor activity. Here, we evaluate the antitumor activity of the LAG-3 antibody favezelimab alone or in combination with pembrolizumab in participants with MSS mCRC. PATIENTS AND METHODS: Eligible participants with MSS PD-1/programmed death-ligand 1 (PD-L1) treatment-naive mCRC that progressed on two or more prior therapies received 800 mg favezelimab, 800 mg favezelimab plus 200 mg pembrolizumab, or 800 mg favezelimab/200 mg pembrolizumab co-formulation, every 3 weeks. The primary endpoint was safety, the secondary endpoint was objective response rate (ORR), and exploratory endpoints included duration of response, progression-free survival (PFS), and overall survival (OS). RESULTS: At the data cut-off date of 23 October 2020, a total of 20 participants received favezelimab alone, 89 received favezelimab plus pembrolizumab (including as favezelimab/pembrolizumab co-formulation); 48 had PD-L1 combined positive score (CPS) ≥1 tumors. At this interim analysis median follow-up was 5.8 months with favezelimab and 6.2 with favezelimab plus pembrolizumab. Treatment-related adverse events (TRAEs) were 65% with favezelimab and 65.2% with favezelimab plus pembrolizumab. Grade ≥3 TRAEs were 15% with favezelimab and 20% with favezelimab plus pembrolizumab. No grade 5 TRAEs occurred. Common TRAEs (≥15%) included fatigue (20.0%), nausea (15.0%) with favezelimab, and fatigue (16.9%) with favezelimab plus pembrolizumab. Confirmed ORR was 6.3% with favezelimab plus pembrolizumab, with median duration of response of 10.6 months (range 5.6-12.7 months), median OS of 8.3 months (95% confidence interval 5.5-12.9 months), and median PFS of 2.1 months (1.9-2.2 months). In an exploratory analysis of PD-L1 CPS ≥1 tumors, the confirmed ORR was 11.1%, median OS was 12.7 months (4.5 to not reached), and median PFS was 2.2 months (1.8-4.2 months) with favezelimab plus pembrolizumab. CONCLUSIONS: Favezelimab with or without pembrolizumab had a manageable safety profile, with no treatment-related deaths. Promising antitumor activity was observed with combination therapy, particularly in participants with PD-L1 CPS ≥1 tumors.

Moving evidence into practice: cost analysis and assessment of macaques' sustained behavioral engagement with videogames and foraging devices.

Environmental enrichment plans for captive nonhuman primates often include provision of foraging devices. The rationale for using foraging devices is to promote species-typical activity patterns that encourage physical engagement and provide multi-sensory stimulation. However, these devices have been shown to be ineffective at sustaining manipulation over long periods of time, and often produce minimal cognitive engagement. Here we use an evidence-based approach to directly compare the amount of object-directed behavior with a foraging device and a computer-based videogame system. We recorded 11 adult male rhesus monkeys' interactions with a foraging device and two tasks within a joystick videogame cognitive test battery. Both techniques successfully produced high levels of engagement during the initial 20 min of observation. After 1 hr the monkeys manipulated the foraging device significantly less than the joystick, F(2,10) = 43.93, P < 0.0001. Subsequent testing showed that the monkeys engaged in videogame play for the majority of a 5 hr period, provided that they received a 94 mg chow pellet upon successful completion of trials. Using a model approach, we developed previously as a basis for standardized cost:benefit analysis to inform facility decisions, we calculated the comprehensive cost of incorporating a videogame system as an enrichment strategy. The videogame system has a higher initial cost compared to widely-used foraging devices, however, the ongoing labor and supply costs are relatively low. Our findings add to two decades of empirical studies by a number of laboratories that have demonstrated the successful use of videogame-based systems to promote sustained non-social cognitive engagement for macaques. The broader significance of the work lies in the application of a systematic approach to compare and contrast enrichment strategies and encourage evidence-based decision making when choosing an enrichment strategy in a manner that promotes meaningful cognitive enrichment to the animals.

Assessment of foraging devices as a model for decision-making in nonhuman primate environmental enrichment.

Continued progress to move evidence-based best practices into community and regulatory animal welfare standards depends in part on developing common metrics to assess cost, benefit, and relative value. Here we describe a model approach to evidence-based evaluation and an example of comprehensive cost-benefit assessment for a common element of environmental enrichment plans for laboratory-housed nonhuman primates. Foraging devices encourage a species-typical activity that dominates the time budget of primates outside captivity and provide inherent cognitive challenges, physical activity demands, and multi-sensory stimulation. However, their implementation is not standard, and is challenged by perception of high costs and labor; nutritional and health concerns; and identification of best practices in implementation (that is, device types, food type, frequency of delivery and rotation). To address these issues, we directly compared monkeys' engagement with different foraging devices and the comprehensive cost of implementing foraging opportunities. We recorded 14 adult male cynomolgus monkeys' interactions with 7 types of devices filled with a range of enrichment foods. All devices elicited foraging behavior, but there were significant differences among them both initially and over subsequent observations. Devices that afforded opportunity for extraction of small food items and that posed manipulative challenge elicited greater manipulation. The cost of providing a foraging opportunity to a single monkey is roughly US$1, with approximately 80% attributable to labor. This study is the first to perform a rigorous cost-benefit analysis and comparison of common foraging devices included in environmental enrichment. Its broader significance lies in its contribution to the development of methods to facilitate improvement in evidence-based practices and common standards to enhance laboratory animal welfare.

The measurement of analyte diffusion from hydrogel microcapsules in model aqueous environments.

Model flavor analytes with varying partition coefficients were successfully encapsulated in approximately 150 microm diameter hydrogel microcapsules containing medium chain triglycerides (MCT). Analyte diffusion from the microcapsules into water and dextrose solutions was measured using a UV-VIS spectrophotometer fitted with a fiber optic probe. The diffusion of acetophenone, considered a hydrophilic analyte, was reduced as the dextrose concentration was increased in the water environment. Diffusion comparison was noted as t(1/2) which is the measure of time when 50% of the analyte concentration is released from the microcapsules. The measured t(1/2) for acetophenone in distilled water was 0.40 min. The addition of 20% dextrose to distilled water increased the t(1/2) to 0.60 min while the 40% dextrose addition extended the t(1/2) to 1.4 min. The same t(1/2) trends were also noted with methyl salicylate which is considered a hydrophobic analyte. The measured t(1/2) for methyl salicylate in distilled water was 2.0 min. The addition of 20% dextrose to distilled water increased the t(1/2) to 4.0 min while the 40% dextrose addition extended the t(1/2) to 9.0 min. The addition of dextrose to the water media effectively changes the partitioning media, increasing the resulting partition coefficient (Log P) values. Large changes in the Log P values were confirmed by the GC/FID analysis. In essence, water becomes a poor solvent for the analytes and diffusion is slowed from the hydrophobic MCT contained within the microcapsules as higher concentrations of dextrose is added to the water phase. Since methyl salicylate has a larger Log P(MCT: Water) value compared to acetophenone, the addition of dextrose extends the t(1/2) to a larger extent.

Mechanochemical ATPases and transcriptional activation.

Transcriptional activator proteins that act upon the sigma54-containing form of the bacterial RNA polymerase belong to the extensive AAA+ superfamily of ATPases, members of which are found in all three kingdoms of life and function in diverse cellular processes, often via chaperone-like activities. Formation and collapse of the transition state of ATP for hydrolysis appears to engender the interaction of the activator proteins with sigma54 and leads to the protein structural transitions needed for RNA polymerase to isomerize and engage with the DNA template strand. The common oligomeric structures of AAA+ proteins and the creation of the active site for ATP hydrolysis between protomers suggest that the critical changes in protomer structure required for productive interactions with sigma54-holoenzyme occur as a consequence of sensing the state of the gamma-phosphate of ATP. Depending upon the form of nucleotide bound, different functional states of the activator are created that have distinct substrate and chaperone-like binding activities. In particular, interprotomer ATP interactions rely upon the use of an arginine finger, a situation reminiscent of GTPase-activating proteins.

Binding of transcriptional activators to sigma 54 in the presence of the transition state analog ADP-aluminum fluoride: insights into activator mechanochemical action.

Conformational changes in sigma 54 (sigma(54)) and sigma(54)-holoenzyme depend on nucleotide hydrolysis by an activator. We now show that sigma(54) and its holoenzyme bind to the central ATP-hydrolyzing domains of the transcriptional activators PspF and NifA in the presence of ADP-aluminum fluoride, an analog of ATP in the transition state for hydrolysis. Direct binding of sigma(54) Region I to activator in the presence of ADP-aluminum fluoride was shown and inferred from in vivo suppression genetics. Energy transduction appears to occur through activator contacts to sigma(54) Region I. ADP-aluminum fluoride-dependent interactions and consideration of other AAA+ proteins provide insight into activator mechanochemical action.

Methylprednisolone does not benefit patients undergoing coronary artery bypass grafting and early tracheal extubation.

OBJECTIVE: We sought to determine whether methylprednisolone, when administered to patients undergoing cardiac surgery, is able to ward off the detrimental hemodynamic and pulmonary alterations associated with cardiopulmonary bypass. METHODS: After institutional review board approval and informed consent was obtained, 90 patients scheduled for elective cardiac surgery were randomized to 1 of 3 groups. Group 30MP patients received 30 mg/kg intravenous methylprednisolone during sternotomy and 30 mg/kg during initiation of cardiopulmonary bypass, group 15MP patients received 15 mg/kg methylprednisolone at the same 2 times, and group NS patients received similar volumes of isotonic sodium chloride solution at the same 2 times. Perioperative care was standardized, and all caregivers were blinded to treatment group. Various hemodynamic and pulmonary measurements were obtained perioperatively, as well as fluid balance, weight, peak postoperative blood glucose level, and tracheal extubation time. RESULTS: Demographic and clinical characteristics of patients and intraoperative data were similar among the 3 groups. Patients receiving methylprednisolone (either dose) exhibited significantly increased cardiac index (P =.0006), significantly decreased systemic vascular resistance (P =.0005), and significantly increased shunt flow (P =.0020) during the immediate postoperative period. All 3 groups exhibited significant increases in alveolar-arterial oxygen gradient (P <.0001), significant decreases in dynamic lung compliance (P <.0001), and significant decreases in static lung compliance (P <.0001) during the immediate postoperative period, with no differences between groups. Perioperative fluid balance and weights were similar between groups. A statistically significant difference in peak postoperative blood glucose level existed (P =.016) among group NS (234 +/- 96 mg/dL), group 15MP (292 +/- 93 mg/dL), and group 30MP (311 +/- 90 mg/dL). In patients extubated within 12 hours of intensive care unit arrival, a statistically significant difference in extubation times existed (P =.025) between group NS (5.7 +/- 2.3 hours), group 15MP (5.9 +/- 2.2 hours), and group 30MP (7.5 +/- 2.7 hours). CONCLUSIONS: Methylprednisolone, as used in this investigation, offers no clinical benefits to patients undergoing elective coronary artery bypass grafting with cardiopulmonary bypass and may in fact be detrimental by initiating postoperative hyperglycemia and possibly hindering early postoperative tracheal extubation for undetermined reasons.

Regulatory sequences in sigma 54 localise near the start of DNA melting.

Transcription initiation by the enhancer-dependent sigma(54) RNA polymerase holoenzyme is positively regulated after promoter binding. The promoter DNA melting process is subject to activation by an enhancer-bound activator protein with nucleoside triphosphate hydrolysis activity. Tethered iron chelate probes attached to amino and carboxyl-terminal domains of sigma(54) were used to map sigma(54)-DNA interaction sites. The two domains localise to form a centre over the -12 promoter region. The use of deletion mutants of sigma(54) suggests that amino-terminal and carboxyl-terminal sequences are both needed for the centre to function. Upon activation, the relationship between the centre and promoter DNA changes. We suggest that the activator re-organises the centre to favour stable open complex formation through adjustments in sigma(54)-DNA contact and sigma(54) conformation. The centre is close to the active site of the RNA polymerase and includes sigma(54) regulatory sequences needed for DNA melting upon activation. This contrasts systems where activators recruit RNA polymerase to promoter DNA, and the protein and DNA determinants required for activation localise away from promoter sequences closely associated with the start of DNA melting.

Protective ventilation attenuates postoperative pulmonary dysfunction in patients undergoing cardiopulmonary bypass.

OBJECTIVE: To ascertain if protective ventilation can attenuate the damaging postoperative pulmonary effects of cardiopulmonary bypass (increases in airway pressure, decreases in lung compliance, and increases in shunt). DESIGN: Prospective, randomized clinical trial. SETTING: Single university hospital. PARTICIPANTS: Twenty-five patients undergoing elective coronary artery bypass graft procedure and early extubation. INTERVENTIONS: Thirteen patients received conventional mechanical ventilation (CV; respiratory rate, 8 breaths/min; tidal volume, 12 mL/kg; fraction of inspired oxygen [FIO2], 1.0; positive end-expiratory pressure [PEEP], +5), and 12 patients received protective mechanical ventilation (PV; respiratory rate, 16 breaths/min; tidal volume, 6 mL/kg; FIO2, 1.0; PEEP, +5). Perioperative anesthetic and surgical management were standardized. Various pulmonary parameters were determined twice perioperatively: 10 minutes after intubation and 60 minutes after arrival in the intensive care unit. MEASUREMENTS AND MAIN RESULTS: The mean postoperative increase in peak airway pressure in group CV was significantly larger than the mean postoperative increase in peak airway pressure in group PV (7.1 v 2.4 cm H2O; p < 0.001). Group CV experienced significant postoperative increases in plateau airway pressure (p = 0.007), but group PV did not (p = 0.644). The mean postoperative decrease in dynamic lung compliance in group CV was significantly larger than the mean postoperative decrease in dynamic lung compliance in group PV (14.9 v 5.5 mL/cm H2O; p = 0.002). Group CV experienced significant postoperative decreases in static lung compliance (p = 0.014), but group PV did not (p = 0.645). Group CV experienced significant postoperative increases in shunt (15.5% to 21.4%; p = 0.021), but group PV did not (18.4% to 21.2%; p = 0.265). CONCLUSIONS: Data indicate that protective ventilation decreases pulmonary damage caused by mechanical ventilation in normal and abnormal lungs. The results of this investigation indicate that protective ventilation may also help attenuate the postoperative pulmonary dysfunction (increases in airway pressure, decreases in lung compliance, and increases in shunt) commonly seen in patients after exposure to cardiopulmonary bypass.

Oligomerizaiton and fibril asssembly of the amyloid-beta protein.

In this chapter, we attempt to analyze the evolution of the amyloid-beta (Abeta) molecular structure from its inception as part of the Abeta precursor protein to its release by the secretases and its extrusion from membrane into an aqueous environment. Biophysical studies suggest that the Abeta peptide sustains a series of transitions from a molecule rich in alpha-helix to a molecule in which beta-strands prevail. It is proposed that initially the extended C-termini of two opposing Abeta dimers form an antiparallel beta-sheet and that the subsequent addition of dimers generates a helical Abeta protofilament. Two or more protofilaments create a strand in which the hydrophobic core of the beta-sheets is shielded from the aqueous environment by the N-terminal polar domains of the Abeta dimers. Once the nucleation has occurred, the Abeta filament grows in length by the addition of dimers or tetramers.

Cellular cofactors potentiating induction of stress and cytotoxicity by amyloid beta-peptide.

Insights into factors underlying causes of familial Alzheimer's disease (AD), such as mutant forms of beta-amyloid precursor protein and presenilins, and those conferring increased risk of sporadic AD, such as isoforms of apolipoprotein E and polymorphisms of alpha2-macroglobulin, have been rapidly emerging. However, mechanisms through which amyloid beta-peptide (Abeta), the fibrillogenic peptide most closely associated with neurotoxicity in AD, exerts its effects on cellular targets have only been more generally outlined. Late in the course of AD, when Abeta fibrils are abundant, non-specific interactions of amyloid with cellular elements are likely to induce broad cytotoxicity. However, early in AD, when concentrations of Abeta are much lower and extracellular deposits are infrequent, mechanisms underlying cellular dysfunction have not been clearly defined. The key issue in elucidating the means through which Abeta perturbs cellular properties early in AD is the possibility that protective therapy at such times may prevent cytotoxicity at a point when damage is still reversible. This brief review focusses on two cellular cofactors for Abeta-induced cellular perturbation: the cell surface immunoglobulin superfamily molecule RAGE (receptor for advanced glycation endproducts) and ABAD (Abeta binding alcohol dehydrogenase). Although final proof for the involvement of these cofactors in cellular dysfunction in AD must await the results of further in vivo experiments, their increased expression in AD brain, as well as other evidence described below, suggests the possibility of specific pathways for Abeta-induced cellular perturbation which could provide future therapeutic targets.

Severe incisional pain and long thoracic nerve injury after port-access minimally invasive mitral valve surgery.

The authors describe the occurrence of severe postoperative pain and long thoracic nerve injury after Port-Access minimally invasive mitral valve surgery. The potential for these events and the impact on postoperative hospitalization and rehabilitation are emphasized.

Port-access minimally invasive cardiac surgery increases surgical complexity, increases operating room time, and facilitates early postoperative hospital discharge.

BACKGROUND: Proposed advantages of port-access cardiac surgery have yet to be substantiated. The authors retrospectively compared patients undergoing port-access cardiac surgery with a matched group undergoing conventional cardiac surgery. METHODS: Forty-six patients who underwent port-access cardiac surgery were matched with 46 who underwent conventional cardiac surgery. Absolute criteria for matching included morning-of-surgery admission, procedure undergone, and care being delivered by one of two surgeons. If possible, matching included care delivered by one of two anesthesiologists. Patients were matched as closely as possible for preoperative demographic and clinical characteristics. RESULTS: All 46 pairs of patients were matched for procedure and admitted the morning of surgery. All 92 operations were performed by one of two surgeons, and 89% were performed by one of two anesthesiologists. Preoperative demographic and clinical characteristics were equivalent between groups. Compared with conventional cardiac surgery, port-access cardiac surgery increased surgical complexity (it almost tripled cardiopulmonary bypass time during coronary artery bypass grafting and increased it almost 40% during mitral valve procedures) and increased total operating room time (P < 0.0001). Port-access cardiac surgery had no beneficial effect on earlier postoperative extubation, decreased incidence of atrial fibrillation, or intensive care unit time, yet it decreased postoperative duration of stay (P = 0.029, all patients), a benefit observed primarily in patients undergoing coronary artery bypass grafting (P = 0.002). CONCLUSIONS: This retrospective analysis revealed that port-access cardiac surgery increases surgical complexity, increases operating room time, has no effect on earlier postoperative extubation or decreased incidence of atrial fibrillation or intensive care unit time, and may facilitate postoperative hospital discharge (primarily in patients undergoing coronary artery bypass grafting). Properly designed prospective investigation is necessary to ascertain whether port-access cardiac surgery truly offers any benefits over conventional cardiac surgery.

Sequences within the DNA cross-linking patch of sigma 54 involved in promoter recognition, sigma isomerization, and open complex formation.

The bacterial RNA polymerase holoenzyme containing the final sigma(54) subunit functions in enhancer-dependent transcription. Mutagenesis has been used to probe the function of a sequence in the final sigma(54) DNA binding domain that includes residues that cross-link to promoter DNA. Several activities of the final sigma and holoenzyme are shown to depend on the cross-linking patch. The patch contributes to promoter binding by final sigma(54), and holoenzyme and is involved in activator-dependent final sigma isomerization. As part of the final sigma(54)-holoenzyme, some residues in the patch limit basal transcription. Other cross-linking patch sequences appear to limit activator-dependent open complex formation. Deletion of 19 residues adjacent to the cross-linking patch resulted in a holoenzyme unable to respond to activator but capable of activator-independent (bypass) transcription in vitro. Overall results are consistent with the cross-linking patch directing interactions to the -12 promoter region to set basal and activated levels of transcription.

Attempting to maintain normoglycemia during cardiopulmonary bypass with insulin may initiate postoperative hypoglycemia.

UNLABELLED: We attempted to develop an insulin administration protocol that maintains normoglycemia in patients undergoing cardiac surgery and to study the effects of intraoperative blood glucose management on serum levels of creatine phosphokinase isoenzyme BB (CK-BB) and S-100 protein. Twenty nondiabetic patients were randomly allocated to receive either "tight control" of blood glucose with a standardized IV insulin infusion intraoperatively (Group TC) or "no control" of blood glucose intraoperatively (Group NC). Perioperative serum levels of glucose, CK-BB, and S-100 protein were determined in all patients. Group TC patients received 90.0 +/- 49.2 units of insulin, whereas Group NC patients received none. Despite insulin, both Group TC (P = 0.00026) and Group NC (P = 0.00003) experienced similar significant increases in blood glucose levels during hypothermic cardiopulmonary bypass. However, mean blood glucose level upon intensive care unit arrival was significantly decreased in Group TC, compared with Group NC (84.7 +/- 41.0 mg/dL, range 32-137 mg/dL vs 201.4 +/- 67.5 mg/dL, range 82-277 mg/dL, respectively; P = 0.0002). Forty percent of Group TC patients required treatment for postoperative hypoglycemia (blood glucose level <60 mg/dL). Substantial interindividual variability existed in regard to insulin resistance. The investigation was terminated after we realized that normoglycemia was unattainable with the study protocol and that postoperative hypoglycemia was unpredictable. All patients in both groups experienced similar significant increases in postoperative serum levels of CK-BB and S-100 protein. These results indicate that "tight control" of intraoperative blood glucose in nondiabetic patients undergoing cardiac surgery was unattainable with the study protocol and may initiate postoperative hypoglycemia. IMPLICATIONS: The appropriate intraoperative management of hyperglycemia and whether it adversely affects neurologic outcome in patients after cardiac surgery remains controversial. This investigation reveals that attempting to maintain normoglycemia in this setting with insulin may initiate postoperative hypoglycemia.

The C-terminal 12 amino acids of sigma(N) are required for structure and function.

The sigma(N) protein is an alternative sigma subunit of bacterial RNA polymerase. We investigated the role of a 12-amino-acid "tail" at the C-terminus of Klebsiella pneumoniae sigma(N), which was predicted to be largely surface-exposed and to be mostly loop (that is not alpha-helical or beta-strand). Deletion of this tail from N-terminal hexahistidine-tagged sigma(N) led to loss of sigma(N)-dependent transcription activity in vivo. We overexpressed and purified this deletion-mutant protein for in vitro characterization. The purified deleted protein showed decreased RNA polymerase core- and DNA-binding activities compared to the full-length protein and transcription activity was greatly impaired. Furthermore, evidence from circular dichroism and protease digestion experiments together suggested that deletion of the C-terminus tail resulted in a loss of conformational constraint in the protein. We discuss a possible structural role for the 12 amino acids at the C-terminus of sigma(N).

Intrathecal morphine for coronary artery bypass graft procedure and early extubation revisited.

OBJECTIVE: To determine the dose of intrathecal (IT) morphine (along with the intraoperative baseline anesthetic) that provides significant analgesia yet does not delay extubation in the immediate postoperative period in patients undergoing cardiac surgery and early extubation. DESIGN: Prospective, randomized, double-blinded, placebo-controlled clinical study. SETTING: Single university hospital. PARTICIPANTS: Forty patients undergoing elective coronary artery bypass graft procedure and early extubation. INTERVENTIONS: Twenty patients received 10 microg/kg of IT morphine, and 20 patients received IT placebo. Perioperative anesthetic management was standardized and included postoperative patient-controlled morphine analgesia. MAIN RESULTS: Of the patients tracheally extubated during the immediate postoperative period, mean time to extubation was similar in patients who received IT morphine (6.8+/-2.8 h) or IT placebo (6.5+/-3.2 h). Four patients who received IT morphine had extubation substantially delayed because of prolonged ventilatory depression. There was no difference between groups in postoperative patient-controlled morphine analgesia use. CONCLUSION: Even when used in conjunction with an intraoperative baseline anesthetic that allows early extubation, IT morphine (10 microg/kg) was unable to provide substantial postoperative analgesia. The risks of using IT morphine in patients undergoing cardiac surgery and early extubation may outweigh the potential benefits.