Induction of antigen specific intrahepatic CD8+ T cell responses by a secreted heat shock protein based gp96-Ig-PfCA malaria vaccine.

Introduction: A highly efficacious and durable vaccine against malaria is an essential tool for global malaria eradication. One of the promising strategies to develop such a vaccine is to induce robust CD8+ T cell mediated immunity against malaria liver-stage parasites. Methods: Here we describe a novel malaria vaccine platform based on a secreted form of the heat shock protein, gp96-immunoglobulin, (gp96-Ig) to induce malaria antigen specific, memory CD8+ T cells. Gp96-Ig acts as an adjuvant to activate antigen presenting cells (APCs) and chaperone peptides/antigens to APCs for cross presentation to CD8+ T cells. Results: Our study shows that vaccination of mice and rhesus monkeys with HEK-293 cells transfected with gp96-Ig and two well-known Plasmodium falciparum CSP and AMA1 (PfCA) vaccine candidate antigens, induces liver-infiltrating, antigen specific, memory CD8+ T cell responses. The majority of the intrahepatic CSP and AMA1 specific CD8+ T cells expressed CD69 and CXCR3, the hallmark of tissue resident memory T cells (Trm). Also, we found intrahepatic, antigen-specific memory CD8+ T cells secreting IL-2, which is relevant for maintenance of effective memory responses in the liver. Discussion: Our novel gp96-Ig malaria vaccine strategy represents a unique approach to induce liver-homing, antigen-specific CD8+ T cells critical for Plasmodium liver-stage protection.

Assessment of 2 Hair Removal Methods in New Zealand White Rabbits (Oryctolagus cuniculus).

Rabbits are frequently used as surgical models in research. However, studies assessing the effects of various hair removal methods on wound healing and surgical site infection (SSI) in rabbits are sparse. Here we evaluated the effects of 2 hair removal methods-clipping with electric clippers and using a commercial depilatory agent-on wound healing and SSI as assessed via wound scoring and histology. Incisions were assigned ASEPSIS scores on days 3 and 7. To assess whether the hair removal methods influenced aseptic preparation, swabs for bacterial culture were obtained just after hair removal on day 0, after aseptic skin preparation on day 0, and on day 1. For histopathologic assessment, full-thickness punch biopsies were obtained on days 0, 1, 3, 7, and 21. Histopathology revealed significant differences between the 2 methods, with the depilatory method having consistently higher scores (that is, more abnormalities). We conclude that for a surgical preparation regimen, clipping is safer, more efficacious, and less traumatic to tissues in rabbits.

PERINATAL CENTRILOBULAR HEPATIC NECROSIS IN GIANT PANDAS (AILUROPODA MELANOLEUCA): A RETROSPECTIVE STUDY.

Between 1983 and 2012, six giant panda cubs (Ailuropoda melanoleuca) born at a zoological institution were stillborn or died between the ages of 3 and 200 h. Two of the six cubs had panhepatic centrilobular hepatic necrosis (CHN), granulocytic extramedullary hematopoiesis (GEM), positive liver culture for Staphylococcus species, and terminal liver failure. Another low-weight cub was administered oxygen therapy immediately after birth and developed hyaline membranes in air spaces and hepatic necrosis restricted to the hilar region. A retrospective analysis of liver and lung lesions, pulmonary microanatomy, blood-gas barrier ultrastructure, and hepatic myofibroblast proliferation was conducted on the six cubs. Neonates with CHN had concurrent severe periportal GEM accompanied by severe myofibroblast proliferation. The pulmonary blood-gas barrier was markedly increased in one cub with CHN. Developmentally, the lungs of all but one cub were at the late saccular stage, and the lowest-weight cub was in early saccular stage, consistent with immaturity, and had pneumonia comparable to neonatal respiratory distress syndrome (RDS). Stage of lung development was eliminated as the primary factor leading to CHN. The pathogenesis of CHN in these neonates is proposed to be transformation of hepatic stellate cells to myofibroblasts initiating blockage and microvascular constriction of hepatic sinusoids, resulting in insufficient perfusion and cellular hypoxia of hepatocytes surrounding central veins in acinar zone 3.

Early local delivery of vancomycin suppresses ectopic bone formation in a rat model of trauma-induced heterotopic ossification.

Heterotopic ossification (HO) is a debilitating sequela of high-energy injuries. It frequently requires surgical excision once symptomatic and there is no practical prophylaxis for combat-injured patients. In this study, we examined the effect of local vancomycin powder on HO formation in a small animal model of blast-related, post-traumatic HO. Male Sprague-Dawley rats were subjected to a polytraumatic extremity injury and amputation with or without methicillin-resistant Staphylococcus aureus infection. Animals were randomized to receive a single local application of vancomycin (20 mg/kg) at the time of injury (POD-0, n = 34) or on postoperative day-3 (POD-3, n = 11). Quantitative volumetric measurement of ectopic bone was calculated at 12-weeks post-injury by micro-CT. Bone marrow and muscle tissues were also collected to determine the bacterial burden. Blood for serum cytokine analysis was collected at baseline and post-injury. Vancomycin treatment on POD-0 suppressed HO formation by 86% and prevented bone marrow and soft tissue infections. We concurrently observed a marked reduction histologically in nonviable tissue, chronic inflammatory cell infiltrates, bone infection, fibrous tissue, and areas of bone necrosis within this same cohort. Delayed treatment was significantly less efficacious. Neither treatment had a marked effect on the production of pro-inflammatory cytokines. Our study demonstrates that local vancomycin treatment at the time of injury significantly reduces HO formation in both the presence and absence of infection, with decreased efficacy if not given early. These findings further support the concept that the therapeutic window for prophylaxis is narrow, highlighting the need to develop early treatment strategies for clinical management. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2397-2406, 2017.

Double-blinded, placebo-controlled study of early tranexamic acid treatment in swine uncontrolled hemorrhage model.

BACKGROUND: Tranexamic acid (TXA) is an antifibrinolytic drug that was shown to increase survival in trauma patients, but the mechanisms remain unclear. The purpose of this double-blinded, randomized placebo-controlled study was to determine if TXA with hypotensive resuscitation with Hextend (HEX) or fresh frozen plasma (FFP) reduced blood loss (BL) and improved survival in a model of uncontrolled hemorrhage. METHODS: Instrumented, anesthetized pigs (n = 11 per group) were subjected to 24-mL/kg controlled hemorrhage, followed by transection of the spleen. After 15 minutes of bleeding, TXA (1.43 mg/kg/min) or normal saline (NS) was given over 10 minutes, and then 15-mL/kg HEX or FFP was administered. At 90 minutes, a second infusion of TXA or NS was given. BL, coagulation status, and 5-hour survival were determined. Tissue plasminogen activator (tPA) was added to blood samples collected before and after TXA administration to confirm that the TXA inhibited fibrinolysis. In addition, a comparison of a dose response to tPA-induced fibrinolysis was made between swine and human plasma in vitro. RESULTS: TXA prevented the rise in d-dimers that occurred after spleen injury. However, there was no significant effect of TXA on survival or BL compared with NS with HEX (HEX + NS, 17 ± 2 mL/kg vs. HEX + TXA, 17 ± 2 mL/kg) or FFP (FFP + NS, 7 ± 2 mL/kg vs. FFP + TXA, 12 ± 3 mL/kg), while FFP significantly reduced BL and increased survival compared with HEX in the NS-treated animals. The tPA-induced fibrinolysis was inhibited in the blood from TXA-treated animals, yet in fibrinolysis sensitivity studies, human plasma was 30 times more sensitive to tPA-induced fibrinolysis than swine plasma. CONCLUSION: TXA did not reduce BL, even though TXA was antifibrinolytic in the pigs. The possibility remains that the pig is highly resistant to fibrinolysis and not a good model to study the effects of antifibrinolytics or that fibrinolysis is not a major factor in bleeding from splenic injury.

Fat Intravasation from Intraosseous Flush and Infusion Procedures.

STUDY HYPOTHESIS: The primary study objective was to delineate the procedural aspects of intraosseous (IO) infusions responsible for fat intravasation by testing the hypothesis that the fat content of effluent blood increases during IO infusions. METHODS: IO cannulas were inserted into the proximal tibiae of 35 anesthetized swine (Sus scrofa, 50.1 ± 3.5 kg) and intravasated fat was assessed using a lipophilic fluoroprobe (Nile red) and by vascular ultrasound imaging. Effluent blood bone marrow fat was assessed at baseline, during flush, and with regimens of controlled infusion pressures (73-300 mmHg) and infusion flow rates (0.3-3.0 mL per second). Fat intravasation was also assessed with IO infusions at different tibial cannulation sites and in the distal femur. In 7 animals, the lipid uptake of alveolar macrophages and lung tissue assessed for fat embolic burden using oil red O stain 24 hours post infusion. Additionally, bone marrow shear-strain was assessed radiographically with IO infusions. RESULTS: Fat intravasation was observed during all IO infusion regimens, with subclinical pulmonary fat emboli persisting 24 hours post infusion. It was noted that initial flush was a significant factor in fat intravasation, low levels of intravasation occurred with infusions ≤300 mmHg, fat intravasation and bone marrow shear-strain increased with IO infusion rates, and intravasation was influenced by cannula insertion site. Ultrasound findings suggest that echogenic particles consistent with fat emboli are carried in fast and slow venous blood flow fields. Echo reflective densities were observed to rise to the nondependent endovascular margins and coalesce in accordance with Stoke's law. In addition, ultrasound findings suggested that intravasated bone marrow fat was thrombogenic. CONCLUSION: Results suggest that in swine the intravasation of bone marrow fat is a common consequence of IO infusion procedures and that its magnitude is influenced by the site of cannulation and infusion forces. Although the efficacy and benefits of IO infusions for emergent care are well established, emergency care providers also should be cognizant that infusion procedures affect bone marrow fat intravasation.

Chlorine-35 solid-state NMR spectroscopy as an indirect probe of germanium oxidation state and coordination environment in germanium chlorides.

Due to the prevalence of Ge-Cl bonds in germanium chemistry and the inherent challenges of germanium-73 NMR spectroscopy, chlorine-35 NMR spectroscopy was investigated as an indirect method of characterization for these ubiquitous and useful compounds. Chlorine-35 NMR parameters were correlated with structural metrics as well as the oxidation state of germanium.

Toxoplasmosis-associated abortion in an alpaca (Vicugna pacos) fetus.

A near-full-term alpaca (Vicugna pacos) was stillborn 2 days before the expected date of delivery; necropsy examination was conducted within 6 hr of delivery. Gross lesions were enlarged liver and hydrocephalus. On histologic examination, mild inflammatory lesions were identified in the placenta, liver, and lungs, although no etiology was recognized. Within the brain, there was a mild nonsuppurative meningoencephalitis, hydrocephalus, and hydromyelia. Both kidneys had inflammatory foci in cortex and medulla associated with protozoal tissue cysts. Protozoa in brain and kidneys were identified as Toxoplasma gondii based on immunoreactivity to T. gondii polyclonal antibodies that stain both tachyzoites and tissue cysts and BAG1 T. gondii antibodies that are bradyzoite specific. The tissue cysts exhibited intense positivity to T. gondii and mild immunoreactivity to Neospora caninum antibodies. The dam had a high antibody titer (1 : 12,800) to T. gondii and low titer (1 : 100) to N. caninum using their respective agglutination tests. This is the first report of toxoplasmosis-associated abortion in alpaca.

In vivo assessment of the Combat Ready Clamp to control junctional hemorrhage in swine.

BACKGROUND: Junctional wounds and associated hemorrhage have become more common and more lethal in the current war. The Combat Ready Clamp (CRoC) has been developed and deployed for treating junctional hemorrhage on the battlefield. This study examined the efficacy of CRoC and its acute effects in an animal model. METHODS: Anesthetized pigs (n = 6) were subjected to laparotomy, splenectomy, and abdominal closure. Next, coagulopathy was induced in animals by hemodilution and hypothermia. The left femoral artery was isolated, punctured (6-mm hole), and allowed to bleed for 15 seconds. The groin wound was packed with gauze, and a CRoC applied and tightened until hemorrhage stopped. It was kept in place for 1 hour (treatment period) and then released for another hour or less (control-period) if animal exsanguinated. Fluid resuscitation was administered, and vascular blood flow was examined by Doppler and CT scans. After death, local tissues were collected for histology. RESULTS: CRoC generated 800 to 900 mm Hg pressure on the wounds, which stopped the hemorrhage and prevented rebleeding during the first hour in all animals. Blood loss was minimal (≤137 mL), and mean arterial pressure remained at or higher than the target level (65 mm Hg) during this period. Removal of the clamp promptly led to rebleeding and exsanguination of five of six pigs during the second hour despite fluid resuscitation. Blood loss, survival, shock indices, and other measures were significantly (p < 0.01) different between the two periods. Doppler tests and CT scans showed no blood flow in the proximal, distal, and collateral arteries of the clamped leg. Minor inflammation was seen on blood vessels (endothelium) and nerves. CONCLUSION: CRoC functioned as an effective hemostatic adjunct for compression and control of groin hemorrhage. Although no acute histological damages were seen in compressed tissues, the short- and long-term effects of CRoC application (e.g., total ischemia) on limb function remain unknown and warrant investigation.

Green ergonomics: challenges and opportunities.

Addressing the causes and consequences of environmental degradation presents significant challenges for humankind. This paper considers what ergonomics/human factors (E/HF) professionals can contribute to understanding and tackling some of the issues that arise through the movement towards a more environmentally sustainable economy. These issues are considered in relation to work in green industries (specifically, sustainable energy production, recycling and organic food production), and there is a need to ensure that these jobs are safe and healthy; the design of products and systems that are 'environmentally friendly' to facilitate their acceptability and use and how E/HF professionals can contribute to understanding and promoting behavioural change relating to environmental choices. The activities of some international organisations in this area are identified and the potential for E/HF involvement is considered. The implications for the E/HF profession are discussed. PRACTITIONER SUMMARY: This paper considers how ergonomics/human factors professionals can contribute to the movement towards more sustainable and 'environmentally friendly' design and work. Potential challenges and opportunities are discussed in relation to jobs in green industries, products and systems and behaviour change.

Negative-pressure wound therapy: a hemostatic adjunct for control of coagulopathic hemorrhage in large soft tissue wounds.

BACKGROUND: Negative-pressure wound therapy has been commonly used for treating chronic wounds and recently applied for treatment of traumatic wounds. We investigated the potential hemostatic benefit of negative-pressure wound therapy for control of refractory hemorrhage in a soft tissue wound model in swine. METHODS: Coagulopathy was induced in pigs (n = 38, 36 kg) by hemodilution and hypothermia. Next, a large soft tissue wound (diameter, approximately 20 cm) was created by slicing the gluteus maximus muscle. Free bleeding was allowed for 1 minute, and wounds were then randomly dressed with either laparotomy gauze (G) alone or TraumaPad (TP, a kaolin-coated dressing) alone or in combination with negative pressure (NP, approximately -500 mm Hg). All wounds were sealed with adhesive drapes. Fluid resuscitation was administered and targeted to mean arterial pressure of 60 mm Hg. Pigs were observed for 150 minutes or until death after which tissues were sampled for histologic examination. RESULTS: Induced coagulopathy as measured by increases in prothrombin time (12%) and activated partial thromboplastin time (22%) and decreases in fibrinogen (48%) were similar in all groups. There were no differences in initial bleeding rates (4.5 mL/kg/min). Dressing the wounds with G or TP produced hemostasis only in one pig (1 of 18 pigs). Addition of NP to these dressings secured hemostasis in 70% (G) and 90% (TP) of animals with average hemostasis time of 34 minutes and 25 minutes, respectively. Blood losses and fluid resuscitation requirements were significantly less, and survival times were significantly longer in NP adjunct groups than in the other groups. Survival rates were 80% (G+NP) and 90% (TP+NP) versus 0% (G) and 10% (TP) in the respective groups. Histologic examination showed similar superficial myofibril damages in all groups. CONCLUSION: To our knowledge, the present data provide the first evidence that NP serves as an effective hemostatic adjunct and when combined with standard hemostatic dressing it is able to stop lethal coagulopathic bleeding in large soft tissue wounds.

Solid-state 73Ge NMR spectroscopy of simple organogermanes.

Germanium-73 is an extremely challenging nucleus to examine by NMR spectroscopy due to its unfavorable NMR properties. Through the use of an ultrahigh (21.1 T) magnetic field, a systematic study of a series of simple organogermanes was carried out. In those cases for which X-ray structural data were available, correlations were drawn between the NMR parameters and structural metrics. These data were combined with DFT calculations to obtain insight into the structures of several compounds with unknown crystal structures.

Cationic cryptand complexes of tin(II).

A series of cationic cryptand complexes of tin(II), [Cryptand[2.2.2]SnX][SnX(3)] (10, X = Cl; 11, X = Br; 12, X = I) and [Cryptand[2.2.2]Sn][OTf](2) (13), were synthesized by the addition of cryptand[2.2.2] to a solution of either tin(II) chloride, iodide, or trifluoromethanesulfonate. The complexes could also be synthesized by the addition of the appropriate trimethylsilyl halide (or pseudohalide) reagent to a solution of tin(II) chloride and cryptand[2.2.2]. The complexes were characterized using a variety of techniques including NMR, Raman, and temperature-dependent Mössbauer spectroscopy, mass spectrometry, and X-ray diffraction.

Comparison of airway pressure release ventilation to conventional mechanical ventilation in the early management of smoke inhalation injury in swine.

OBJECTIVE: The role of airway pressure release ventilation in the management of early smoke inhalation injury has not been studied. We compared the effects of airway pressure release ventilation and conventional mechanical ventilation on oxygenation in a porcine model of acute respiratory distress syndrome induced by wood smoke inhalation. DESIGN: Prospective animal study. SETTING: Government laboratory animal intensive care unit. PATIENTS: Thirty-three Yorkshire pigs. INTERVENTIONS: Smoke inhalation injury. MEASUREMENTS AND MAIN RESULTS: Anesthetized female Yorkshire pigs (n = 33) inhaled room-temperature pine-bark smoke. Before injury, the pigs were randomized to receive conventional mechanical ventilation (n = 15) or airway pressure release ventilation (n = 12) for 48 hrs after smoke inhalation. As acute respiratory distress syndrome developed (PaO2/Fio2 ratio <200), plateau pressures were limited to <35 cm H2O. Six uninjured pigs received conventional mechanical ventilation for 48 hrs and served as time controls. Changes in PaO2/Fio2 ratio, tidal volume, respiratory rate, mean airway pressure, plateau pressure, and hemodynamic variables were recorded. Survival was assessed using Kaplan-Meier analysis. PaO2/Fio2 ratio was lower in airway pressure release ventilation vs. conventional mechanical ventilation pigs at 12, 18, and 24 hrs (p < .05) but not at 48 hrs. Tidal volumes were lower in conventional mechanical ventilation animals between 30 and 48 hrs post injury (p < .05). Respiratory rates were lower in airway pressure release ventilation at 24, 42, and 48 hrs (p < .05). Mean airway pressures were higher in airway pressure release ventilation animals between 6 and 48 hrs (p < .05). There was no difference in plateau pressures, hemodynamic variables, or survival between conventional mechanical ventilation and airway pressure release ventilation pigs. CONCLUSIONS: In this model of acute respiratory distress syndrome caused by severe smoke inhalation in swine, airway pressure release ventilation-treated animals developed acute respiratory distress syndrome faster than conventional mechanical ventilation-treated animals, showing a lower PaO2/Fio2 ratio at 12, 18, and 24 hrs after injury. At other time points, PaO2/Fio2 ratio was not different between conventional mechanical ventilation and airway pressure release ventilation.

Exploring the limits of (73)Ge solid-state NMR spectroscopy at ultrahigh magnetic field.

The ultrahigh field natural abundance (73)Ge solid-state wide-line NMR study of germanium dichloride complexed with 1,4-dioxane and tetraphenylgermane yields the largest (73)Ge quadrupolar coupling constant determined by NMR spectroscopy to date and the first direct observation of (73)Ge chemical shift anisotropy.