Implementation of a pharmacist and student pharmacist-led primary care service to identify and treat rural veterans at risk for osteoporotic fracture.

OBJECTIVE: To develop and implement a pharmacist and student pharmacist-led osteoporosis service to increase dual-energy x-ray absorptiometry (DXA) screening rates among rural veterans and treat those at high risk of osteoporotic fractures. PRACTICE DESCRIPTION: Pharmacists are uniquely positioned to provide direct patient care in the Department of Veterans Affairs ambulatory care setting owing to their broad scope of practice. Clinical Pharmacy Specialists (CPSs) have the authority to order laboratory tests and imaging, prescribe medications, refer patients to specialty services, and monitor patients along with the primary care team. PRACTICE INNOVATION: The implementation of a pharmacist-led osteoporosis primary prevention service using student pharmacists to identify and treat patients has not been previously described in the literature to the authors' knowledge. EVALUATION METHODS: Student pharmacists in their third year contacted veterans who met the inclusion criteria for osteoporosis screening. The veterans were offered DXA scans and provided education on the risk factors for osteoporosis. After the DXA scans were completed, the students and the CPS reviewed the results to determine treatment strategies. The primary objective was evaluated by comparing the pre- and post-implementation rates of DXA screening. The other process markers that were evaluated included (1) completed DXA scans, (2) new diagnoses of osteoporosis or osteopenia, (3) patients eligible for treatment on the basis of the DXA screening results, and (4) patients who started oral bisphosphonate therapy. RESULTS: Of the 232 rural veterans evaluated, 36 had completed DXA scans before this service was implemented. After the service was implemented, 115 veterans completed DXA scans. A total of 57 patients received a new diagnosis, 33 were eligible for therapy, and 12 started oral bisphosphonate therapy after intervention by the CPS. CONCLUSION: The implementation of a pharmacist-driven osteoporosis screening and treatment service demonstrated an increase in the rate of DXA screening among rural veterans.

Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts.

During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. SB431542, an inhibitor of transforming growth factor-ß1 (TGF-ß1)-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-ß1 at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-ß1 is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia.

Encapsulated mesenchymal stromal cells for in vivo transplantation.

Immunomodulatory human mesenchymal stromal cells (hMSC) have been incorporated into therapeutic protocols to treat secondary inflammatory responses post-spinal cord injury (SCI) in animal models. However, limitations with direct hMSC implantation approaches may prevent effective translation for therapeutic development of hMSC infusion into post-SCI treatment protocols. To circumvent these limitations, we investigated the efficacy of alginate microencapsulation in developing an implantable vehicle for hMSC delivery. Viability and secretory function were maintained within the encapsulated hMSC population, and hMSC secreted anti-inflammatory cytokines upon induction with the pro-inflammatory factors, TNF-α and IFN-γ. Furthermore, encapsulated hMSC modulated inflammatory macrophage function both in vitro and in vivo, even in the absence of direct hMSC-macrophage cell contact and promoted the alternative M2 macrophage phenotype. In vitro, this was evident by a reduction in macrophage iNOS expression with a concomitant increase in CD206, a marker for M2 macrophages. Finally, Sprague-Dawley rat spinal cords were injured at vertebra T10 via a weight drop model (NYU model) and encapsulated hMSC were administered via lumbar puncture 24 h post-injury. Encapsulated hMSC localized primarily in the cauda equina of the spinal cord. Histological assessment of spinal cord tissue 7 days post-SCI indicated that as few as 5 × 10(4) encapsulated hMSC yielded increased numbers of CD206-expressing macrophages, consistent with our in vitro studies. The combined findings support the inclusion of immobilized hMSC in post-CNS trauma tissue protective therapy, and suggest that conversion of macrophages to the M2 subset is responsible, at least in part, for tissue protection.

Liaison psychiatry services in Wales.

Aims and methodRecent funding from Welsh Government for mental health has helped to develop liaison psychiatry services in Wales. Systematic data collection was undertaken to map the liaison psychiatry services in Wales in collaboration with the Royal College of Psychiatrists in Wales and Public Health Wales 1000 Lives Improvement. A questionnaire was designed and circulated to all the health boards in Wales to gather information to map liaison psychiatry services in Wales. Up-to-date information was confirmed in January 2018, via email. RESULTS: Over the past 2 years, liaison psychiatry services have been set up in six out of seven health boards in Wales. Staffing levels have increased and the remit of services has broadened.Clinical implicationsMapping has highlighted that liaison psychiatry services in Wales continue to evolve. It will be important to continue to monitor these developments and their effects. Comparison with services in England will provide a useful comparison of service provision. A particular challenge will be to establish and monitor liaison psychiatry standards in Wales.Declaration of interestNone.

Donor variability among anti-inflammatory pre-activated mesenchymal stromal cells.

Therapeutic mesenchymal stromal cells (MSCs) are attractive in part due to their immunomodulatory properties, achieved by their paracrine secretion of factors including prostaglandin E2 (PGE2). Despite promising pre-clinical data, demonstrating clinical efficacy has proven difficult. The current studies were designed to develop approaches to pre-induce desired functions from naïve MSCs and examine MSC donor variability, two factors contributing to this disconnect. MSCs from six human donors were pre-activated with interleukin 1 beta (IL-1ß) at a concentration and duration identified as optimal or interferon gamma (IFN-γ) as a comparator. Their secretion of PGE2 after pre-activation and secondary exposure to pro-inflammatory molecules was measured. Modulation of tumor necrosis factor alpha (TNF-α) secretion from M1 pro-inflammatory macrophages by co-cultured pre-activated MSCs was also measured. Our results indicated that pre-activation of MSCs with IL-1ß resulted in upregulated PGE2 secretion post exposure. Pre-activation with IL-1ß or IFN-γ resulted in higher sensitivity to induction by secondary stimuli compared to no pre-activation. While IL-1ß pre-activation led to enhanced MSC-mediated attenuation of macrophage TNF-α secretion, IFN-γ pre-activation resulted in enhanced TNF-α secretion. Donor variability was noted in PGE2 secretion and upregulation and the level of improved or impaired macrophage modulation.

The effect of local anesthetic on pro-inflammatory macrophage modulation by mesenchymal stromal cells.

Administering local anesthetics (LAs) peri- and post-operatively aims to prevent or mitigate pain in surgical procedures and after tissue injury in cases of osteoarthritis (OA) and other degenerative diseases. Innovative tissue protective and reparative therapeutic interventions such as mesenchymal stromal cells (MSCs) are likely to be exposed to co-administered drugs such as LAs. Therefore, it is important to determine how this exposure affects the therapeutic functions of MSCs and other cells in their target microenvironment. In these studies, we measured the effect of LAs, lidocaine and bupivacaine, on macrophage viability and pro-inflammatory secretion. We also examined their effect on modulation of the macrophage pro-inflammatory phenotype in an in vitro co-culture system with MSCs, by quantifying macrophage tumor necrosis factor (TNF)-α secretion and MSC prostaglandin E2 (PGE2) production. Our studies indicate that both LAs directly attenuated macrophage TNF-α secretion, without significantly affecting viability, in a concentration- and potency-dependent manner. LA-mediated attenuation of macrophage TNF-α was sustained in co-culture with MSCs, but MSCs did not further enhance this anti-inflammatory effect. Concentration- and potency-dependent reductions in macrophage TNF-α were concurrent with decreased PGE2 levels in the co-cultures further indicating MSC-independent macrophage attenuation. MSC functional recovery from LA exposure was assessed by pre-treating MSCs with LAs prior to co-culture with macrophages. Both MSC attenuation of TNF-α and PGE2 secretion were impaired by pre-exposure to the more potent bupivacaine and high dose of lidocaine in a concentration-dependent manner. Therefore, LAs can affect anti-inflammatory function by both directly attenuating macrophage inflammation and MSC secretion and possibly by altering the local microenvironment which can secondarily reduce MSC function. Furthermore, the LA effect on MSC function may persist even after LA removal.

Lipid composition influences the shape of human low density lipoprotein in vitreous ice.

Earlier cryo-electron microscopic studies have indicated that the normal low density lipoprotein (N-LDL) has a discoid shape when its core is in the liquid-crystalline state. In the present study, we investigated whether the shape of LDL depends on the physical state and/or the lipid composition of the lipoprotein core. Using a custom-built freezing device, we vitrified NLDL samples from either above or below the phase-transition temperature of the core (42 and 24 degrees C, respectively). Cryo-electron microscopy revealed no differences between these samples and indicated a discoid shape of the N-LDL particle. In contrast, TG-enriched LDL (T-LDL) did not have discoid features and appeared to be quasi-spherical in preparations that were vitrified from either 42 or 24 degrees C. These results suggest that the shape of NLDL is discoid, regardless of the physical state of its core, whereas T-LDL is more spherical. Aspects that may influence the shape of LDL are discussed.

Identification of IL-1ß and LPS as optimal activators of monolayer and alginate-encapsulated mesenchymal stromal cell immunomodulation using design of experiments and statistical methods.

Induction of therapeutic mesenchymal stromal cell (MSC) function is dependent upon activating factors present in diseased or injured tissue microenvironments. These functions include modulation of macrophage phenotype via secreted molecules including prostaglandin E2 (PGE2). Many approaches aim to optimize MSC-based therapies, including preconditioning using soluble factors and cell immobilization in biomaterials. However, optimization of MSC function is usually inefficient as only a few factors are manipulated in parallel. We utilized fractional factorial design of experiments to screen a panel of 6 molecules (lipopolysaccharide [LPS], polyinosinic-polycytidylic acid [poly(I:C)], interleukin [IL]-6, IL-1ß, interferon [IFN]-ß, and IFN-γ), individually and in combinations, for the upregulation of MSC PGE2 secretion and attenuation of macrophage secretion of tumor necrosis factor (TNF)-α, a pro-inflammatory molecule, by activated-MSC conditioned medium (CM). We used multivariable linear regression (MLR) and analysis of covariance to determine differences in functions of optimal factors on monolayer MSCs and alginate-encapsulated MSCs (eMSCs). The screen revealed that LPS and IL-1ß potently activated monolayer MSCs to enhance PGE2 production and attenuate macrophage TNF-α. Activation by LPS and IL-1ß together synergistically increased MSC PGE2, but did not synergistically reduce macrophage TNF-α. MLR and covariate analysis revealed that macrophage TNF-α was strongly dependent on the MSC activation factor, PGE2 level, and macrophage donor but not MSC culture format (monolayer versus encapsulated). The results demonstrate the feasibility and utility of using statistical approaches for higher throughput cell analysis. This approach can be extended to develop activation schemes to maximize MSC and MSC-biomaterial functions prior to transplantation to improve MSC therapies.

Effect of Local Anesthetics on Human Mesenchymal Stromal Cell Secretion.

Anti-fibrotic and tissue regenerative mesenchymal stromal cell (MSC) properties are largely mediated by secreted cytokines and growth factors. MSCs are implanted to augment joint cartilage replacement and to treat diabetic ulcers and burn injuries simultaneously with local anesthetics, which reduce pain. However, the effect of anesthetics on therapeutic human MSC secretory function has not been evaluated. In order to assess the effect of local anesthetics on the MSC secretome, a panel of four anesthetics with different potencies - lidocaine, procaine, ropivacaine and bupivacaine - was evaluated. Since injured tissues secrete inflammatory cytokines, the effects of anesthetics on MSCs stimulated with tumor necrosis factor (TNF)-α and interferon (IFN)-γ were also measured. Dose dependent and anesthesia specific effects on cell viability, post exposure proliferation and secretory function were quantified using alamar blue reduction and immunoassays, respectively. Computational pathway analysis was performed to identify upstream regulators and molecular pathways likely associated with the effects of these chemicals on the MSC secretome. Our results indicated while neither lidocaine nor procaine greatly reduced unstimulated cell viability, ropivacaine and bupivacaine induced dose dependent viability decreases. This pattern was exaggerated in the simulated inflammatory environment. The reversibility of these effects after withdrawal of the anesthetics was attenuated for TNF-α/IFN-γ-stimulated MSCs exposed to ropivacaine and bupivacaine. In addition, secretome analysis indicated that constitutive secretion changes were clearly affected by both anesthetic alone and anesthetic plus TNFα/IFNγ cell stimulation, but the secretory pattern was drug specific and did not necessarily coincide with viability changes. Pathway analysis identified different intracellular regulators for stimulated and unstimulated MSCs. Within these groups, ropivacaine and bupivacaine appeared to act on MSCs similarly via the same regulatory mechanisms. Given the variable effect of local anesthetics on MSC viability and function, these studies underscore the need to evaluate MSC in the presence of medications, such as anesthetics, that are likely to accompany cell implantation.

Sizes and Sufficient Quantities of MSC Microspheres for Intrathecal Injection to Modulate Inflammation in Spinal Cord Injury.

Microencapsulation of mesenchymal stem cells (MSC) in alginate facilitates cell delivery, localization and survival, and modulates inflammation in vivo. However, we found that delivery of the widely used ~0.5 mm diameter encapsulated MSC (eMSC) by intrathecal injection into spinal cord injury (SCI) rats was highly variable. Injections of smaller (~0.2 mm) diameter eMSC into the lumbar spine were much more reproducible and they increased the anti-inflammatory macrophage response around the SCI site. We now report that injection of small eMSC >2 cm caudal from the rat SCI improved locomotion and myelin preservation 8 weeks after rat SCI versus control injections. Because preparation of sufficient quantities of small eMSC for larger studies was not feasible and injection of the large eMSC is problematic, we have developed a procedure to prepare medium-sized eMSC (~0.35 mm diameter) that can be delivered more reproducibly into the lumbar rat spine. The number of MSC incorporated/capsule in the medium sized capsules was ~5-fold greater than that in small capsules and the total yield of eMSC was ~20-fold higher than that for the small capsules. Assays with all three sizes of eMSC capsules showed that they inhibited TNF-α secretion from activated macrophages in co-cultures, suggesting no major difference in their anti-inflammatory activity in vitro. The in vivo activity of the medium-sized eMSC was tested after injecting them into the lumbar spine 1 day after SCI. Histological analyses 1 week later showed that eMSC reduced levels of activated macrophages measured by IB4 staining and increased white matter sparing in similar regions adjacent to the SCI site. The combined results indicate that ~0.35 mm diameter eMSC reduced macrophage inflammation in regions where white matter was preserved during critical early phases after SCI. These techniques enable preparation of eMSC in sufficient quantities to perform pre-clinical SCI studies with much larger numbers of subjects that will provide functional analyses of several critical parameters in rodent models for CNS inflammatory injury.

The physical state of the LDL core influences the conformation of apolipoprotein B-100 on the lipoprotein surface.

We assessed the influence of temperature on the secondary structure of apolipoprotein B-100 (apoB) in normal low-density lipoprotein (N-LDL) and triglyceride-rich LDL (T-LDL). Gradual heating from 7 degrees C to the phase-transition temperature of the lipoprotein core ( approximately 28 degrees C and approximately 15 degrees C for N-LDL and T-LDL, respectively) gradually altered the secondary structure of apoB, while further heating from the phase-transition temperature to 45 degrees C had no additional effect. Above the phase-transition temperature of the core, the apoBs of N-LDL and T-LDL had a similar secondary structure. These results indicate that the conformation of apoB on the LDL surface depends strongly on the physical state of the lipoprotein core, and less on the lipid composition of the core per se.

Bartonella henselae infection associated with neuroretinitis, central retinal artery and vein occlusion, neovascular glaucoma, and severe vision loss.

PURPOSE: To report a case of Bartonella henselae infection. DESIGN: Observational case report. METHODS: Review of the clinical, laboratory, photographic, and angiographic records of a patient with cat scratch disease associated with central retinal artery and vein occlusion, neovascular glaucoma, and severe vision loss. RESULTS: A 21-year-old man had no light perception in the left eye secondary to concurrent central retinal artery and vein occlusion believed to have resulted from infection with Bartonella henselae. Forty days later, he developed neovascular glaucoma in the left eye. CONCLUSION: Ocular complications associated with Bartonella henselae infection may include central retinal artery and vein occlusion, neovascular glaucoma, and severe vision loss.

Neuroretinitis associated with serologies positive for bartonella henselae and borrelia burgdorferi.

PURPOSE: To describe a patient with neuroretinitis with features of both cat-scratch disease and Lyme disease who had serologies positive for both Bartonella henselae and Borrelia burgdorferi. METHODS: Case report of a single individual undergoing diagnostic testing and treatment for neuroretinitis. RESULTS: A 47-year-old woman developed acute painless loss of vision and was found to have neuroretinitis. Diagnostic workup yielded serologies positive for both B. henselae and B. burgdorferi. The patient was treated with oral antibiotics for coverage of both etiologies, and her condition improved. CONCLUSION: Serologies positive for both B. henselae and B. burgdorferi can be obtained in the workup of neuroretinitis. Clinicians should be aware and use clinical judgment in guiding their diagnosis and treatment of neuroretinitis.