Effects of Liposome‐Encapsulated Hemoglobin on Gastric Wound Healing in the Rat

Liposome‐encapsulated hemoglobin (LEH) may improve microcirculation and oxygen (O₂) metabolism at a surgical wound to accelerate its healing. Ten mL/kg of LEH with high (h‐LEH) or low O₂‐affinity (l‐LEH), homologous red blood cells (RBC), empty liposome or saline as a control was infused before a 10‐mm incision and interrupted suture closure of the gastric wall in a total of 110 rats. Two and 4 days later, the stomach was excised for bursting pressure determination and histological sampling. The dose–response relationship was examined in 70 additional rats receiving progressively reduced doses of h‐LEH. Hypoxia‐inducible factor‐1α (HIF‐1α) was stained immunohistochemically in 54 other rats to examine its accumulation at the anastomotic sites. Bursting pressure of the surgical wound was significantly higher 2 days after surgery only in the h‐LEH‐treated rats (P < 0.05), but not at 4 days after surgery, when other rats showed increased bursting pressure to a nonsignificant level. Histological examination revealed less granulocyte infiltration, better granulation, and more macrophage infiltration in h‐LEH‐treated rats at 2 days, but no longer at 4 days postsurgery. Dose–response study revealed that 0.4 mL/kg of h‐LEH (hemoglobin 24 mg/kg) was effective for elevating bursting pressure at 2 days. h‐LEH‐treated rats had significantly suppressed HIF‐1α accumulation in the wound 6, 24, and 48 h after surgery as compared with control animals treated with homologous RBC or saline. In conclusion, the results suggest that h‐LEH, but not l‐LEH or homologous transfusion, may accelerate wound healing early after gastric incision and anastomosis in the rat. The mechanism(s) appears to be related to improved O₂ supply, aerobic metabolism, and suppressed inflammation in the wound.     

Liposome‐Encapsulated Hemoglobin Improves Tumor Oxygenation as Detected by Near‐Infrared Spectroscopy in Colon Carcinoma in Mice

Liposome‐encapsulated hemoglobin (LEH) with high (h‐LEH, P₅₀O₂ = 10 mm Hg) or low O₂ affinity (l‐LEH, P₅₀O₂ = 40 mm Hg) may improve O₂ delivery to sensitize tumor tissues for radiotherapy. A total of 10 mL/kg of h‐LEH, l‐LEH, red blood cells (RBCs), or saline was infused in mice transplanted with murine colon carcinoma with near‐infrared spectroscopy (NIRS) detectors set at the tumor (right leg) and intact muscle (left leg). NIRS recorded changes in the amount of oxyhemoglobin (oxyHb), deoxyhemoglobin (deoxyHb), and their sum (tHb) with the animals spontaneously breathing room air (10 min), pure O₂ (5 min), and then back to room air. The tumor was finally excised for histological examination. In mice treated with h‐LEH, tHb significantly increased compared to mice receiving other solutions. The magnitude was significantly attenuated in the tumor compared to the intact muscle under room air. Reciprocal changes in oxyHb and deoxyHb between intact muscle and tumor in response to infused solutions allowed assumption of average tissue PO₂ between 30 and 40 mm Hg in muscle and at around 10 mm Hg in tumor. While O₂ respiration increased oxyHb and decreased deoxyHb both in muscle and tumor, their sum or tHb consistently decreased in muscle and increased in tumor regardless of preceding infusion. Such responses were totally reversed when mice were placed under hypoxia (10% O₂), suggesting that a lack of physiological circulatory regulation in tumor may account for heavier immunohistochemical staining for human hemoglobin in tumors of mice treated with h‐LEH than with l‐LEH. The results suggest that h‐LEH may cause significant tumor oxygenation compared to RBC, l‐LEH, or saline probably due to its nanometer size (vs. RBC) and high O₂ affinity (vs. l‐LEH) without increasing O₂ content in the intact tissue (vs. O₂ respiration) probably due to a lack of physiological circulatory regulation.     

Liposome‐Encapsulated Hemoglobin Accelerates Bronchial Healing After Pneumonectomy in the Rat With or Without Preoperative Radiotherapy

Liposome‐encapsulated hemoglobin (LEH) has been reported to accelerate wound healing in the stomach and skin in an experimental setting. LEH was tested in bronchial anastomotic healing after radiation and pneumonectomy in the rat. Sprague‐Dawley rats (n = 61) received preoperative radiation (20 Gy) to the chest and underwent left pneumonectomy with bronchial stump closure using the Sweet method 4 days later, when they were randomized to receive intravenous infusion of LEH with high O₂ affinity (P₅₀O₂ = 17 mm Hg, 10 mL/kg, n = 32) or saline (n = 29). Additional rats (n = 18) were treated in the same way without preoperative radiation. Bronchial anastomotic healing was evaluated 2 days after surgery by determining the bursting pressure and infiltration of neutrophils, monocytes, and macrophages. Bronchial bursting pressure was elevated in the rats receiving LEH both in the unirradiated group (LEH 212 ± 78 vs. saline 135 ± 63 mm Hg, P < 0.05) and in rats with preoperative radiation (LEH 162 ± 48 vs. saline 116 ± 56 mm Hg, P < 0.01). Moreover, the percentage of rats with bursting pressure <100 mm Hg tended to be smaller in the unirradiated group (LEH 1/9 [11.1%] vs. saline 4/9 [44.4%], NS) and was significantly reduced in irradiated animals (LEH 3/32 [9.4%] vs. saline 11/29 [38%], P < 0.05). There were no morphological differences except for macrophage infiltration to the anastomotic area, which was significantly prominent in the LEH‐treated rats (P < 0.05) regardless of the presence or absence of preoperative irradiation (IR). The results suggest that LEH with high O₂ affinity may improve mechanical strength and morphological findings in bronchial anastomosis in rats regardless of the presence or absence of preoperative IR. The irradiated rats later treated with LEH had equivalent or better bronchial healing than that of saline‐treated naïve animals undergoing pneumonectomy alone.     

Effect of liposome-encapsulated hemoglobin on antigen-presenting cells in mice

Liposome‐encapsulated hemoglobin (LEH) is removed from the circulation and degraded in the reticuloendothelial system, including dendritic cells (DCs) and macrophages. Therefore, LEH at a large dose may overload the system, cause a competitive inhibition in antigen‐presenting activity, and impair the immune response of the host. Changes in cellularity of immunocompetent cells were monitored serially up to 4 weeks by flow cytometry in wild‐type mice receiving 20 mL/kg of LEH, syngeneic red blood cells (RBCs), or saline. DCs were collected from the host spleen 1, 7, and 28 days after receiving the solution and were cocultured with naïve cluster of differentiation 4 T cells from T‐cell receptor transgenic mice in the absence or presence of third‐party antigens. After LEH administration, the cellularity of DCs and macrophages in the recipient spleen remained unchanged from control mice receiving RBCs or saline. While subset populations and costimulatory molecule expressions were different, DCs from LEH‐administered mice expressed high levels of interleukin‐2 production and helper T‐cell activation in response to a third‐party antigen and superantigens, as did the DCs from control mice receiving RBCs or saline. The results suggest that 20 mL/kg of LEH does not greatly alter antigen‐presenting activity to third‐party antigens.     

Liposome‐Encapsulated Hemoglobin Ameliorates Ischemic Stroke in Nonhuman Primates: Longitudinal Observation

Liposome‐encapsulated hemoglobin (LEH) is protective early after brain ischemia in rats and nonhuman primates, but it remains unclear whether the protection persists and confers any benefits beyond the acute phase of brain ischemia and reperfusion. Ten monkeys underwent middle cerebral artery occlusion, received LEH (2 mL/kg, n = 5) or saline (2 mL/kg, n = 5) 5 min later, and reperfusion 3 h later. Positron emission tomography studies were repeated for the cerebral metabolic rate of O₂ (CMRO₂) as well as glucose (CMRglc) up to 8 days after reperfusion, when the animals were euthanized for morphological studies. There was no difference in O₂ metabolism until 3 h after reperfusion, when CMRO₂ was significantly better preserved in the cortex, but not in basal ganglia, on Day 0 in LEH‐treated monkeys. The extent of cortical infarction (saline 68 ± 10% vs. LEH 38 ± 9%, P < 0.05) and CMRO₂ (mild suppression: saline 34 ± 10% vs. LEH 14 ± 4%, P < 0.05) remained significantly better preserved 8 days later, when CMRglc showed a similar pattern of cortical protection (mild suppression: saline 49 ± 15% vs. LEH 37 ± 4%, P < 0.05) in LEH‐treated monkeys, together with regained body weight. Somatic weight control, morphological integrity, CMRO₂, and CMRglc were better preserved immediately, as well as 8 days after occlusion and reperfusion of the middle cerebral artery in monkeys receiving LEH early after onset of ischemia.     

Liposome‐Encapsulated Hemoglobin Enhances Chemotherapy to Suppress Metastasis in Mice

Liposome‐encapsulated hemoglobin with high O₂‐affinity (P₅₀O₂ = 10 mm Hg, h‐LEH) was reported to enhance tumor radiosensitivity. We hypothesize that targeted O₂ delivery to tumor hypoxia by h‐LEH may also enhance chemotherapy to suppress tumor growth and metastasis in mice. Doxorubicin (DXR; 0.5 or 2 mg/kg i.p.) or S‐1 (4 or 8 mg/kg orally) alone or in combination with h‐LEH (5 mL/kg i.v.) was administered for 2 weeks to C57BL/6N mice inoculated with Lewis Lung Carcinoma (LLC) in the leg. After the 2‐week therapy in six treatment groups, mice were sacrificed for quantitative assessment of tumor growth and lung metastasis. The tumor was then evaluated for its expression of hypoxia‐inducible factor‐1α (HIF‐1α) and matrix metallopoteinase‐2 (MMP‐2) activity. Combined use of h‐LEH and chemotherapeutic agents (DXR or S‐1) showed no additional enhancement on suppression of the tumor growth over the chemotherapeutic agent alone. However, the combination use of h‐LEH significantly suppressed the number and total area of metastatic colonies in the lung compared with each chemotherapeutic agent alone. Although HIF‐1α expression and MMP‐2 activity in the original tumor was significantly suppressed in the groups of mice treated with either DXR or S‐1 alone, the addition of h‐LEH to either agent showed further enhancement of oxygen‐mediated degradation of HIF‐1α and suppression of MMP‐2 activity. Although the addition of h‐LEH to DXR or S‐1 had little effect on original LLC tumor growth, it significantly enhanced suppression of lung metastasis in mice.     

Biological Evaluation of Liposome‐Encapsulated Hemoglobin Surface‐Modified With a Novel PEGylated Nonphospholipid Amphiphile

Traumatic injury is often associated with hemorrhagic shock. Liposome‐encapsulated hemoglobin (LEH) is being developed as an artificial oxygen carrier to address post‐hemorrhage oxygen and volume deficit. Here, we report a new composition of LEH based on the use of polyethylene glycol (PEG_2K) conjugated with nonphospholipid hexadecylcarbamoylmethylhexadecanoate (HDAS) to modify the surface of LEH particles. LEH was manufactured by the high‐pressure homogenization method using dipalmitoylphosphatidylcholine (～38 mol%), cholesterol (～38 mol%), HDAS (～20 mol%), and highly purified stroma‐free human hemoglobin. HDAS‐PEG_2K was postinserted into the resultant LEH to generate HDAS‐PEG_2K‐LEH. We investigated the potential immune response to HDAS‐PEG_2K‐LEH in a mice model. At the same time, the preparation was tested in a rat model to study the effect of repeated HDAS‐PEG_2K‐LEH injection over 4 weeks. We found that HDAS‐PEG_2K modification substantially reduced the circulating levels of anaphylatoxins C3a and C5a, as well as plasma levels of thromboxane B2, in mice. Repeated injections of HDAS‐PEG_2K‐LEH in rats did not appear to alter its clearance profile after 4 weeks of treatment. No antibody response against human hemoglobin or PEG was detected in rat plasma. Histological observations of lung, liver, spleen, and kidney were not significantly different between saline‐treated rats and HDAS‐PEG_2K‐LEH‐treated rats. Immunohistochemical staining for rat heme oxygenase‐1 (HO‐1) did not show induced expression of HO‐1 in these organs. These results suggest that the new surface modification of LEH is immune‐neutral and does not adversely affect histology even after repeated administration.     

Effect of Oxygen Affinity of Liposome‐Encapsulated Hemoglobin on Cerebral Ischemia and Reperfusion as Detected by Positron Emission Tomography in Nonhuman Primates

We tested a hypothesis that liposome‐encapsulated hemoglobin (LEH) with high oxygen (O₂) affinity (h‐LEH, P₅₀O₂ = 10 mm Hg) may work better than LEH with low O₂ affinity (l‐LEH, P₅₀O₂ = 40 mm Hg) in cerebral ischemia and reperfusion injury using positron emission tomography (PET) in primates undergoing middle cerebral artery (MCA) occlusion and reperfusion. Cerebral blood flow (CBF), O₂ extract fraction (OEF), and cerebral metabolic rate of O₂ (CMRO₂) were successively determined by PET before ischemia, at 2 h of ischemia, immediately after reperfusion, and 3 h after reperfusion. Five minutes after MCA occlusion, 10 mL/kg of h‐LEH (n = 6) was intravenously infused and compared with the results from previous data of monkeys treated with l‐LEH (n = 6), empty liposome (n = 4), or saline (n = 8) as control. After the series of PET studies, the integrated area of cerebral infarction was determined histologically in 12 coronal brain slices. There was no significant difference in CBF, OEF, or CMRO₂ up to 2 h of ischemia. A high CBF with a low OEF tended to be suppressed after reperfusion in LEH‐treated monkeys. Three hours after reperfusion, the area of mild CMRO₂ reduction (down to ?30%) decreased (P < 0.05) and the area of mild CMRO₂ increase (up to 30%) expanded in LEH‐treated monkeys (P < 0.05) regardless of O₂ affinity with no difference in the area of moderate‐to‐severe reduction (2 compared to animals treated with empty liposome or saline. Distribution of CMRO₂ reduction and histological damages showed that LEH mainly protected the cerebral cortex rather than basal ganglia where neuronal dendritic processes were severely lost. There was little difference between the animals treated with l‐LEH or h‐LEH both at 10 mL/kg or between treatment with empty liposome or saline. In conclusion, LEH was effective regardless of O₂ affinity in preserving CMRO₂ and in reducing the area of histological damage in the cerebral cortex, but not in basal ganglia, shortly after occlusion/reperfusion of MCA in monkey.     

Liposome‐Encapsulated Hemoglobin Accelerates Skin Wound Healing in Diabetic dB/dB Mice

Since liposome‐encapsulated hemoglobin with high O₂ affinity (h‐LEH, P₅₀O₂ = 10 mm Hg) has been reported to accelerate skin wound healing in normal mice, it was tested in dB/dB mice with retarded wound healing, as seen in human diabetics. Two full‐thickness dorsal wounds 6 mm in diameter encompassed by silicone stents were created in dB/dB mice. Two days later (day 2), the animals were randomly assigned to receive intravenous h‐LEH (2 mL/kg, n = 7) or saline (2 mL/kg, n = 7). The same treatment was repeated 4 days after wounding (day 4), and the size of the skin lesions was analyzed by photography, surface perfusion was detected by Laser‐Doppler imager, and plasma cytokines and chemokines were determined on days 0, 2, 4, and 7, when all animals were euthanized for morphological studies. The size of the ulcer compared to the skin defect or silicone stent became significantly reduced on days 4 and 7 in mice treated with h‐LEH (47 ± 8% of original size), similar to the level in wild‐type mice, compared to saline‐treated dB/dB mice (68 ± 18%, P < 0.01). Mice treated with h‐LEH had significantly attenuated inflammatory cytokines, increased surface perfusion, and increased Ki67 expression on day 7 in accordance with the ulcer size reduction, while there was no significant difference in chemokines, histological granulation, epithelial thickness, and granulocyte infiltration detected by immunohistochemical staining in the ulcer between the treatment groups. The results suggest that h‐LEH (2 mL/kg) early after wounding may accelerate skin wound healing in dB/dB mice to levels equivalent to wild‐type mice probably via mechanism(s) involving reduced hypoxia, increased surface perfusion, suppressed inflammation, accelerated in situ cell proliferation and protein synthesis.     

Liposome-encapsulated hemoglobin alleviates hearing loss after transient cochlear ischemia and reperfusion in the gerbil

To test liposome‐encapsulated hemoglobin (LEH) in transient cochlear ischemia/reperfusion as a model of sudden deafness, Mongolian gerbils were randomly assigned to receive 2 mL/kg of either low‐affinity LEH (l‐LEH, P₅₀O₂ = 40 mm Hg), high‐affinity LEH (h‐LEH, P₅₀O₂ = 10 mm Hg), homologous red blood cells (RBCs), or saline (each group n = 6) 30 min before 15‐min occlusion of the bilateral vertebral arteries and reperfusion. Sequential changes in hearing were assessed by auditory brain response 1, 4, and 7 days after ischemia/reperfusion, when the animals were sacrificed for pathological studies. h‐LEH was significantly more protective than l‐LEH in suppressing hearing loss, in contrast to RBC or saline treatment, at 8, 16, and 32 kHz, where hearing loss was most severe (P < 0.05 between any two groups) on the first day after cochlear ischemia/reperfusion. Thereafter, hearing loss improved gradually in all groups, with a significant difference among groups up to 7 days, when morphological studies revealed that the inner hair cells but not the outer hair cells, were significantly lost in the groups in the same order. The results suggest that pretreatment with h‐LEH is significantly more protective than l‐LEH in mitigating hearing loss and underlying pathological damage, in contrast to transfusion or saline infusion 7 days after transient cochlear ischemia/reperfusion.     

Effects of varicocele on DNA methylation pattern of H19 and Snrpn gene in spermatozoa and behavioural characteristics of adult rat offspring

The aim of this study was to explore the effects of varicocele on DNA methylation pattern of H19 and Snrpn gene in spermatozoa and behavioural characteristics of adult rat offspring. Wistar rats with experimentally induced varicocele were used. The status of promoter methylation of H19 and Snrpn gene in spermatozoa of rats with varicocele or without varicocele was investigated. In addition, the Morris water maze test, elevated plus maze test and forced swimming test were employed to evaluate the learning and memory capability, and emotional behaviour of adult rat offspring. There were no significant differences in DNA methylation levels of H19 and Snrpn gene in spermatozoa among the control group, sham operation group and varicocele‐induced group. Furthermore, no significant differences were found in Morris water maze test and elevated plus maze test among the offspring in the three groups. However, in forced swimming test, the immobility time of offspring (both male and female) in the varicocele‐induced group was significantly longer than that in the control group and the sham operation group. Varicocele does not alter the methylation profiles of H19 and Snrpn gene, but exerts depressant‐like effect on the adult rat offspring.     

Liposome-encapsulated hemoglobin ameliorates tumor hypoxia and enhances radiation therapy to suppress tumor growth in mice

We hypothesize that liposome‐encapsulated hemoglobin with high O₂ affinity (P₅₀0₂ = 12 mm Hg, h‐LEH) may increase O₂ delivery to hypoxic tumors and enhance radiation therapy synergistically to suppress tumor growth. First, h‐LEH (5, 10, and 20 mL/kg) was intravenously infused 30 min before radiation (20 Gy) of SCCVII tumor grown in C3H/HeN mice. Second, 10 mL/kg of h‐LEH was administered 30, 60, 90, and 120 min prior to radiation to determine optimal timing. Tumor size was monitored thereafter to titrate tumor growth suppression. Third, additional mice with SCCVII tumor were infused with h‐LEH or empty liposome (EL), and tumors were excised at various time points for immunohistochemical examination of h‐LEH and hypoxia‐inducible factor‐1α (HIF‐1α). h‐LEH was most effective at 10 mL/kg in comparison to 5 or 20 mL/kg of h‐LEH or EL. Tumor growth was most suppressed when the interval between h‐LEH infusion and radiation was shortest, 30 min. As a result, 10 mL/kg of h‐LEH infusion 30 min prior to radiation prolonged 5‐fold tumor‐growth time from 20.0 days (radiation and EL) to 26.5 days, P < 0.01, synergy ratio 1.42. While human hemoglobin (h‐LEH) was detected in tumors 0.5 to 24 h after administration, HIF‐1α accumulation was sparse and became significantly reduced compared to controls 48 and 72 h after h‐LEH infusion. h‐LEH (10 mL/kg) was highly effective in enhancing radiation therapy synergistically under ambient respiration against tumor growth in mice. Decreased accumulation of HIF‐1α in h‐LEH‐treated tumor may suggest targeted tumor oxygenation as a potential mechanism.     

Daily Running Promotes Spatial Learning and Memory in Rats

Previous studies have shown that physical activity improves learning and memory. Present study was performed to determine the effects of acute, chronic and continuous exercise with different periods on spatial learning and memory recorded as the latency and length of swim path in the Morris water maze testing in subsequent 8 days. Four rat groups were included as follows: 1- Group C (controls which did not exercise). 2- Group A (30 days treadmill running before and 8 days during the Morris water maze testing period). 3- Group B (30 days exercise before the Morris water maze testing period only) and 4- Group D (8 days exercise only during the Morris water maze testing period). The results showed that chronic (30 days) and continuous (during 8 days of Morris water maze testing days) treadmill training produced a significant enhancement in spatial learning and memory which was indicated by decreases in path length and latency to reach the platform in the Morris water maze test (p < 0.05). The benefits in these tests were lost in three days, if the daily running session was abandoned. In group D with acute treadmill running (8 days exercise only) the difference between the Group A disappeared in one week and benefit seemed to be obtained in comparison with the controls without running program. In conclusion the chronic and daily running exercises promoted learning and memory in Morris water maze, but the benefits were lost in few days without daily running sessions in adult rats.

Key points

• Daily running influence on spatial memory.
• The velocity of learning can be influenced by running activity.
• Path length is important parameter for measuring the speed of learning.

Key words: Exercise, spatial memory, hippocampus, Morris water maze     

Liposome-encapsulated hemoglobin accelerates skin wound healing in mice

Effects of liposome‐encapsulated hemoglobin with high O₂ affinity (m‐LEH, P₅₀O₂ = 17 mm Hg) on skin wound healing in mice were examined. Two full‐thickness dorsal wounds 6 mm in diameter encompassed by silicone stents were created in Balb/c mice. Two days later (day 2), the animals randomly received intravenous m‐LEH (2 mL/kg, n = 12), homologous blood transfusion (red blood cell [RBC], n = 11), or saline (n = 12). The same treatment was repeated 4 days after wounding (day 4), and the sizes of the skin defects and ulcers were monitored on days 0, 2, 4, and 7, when all animals were euthanized for morphological studies. While the size of the skin defect in relation to the stent ring remained the same in all groups, the size of the ulcer compared with the skin defect (or silicone stent) became significantly reduced on days 4 and 7 in mice treated with m‐LEH (46 ± 10% of pretreatment size, P < 0.01) compared with mice treated with RBC transfusion (73 ± 6%) or saline (76 ± 7%). m‐LEH treatment significantly accelerated granulation, increased epithelial thickness, suppressed early granulocyte infiltration, and increased Ki67 expression in accordance with the ulcer size reduction, while there was no difference in surface blood flow or CD31 expression among the groups. The results suggest that m‐LEH (2 mL/kg) may accelerate skin wound healing in Balb/c mice via mechanism(s) involving reduced inflammation and increased metabolism, but not by improved hemodynamics or endothelial regeneration.     

Subjective effects of Lepidium meyenii (Maca) extract on well‐being and sexual performances in patients with mild erectile dysfunction: a randomised,double‐blind clinical trial

Lepidium meyenii (Maca) is a cultivated root belonging to the brassica family used in the Andean region for its supposed aphrodisiac properties. We carried out a double‐blind clinical trial on 50 Caucasian men affected by mild erectile dysfunction (ED), randomised to treatment with Maca dry extract, 2400 mg, or placebo. The treatment effect on ED and subjective well‐being was tested administrating before and after 12 weeks the International Index of Erectile Function (IIEF‐5) and the Satisfaction Profile (SAT‐P). After 12 weeks of treatment, both Maca‐ and placebo‐treated patients experienced a significant increase in IIEF‐5 score (P < 0.05 for both). However, patients taking Maca experienced a more significant increase than those taking placebo (1.6 ± 1.1 versus 0.5 ± 0.6, P < 0.001). Both Maca‐ and placebo‐treated subjects experienced a significant improvement in psychological performance‐related SAT‐P score, but the Maca group higher than that of placebo group (+9 ± 6 versus +6 ± 5, P < 0.05). However, only Maca‐treated patients experienced a significant improvement in physical and social performance‐related SAT‐P score compared with the baseline (+7 ± 6 and +7 ± 6, both P < 0.05). In conclusion, our data support a small but significant effect of Maca supplementation on subjective perception of general and sexual well‐being in adult patients with mild ED.     

Characterization of transboundary foot‐and‐mouth disease viruses in Nigeria and Cameroon during 2016

Continuous surveillance for foot‐and‐mouth disease (FMD) in endemic settings such as West Africa is imperative to support improved local and regional control plans, with the long‐term goal of regional eradication. This paper describes the genetic characterization of FMD viruses (FMDV) obtained from outbreaks in Nigeria (n = 45) and Cameroon (n = 15) during 2016 and from archival samples (n = 3) retrieved from a 2014 outbreak in Nigeria. These viruses were analysed in the context of previously published FMDV sequences from the region. Four FMDV serotypes: O, A, SAT1 and SAT2, were detected. Phylogenetic analyses of the VP1 coding sequences indicate the continuity of FMDV serotype O East Africa‐3 (O/EA‐3), serotype A AFRICA genotype G‐IV (A/AFRICA/G‐IV) and serotype South African Territories (SAT) 2 lineage VII (SAT2/VII). The FMDV SAT1 topotype X (SAT1/X), which emerged in Nigeria in 2015, continued to be associated with outbreaks in the region during 2016, and SAT1 is reported for the first time from Cameroon. Additionally, a re‐emergence or re‐introduction of the serotype O West Africa (O/WA) topotype in Nigeria is described herein. Our findings indicate a consistent, pan‐serotypic relationship between FMDV strains detected in Cameroon and Nigeria. Additionally, FMDV strains from West Africa obtained in this study were genetically related to those occurring in East and North Africa. These phylogenetic relationships suggest that animal movements (pastoralism and/or trade) are important factors for virus spread across the African continent. These data provide critical baselines which are a necessary component of Stages 0 and 1 of the Progressive Control Pathway of FMD (PCP‐FMD). Specifically, characterizing the existing virus strains (risk) provides the basis for the comprehensive risk‐based control plan which is the requisite criteria for Nigeria's transition to Stage 2 of PCP‐FMD, and for coordinated regional control of FMD.     

High‐fat high‐sucrose diet leads to dynamic structural and inflammatory alterations in the rat vastus lateralis muscle

The influence of obesity on muscle integrity is not well understood. The purpose of this study was to quantify structural and molecular changes in the rat vastus lateralis (VL) muscle as a function of a 12‐week obesity induction period and a subsequent adaptation period (additional 16‐weeks). Male Sprague–Dawley rats consumed a high‐fat, high‐sucrose (DIO, n = 40) diet, or a chow control‐diet (n = 14). At 12‐weeks, DIO rats were grouped as prone (DIO‐P, top 33% of weight change) or resistant (DIO‐R, bottom 33%). Animals were euthanized at 12‐ or 28‐weeks on the diet. At sacrifice, body composition was determined and VL muscles were collected. Intramuscular fat, fibrosis, and CD68+ cells were quantified histologically and relevant molecular markers were evaluated using RT‐qPCR. At 12‐ and 28‐weeks post‐obesity induction, DIO‐P rats had more mass and body fat than DIO‐R and chow rats (p < 0.05). DIO‐P and DIO‐R rats had similar losses in muscle mass, which were greater than those in chow rats (p < 0.05). mRNA levels for MAFbx/atrogin‐1 were reduced in DIO‐P and DIO‐R rats at 12‐ and 28‐weeks compared to chow rats (p < 0.05), while expression of MuRF1 was similar to chow values. DIO‐P rats demonstrated increased mRNA levels for pro‐inflammatory mediators, inflammatory cells, and fibrosis compared to DIO‐R and chow animals, despite having similar levels of intramuscular fat. The down‐regulation of MAFbx/atrogin‐1 may suggest onset of degenerative changes in VL muscle integrity of obese rats. DIO‐R animals exhibited fewer inflammatory changes compared to DIO‐P animals, suggesting a protective effect of obesity resistance on local inflammation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2069–2078, 2016.     

Challenges for Serology‐Based Characterization of Foot‐and‐Mouth Disease Outbreaks in Endemic Areas; Identification of Two Separate Lineages of Serotype O FMDV in Uganda in 2011

Control of foot‐and‐mouth disease (FMD) in Uganda by ring vaccination largely depends on costly trivalent vaccines, and use of monovalent vaccines could improve the cost effectiveness. This, however, requires application of highly specific diagnostic tests. This study investigated outbreaks of FMD in seven Ugandan districts, during 2011, using the PrioCHECK^® FMDV NS ELISA, solid‐phase blocking ELISAs (SPBEs) and virus neutralization tests (VNTs), together with virological analyses for characterization of the responsible viruses. Two hundred and eighteen (218) cattle and 23 goat sera as well as 82 oropharyngeal fluid/epithelial tissue samples were collected. Some 50% of the cattle and 17% of the goat sera were positive by the PrioCHECK^® FMDV NS ELISA, while SPBEs identified titres ≥80 for antibodies against serotype O FMD virus (FMDV) in 51% of the anti‐NSP positive cattle sera. However, 35% of the anti‐NSP positive cattle sera had SPBE titres ≥80 against multiple serotypes, primarily against serotypes O, SAT 1 and SAT 3. Comparison of SPBEs and VNTs for the detection of antibodies against serotypes O, SAT 1 and SAT 3 in 72 NSP positive cattle sera showed comparable results against serotype O (P = 0.181), while VNTs detected significantly fewer samples positive for antibodies against SAT 1 and SAT 3 than the SPBEs (P < 0.001). Detection of antibodies against serotype O was consistent with the isolation of serotype O FMDVs from 13 samples. Four of these viruses were sequenced and belonged to two distinct lineages within the East Africa‐2 (EA‐2) topotype, each differing from the currently used vaccine strain (EA‐1 topotype). The relationships of these lineages to other serotype O viruses in the Eastern Africa region are discussed. To enhance the control of FMD in Uganda, there is need to improve the specificity of the SAT‐SPBEs, perform vaccine matching and implement improved regional FMD control.     

6‐Gingerol‐rich fraction from Zingiber officinale ameliorates carbendazim‐induced endocrine disruption and toxicity in testes and epididymis of rats

This study evaluated the protective effects of 6‐gingerol‐rich fraction (6‐GRF) from Zingiber officinale on carbendazim (CBZ)‐induced reproductive toxicity in rats. Adult male rats were treated with either CBZ (50 mg/kg) alone or in combination with 6‐GRF (50, 100 and 200 mg/kg) for 14 consecutive days. Gas chromatography–mass spectrometry (GCMS) analysis revealed that 6‐GRF consists of ten bioactive chemical components with 6‐gingerol being the most abundant (30.76%). Administration of 6‐GRF significantly (p < .05) prevented CBZ‐mediated increase in absolute and relative testes weights as well as restored the sperm quantity and quality in the treated rats to near control. In testes and epididymis, 6‐GRF significantly abolished CBZ‐mediated increase in oxidative damage as well as augmented antioxidant enzymes activities and glutathione level in the treated rats. Moreover, CBZ administration alone significantly decreased plasma levels of testosterone, thyrotropin, triiodothyronine and tetraiodothyronine, whereas follicle‐stimulating hormone was significantly elevated without affecting luteinising hormone and prolactin levels when compared with the control. Conversely, 6‐GRF ameliorated the disruption in the hormonal levels and restored their levels to near normalcy in CBZ‐treated rats. Collectively, 6‐GRF inhibited the adverse effects of CBZ on the antioxidant defence systems, hormonal balance and histology of the testes and epididymis in rats.     

Characteristics of Foot‐and‐Mouth Disease Viral Strains Circulating at the Wildlife/livestock Interface of the Great Limpopo Transfrontier Conservation Area

Foot‐and‐mouth disease (FMD) inflicts severe economic losses within infected countries and is arguably the most important trade‐restricting livestock disease in the world. In southern Africa, infected African buffaloes (Syncerus caffer) are the major reservoir of the South African Territories (SAT) types of the virus. With the progressive expansion of transfrontier conservation areas (TFCAs), the risk of FMD outbreaks is expected to increase due to a higher probability of buffalo/livestock contacts. To investigate the dynamics of FMD within and around the Great Limpopo TFCA (GLTFCA), 5 herds of buffaloes were sampled in June 2010 to characterize circulating viruses in South Africa and Zimbabwe. Three SAT‐2 and three SAT‐3 viral strains were isolated in both countries, including one that was genetically linked with a recent SAT‐2 outbreak in Mozambique in 2011. In addition, two groups of unvaccinated cattle (n = 192) were serologically monitored for 1 year at the wildlife/livestock interface of Gonarezhou National Park (GNP) in Zimbabwe between April 2009 and January 2010, using the liquid‐phase blocking ELISA (LPBE) and a test for antibodies directed against non‐structural proteins (NSP). Neither clinical signs nor vaccination of cattle were reported during the study, yet a high proportion of the monitored cattle showed antibody responses against SAT‐3 and SAT‐1. Antibodies against NSP were also detected in 10% of the monitored cattle. The results of this study suggest that cattle grazing in areas adjacent to the GLTFCA can be infected by buffalo or other infected livestock and that cattle trade movements can act as efficient disseminators of FMD viruses to areas several hundred kilometres from the virus source. Current methods of surveillance of FMD at the GLTFCA interface seem insufficient to control for FMD emergence and dissemination and require urgent reassessment and regional coordination.