TALEN-mediated intron editing of HSPCs enables transgene expression restricted to the myeloid lineage.

Gene therapy in hematopoietic stem and progenitor cells (HSPCs) shows great potential for the treatment of inborn metabolic diseases. Typical HSPC gene therapy approaches rely on constitutive promoters to express a therapeutic transgene, which is associated with multiple disadvantages. Here, we propose a novel promoterless intronic gene editing approach that triggers transgene expression only after cellular differentiation into the myeloid lineage. We integrated a splicing-competent eGFP cassette into the first intron of CD11b and observed expression of eGFP in the myeloid lineage but minimal to no expression in HSPCs or differentiated non-myeloid lineages. In vivo, edited HSPCs successfully engrafted in immunodeficient mice and displayed transgene expression in the myeloid compartment of multiple tissues. Using the same approach, we expressed alpha-L-iduronidase (IDUA), the defective enzyme in Mucopolysaccharidosis type I, and observed a 10-fold supraendogenous IDUA expression exclusively after myeloid differentiation. Edited cells efficiently populated bone marrow, blood, and spleen of immunodeficient mice, and retained the capacity to secrete IDUA ex vivo. Importantly, cells edited with the eGFP and IDUA transgenes were also found in the brain. This approach may unlock new therapeutic strategies for inborn metabolic and neurological diseases that require the delivery of therapeutics in brain.

Mysterious Arm Swelling in a Patient With Non-Hodgkin Lymphoma Complicated by Superior Vena Cava Syndrome.

Hematologic malignancies often create difficult venous complications. Specifically, with lymphoma, the risk of venous thromboembolism (VTE) is high, often requiring highly specialized accuracy in balancing this coagulopathy. This case study demonstrates a situation where the advanced practitioner participated in the differential diagnosis of VTEs, management, and workup of subsequent central venous complications in a patient with non-Hodgkin lymphoma and superior vena cava syndrome.

Nonoperative Treatment of Periprosthetic Humeral Shaft Fractures After Reverse Total Shoulder Arthroplasty.

Few studies report on periprosthetic humeral shaft fractures after reverse total shoulder arthroplasty (RTSA). The authors evaluated outcomes of 5 patients with this complication who were initially treated nonoperatively. Of 152 patients who underwent RTSA at the authors' institution from 2012 to 2017, 4 experienced periprosthetic humeral shaft fractures. One patient was referred to the authors for fracture treatment. All 5 patients were initially treated nonoperatively. The mean duration of follow-up was 11.5 months (range, 1.5-26 months). The authors analyzed time to fracture union, Single Assessment Numeric Evaluation (SANE) score, visual analog scale (VAS) score for pain, and active shoulder range of motion. Fracture union occurred in 4 patients treated nonoperatively at a mean of 4.4 months. Mean SANE score was 55 of 100 (range, 20-85). Mean VAS score was 3.4 of 10 (range, 0-8). Mean forward elevation was 83° (range, 45°-110°); mean abduction was 65° (range, 45°-80°); and mean external rotation with the arm at the side was 15° (range, 0°-30°). Many factors must be considered when customizing treatment for patients with periprosthetic fracture after RTSA. This case series indicates that nonoperative treatment of postoperative periprosthetic humeral shaft fractures can be successful. [Orthopedics. 2020;43(6):e553-e560.].

Association Between Delays in Radiography and Surgery With Hip Fracture Outcomes in Elderly Patients.

Appropriate waiting time for hip fracture surgery is disputed. The American Academy of Orthopaedic Surgeons recommends surgery within 48 hours of presentation, although evidence suggests that earlier surgery (within 24 hours) reduces the risks of complications and death. The authors asked: (1) Do patients who receive earlier radiographic evaluation of hip fracture undergo surgery earlier? and (2) Is "surgery delay" (time from presentation to surgery) associated with postoperative opioid use, duration of hospital stay, and 30-day and 1-year mortality rates? The authors identified 511 adults 60 years or older who were admitted to their emergency department with hip fractures from 2015 through 2017. Patients were divided into 6 cohorts according to length of surgery delay and 3 cohorts according to length of radiography delay (time from presentation to first hip radiograph). The authors found that medium radiography delay (>2 to 4 hours) was associated with an additional 11 hours of surgery delay compared with short radiography delay (≤2 hours; P=.026). Longer surgery delay (>12 hours) was associated with use of 9.6 more morphine equivalents (95% confidence interval, 0.7 to 8.6) during the first 24 hours postoperatively compared with shorter surgery delay (≤12 hours). Surgery delay of greater than 36 hours was an independent risk factor for longer hospital stay (odds ratio, 2.8; 95% confidence interval, 1.7 to 4.8). Thirty-day and 1-year mortality rates were significantly higher among patients who experienced a surgery delay of greater than 36 hours compared with those who experienced a surgery delay of 36 hours or less. [Orthopedics. 2020;43(6):e609-e615.].

Racial Disparities are Present in the Timing of Radiographic Assessment and Surgical Treatment of Hip Fractures.

BACKGROUND: Hip fractures are associated with 1-year mortality rates as high as 19% to 33%. Nonwhite patients have higher mortality and lower mobility rates at 6 months postoperatively than white patients. Studies have extensively documented racial disparities in hip fracture outcomes, but few have directly assessed racial disparities in the timing of hip fracture care. QUESTIONS/PURPOSES: Our purpose was to assess racial disparities in the care provided to patients with hip fractures. We asked, (1) do racial disparities exist in radiographic timing, surgical timing, length of hospital stay, and 30-day hospital readmission rates? (2) Does the hospital type modify the association between race and the outcomes of interest? METHODS: We retrospectively reviewed the records of 1535 patients aged 60 years or older who were admitted to the emergency department and treated surgically for a hip fracture at one of five hospitals (three community hospitals and two tertiary hospitals) in our health system from 2015 to 2017. Multivariable generalized linear models were used to assess associations between race and the outcomes of interest. RESULTS: After adjusting for patient characteristics, we found that black patients had a longer mean time to radiographic evaluation (4.2 hours; 95% confidence interval, -0.6 to 9.0 versus 1.2 hours; 95% CI, 0.1-2.3; p = 0.01) and surgical fixation (41 hours; 95% CI, 34-48 versus 34 hours 95% CI, 32-35; p < 0.05) than white patients did. Hospital type only modified the association between race and surgical timing. In community hospitals, black patients experienced a 51% (95% CI, 17%-95%; p < 0.01) longer time to surgery than white patients did; however, there were no differences in surgical timing between black and white patients in tertiary hospitals. No race-based differences were observed in the length of hospital stay and 30-day hospital readmission rates. CONCLUSIONS: After adjusting for patient characteristics, we found that black patients experienced longer wait times to radiographic evaluation and surgical fixation than white patients. Hospitals should consider evaluating racial disparities in the timing of hip fracture care in their health systems. Raising awareness of these disparities and implementing unconscious bias training for healthcare providers may help mitigate these disparities and improve the timing of care for patients who are at a greater risk of delay. LEVEL OF EVIDENCE: Level III, therapeutic study.

Three- and 4-part proximal humeral fracture fixation with an intramedullary cage: 1-year clinical and radiographic outcomes.

BACKGROUND: Reverse total shoulder arthroplasty is a treatment option for 3- and 4-part proximal humeral fractures in elderly patients. However, arthroplasty has drawbacks in younger patients because of their greater activity level and more likely need for revision surgery. In such patients, an intramedullary cage may allow for reconstruction of the proximal humerus. METHODS: We reviewed the outcomes of patients with proximal humeral fractures treated with expandable intramedullary cages from 2016 to 2017. We included patients with closed 3- or 4-part fractures (Neer classification), no osteoarthritis of the glenohumeral joint, and minimum 12-month follow-up. We assessed range of motion, pain, the American Shoulder and Elbow Surgeons score, and the Subjective Shoulder Value. RESULTS: Eleven patients (mean follow-up, 54 weeks [range, 49-61 weeks]) were included. Two patients had 3-part fractures, and nine had 4-part fractures. At final follow-up, the mean visual analog scale score for pain was 1.4 (range, 0-6), the mean Subjective Shoulder Value was 69 (range, 20-90), and the mean American Shoulder and Elbow Surgeons score was 80 (range, 27-98). Mean forward flexion was 123° (range, 45°-160°), mean abduction was 82° (range, 30°-90°), and mean external rotation in 90° of abduction was 71° (range, 30°-90°). At final radiographic evaluation, all fractures were healed. No wound-related or neurologic complications were noted. Avascular necrosis developed in 1 patient; conversion to shoulder arthroplasty was performed. CONCLUSION: Treatment of 3- and 4-part proximal humeral fractures with an expandable intramedullary cage produced good clinical outcomes and a 100% union rate.

Host- and Helminth-Derived Endocannabinoids That Have Effects on Host Immunity Are Generated during Infection.

Helminths have coevolved with their hosts, resulting in the development of specialized host immune mechanisms and parasite-specific regulatory products. Identification of new pathways that regulate helminth infection could provide a better understanding of host-helminth interaction and may identify new therapeutic targets for helminth infection. Here we identify the endocannabinoid system as a new mechanism that influences host immunity to helminths. Endocannabinoids are lipid-derived signaling molecules that control important physiologic processes, such as feeding behavior and metabolism. Following murine infection with Nippostrongylus brasiliensis, an intestinal nematode with a life cycle similar to that of hookworms, we observed increased levels of endocannabinoids (2-arachidonoylglycerol [2-AG] or anandamide [AEA]) and the endocannabinoid-like molecule oleoylethanolamine (OEA) in infected lung and intestine. To investigate endocannabinoid function in helminth infection, we employed pharmacological inhibitors of cannabinoid subtype receptors 1 and 2 (CB1R and CB2R). Compared to findings for vehicle-treated mice, inhibition of CB1R but not CB2R resulted in increased N. brasiliensis worm burden and egg output, associated with significantly decreased expression of the T helper type 2 cytokine interleukin 5 (IL-5) in intestinal tissue and splenocyte cultures. Strikingly, bioinformatic analysis of genomic and transcriptome sequencing (RNA-seq) data sets identified putative genes encoding endocannabinoid biosynthetic and degradative enzymes in many parasitic nematodes. To test the novel hypothesis that helminth parasites produce their own endocannabinoids, we measured endocannabinoid levels in N. brasiliensis by mass spectrometry and quantitative PCR and found that N. brasiliensis parasites produced endocannabinoids, especially at the infectious larval stage. To our knowledge, this is the first report of helminth- and host-derived endocannabinoids that promote host immune responses and reduce parasite burden.

Hematopoietic cell-derived RELMα regulates hookworm immunity through effects on macrophages.

Resistin-like molecule α (RELMα) is a highly secreted protein in type 2 (Th2) cytokine-induced inflammation including helminth infection and allergy. In infection with Nippostrongylus brasiliensis (Nb), RELMα dampens Th2 inflammatory responses. RELMα is expressed by immune cells, and by epithelial cells (EC); however, the functional impact of immune versus EC-derived RELMα is unknown. We generated bone marrow (BM) chimeras that were RELMα deficient (RELMα-/- ) in BM or non BM cells and infected them with Nb. Non BM RELMα-/- chimeras had comparable inflammatory responses and parasite burdens to RELMα+/+ mice. In contrast, both RELMα-/- and BM RELMα-/- mice exhibited increased Nb-induced lung and intestinal inflammation, correlated with elevated Th2 cytokines and Nb killing. CD11c+ lung macrophages were the dominant BM-derived source of RELMα and can mediate Nb killing. Therefore, we employed a macrophage-worm co-culture system to investigate whether RELMα regulates macrophage-mediated Nb killing. Compared to RELMα+/+ macrophages, RELMα-/- macrophages exhibited increased binding to Nb and functionally impaired Nb development. Supplementation with recombinant RELMα partially reversed this phenotype. Gene expression analysis revealed that RELMα decreased cell adhesion and Fc receptor signaling pathways, which are associated with macrophage-mediated helminth killing. Collectively, these studies demonstrate that BM-derived RELMα is necessary and sufficient to dampen Nb immune responses, and identify that one mechanism of action of RELMα is through inhibiting macrophage recruitment and interaction with Nb. Our findings suggest that RELMα acts as an immune brake that provides mutually beneficial effects for the host and parasite by limiting tissue damage and delaying parasite expulsion.

Continuous Inhalation Exposure to Fungal Allergen Particulates Induces Lung Inflammation While Reducing Innate Immune Molecule Expression in the Brainstem.

Continuous exposure to aerosolized fine (particle size ≤2.5 µm) and ultrafine (particle size ≤0.1 µm) particulates can trigger innate inflammatory responses in the lung and brain depending on particle composition. Most studies of manmade toxicants use inhalation exposure routes, whereas most studies of allergens use soluble solutions administered via intranasal or injection routes. Here, we tested whether continuous inhalation exposure to aerosolized Alternaria alternata particulates (a common fungal allergen associated with asthma) would induce innate inflammatory responses in the lung and brain. By designing a new environmental chamber able to control particle size distribution and mass concentration, we continuously exposed adult mice to aerosolized ultrafine Alternaria particulates for 96 hr. Despite induction of innate immune responses in the lung, induction of innate immune responses in whole brain samples was not detected by quantitative polymerase chain reaction or flow cytometry. However, exposure did trigger decreases in Arginase 1, inducible nitric oxide synthase, and tumor necrosis factor alpha mRNA in the brainstem samples containing the central nervous system respiratory circuit (the dorsal respiratory group, ventral respiratory group, and the pre-Bötzinger and Bötzinger complexes). In addition, a significant decrease in the percentage of Toll-like receptor 2-expressing brainstem microglia was detected by flow cytometry. Histologic analysis revealed a significant decrease in Iba1 but not glial fibrillary acidic protein immunoreactivity in both the brainstem and the hippocampus. Together these data indicate that inhalation exposure to a natural fungal allergen under conditions sufficient to induce lung inflammation surprisingly causes reductions in baseline expression of select innate immune molecules (similar to that observed during endotoxin tolerance) in the region of the central nervous system controlling respiration.

Human resistin protects against endotoxic shock by blocking LPS-TLR4 interaction.

Helminths trigger multiple immunomodulatory pathways that can protect from sepsis. Human resistin (hRetn) is an immune cell-derived protein that is highly elevated in helminth infection and sepsis. However, the function of hRetn in sepsis, or whether hRetn influences helminth protection against sepsis, is unknown. Employing hRetn-expressing transgenic mice (hRETNTg+) and recombinant hRetn, we identify a therapeutic function for hRetn in lipopolysaccharide (LPS)-induced septic shock. hRetn promoted helminth-induced immunomodulation, with increased survival of Nippostrongylus brasiliensis (Nb)-infected hRETNTg+ mice after a fatal LPS dose compared with naive mice or Nb-infected hRETNTg- mice. Employing immunoprecipitation assays, hRETNTg+Tlr4-/- mice, and human immune cell culture, we demonstrate that hRetn binds the LPS receptor Toll-like receptor 4 (TLR4) through its N terminal and modulates STAT3 and TBK1 signaling, triggering a switch from proinflammatory to anti-inflammatory responses. Further, we generate hRetn N-terminal peptides that are able to block LPS proinflammatory function. Together, our studies identify a critical role for hRetn in blocking LPS function with important clinical significance in helminth-induced immunomodulation and sepsis.

CD103+ CD8 T Cells in the Toxoplasma-Infected Brain Exhibit a Tissue-Resident Memory Transcriptional Profile.

During chronic infection, memory T cells acquire a unique phenotype and become dependent on different survival signals than those needed for memory T cells generated during an acute infection. The distinction between the role of effector and memory T cells in an environment of persistent antigen remains unclear. Here, in the context of chronic Toxoplasma gondii infection, we demonstrate that a population of CD8 T cells exhibiting a tissue-resident memory (TRM) phenotype accumulates within the brain. We show that this population is distributed throughout the brain in both parenchymal and extraparenchymal spaces. Furthermore, this population is transcriptionally distinct and exhibits a transcriptional signature consistent with the TRM observed in acute viral infections. Finally, we establish that the CD103+ TRM population has an intrinsic capacity to produce both IFN-γ and TNF-α, cytokines critical for parasite control within the central nervous system (CNS). The contribution of this population to pro-inflammatory cytokine production suggests an important role for TRM in protective and ongoing immune responses in the infected CNS. Accession number: GSE95105.

Comparison of RELMα and RELMß Single- and Double-Gene-Deficient Mice Reveals that RELMα Expression Dictates Inflammation and Worm Expulsion in Hookworm Infection.

Resistin-like molecules (RELMs) are highly expressed following helminth infection, where they impact both the host and helminth. While RELMα (Retnla) impairs helminth expulsion by inhibiting protective Th2 immunity, RELMß (Retnlb) can promote its expulsion. We employed Retnla(-/-) and Retnlb(-/-) mice to delineate the function of both proteins following infection with Nippostrongylus brasiliensis, a hookworm that infects the lung and intestine. Whereas wild-type (WT) and Retnlb(-/-)mice exhibited equivalent infection-induced inflammation, Retnla(-/-) mice suffered a heightened inflammatory response, including increased mortality, weight loss, and lung inflammation. In the intestine, Retnla(-/-)mice had low parasite egg burdens compared to those of WT mice, while Retnlb(-/-) mice exhibited high egg burdens, suggesting that RELMα and RELMß have functionally distinct effects on immunity and inflammation to N. brasiliensis To test the importance of both proteins, we generated Retnla(-/-) Retnlb(-/-) mice. Infected Retnla(-/-)Retnlb(-/-) mice exhibited similar responses to Retnla(-/-) mice, including increased mortality and lung inflammation. This inflammatory response in Retnla(-/-) Retnlb(-/-) mice negatively impacted N. brasiliensis fitness, as demonstrated by significantly lower worm ATP levels and decreased intestinal worm burden and fecundity. Lung cytokine analysis revealed that Retnla(-/-) and Retnla(-/-) Retnlb(-/-) mice expressed significantly increased levels of interleukin-4 (IL-4). Finally, we generated Retnla(-/-) mice on the Rag(-/-) background and observed that the effects of RELMα were abrogated in the absence of adaptive immunity. Together, these data demonstrate that RELMα but not RELMß significantly impacts the immune response toN. brasiliensis infection by downregulating the Th2 adaptive immune response in the lung, which protects the host but allows improved parasite fitness.

Macrophage-derived human resistin is induced in multiple helminth infections and promotes inflammatory monocytes and increased parasite burden.

Parasitic helminth infections can be associated with lifelong morbidity such as immune-mediated organ failure. A better understanding of the host immune response to helminths could provide new avenues to promote parasite clearance and/or alleviate infection-associated morbidity. Murine resistin-like molecules (RELM) exhibit pleiotropic functions following helminth infection including modulating the host immune response; however, the relevance of human RELM proteins in helminth infection is unknown. To examine the function of human resistin (hResistin), we utilized transgenic mice expressing the human resistin gene (hRetnTg+). Following infection with the helminth Nippostrongylus brasiliensis (Nb), hResistin expression was significantly upregulated in infected tissue. Compared to control hRetnTg- mice, hRetnTg+ mice suffered from exacerbated Nb-induced inflammation characterized by weight loss and increased infiltration of inflammatory monocytes in the lung, along with elevated Nb egg burdens and delayed parasite expulsion. Genome-wide transcriptional profiling of the infected tissue revealed that hResistin promoted expression of proinflammatory cytokines and genes downstream of toll-like receptor signaling. Moreover, hResistin preferentially bound lung monocytes, and exogenous treatment of mice with recombinant hResistin promoted monocyte recruitment and proinflammatory cytokine expression. In human studies, increased serum resistin was associated with higher parasite load in individuals infected with soil-transmitted helminths or filarial nematode Wuchereria bancrofti, and was positively correlated with proinflammatory cytokines. Together, these studies identify human resistin as a detrimental factor induced by multiple helminth infections, where it promotes proinflammatory cytokines and impedes parasite clearance. Targeting the resistin/proinflammatory cytokine immune axis may provide new diagnostic or treatment strategies for helminth infection and associated immune-mediated pathology.

Polarizing the T helper 17 response in Citrobacter rodentium infection via expression of resistin-like molecule α.

Citrobacter rodentium infection is a murine model of pathogenic Escherichia coli infection that allows investigation of the cellular and molecular mechanisms involved in host-protective immunity and bacterial-induced intestinal inflammation. We recently demonstrated that following C. rodentium infection, the absence of Resistin-Like Molecule (RELM) α resulted in attenuated Th17 cell responses and reduced intestinal inflammation with minimal effects on bacterial clearance. In this addendum, we investigated the cytokine modulatory effects of RELMα and RELMα expression in the intestinal mucosa following C. rodentium infection. We show that in addition to promoting Th17 cytokine responses, RELMα inhibits Th2 cytokine expression and Th2-cytokine effector macrophage responses in the C. rodentium-infected colons. Second, utilizing reporter C. rodentium, we examined RELMα expression and macrophage recruitment at the host pathogen interface. We observed infection-induced macrophage infiltration and RELMα expression by intestinal epithelial cells. The influence of infection-induced RELMα on macrophage recruitment in the intestine is discussed.

Current state of palliative and end-of-life care in home versus inpatient facilities and urban versus rural settings in Africa.

OBJECTIVE: Because palliative care in sub-Saharan Africa may not fit the style of delivery of palliative care in the global north, exploring the evidence can serve to reduce existing barriers and help streamline national policies that determine the optimal setting to implement formal palliative and end-of-life services. METHOD: A search was conducted in Ovid MEDLINE®, PubMed, and Google Scholar databases using the search terms nursing care, terminal care, end of life care, palliative care, dying, death, hospice, opioids, morphine, Africa, sub-Saharan Africa, caregivers, and place of death. Eighty-seven relevant articles were found using the search terms. Of these, 22 matched inclusion criteria and were reviewed. RESULTS: Opioid availability and distribution is best accessed at the inpatient level, and hence, pain and end-of-life symptoms are best managed at the inpatient level. Despite the great need, nurses' lack of prescription power in the home-based setting is a shortcoming. Home deaths have not been adequately studied, but research suggests that palliative care generally causes economic strain, psychosocial distress on family members, and increased risk of transmission of communicable disease. Hospice is understudied but shows favorable outcomes. SIGNIFICANCE OF RESULTS: Funding and research need to focus on development of inpatient palliative and hospice care units in urban areas. In rural areas, the priority should be a home-based care model that involves nurses who are privileged to prescribe opioids and adjunctive medication therapies.

Alternatively Activated Macrophages Revisited: New Insights into the Regulation of Immunity, Inflammation and Metabolic Function following Parasite Infection.

The role of macrophages in homeostatic conditions and the immune system range from clearing debris to recognizing and killing pathogens. While classically activated macrophages (CAMacs) are induced by T helper type 1 (Th1) cytokines and exhibit microbicidal properties, Th2 cytokines promote alternative activation of macrophages (AAMacs). AAMacs contribute to the killing of helminth parasites and mediate additional host-protective processes such as regulating inflammation and wound healing. Yet, other parasites susceptible to Th1 type responses can exploit alternative activation of macrophages to diminish Th1 immune responses and prolong infection. In this review, we will delineate the factors that mediate alternative activation (e.g. Th2 cytokines and chitin) and the resulting downstream signaling events (e.g. STAT6 signaling). Next, the specific AAMac-derived factors (e.g. Arginase1) that contribute to resistance or susceptibility to parasitic infections will be summarized. Finally, we will conclude with the discussion of additional AAMac functions beyond immunity to parasites, including the regulation of inflammation, wound healing and the regulation of metabolic disorders.