Army and Navy ECHO Pain Telementoring Improves Clinician Opioid Prescribing for Military Patients: an Observational Cohort Study.

BACKGROUND: Opioid overdose deaths occur in civilian and military populations and are the leading cause of accidental death in the USA. OBJECTIVE: To determine whether ECHO Pain telementoring regarding best practices in pain management and safe opioid prescribing yielded significant declines in opioid prescribing. DESIGN: A 4-year observational cohort study at military medical treatment facilities worldwide. PARTICIPANTS: Patients included 54.6% females and 46.4% males whose primary care clinicians (PCCs) opted to participate in ECHO Pain; the comparison group included 39.9% females and 60.1% males whose PCCs opted not to participate in ECHO Pain. INTERVENTION: PCCs attended 2-h weekly Chronic Pain and Opioid Management TeleECHO Clinic (ECHO Pain), which included pain and addiction didactics, case-based learning, and evidence-based recommendations. ECHO Pain sessions were offered 46 weeks per year. Attendance ranged from 1 to 3 sessions (47.7%), 4-19 (32.1%, or > 20 (20.2%). MAIN MEASURES: This study assessed whether clinician participation in Army and Navy Chronic Pain and Opioid Management TeleECHO Clinic (ECHO Pain) resulted in decreased prescription rates of opioid analgesics and co-prescribing of opioids and benzodiazepines. Measures included opioid prescriptions, morphine milligram equivalents (MME), and days of opioid and benzodiazepine co-prescribing per patient per year. KEY RESULTS: PCCs participating in ECHO Pain had greater percent declines than the comparison group in (a) annual opioid prescriptions per patient (- 23% vs. - 9%, P < 0.001), (b) average MME prescribed per patient/year (-28% vs. -7%, p < .02), (c) days of co-prescribed opioid and benzodiazepine per opioid user per year (-53% vs. -1%, p < .001), and (d) the number of opioid users (-20.2% vs. -8%, p < .001). Propensity scoring transformation-adjusted results were consistent with the opioid prescribing and MME results. CONCLUSIONS: Patients treated by PCCs who opted to participate in ECHO Pain had greater declines in opioid-related prescriptions than patients whose PCCs opted not to participate.

Persistent hepatitis C viral replication despite priming of functional CD8+ T cells by combined therapy with a vaccine and a direct-acting antiviral.

UNLABELLED: Exhaustion of antiviral CD8(+) T cells contributes to persistence of hepatitis C viral (HCV) infection. This immune response has proved difficult to restore by therapeutic vaccination, even when HCV replication is suppressed using antiviral regimens containing type I interferon. Because immunomodulatory effects of type I interferon may be a factor in poor T-cell priming, we undertook therapeutic vaccination in two chronically infected chimpanzees during treatment with a direct-acting antiviral (DAA) targeting the HCV NS5b polymerase protein. Immunization with genetic vaccines encoding the HCV NS3-NS5b nonstructural proteins during DAA treatment resulted in a multifunctional CD8(+) T-cell response. However, these antiviral CD8(+) T cells did not prevent persistent replication of DAA-resistant HCV variants that emerged during treatment. Most vaccine-induced CD8(+) T cells targeted class I epitopes that were not conserved in the circulating virus. Exhausted intrahepatic CD8(+) T-cell targeting-conserved epitopes did not expand after vaccination, with a notable exception. A sustained, multifunctional CD8(+) T-cell response against at least one intact class I epitope was detected in blood after vaccination. Persistence of HCV was not due to mutational escape of this epitope. Instead, failure to control HCV replication was likely caused by localized exhaustion in the liver, where CD8(+) T-cell expression of the inhibitory receptor programmed cell death 1 increased 25-fold compared with those in circulation. CONCLUSION: Treatment with a DAA during therapeutic vaccination provided transient control of HCV replication and a multifunctional T-cell response, primarily against nonconserved class I epitopes; exhaustion of liver-infiltrating CD8(+) T cells that target conserved epitopes may not be averted when DAA therapy fails prematurely due to emergence of resistant HCV variants.

Immunotherapy of chronic hepatitis C virus infection with antibodies against programmed cell death-1 (PD-1).

Hepatitis C virus (HCV) persistence is facilitated by exhaustion of CD8+ T cells that express the inhibitory receptor programmed cell death 1 (PD-1). Blockade of PD-1 signaling improves in vitro proliferation of HCV-specific T lymphocytes, but whether antiviral function can be restored in infected individuals is unknown. To address this question, chimpanzees with persistent HCV infection were treated with anti-PD-1 antibodies. A significant reduction in HCV viremia was observed in one of three treated animals without apparent hepatocellular injury. Viremia rebounded in the responder animal when antibody treatment was discontinued. Control of HCV replication was associated with restoration of intrahepatic CD4+ and CD8+ T-cell immunity against multiple HCV proteins. The responder animal had a history of broader T-cell immunity to multiple HCV proteins than the two chimpanzees that did not respond to PD-1 therapy. The results suggest that successful PD-1 blockade likely requires a critical threshold of preexisting virus-specific T cells in liver and warrants consideration of therapeutic vaccination strategies in combination with PD-1 blockade to broaden narrow responses. Anti-PD-1 immunotherapy may also facilitate control of other persistent viruses, notably the hepatitis B virus where options for long-term control of virus replication are limited.

T-cell immunity and hepatitis C virus reinfection after cure of chronic hepatitis C with an interferon-free antiviral regimen in a chimpanzee.

UNLABELLED: Memory CD8+ T cells generated by spontaneous resolution of hepatitis C virus (HCV) infection rapidly control secondary infections and reduce the risk of virus persistence. Here, CD8+ T-cell immunity and response to reinfection were assessed in a chimpanzee cured of an earlier chronic infection with an interferon (IFN)-free antiviral regimen. CD8+ T cells expanded from liver immediately before and 2 years after cure of chronic infection with two direct-acting antivirals (DAAs) targeted epitopes in the E2, nonstructural (NS)5a, and NS5b proteins. A second infection to assess CD8+ T-cell responsiveness resulted in rapid suppression of HCV replication by week 2, but viremia rebounded 3 weeks later and the infection persisted. The E2, NS5a, and NS5b proteins remained dominant CD8+ T-cell targets after reinfection. Resurgent HCV replication was temporally associated with mutational escape of NS5a and NS5b class I epitopes that had also mutated during the first chronic infection. Two epitopes in E2 remained intact throughout both persistent infections. Intrahepatic CD8+ T cells targeting intact and escape-prone epitopes differed in expression of phenotypic markers of functional exhaustion 2 years after successful DAA therapy and in the capacity to expand in liver upon reinfection. CONCLUSIONS: The intrahepatic HCV-specific CD8+ T-cell repertoire established during chronic infection was narrowly focused, but very stable, after cure with DAA. Existing intrahepatic CD8+ T cells targeting dominant epitopes of the challenge virus failed to prevent persistence. Vaccination after DAA cure may be necessary to broaden T-cell responses and reduce the risk of a second persistent infection.

Novel infectious cDNA clones of hepatitis C virus genotype 3a (strain S52) and 4a (strain ED43): genetic analyses and in vivo pathogenesis studies.

Previously, RNA transcripts of cDNA clones of hepatitis C virus (HCV) genotypes 1a (strains H77, HCV-1, and HC-TN), 1b (HC-J4, Con1, and HCV-N), and 2a (HC-J6 and JFH1) were found to be infectious in chimpanzees. However, only JFH1 was infectious in human hepatoma Huh7 cells. We performed genetic analysis of HCV genotype 3a (strain S52) and 4a (strain ED43) prototype strains and generated full-length consensus cDNA clones (pS52 and pED43). Transfection of Huh7.5 cells with RNA transcripts of these clones did not yield cells expressing HCV Core. However, intrahepatic transfection of chimpanzees resulted in robust infection with peak HCV RNA titers of approximately 5.5 log(10) international units (IU)/ml. Genomic consensus sequences recovered from serum at the times of peak viral titers were identical to the sequences of the parental plasmids. Both chimpanzees developed acute hepatitis with elevated liver enzymes and significant necroinflammatory liver changes coinciding with detection of gamma interferon-secreting, intrahepatic T cells. However, the onset and broadness of intrahepatic T-cell responses varied greatly in the two animals, with an early (week 4) multispecific response in the ED43-infected animal (3 weeks before the first evidence of viral control) and a late (week 11) response with limited breadth in the S52-infected animal (without evidence of viral control). Autologous serum neutralizing antibodies were not detected during the acute infection in either animal. Both animals became persistently infected. In conclusion, we generated fully functional infectious cDNA clones of HCV genotypes 3a and 4a. Proof of functionality of all genes might further the development of recombinant cell culture systems for these important genotypes.

Challenge pools of hepatitis C virus genotypes 1-6 prototype strains: replication fitness and pathogenicity in chimpanzees and human liver-chimeric mouse models.

Chimpanzees represent the only animal model for studies of the natural history of hepatitis C virus (HCV). To generate virus stocks of important HCV variants, we infected chimpanzees with HCV strains of genotypes 1-6 and determined the infectivity titer of acute-phase plasma pools in additional animals. The courses of first- and second-passage infections were similar, with early appearance of viremia, HCV RNA titers of >10(4.7) IU/mL, and development of acute hepatitis; the chronicity rate was 56%. The challenge pools had titers of 10(3)-10(5) chimpanzee infectious doses/mL. Human liver-chimeric mice developed high-titer infections after inoculation with the challenge viruses of genotypes 1-6. Inoculation studies with different doses of the genotype 1b pool suggested that a relatively high virus dose is required to consistently infect chimeric mice. The challenge pools represent a unique resource for studies of HCV molecular virology and for studies of pathogenesis, protective immunity, and vaccine efficacy in vivo.

Previously infected chimpanzees are not consistently protected against reinfection or persistent infection after reexposure to the identical hepatitis C virus strain.

Protective immunity after resolved hepatitis C virus (HCV) infection has been reported. However, the breadth of this immunity has remained controversial, and the role of neutralizing antibodies has not been well-defined. In the present study, two chimpanzees (CH96A008 and CH1494) with resolved monoclonal H77C (genotype 1a) infection were rechallenged with low-dose homologous H77C virus about 12 months after viral clearance; CH96A008 became persistently infected, and CH1494 had transient viremia lasting 2 weeks. CH1494 was subsequently either partially or completely protected following five homologous rechallenges with monoclonal H77C or polyclonal H77 and after six heterologous rechallenges with HC-J4 (genotype 1b) or HC-J6 (genotype 2a) viruses. Subsequently, a final challenge with H77C resulted in persistent HCV infection. In both chimpanzees, serum neutralizing antibodies against retroviral pseudoparticles bearing the H77C envelope proteins were not detected during the initial infection or during rechallenge. However, anamnestic cellular immune responses developed during the initial homologous rechallenge, in particular in CH96A008, which developed a persistent infection. Polyprotein sequences of viruses recovered from CH1494 after the two homologous rechallenges that resulted in transient viremia were identical with the H77C virus. In contrast, the polyprotein sequences of viruses recovered from both chimpanzees after homologous rechallenge resulting in persistent infection had numerous changes. These findings have important implications for our understanding of immunity against HCV; even in the best-case scenario with autologous rechallenge, low-level viral persistence was seen in the presence of primed T-cell responses.

Sleep disturbance during military deployment.

This preliminary investigation evaluated symptoms of sleep disturbance and insomnia in a group of 156 deployed military personnel. A 21-item Military Deployment Survey of Sleep was administered to provide self-reported estimates of a variety of sleep parameters. The results indicated that 74% of participants rated their quality of sleep as significantly worse in the deployed environment, 40% had a sleep efficiency of < 85%, and 42% had a sleep onset latency of > 30 minutes. Night-shift workers had significantly worse sleep efficiency and more problems getting to sleep and staying asleep as compared to day-shift workers. The results of the study indicate the need for programs to help deployed military members get more and better sleep.

Sheep stromal-epithelial cell interactions and ovarian tumor progression.

Previous studies suggest that underlying ovarian stromal cues may regulate the ovarian surface epithelium. However, little is known about the interaction between ovarian stromal cells (OSC) and ovarian surface epithelial cells (OSE) under normal physiologic and pathologic conditions, largely because of the lack of a suitable model. In the current study, the OSC obtained from a sheep were immortalized with SV-40 T/t antigen (designated IOSC) and telomerase reverse transcriptase (designated IOSCH), followed by transfection with the oncogenic allele of the human H-Ras oncogene (designated IOSChR). IOSC cells transfected with H-Ras before immortalization with telomerase were designated IOSCRH. These sheep OSCs were used in both in vitro and in vivo model systems to evaluate mechanisms by which OSCs influence ovarian tumor progression. Normal sheep OSCs were found to inhibit the growth of SKOV3 and OVCAR3 human ovarian cancer cells, but not normal sheep OSE and human OSE cells (hOSE137 cells). In contrast, IOSChR and IOSCRH cells stimulated the growth of normal sheep and human OSE cells, as well as cancer cells. These findings were confirmed by in vivo studies. Our data provide compelling support for the importance of stromal-epithelial cell interactions during tumor progression, and show for the first time that immortalized and transformed OSCs promote growth of ovarian epithelial tumors.

Combination of 4-HPR and oral contraceptive in monkey model of chemoprevention of ovarian cancer.

4-(N-hydroxyphenyl) retinamide (4-HPR) and the oral contraceptives (OCP) are currently being used alone, and in combination, for the prevention of ovarian cancer. However, the mechanism of their effects has not been studied. Non-human primate models are ideal for studying the role of these and other drugs for cancer chemoprevention because of the genetic similarity between primates and humans in respect to hormone regulation and menstrual cycle. 4-HPR and OCP were administered to sixteen female adult Macacca mulatta (Rhesus macaques) for three months alone and in combination. Laparotomy was performed before and after treatment, and ovarian biopsies were obtained to evaluate the expression of retinoid and hormone receptors, and apoptosis. ER alpha was undetectable, but ER beta, PR, RXR alpha, and RXR gamma were constitutively expressed in the ovaries. 4-HPR induced RXR alpha and RXR gamma expression at a low level and, OCP induced expression of ER beta. However, the combination of 4-HPR with OCP had a larger effect on expression of retinoid receptors. Apoptosis was detected in the 4-HPR group (equivalent dose: 200 mg/day).

Assessment of cyclic changes of microvessels in ovine ovaries using Sonovue contrast-enhanced ultrasound.

Our objective was to detect variations of ovarian vascularization that occur in the estrus cycle of the ewe using an IV contrast agent. Five ewes were investigated using contrast-enhanced power Doppler after Sonovue injection at day 0, 3, 5, 10 and 13 of the cycle in two successive estrus cycles. Transvaginal ultrasound monitoring of each ewe was performed after a dose of Sonovue. Parameters derived from the time-intensity curves were compared. Quantification of the enhancement and wash-in period parameters changed significantly between ovaries and between the follicular and luteal periods of the cycle. Uptake time, wash-out time, total time of enhancement and area under the curve were the parameters with the smallest variation between ovaries. Wash-out time and area under the curve are two contrast parameters that did not change with cyclic changes. Thus, using these parameters in premenopausal women will allow more accurate detection of vascular modifications that may be associated with ovarian cancer.

Ovine model for engineering bone segments.

We propose a large animal model for bone tissue engineering that yields quantitative data and simulates clinical methods and tissue needs. Skeletally mature domestic sheep (n = 20) were each implanted with three rectangular (1 x 1 x 4 cm), hollow tissue-molding chambers that were empty (control) or filled with equal weights (6.71-6.78 g) of particulate autologous bone graft (MBG) or bone graft that was autoclaved to denature stored growth factors (DeMBG). MBG provided scaffold and bioactive factors, and DeMBG provided only scaffold. The chambers were enclosed on five sides and securely implanted so that the open face was apposed to the osteogenic (i.e., cambium) layer of the rib periosteum for 3, 6, 9, 12, or 24 weeks, after which the chambers were harvested and the contents analyzed. Each chamber contained osseous and fibrovascular tissue. MBG-containing chambers had the best maintenance of tissue volume compared with DeMBG-containing or empty chambers, but it still decreased steadily over time. Despite this, the MBG-containing chambers showed continuous active bone formation. There was increasing calcified tissue with penetration of osteogenesis up to a mean of 0.75 +/- 0.15 cm from the periosteum by 9 weeks, and the osteogenic area peaked at 0.59 +/- 0.13 cm2 by 12 weeks. Using quantitative measures that reflect clinical needs (i.e., tissue volume, shape, and quality), it was possible to distinguish differences in performance associated with manipulation of implanted scaffold and bioactive factors. This ovine model may serve as a useful tool to develop clinical osseous tissue-engineering strategies.

Ovine model to evaluate ovarian vascularization by using contrast-enhanced sonography.

This project was designed to evaluate an ovine model for the use of contrast agent to visualize the microcirculation of normal ovaries. Intraovarian vascularization was investigated in eight ewes by using contrast-enhanced power Doppler after intravenous injection of Sonovue at five different times during each of ten normal estrous cycles. Sheep under general anesthesia underwent transvaginal B mode and power Doppler ultrasound examination of both ovaries, then received two successive doses of 5 ml of Sonovue, one dose for each ovary. Each ovary was monitored after the contrast injection, and a 3-min video clip was stored for each side. The video clip was used to derive time-intensity curves, which were then used to derive the contrast parameters. A total of 108 doses of contrast agent were used; 93% of the injections were available for contrast enhancement analysis. The optimal dose was determined on the first two sheep. Enhancement was strongest and longest with the 5-ml dose. In one sheep, enhancement of both ovaries remained weak irrespective of the period of the cycle. No adverse side effects of Sonovue were seen in the sheep. Contrast injection improved visualization of ovarian microcirculation by 248% (95% confidence interval [CI], 210 to 285%) for 74.2 sec (95% CI, 68.2 to 80.2 sec). Ovaries on the side of ovulation had a stronger enhancement compared with the ovary with no ovulation (368% versus 175%, P < 0.001), but the enhancement time was the same. We concluded that the sheep is an excellent animal model to illustrate microcirculation enhancement by using Sonovue to demonstrate ovarian vascular changes.

Arthroscopic patellar "bankart" repair after acute dislocation.

We present a case of acute patellar dislocation in a skeletally immature patient treated with arthroscopic medial patellofemoral ligamentous complex repair using suture anchors with a horizontal mattress suture technique. Patellar dislocation is a common problem in the skeletally immature. Treatment is controversial for first-time dislocators because of the high rate of recurrent instability and functional disability in these patients. Surgical repair of the medial restraints may decrease recurrent instability, although anterior knee pain and crepitus is a common finding after open surgical techniques. Arthroscopic medial retinacular imbrication has been described for patients with persistent instability with good short-term results. Acute dislocations may cause avulsion of the medial patellofemoral ligamentous complex from the patella; this is amenable to direct primary repair to prevent recurrent instability and avoid the morbidity of open surgery. This technique recreates normal anatomy and function with minimally invasive surgery.

Fluorescence spectroscopy as a biomarker in a cell culture and in a nonhuman primate model for ovarian cancer chemopreventive agents.

OBJECTIVE: The objective of this study was to compare the effects of chemopreventive agents on natural fluorescence emission of ovarian cells in a cell culture and in a primate model as a feasibility trial to monitor drug activity. METHODS: Fluorescence emission spectra were collected from normal (NOE) and immortalized ovarian surface epithelial cells at 290, 360, and 450 nm excitation. Redox potentials were calculated and compared to % apoptosis and cell survival. Fluorescence emission spectra were collected from 18 female rhesus macaques receiving fenretinide [N-(-hydroxyphenyl)retinamide (4-HPR)] orally and/or oral contraceptive pills (OCP) or no medication. Fluorescence intensities and redox ratios were compared using a two-tailed Student's t test. RESULTS: Apoptosis and cell survival correlated with fluorescence emission consistent with metabolically active proteins [flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD(P)H)] and the resulting redox ratio in cells grown with 4-HPR. The 4-HPR consistently inhibited cell survival in a dose dependent manner. Degree of correlation varied between different cell lines. In primates receiving 4-HPR, fluorescence emission was increased at 450 nm excitation, 550 nm emission consistent with FAD presence, whereas those receiving OCP showed decreased emission at 350 nm excitation, 450 nm emission consistent with decreased NAD(P)H presence. Redox ratios were increased by both drugs. CONCLUSIONS: Fluorescence intensity and redox ratio appear to be altered by 4-HPR treatment in vivo and in cell culture and by OCP in vivo. Fluorescence intensity may be useful to monitor chemopreventive agents in clinical trials.

Obstructive uropathy secondary to uterine leiomyoma in a chimpanzee (Pan troglodytes).

Complications due to uterine leiomyomata in chimpanzees have rarely been documented. Here we describe a female chimpanzee that developed severe hydronephrosis in the right kidney due to leiomyoma. Because hysterectomy did not alleviate the hydronephrosis, nephrectomy was elected. After these procedures, the chimpanzee is doing well. Leiomyomata screening programs with treatment algorithms are a useful component of a comprehensive chimpanzee program.

In vivo bone formation in silk fibroin and chitosan blend scaffolds via ectopically grafted periosteum as a cell source: a pilot study.

Reconstruction of a critical size bone defect in the head and neck after trauma or tumor resection remains challenging. While certain defects, such as isolated orbital floor fractures, may be reconstructed with alloplastic biomaterials, larger defects or those involving load bearing bones usually require autologous tissue reconstruction. Vascularized bone free flaps remain the gold standard for large bone defects of the head and neck. These are generally lengthy, complicated, multi-step procedures that require subspecialty expertise to assure optimal outcomes.1 Invariably any procedure where autologous bone is harvested carries with it donor site morbidity.2 To spare the patient this additional morbidity and avoid potential complications associated with the harvest of this tissue, an alternative source for bone that would be sufficient to fill a critical-sized defect is needed.

Use of vascularized periosteum or bone to improve healing of segmental allografts after tumor resection: an ovine rib model.

BACKGROUND: Despite technical advances, nonunion or delayed union remains an important clinical problem when segmental allografts are used to repair diaphyseal defects after bone tumor resection. Using an ovine rib model, the authors studied whether the addition of a vascularized periosteum or bone flap improved healing compared with a segmental allograft alone. METHODS: A 4-cm segment of rib was resected from four consecutive ribs of 15 sheep. Three different reconstructions were compared within the same sheep: allograft alone, allograft and vascularized periosteum, and allograft and vascularized bone. One defect was not reconstructed and served as a control. Five sheep were humanely killed at each of the following time points: 9, 12, and 15 weeks. The host-allograft junctions were analyzed using plain radiographs, micro-computed tomography, and histologic examination. RESULTS: Micro-computed tomographic analysis showed significant improvement with each reconstruction technique over time. Plain radiographs and histologic analyses demonstrated earlier bridging of the host-allograft junction when either vascularized periosteum or vascularized bone was used compared with allograft alone. CONCLUSION: Use of vascularized periosteum or bone may facilitate healing of the host-allograft junction after intercalary allograft reconstruction.

Comparison of guided bone formation from periosteum and muscle fascia.

BACKGROUND: Muscle fascia and periosteum have been used clinically to guide prefabrication of vascularized bone flaps for reconstruction of complex three-dimensional tissues. Although it seems that both locations have the capacity to generate vascularized bone, there have been no studies that directly compare different implantation sites. The authors performed a rigorous, quantitative, histomorphometric comparison of bone prefabrication in a large-animal model comparing graft implanted against muscle fascia and periosteum. METHODS: Twenty skeletally mature domestic sheep were implanted with rectangular chambers containing equal weights of morcellized bone graft. Two chambers were implanted into each sheep, one with the open face apposed to the cambium layer of the rib periosteum and the other with the open face apposed to the fascia of the latissimus dorsi muscle. Animals were euthanized at 3, 6, 9, 12, and 24 weeks and the chambers were harvested. Tissue inside the chambers was analyzed for shape conformation to chamber geometry, gross tissue volume, and bone histomorphology. RESULTS: There were no differences in volume or shape of tissue formed in the chambers. However, chambers in contact with fascia consisted almost entirely of fibrovascular tissue, with progressive resorption of the morcellized bone graft and little evidence of new bone. Chambers in contact with periosteum showed active endochondral, direct, and appositional bone formation over time, with increasing calcified tissue area and new bone formation. CONCLUSIONS: Both periosteum and muscle fascia were able to vascularize bone grafts, but bone formation was higher in the periosteum. The periosteum appears to be a more suitable foundation from which to promote flap prefabrication.