Chemokine receptor hetero-oligomers regulate monocyte chemotaxis.

It is known that stress influences immune cell function. The underlying molecular mechanisms are unclear. We recently reported that many chemokine receptors (CRs) heteromerize with α1-adrenoceptors (α1-ARs) through which CRs are regulated. Here, we show that arginine vasopressin receptor 1A (AVPR1A) heteromerizes with all human CRs, except chemokine (C-X-C motif) receptor (CXCR)1, in recombinant systems and that such heteromers are detectable in THP-1 cells and human monocytes. We demonstrate that ligand-free AVPR1A differentially regulates the efficacy of CR partners to mediate chemotaxis and that AVPR1A ligands disrupt AVPR1A:CR heteromers, which enhances chemokine (C-C motif) receptor (CCR)1-mediated chemotaxis and inhibits CCR2-, CCR8-, and CXCR4-mediated chemotaxis. Using bioluminescence resonance energy transfer to monitor G protein activation and CRISPR/Cas9 gene-edited THP-1 cells lacking AVPR1A or α1B-AR, we show that CRs that share the propensity to heteromerize with α1B/D-ARs and AVPR1A exist and function within interdependent hetero-oligomeric complexes through which the efficacy of CRs to mediate chemotaxis is controlled. Our findings suggest that hetero-oligomers composed of CRs, α1B/D-ARs, and AVPR1A may enable stress hormones to regulate immune cell trafficking.

Examining surgeon stress in robotic and laparoscopic surgery.

Robotic surgery may decrease surgeon stress compared to laparoscopic. To evaluate intraoperative surgeon stress, we measured salivary alpha-amylase and cortisol. We hypothesized robotic elicited lower increases in surgeon salivary amylase and cortisol than laparoscopic. Surgical faculty (n = 7) performing laparoscopic and robotic operations participated. Demographics: age, years in practice, time using laparoscopic vs robotic, comfort level and enthusiasm for each. Operative data included operative time, WRVU (surgical "effort"), resident year. Saliva was collected using passive drool collection system at beginning, middle and end of each case; amylase and cortisol measured using ELISA. Standard values were created using 7-minute exercise (HIIT), collecting saliva pre- and post-workout. Linear regression and Student's t test used for statistical analysis; p values < 0.05 were significant. Ninety-four cases (56 robotic, 38 laparoscopic) were collected (April-October 2022). Standardized change in amylase was 8.4 ± 4.5 (p < 0.001). Among operations, raw maximum amylase change in laparoscopic and robotic was 23.4 ± 11.5 and 22.2 ± 13.4; raw maximum cortisol change was 44.21 ± 46.57 and 53.21 ± 50.36, respectively. Values normalized to individual surgeon HIIT response, WRVU, and operative time, showing 40% decrease in amylase in robotic: 0.095 ± 0.12, vs laparoscopic: 0.164 ± 0.16 (p < 0.02). Normalized change in cortisol was: laparoscopic 0.30 ± 0.44, robotic 0.22 ± 0.4 (p = NS). On linear regression (p < 0.001), surgeons comfortable with complex laparoscopic cases had lower change in normalized amylase (p < 0.01); comfort with complex robotic was not significant. Robotic may be less physiologically stressful, eliciting less increase in salivary amylase than laparoscopic. Comfort with complex laparoscopic decreased stress in robotic, suggesting laparoscopic experience is valuable prior to robotic.

Effects of chemokine (C-C motif) receptor 2 and 3 antagonists in rat models of hemorrhagic shock.

Systemic concentrations of chemokine CCL2, an agonist at chemokine receptors CCR2/3/5, have been associated with hemodynamic instability after traumatic-hemorrhagic shock. We reported previously that the CCR2 antagonist INCB3284 prevents cardiovascular collapse and reduces fluid requirements after 30min of hemorrhagic shock (HS), whereas the CCR5 antagonist Maraviroc was ineffective. The effects of CCR3 blockade after HS are unknown and information on the therapeutic potential of INCB3284 after longer periods of HS and in HS models in the absence of fluid resuscitation (FR) is lacking. The aims of the present study were to assess the effects of CCR3 blockade with SB328437 and to further define the therapeutic efficacy of INCB3284. In series 1-3, Sprague-Dawley rats were hemorrhaged to a mean arterial blood pressure (MAP) of 30mmHg, followed by FR to MAP of 60mmHg or systolic blood pressure of 90mmHg. Series 1: 30min HS and FR until t = 90min. SB328437 at t = 30min dose-dependently reduced fluid requirements by >60%. Series 2: 60min HS and FR until t = 300min. INCB3284 and SB328437 at t = 60min reduced fluid requirements by more than 65% (p<0.05 vs. vehicle) and 25% (p>0.05 vs. vehicle), respectively, until t = 220min. Thereafter, all animals developed a steep increase in fluid requirements. Median survival time was 290min with SB328437 and >300min after vehicle and INCB3284 treatment (p<0.05). Series 3: HS/FR as in series 2. INCB3284 at t = 60min and t = 200min reduced fluid requirements by 75% until t = 300min (p<0.05 vs. vehicle). Mortality was 70% with vehicle and zero with INCB3284 treatment (p<0.05). Series 4: INCB3284 and SB328437 did not affect survival time in a lethal HS model without FR. Our findings further support the assumption that blockade of the major CCL2 receptor CCR2 is a promising approach to improve FR after HS and document that the dosing of INCB3284 can be optimized.

Causal analysis of socioeconomic influence on cost of care: The emergency general surgery model.

BACKGROUND: This study characterizes the relationship between SES and cost of emergency general surgery (EGS). METHODS: Utilizing Florida AHCA (2016-2020), patients undergoing the 7 most common EGS were identified. Distressed Community Index (DCI) was linked, which quantifies SES through unemployment, poverty, and other factors. Zipcodes are assigned DCI 0 (no distress) to 100 (severe distress). Linear regression with stepwise elimination was conducted. Top and bottom DCI quintiles were propensity matched for demographics, comorbidities, and procedure. RESULTS: 144,924 admissions were included. Linear regression eliminated 5 of 28 variables, including DCI. Top cost contributors were discharge-43%; comorbidities-14%; age-9%. Distressed patients received less home health and inpatient rehab. Distressed patients utilized 4-/5-star hospitals less and had higher odds of mortality. CONCLUSION: Discharge, mortality, and hospital characteristics differ significantly between DCI communities. Total cost was similar, and is strongly influenced by discharge status, while DCI had no effect.

α1-adrenoceptor ligands inhibit chemokine receptor heteromerization partners of α1B/D-adrenoceptors via interference with heteromer formation.

We reported previously that α1-adrenoceptor (α1-AR) ligands inhibit chemokine receptor (CR) heteromerization partners of α1B/D-AR. The underlying mechanisms are unknown and in vivo evidence for such effects is missing. Utilizing CCR2 and α1B-AR as prototypical partners, we observed in recombinant systems and THP-1 cells that α1B-AR enhanced whereas its absence inhibited Gαi signaling of CCR2. Phenylephrine and phentolamine reduced the CCR2:α1B-AR heteromerization propensity and inhibited Gαi signaling of CCR2. Phenylephrine cross-recruited ß-arrestin-2 to CCR2, and reduced expression of α1B/D-AR, CR partners (CCR1/2, CXCR4) and corresponding heteromers. Phentolamine reduced CR:α1B/D-AR heteromers without affecting ß-arrestin-2 recruitment or receptor expression. Phenylephrine/phentolamine prevented leukocyte infiltration mediated via CR heteromerization partners in a murine air pouch model. Our findings document that α1-AR ligands inhibit leukocyte migration mediated by CR heteromerization partners in vivo and suggest interference with α1B-AR:CR heteromerization as a mechanism by which CR partners are inhibited. These findings provide new insights into the pharmacology of GPCR heteromers and indicate that an agonist and antagonist at one GPCR can act as antagonists at heteromerization partners of their target receptors.

Heteromerization between α1B -adrenoceptor and chemokine (C-C motif) receptor 2 biases α1B -adrenoceptor signaling: Implications for vascular function.

Previously, we reported that chemokine (C-C motif) receptor 2 (CCR2) heteromerizes with α1B -adrenoceptor (α1B -AR) in leukocytes, through which α1B -AR controls CCR2. Whether such heteromers are expressed in human vascular smooth muscle cells (hVSMCs) is unknown. Bioluminescence resonance energy transfer confirmed formation of recombinant CCR2:α1b -AR heteromers. Proximity ligation assays detected CCR2:α1B -AR heteromers in hVSMCs and human mesenteric arteries. CCR2:α1B -AR heteromerization per se enhanced α1B -AR-mediated Gαq -coupling. Chemokine (C-C motif) ligand 2 (CCL2) binding to CCR2 inhibited Gαq activation via α1B -AR, cross-recruited ß-arrestin to and induced internalization of α1B -AR in recombinant systems and in hVSMCs. Our findings suggest that CCR2 within CCR2:α1B -AR heteromers biases α1B -AR signaling and provide a mechanism for previous observations suggesting a role for CCL2/CCR2 in the regulation of cardiovascular function.

The Chemokine (C-C Motif) Receptor 2 Antagonist INCB3284 Reduces Fluid Requirements and Protects From Hemodynamic Decompensation During Resuscitation From Hemorrhagic Shock.

Clinical correlations suggest that systemic chemokine (C-C motif) ligand (CCL) 2 release may contribute to blood pressure regulation and the development of hemodynamic instability during the early inflammatory response to traumatic-hemorrhagic shock. Thus, we investigated whether blockade of the principal CCL2 receptor chemokine (C-C motif) receptor (CCR) 2 affects blood pressure in normal animals, and hemodynamics and resuscitation fluid requirements in hemorrhagic shock models. DESIGN: Randomized prospective treatment study. SETTING: University laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: First, treatment of healthy anesthetized rats with increasing doses of INCB3284 or vehicle. Second, rats were hemorrhaged for 30 minutes, followed by treatment with the CCR2 antagonist INCB3284 (1.1 and 5.5 µmol/kg), the CCR5 antagonist Maraviroc (=control, 5.5 µmol/kg) or vehicle, and subsequent fluid resuscitation to maintain blood pressure until t = 90 minutes. Third, treatment of rats with 5 µmol/kg INCB3284 or vehicle after hemorrhage and fluid resuscitation until t = 300 minutes. MEASUREMENTS AND MAIN RESULTS: INCB3284 did not affect intrinsic function of isolated rat resistance arteries in pressure myography experiments. Blood pressure in anesthetized vehicle-treated animals continuously decreased by 0.09 ± 0.01 mm Hg/min (p < 0.001) but remained constant after INCB3284 injections. Systemic concentrations of the CCR2 agonists CCL2, CCL5, and CCL11 increased during hemorrhage and fluid resuscitation. INCB3284 dose-dependently reduced fluid requirements by 58% ± 11% in short-term experiments, whereas Maraviroc and vehicle-treated animals were indistinguishable. When resuscitation was performed until t = 300 minutes, INCB3284 reduced fluid requirements by 62% ± 6%, prevented from hemodynamic decompensation, reduced mortality from 50% with vehicle treatment to zero, and reduced overall tissue wet-weight/dry-weight ratios. CONCLUSIONS: Our findings suggest that CCR2 is involved in the regulation of normal cardiovascular function and during the cardiovascular stress response to hemorrhagic shock and fluid resuscitation. The present study identifies CCR2 as a drug target to reduce fluid requirements and to prevent death from hemodynamic decompensation during resuscitation from hemorrhagic shock.

α1B/D-adrenoceptors regulate chemokine receptor-mediated leukocyte migration via formation of heteromeric receptor complexes.

It is known that catecholamines regulate innate immune functions. The underlying mechanisms, however, are not well understood. Here we show that at least 20 members of the human chemokine receptor (CR) family heteromerize with one or more members of the α1-adrenergic receptor (AR) family in recombinant systems and that such heteromeric complexes are detectable in human monocytes and the monocytic leukemia cell line THP-1. Ligand binding to α1-ARs inhibited migration toward agonists of the CR heteromerization partners of α1B/D-ARs with high potency and 50 to 77% efficacy but did not affect migration induced by a noninteracting CR. Incomplete siRNA knockdown of α1B/D-ARs in THP-1 cells partially inhibited migration toward agonists of their CR heteromerization partners. Complete α1B-AR knockout via CRISPR-Cas9 gene editing in THP-1 cells (THP-1_ADRA1BKO) resulted in 82% reduction of α1D-AR expression and did not affect CR expression. Migration of THP-1_ADRA1BKO cells toward agonists of CR heteromerization partners of α1B/D-ARs was reduced by 82 to 95%. Our findings indicate that CR:α1B/D-AR heteromers are essential for normal function of CR heteromerization partners, provide a mechanism underlying neuroendocrine control of leukocyte trafficking, and offer opportunities to modulate leukocyte and/or cancer cell trafficking in disease processes.

Association of individual surgeon volume and postoperative outcome in esophagomyotomy for achalasia.

BACKGROUND: Many surgical disciplines have demonstrated superior outcomes when procedures are performed at "high-volume". Esophagomyotomy is commonly performed for achalasia, however it's unclear what constitutes "high-volume" for this procedure, and if individual procedure volume and outcome are related. We identified physicians performing esophagomyotomy, stratified them by individual case volume, and examined their outcomes with the hypothesis that high-volume surgeons will be associated with improved outcomes as compared to low-volume surgeons. METHODS: The 2015-2019 Florida Agency for Health Care Administration (AHCA) inpatient dataset was queried for esophagomyotomy. Surgeons who performed ≥ 10 procedures during the study period were placed into the high-volume cohort, and those performing < 10 into the low-volume cohort. Groups were compared by length of stay, discharge disposition, and postoperative complications. Patient demographics were evaluated using student's t test and chi square test, p < 0.05 considered significant. RESULTS: Six hundred and sixty-two procedures performed by 135 surgeons were identified. The mean number of esophagomyotomies per surgeon was 4.9 (Range 1-147). The high-volume group (n = 12) performed 362 of the 662 procedures (55%), while the low-volume group (n = 123) performed the remaining 300 (45%). Patients of high-volume physicians had decreased length of stay (1.4 ± 0.8 days vs 4.9 ± 6.7 days, p = 0.01) and were more likely to be discharged to home following surgery (92.8% vs 86.0, p = 0.04). High volume physicians also had statistically significant differences in rates of urinary tract infection (1.4% vs 4.0%, p = 0.034), postoperative malnutrition (5.8% vs 11.0%, p = 0.015), and postoperative fluid and electrolyte disorders (5.5% vs 13.3%, p < 0.0001). CONCLUSION: Surgeons who perform higher volumes of esophagomyotomies are associated with decreased length of stay, higher likelihood of patient discharge to home, and decreased rates of some postoperative complications. This research should prompt further inquiry into defining what constitutes a high-volume center in foregut surgery and their role in improving patient outcomes.

Revisional Procedures for Recurrent Symptoms After Heller Myotomy and Per-Oral Endoscopic Myotomy.

Background: Achalasia and other esophageal motility disorders are incurable diseases for which palliation and symptom relief are the goals. One of the many ways these diseases are treated is with either a Heller myotomy or, now more commonly, per-oral endoscopic myotomy (POEM). Unfortunately, symptoms persistence or recurrence is common. This review presents our current approach to these complex patients. Methods: Review of the literature pertaining to approaches to recurrent or persistent symptoms after myotomy for esophageal motility disorders and elucidation of our multidisciplinary approach to this patient group. Results: There are a myriad of causes of recurrent or persistent symptoms. These include incomplete myotomy, periesophageal scarring, reflux-induced stricture, obstructing fundoplication, functional dysphagia, and end-stage achalasia. Therapeutic options include redo myotomy (either Heller or POEM), botulinum toxin injection, pneumatic, balloon or Savary dilation, adhesiolysis, and fundoplication reversal or esophagectomy. Choice of approach is best done through multidisciplinary consensus. Conclusions: A multidisciplinary approach to patients with persistent and recurrent symptoms after myotomy can best tailor the therapeutic approach based on symptom causation.