Preemptive Intravenous Human Immunoglobulin G Suppresses BK Polyomavirus Replication and Spread of Infection In Vitro.

BK polyomavirus (BKPyV) infection causes various diseases in immunocompromised patients. Cells from human lung and kidney were infected with BKPyV and treated with commercially available intravenous immunoglobulin G (IVIG). Its effects on BKPyV replication and spread of infection were investigated, focusing on administration timing. IVIG treatment 3 hours after infection suppressed BKPyV replication assessed by real-time PCR and expression of the viral capsid protein 1 and large T-antigen. IVIG effectively reduced the number of BKPyV-infected cells 2 weeks after infection in an antibody titer-dependent manner. Virus release in the culture supernatants was not influenced by IVIG treatment 6-80 hours and 3-9 days after infection. Collectively, IVIG did not affect viral release from infected cells but inhibited the spread of infection by neutralizing the released virus and blocking the new infected cell formation, indicating greater efficacy in early localized infection. BKPyV replication resumed in IVIG-treated cultures at 7 days after IVIG removal. Early prophylactic administration of IVIG is expected to reduce the growth and spread of BKPyV infection, resulting in the reduction of infected cell lesions and prevention of BKPyV-associated diseases.

Ubiquitination of the µ-opioid receptor regulates receptor internalization without affecting Gi/o-mediated intracellular signaling or receptor phosphorylation.

Opioids are highly potent analgesics but develop tolerance. Previous studies have focused on phosphorylation of the µ-opioid receptor as it is involved in maintaining cellular sensitivity via desensitization, recycling, and degradation of the activated receptor. Recently, ubiquitination, another form of posttranslational modification has attracted attention in terms of triggering intracellular signaling and regulation of the activated receptor. Here, we generated a ubiquitination-deficient mutant of the µ-opioid receptor to investigate whether ubiquitination is involved in driving Gi/o-mediated analgesic signaling, receptor desensitization or subsequent receptor internalization. Our study shows that the Gi/o pathway and receptor phosphorylation do not require ubiquitination. Instead, ubiquitination regulates the internalization efficiency and might help in promoting internalization of the desensitized MOP.

The molecular associations in clathrin-coated pit regulate ß-arrestin-mediated MAPK signaling downstream of µ-opioid receptor.

It has been thought that µ-opioid receptors (MOPs) activate the G protein-mediated analgesic pathway and ß-arrestin 2-mediated side effect pathway; however, ligands that only minimally recruit ß-arrestin 2 to MOPs may also cause opioid side effects. Moreover, such side effects have been induced in mutant mice lacking ß-arrestin 2 or expressing phosphorylation-deficient MOPs that do not recruit ß-arrestin 2. These findings raise the critical question of whether ß-arrestin 2 recruitment to MOP triggers side effects. Here, we show that ß-arrestin 1 and 2 are essential in the efficient activation of the Gi/o-mediated MAPK signaling at MOP. Moreover, the magnitude of ß-arrestin-mediated signals is not correlated with the magnitude of phosphorylation of the carboxyl-terminal of MOP, which is used to evaluate the ß-arrestin bias of a ligand. Instead, the molecular association with ß2-adaptin and clathrin heavy chain in the formation of clathrin-coated pits is essential for ß-arrestin to activate MAPK signaling. Our findings provide insights into G protein-coupled receptor-mediated signaling and further highlight a concept that the accumulation of molecules required for endocytosis is critical for activating intracellular signaling.

Everolimus reduces BK polyomavirus infection by suppressing its replication and spread of infection.

BK polyomavirus-associated nephropathy is one of serious complications in transplant recipients. Everolimus-a mammalian target of rapamycin inhibitor-has been shown to reduce the incidence of BK polyomavirus infection in transplant recipients. In this study, the effects of everolimus were examined on viral replication and the spread of infection in BK polyomavirus-infected cultures. BK polyomavirus replicated in renal and pulmonary cells, contrary to that in hepatocytes, and spread as diffusely scattered patterns of infected cells, unlike plaque formation through the cell-to-cell mode. BK polyomavirus is stable to heat up to 65 °C with a particle per infectivity ratio of 5000, and the replication cycle was for approximately 34 h. Everolimus administration remarkably reduced the viral replication to 20% in cells treated with 0.1-10 ng/mL, the concentration at which everolimus reached the serum of transplant recipients. In addition, it reduced the amount of viral capsid protein 1 at 5 ng/mL without reducing the ratio of viral capsid protein 1 versus ß-actin, and it also retained the pattern of viral capsid protein 1 localization in the nuclei. Everolimus suppressed the number of infected cells to 32.8% during a 14-day treatment, indicating the reduction of BK polyomavirus-infected cell mass to 18.8% of untreated cultures by modifying cellular functions. The reduction in the total number of BK polyomavirus infected cells by everolimus indicates that everolimus alleviates BK polyomavirus infection, including nephropathy in transplant recipients.

A Nationwide Questionnaire Survey on Awake Craniotomy in Japan.

The number of awake craniotomies is increasing because of its beneficial features. However, not enough information is available regarding the current status of awake craniotomy in Japan. To evaluate the current status of awake craniotomy in institutes, a nationwide questionnaire survey was conducted. From June to August 2019, we conducted a questionnaire survey on awake craniotomy in the neurosurgery department of 45 institutes that perform awake craniotomies in Japan. Responses were obtained from 39 institutes (response rate, 86.7%). The main methods of awake craniotomy were almost the same in all institutes. Twenty-six institutes (66.7%) had fewer than 10 awake craniotomies (low-volume institutes) per year, and 13 high-volume institutes (33.3%) performed more than 10 awake craniotomies annually. Some institutes experienced a relatively high frequency of adverse events. In 11 institutes (28.2%), the frequency of intraoperative seizures was more than 10%. An intraoperative seizure frequency of 1%-9%, 10%-29%, and over 30% was identified in 12 (92%), 0 (0%), and 1 (8%) of the high-volume institutes, which was significantly less than in 16 (62%), 10 (38%), and 0 (0%) of the low-volume institutes (p = 0.0059). The routine usage of preoperative antiepileptic drugs was not different between them, but the old type was used more often in the low-volume institutes (p = 0.0022). Taken together, the annual number of awake craniotomies was less than 10 in over two-thirds of the institutes. Fewer intraoperative seizures were reported in the high-volume institutes, which tend not to preoperatively use the old type of antiepileptic drugs.

CRISPR/Cas9 unveils the dynamics of the endogenous µ-opioid receptors on neuronal cells under continuous opioid stimulation.

Long-term opioid use develops tolerance and attenuates analgesic effects. Upon activation, µ-opioid receptors (MOPs) are internalized and directed to either recycling or degradation pathway. Ligand stimulation also promotes de novo MOP synthesis. These processes collaboratively regulate MOP expression and play critical roles in tolerance development. However, there is limited understanding of how the endogenous MOP expression changes after prolonged opioid administration because previous analyses have focused on individual processes using overexpression systems, which ignored physiological regulation. Another fundamental problem is the unavailability of commercial antibodies to detect the low expression of endogenous MOP in neuronal systems. Here, we established a neuronal cell line to detect endogenous MOP with sufficient sensitivity using CRISPR/Cas9 technology. We incorporated the hemagglutinin sequence into the MOP gene of the SH-SY5Y cell. The genome-editing did not significantly impair MOP functions such as MOP internalization or the downstream signaling. The clone was differentiated into a state similar to the primary culture undergoing treatment with all-trans retinoic acid, followed by brain-derived neurotrophic factor. Upon continuous stimulation with MOP ligands, endogenous MOP constantly decreased up to 48 h. The expression level was maintained at a certain level following this period, depending on the ligand properties. DAMGO reduced MOP from the cell surface by about 70%, while morphine did so by 40%. Our results indicate that even a few days of opioid administration could significantly reduce the MOP expression level. Our cell line could be a potential tool to investigate the molecular mechanisms underlying the problems caused by long-term opioid use.

Factors associated with somnolence during brain function mapping in awake craniotomy.

Somnolence during brain function mapping is one of the factors that inhibit the accomplishment of the goals of awake craniotomy. We examined the effect of anesthesia depth measured by bispectral index (BIS) during pre-awake phase on somnolence during brain function mapping and also explored the factors associated with somnolence. We examined the association between BIS values during pre-awake phase and somnolence during the first 30 min of brain function mapping in 55 patients who underwent awake craniotomy at Kyoto University Hospital from 2015 to 2018. The pre-awake BIS value was defined as the mean BIS value for 60 min before the removal of the airway. Somnolence during brain function mapping was the primary outcome, defined as either of the following conditions: inability to follow up, disorientation, or inability to assess speech function. Additionally, we compared patient or perioperative variables between patients with/without somnolence. Somnolence occurred in 14 patients (25.5%), of which 6 patients (10.9%) were unable to complete brain function mapping. There was no significant difference in the pre-awake BIS value between patients with/without somnolence (median: 46 vs. 49, P = 0.192). Somnolence was not significantly associated with age, gender, and the number of preoperative anticonvulsive drugs, but patients with somnolence had a significantly lower preoperative Western Aphasia Battery (WAB) aphasia quotient score (median 93.8 vs. 98.6, P = 0.011). We did not find an association between pre-awake BIS value and somnolence during brain function mapping. Somnolence likely occurs in patients with a low preoperative WAB aphasia quotient score.

Effects of low-dose remifentanil infusion on analgesic or antiemetic requirement during brain function mapping: A retrospective cohort study.

BACKGROUND: Pain and discomfort during the awake phase in awake craniotomy should be relieved to facilitate brain mapping. Although some anaesthesiologists use low-dose (0.01-0.05 µg/kg/min) remifentanil infusion to provide analgesia during this phase, its efficacy and side effects have never been evaluated. Therefore, this study primarily aimed to investigate the effects of low-dose remifentanil infusion on the need for antiemetic treatment during brain mapping and secondarily aimed to determine its effects on the need for additional analgesic treatment. METHODS: This retrospective study included 218 patients who underwent awake craniotomy at our centre from 2008 to 2018. The relationship between low-dose remifentanil infusion during the awake phase and the requirement for analgesic or antiemetic treatment was examined. A multivariable competing risk regression analysis was performed to adjust for patient and operative variables. RESULTS: Sixty-six patients (30.3%) received low-dose (median rate: 0.01 µg/kg/min) remifentanil infusion during the awake phase. Forty-nine patients (22.5%) received an antiemetic and 99 (45.4%) received additional analgesic treatment. The difference in additional analgesic treatment was not significant between patients who received low-dose remifentanil infusion and those who did not (adjusted hazard ratio: 1.13; 95% confidence interval: 0.75-1.70; P = .570); however, the use of antiemetics significantly increased in patients who received remifentanil (adjusted hazard ratio: 1.78; 95% confidence interval: 1.01-3.15; P = .047). CONCLUSION: Low-dose remifentanil infusion during the awake phase in awake craniotomy significantly increased the need for antiemetics but did not decrease the need for additional analgesic treatment.

Derivation and validation of an equation to determine the optimal ventilator setting in children undergoing intracranial revascularization surgery: A single-center retrospective study.

BACKGROUND: It can be difficult to determine the appropriate ventilator settings to maintain normocapnia in children undergoing general anesthesia for surgery for moyamoya disease, especially immediately following anesthesia induction. AIM: We conducted this study to attempt to derive an equation to predict the appropriate ventilator settings and subsequently validated the accuracy of the equation. METHODS: A retrospective study of 91 pediatric patients less than 18 years of age who underwent cerebral revascularization for moyamoya disease at our institution. Fifty-eight patients were used to derive the equation, and the subsequent 33 patients were used to validate the equation. We calculated the required respiratory rate to attain normocapnia based on the median of all values of the minute volume during normocapnia (estimated partial pressure of arterial carbon dioxide of 38-42 mm Hg) and the assumption that the tidal volume was 8 mL/kg body weight. We derived the regression equation from the derivation data set where the required respiratory rate to attain normocapnia was represented by age. We simplified the equation by rounding coefficients to the nearest integer. The level of agreement between the respiratory rate predicted from the equation and the actual required respiratory rate was assessed in the validation group using Bland-Altman analysis. RESULTS: The derived equation is tidal volume = 8 mL/kg body weight, respiratory rate = 24-age/min. Bland-Altman analysis in the validation group revealed that the mean bias between the predicted and actual respiratory rate was 0.29 (standard deviation, 3.67). The percentage of cases where the predicted rate was within ± 10% and ± 20% of the actual rate was 42.4% and 66.7%, respectively. CONCLUSIONS: We derived and validated a simple and easily applicable equation to predict the ventilator settings required to attain normocapnia during general anesthesia in children with moyamoya disease.

Validation of the Radford Nomogram to Estimate the Minute Volume Required to Attain Normocapnia in Patients Undergoing General Anesthesia: A Single-Center Retrospective Study

Objective: The Radford nomogram, an old mathematical chart device to estimate the required ventilation for maintaining normocapnia, remains unvalidated in patients undergoing modern, balanced anesthesia. This study aims to investigate the performance of the Radford nomogram in patients undergoing general anesthesia and derive a simple equation to estimate the minute volume required to attain normocapnia (MVnorm). Methods: This single-center retrospective study enrolled 78 patients (age ≥ 18 years) undergoing cerebral revascularization for Moyamoya disease. We defi ned MVnorm as the median of all values of the minute volume during normocapnia (estimated PaCO2: 38­42 mmHg). We examined the agreement level between the estimated minute volume using the Radford nomogram and MVnorm using the Bland­Altman analysis. Furthermore, we developed and validated a simple equation predicting MVnorm based on gender and a multiple of body weight, using a split-sample validation technique. Results: The Radford nomogram tended to overestimate MVnorm with a mean bias of 560 mL/min (95% limits of agreement, -848­1,968 mL/min). The equation developed using data from the development group (n = 52): required minute volume (mL/min) = 85 × body weight (kg) in male patients and 70 × body weight (kg) in female patients. In the validation group (n = 26), the mean bias of this simple equation was 224 mL/min (95% limits of agreement, -1,264­1,712 mL/min). Conclusion: The Radford nomogram overestimates MVnorm in modern, balanced anesthesia. The simple equation using gender and a multiple of body weight yields similar predictive performance to the Radford nomogram.

Invasive Respiratory or Vasopressor Support and/or Death as a Proposed Composite Outcome Measure for Perioperative Care Research.

BACKGROUND: There is a need for a clinically relevant and feasible outcome measure to facilitate clinical studies in perioperative care medicine. This large-scale retrospective cohort study proposed a novel composite outcome measure comprising invasive respiratory or vasopressor support (IRVS) and death. We described the prevalence of IRVS in patients undergoing major abdominal surgery and assessed the validity of combining IRVS and death to form a composite outcome measure. METHODS: We retrospectively collected perioperative data for 2776 patients undergoing major abdominal surgery (liver, colorectal, gastric, pancreatic, or esophageal resection) at Kyoto University Hospital. We defined IRVS as requirement for mechanical ventilation for ≥24 hours postoperatively, postoperative reintubation, or postoperative vasopressor administration. We evaluated the prevalence of IRVS within 30 postoperative days and examined the association between IRVS and subsequent clinical outcomes. The primary outcome of interest was long-term survival. Multivariable Cox proportional regression analysis was performed to adjust for the baseline patient and operative characteristics. The secondary outcomes were length of hospital stay and hospital mortality. RESULTS: In total, 85 patients (3.1%) received IRVS within 30 postoperative days, 15 of whom died by day 30. Patients with IRVS had a lower long-term survival rate (1- and 3-year survival probabilities, 66.1% and 48.5% vs 95.2% and 84.0%, respectively; P < .001, log-rank test) compared to those without IRVS. IRVS was significantly associated with lower long-term survival after adjustment for the baseline patient and operative characteristics (adjusted hazard ratio, 2.72; 95% confidence interval, 1.97-3.77; P < .001). IRVS was associated with a longer hospital stay (median [interquartile range], 65 [39-326] vs 15 [12-24] days; adjusted P < .001) and a higher hospital mortality (24.7% vs 0.5%; adjusted P < .001). Moreover, IRVS was adversely associated with subsequent clinical outcomes including lower long-term survival (adjusted hazard ratio, 1.78; 95% confidence interval, 1.21-2.63; P = .004) when the analyses were restricted to 30-day survivors. CONCLUSIONS: Patients with IRVS can experience ongoing risk of serious morbidity and less long-term survival even if alive at postoperative day 30. Our findings support the validity of using IRVS and/or death as a composite outcome measure for clinical studies in perioperative care medicine.

Transient acute kidney injury after major abdominal surgery increases chronic kidney disease risk and 1-year mortality.

PURPOSE: We conducted a retrospective cohort study to determine incidences of transient and persistent acute kidney injury (AKI) after major abdominal surgery and their impacts on long-term outcome. MATERIALS AND METHODS: We enrolled 3751 patients undergoing major abdominal surgery. Postoperative AKI was classified as transient or persistent based on the return of serum creatinine to the non-AKI range within 7 days post-surgery. Primary outcome was mortality within 1 year. We used multivariable Cox proportional hazard regression analysis to assess independent associations between AKI type and mortality. RESULTS: Most patients with AKI were classified as transient (84%). Compared to patients without AKI, both patients with transient and persistent AKI demonstrated elevated 1-year mortality rates [adjusted hazard ratio (95% confidence interval): 2.01 (1.34-2.93); P = 0.001, and 6.20 (3.00-11.43); P < 0.001, respectively] and greater risk of chronic kidney disease progression at 1 year [adjusted odds ratio (95% confidence interval): 3.87 (2.12-7.08) and 23.70 (9.64-58.22), respectively; both P < 0.001]. CONCLUSIONS: Although most AKI cases after major abdominal surgery recover completely within 7 days, even these patients with transient AKI are at higher risk for 1-year mortality and chronic kidney disease progression compared to patients without AKI.

Everolimus delayed and suppressed cytomegalovirus DNA synthesis, spread of the infection, and alleviated cytomegalovirus infection.

Everolimus is an inhibitor of mammalian target of rapamycin (mTOR) and reduces the risk of cytomegalovirus (CMV) infection in transplant recipients. Everolimus inhibits mTOR complex 1, which regulates factors involved in several crucial cellular functions and is required for CMV replication. However, it is not clear how everolimus regulates CMV replication and prevents and alleviates CMV infection. Effects of everolimus on CMV infection, spread, and DNA synthesis and release from infected cells were assessed by plaque formation, infectious centre assay, real-time PCR of infected cells, and culture supernatant in CMV-infected cultures with and without everolimus. Everolimus enhanced plaque formation by 3.6 times, but the size of the plaques was reduced to 36.4% of untreated cultures in the absence of a pretreatment period. Everolimus reduced viral adsorption but enhanced the replication efficiency of inoculated virus, resulting in an increase in plaque number in the early phase of infection. Preinfection treatment of cells with everolimus efficiently exhibited its antiviral efficacy, and everolimus delayed and suppressed viral DNA synthesis and release from infected cells. Everolimus had suppressed the spread of infection and reduced the number of total infected cells to 40% of untreated cells on day 9, indicating reduction of the size of CMV lesions to one-sixth in 2-3 replication cycles. Preinfection treatment of the cells with everolimus augmented its suppressive effect on CMV infection and replication. Everolimus reduced the total number of infected cells and limited the CMV lesions, and this reduction in the spread of CMV infection would alleviate CMV infection in transplant recipients.

Age-dependent trigeminal and female-specific lumbosacral increase in herpes zoster distribution in the elderly.

BACKGROUND: Varicella-zoster virus causes herpes zoster (HZ) along specific dermatomes, but the effects of age and sex on HZ distribution are unclear. OBJECTIVE: We investigated the age- and sex-dependent distribution characteristics of HZ. METHODS: Patients with HZ were monitored by members of the Miyazaki Dermatologist Society. Questionnaires containing information on age, sex, and dermatome distribution and lesion specimens from 2730 patients were collected, and 2508 PCR-diagnosed cases were analyzed. RESULTS: The ratio of lesions in the thoracic area to lesions in the whole body decreased with age, whereas those of other areas increased. HZ incidence increased with age to about four times that of the basic incidence in the dermatome areas at age 0-29 years; the incidence in the trigeminal area in both sexes increased 11-fold, and the incidence in the thoracic and lumbosacral areas increased in females more than in males. Furthermore, the fact that the highest incidence was found along the first branch of the trigeminal nerve suggests an association with long-term ultraviolet ray exposure. Segmental dermatomes comprising thoracic 10-lumbar 1/sacral 2-4 and thoracic 5-6 were significantly more frequently affected in female patients at age 50-59 years and are consistent with areas of obstetric anesthesia for childbirth and of breastfeeding, respectively. CONCLUSIONS: HZ incidence increased with age; moreover, exposure to ultraviolet rays, childbirth, and breastfeeding might increase the incidence at specific dermatomes in older individuals. This study provides important information on the etiology of HZ.

Interaction of Immunoglobulin with Cytomegalovirus-Infected Cells.

Intravenous immunoglobulin (IVIG) is used to treat or prevent severe viral infection, especially cytomegalovirus (CMV) infections. IVIG was characterized to understand its interaction with CMV-infected cells. IVIG retarded CMV spread and reduced virus yields depending on the neutralizing (NT) antibody titer. Immediate early protein synthesis was reduced by IVIG in 3 to 15 h, and IVIG specifically reduced the ratio of 66/68k protein synthesis among immediate early proteins in an NT antibody-dependent manner between 4 and 8 h after infection, indicating that antigenic modulation of CMV-infected cells by IVIG reduced viral protein synthesis and virus production. The half-life of antibody bound to CMV-infected cells was 3.8 h. NT antibody titers to varicella-zoster virus (VZV) and CMV in IVIG were dose dependently absorbed by cells infected with VZV and CMV, respectively, but the antibody titers to CMV and VZV, respectively, were not affected. NT antibody in 0.3 mL of IVIG (15 mg) was specifically absorbed by 108 CMV-infected cells and 107 VZV-infected cells, suggesting that the NT antibody in IVIG might be inactivated by one-tenth of a similar volume of CMV-infected or VZV-infected cells. Various antiviral activities of IVIG may contribute to control and alleviation of CMV infection.

Functional differences between antiviral activities of sulfonated and intact intravenous immunoglobulin preparations toward varicella-zoster virus and cytomegalovirus.

Intravenous immunoglobulin (IVIG) is used to treat severe viral infection, especially varicella-zoster virus (VZV) and cytomegalovirus (CMV) infections. The neutralization antibody titers of eleven IVIG preparations from four companies were examined using VZV and CMV with and without complement. The neutralizing antibody titers of intact IgG preparations were three to six times higher after addition of complement. The effectiveness of the sulfonated IgG preparation was not enhanced by complement, but desulfonated IgG regained enhanced neutralization activity with complement. Antibody-dependent cellular cytotoxicity (ADCC) toward VZV-infected cells was observed with both intact and sulfonated IVIG and guinea pig splenocytes, but ADCC toward CMV-infected cells was not, although NK cell activity toward cells infected with VZV or CMV was detected by splenocytes. Sulfonated IVIG had no complement-activated neutralization of VZV and CMV but retained ADCC toward VZV with less activity after dilution than with intact IVIG. Because sulfonated IVIG is converted to the intact form after intravenous administration, it would show complement-enhanced neutralization and ADCC activity similar to that of intact IVIG in vivo. In this study we showed the effects of intact and sulfonated IgG on the functional activity of IgG against VZV and CMV.