Enhanced egress of intracellular Eimeria tenella sporozoites by splenic lymphocytes from coccidian-infected chickens.

Egress, which describes the mechanism that some intracellular parasites use to exit from parasitophorous vacuoles and host cells, plays a very important role in the parasite life cycle and is central to Eimeria propagation and pathogenesis. Despite the importance of egress in the intracellular parasite's life cycle, very little information is known on this process compared to other steps, e.g., invasion. The present study was conducted to investigate the interplay between the host adaptive immune system and Eimeria egression. Splenic lymphocytes or soluble immune factors were incubated with parasite-infected host cells for 3 or 5 h, and the percentage of egress was calculated according to an established formula. Viability of egressed parasites and host cells was tested using trypan blue exclusion and annexin V and propidium iodide staining, respectively. We found that premature egression of sporozoites from Eimeria tenella-infected primary chicken kidney cells or from chicken peripheral blood mononuclear cells occurred when the cells were cocultured in vitro with spleen lymphocytes from E. tenella-infected chickens but not when they were cocultured with splenocytes from uninfected chickens. Eimeria-specific antibodies and cytokines (gamma interferon [IFN-γ], interleukin-2 [IL-2], and IL-15), derived from E. tenella-primed B and T lymphocytes, respectively, were capable of promoting premature egress of sporozoites from infected host cells. Both egressed parasites and host cells were viable, although the latter showed reduced reinvasion ability. These results suggest a novel, immune-mediated mechanism that the host exploits to interrupt the normal Eimeria life cycle in vivo and thereby block the release of mature parasites into the environment.

The lateral ligament is injured preferentially in posterolateral dislocation of the elbow joint.

AIMS: The purpose of this study was to evaluate the relationships between the degree of injury to the medial and lateral collateral ligaments (MCL and LCL) and associated fractures in patients with a posterolateral dislocation of the elbow, using CT and MRI. METHODS: We retrospectively reviewed 64 patients who presented between March 2009 and March 2018 with a posterolateral dislocation of the elbow and who underwent CT and MRI. CT revealed fractures of the radial head, coronoid process, and medial and lateral humeral epicondyles. MRI was used to identify contusion of the bone and collateral ligament injuries by tear, partial or complete tear. RESULTS: A total of 54 patients had a fracture; some had more than one. Radial head fractures were found in 25 patients and coronoid fractures in 42. Lateral and medial humeral epicondylar fractures were found in eight and six patients, respectively. Contusion of the capitellum was found in 43 patients and rupture of the LCL was seen in all patients (partial in eight and complete in 56), there was complete rupture of the MCL in 37 patients, partial rupture in 19 and eight had no evidence of rupture. The LCL tear did not significantly correlate with the presence of fracture, but the MCL rupture was complete in patients with a radial head fracture (p = 0.047) and there was significantly increased association in those without a coronoid fracture (p = 0.015). CONCLUSION: In posterolateral dislocation of the elbow, LCL ruptures are mostly complete, while the MCL exhibits various degrees of injury, which are significantly associated with the associated fractures. Cite this article: Bone Joint J 2020;102-B(2):227-231.

Engineering N-glycosylation mutations in IL-12 enhances sustained cytotoxic T lymphocyte responses for DNA immunization.

Interleukin-12 (IL-12), consisting of p40 and p35 subunits, produces both p70 heterodimer and free p40. p70 is essential for the induction of T-helper 1 (Th1) and cytotoxic T-cell (CTL) immunity, whereas p40 inhibits p70-mediated function. Here, we found that mutations introduced into N-glycosylation sites (N220 of murine p40 and N222 of human p40) reduced secretion of p40 but not p70. Co-immunization of N220 mutant mIL-12 gene with hepatitis C virus (HCV) E2 DNA significantly enhanced long-term E2-specific CD8+ T-cell response and protection against tumor challenge compared with that of wild type. Our results indicate that the ratio of p70 to p40 is important for generating sustained long-term cell-mediated immunity. Thus, the mutant IL-12 could be utilized for the development of DNA vaccines as an adjuvant for the generation of long-term memory T-cell responses.

Calcium Montmorillonite-Based Dietary Supplement Attenuates Necrotic Enteritis Induced by Eimeria maxima and Clostridium perfringens in Broilers.

Necrotic enteritis (NE) is a poultry disease caused by Clostridium perfringens and characterized by severe intestinal necrosis. The incidence of avian NE has been progressively increasing following the removal of antibiotics from poultry feed. We evaluated the effect of diets supplemented with the thermally-processed clays, calcium montmorillonite (CaMM) on clinical signs, immunopathology, and cytokine responses in broiler chickens using an experimental model of NE consisting of co-infection with Eimeria maxima and C. perfringens. In Trial 1, Ross/Ross chickens were fed from hatch with a normal basal diet or a CaMM-supplemented diet with or without a fermentable fiber, an organic acid, and/or a plant extract, and co-infected with E. maxima and C. perfringens under conditions simulating clinical infection in the field. Chickens fed a diet supplemented with CaMM plus a fermentable fiber and an organic acid had increased body weight gain, reduced gut lesions, and increased serum antibody levels to C. perfringens α-toxin and NetB toxin compared with chickens fed the basal diet alone. Levels of transcripts for interleukin-1ß (IL-1ß), IL-6, inducible nitric oxide synthase, and tumor necrosis factor-α superfamily-15 were significantly altered in the intestine and spleen of CaMM-supplemented chickens compared with unsupplemented controls (p<0.05). In Trial 2, Cobb/Cobb chickens were fed an unsupplemented diet or a diet supplemented with CaMM or Varium®, each with a fermentable fiber and an organic acid, and co-infected with E. maxima and C. perfringens under subclinical infection conditions. Compared with unsupplemented controls, broilers fed with CaMM plus a fermentable fiber and an organic acid had increased body weight gain, and reduced feed conversion ratio, mortality, and intestinal lesions, compared with chickens fed an unsupplemented diet (p<0.05). Dietary supplementation of broiler chickens with CaMM plus a fermentable fiber and an organic acid might be useful to control avian NE in the field.

Sudden coma from acute bilateral internal carotid artery territory infarction.

Six patients with bilateral internal carotid artery occlusion who presented with sudden loss of consciousness, quadriplegia, and initially intact brainstem reflexes are described. They soon lost brainstem reflexes and died within 3 days. The presumed causes of internal carotid artery occlusion were atherothrombosis in three patients and cardiogenic embolism in the others. This catastrophic stroke syndrome mimics severe brainstem stroke and has an extremely poor prognosis.

Effects of dietary plant-derived phytonutrients on the genome-wide profiles and coccidiosis resistance in the broiler chickens.

BACKGROUND: The present study was conducted to investigate the effects of dietary plant-derived phytonutrients, carvacrol, cinnamaldehyde and Capsicum oleoresin, on the translational regulation of genes associated with immunology, physiology and metabolism using high-throughput microarray analysis and in vivo disease challenge model of avian coccidiosis. METHODS: In this study, we used nutrigenomics technology to investigate the molecular and genetic mechanisms of dietary modulation of host innate immunity and metabolism by three phytonutrients. To validate their immunomodulatory effects in a disease model, young broiler chickens fed a standard diet supplemented with three phytochemicals (carvacrol, cinnamaldehyde, and Capsicum oleoresin) from one day post-hatch were orally challenged with E. acervulina. The body weight gain and fecal oocyst production were used to evaluate coccidiosis disease parameters. RESULTS: Analysis of global gene expression profiles of intestinal tissues from phytonutrient-fed birds indicated that Capsicum oleoresin induced the most gene changes compared to the control group where many of these genes were associated with those of metabolism and immunity. The most reliable network induced by dietary cinnamaldehyde treatment was related with the functions of antigen presentation, humoral immune response, and inflammatory disease. Furthermore, dietary supplementation with these phytonutrients significantly protected broiler chickens against live coccidiosis challenge infection based on body weight and parasite fecundity. CONCLUSIONS: The results of this study provide clear evidence to support the idea that plant-derived phytochemicals possess immune-enhancing properties in chickens and these new findings create a new possibility to develop effective drug-free alternative strategies for disease control for poultry infectious diseases.

Membrane-bound proteinase 3 and PAR2 mediate phagocytosis of non-opsonized bacteria in human neutrophils.

The molecular mechanisms underlying the non-opsonic phagocytosis of bacteria by neutrophils are poorly understood. We previously reported the efficient uptake of Streptococcus sanguinis by human neutrophils in the absence of opsonins. To characterize the phagocytosis receptor, protein lysates from neutrophils and HL-60 cells were subjected to affinity chromatography using epoxy beads coated with S. sanguinis. Denaturing electrophoresis of the eluted proteins and subsequent mass spectrometry revealed that one of the proteins eluted from neutrophils was proteinase 3 (PR3). Enzymatic cleavage of the glycosylphosphatidylinositol linker of NB1, a co-receptor for membrane-bound PR3 (mPR3), significantly reduced the phagocytosis of S. sanguinis. In addition, the neutralization of mPR3 with antibody reduced both binding and phagocytosis of S. sanguinis. Treatment of neutrophils with a serine proteinase inhibitor indicated that protease activity is required for phagocytosis. Thus, we studied whether protease-activated receptor 2 (PAR2) is involved in signal transmission from mPR3 during this process. Indeed, neutralizing antibodies against PAR2 inhibited phagocytosis and S. sanguinis-induced calcium mobilization desensitized PAR2. Furthermore, the phagocytosis of S. sanguinis and the concomitant activation of Rho family GTPases were inhibited by the intracellular calcium chelator, BAPTA-AM. Collectively, mPR3 acts as a non-opsonic phagocytosis receptor for bacteria probably by activating PAR2 in neutrophils.

Cortisol, estradiol-17ß, and progesterone secretion within the first hour after awakening in women with regular menstrual cycles.

Cortisol concentration in both serum and saliva sharply increases and reaches a peak within the first hour after waking in the morning. This phenomenon is known as the cortisol awakening response (CAR) and is used as an index of hypothalamus-pituitary-adrenal (HPA) axis function. We examined whether ovarian steroid concentrations increased after awakening as with the CAR in the HPA axis. To do this, cortisol, estradiol-17ß (E(2)), and progesterone (P(4)) concentrations were determined in saliva samples collected immediately upon awakening and 30 and 60 min after awakening in women with regular menstrual cycles and postmenopausal women. We found that both E(2) and P(4) concentrations increased during the post-awakening period in women with regular menstrual cycles, but these phenomena were not seen in any postmenopausal women. The area under the E(2) and P(4) curve from the time interval immediately after awakening to 60 min after awakening (i.e. E(2)auc and P(4)auc) in women with regular menstrual cycles were greater than those in the postmenopausal women. E(2) and P(4) secretory activity during the post-awakening period was influenced by the phase of the menstrual cycle. E(2)auc in the peri-ovulatory phase and P(4)auc in the early to mid-luteal phase were greater than in the menstrual phase. Meanwhile, cortisol secretory activity during the post-awakening period was not influenced by menstrual status or the phase of menstrual cycle. These findings indicate that, as with the CAR in the HPA axis function, ovarian steroidogenic activity increased after awakening and is closely associated with menstrual status and phase of menstrual cycle.

Chemoattractant-mediated Rap1 activation requires GPCR/G proteins.

Rap1 is rapidly activated upon chemoattractant stimulation and plays an important role in cell adhesion and cytoskeletal reorganization during chemotaxis. Here, we demonstrate that G-protein coupled receptors and G-proteins are essential for chemoattractant-mediated Rap1 activation in Dictyostelium. The rapid Rap1 activation upon cAMP chemoattractant stimulation was absent in cells lacking chemoattractant cAMP receptors cAR1/cAR3 or a subunit of the heterotrimeric G-protein complex Gα2. Loss of guanylyl cyclases GCA/SGC or a cGMP-binding protein GbpC exhibited no effect on Rap1 activation kinetics. These results suggest that Rap1, a key regulator for the regulation of cytoskeletal reorganization during cell movement, is activated through the G-protein coupled receptors cAR1/cAR3 and Gα2 proteins in a way independent of the cGMP signaling pathway.

Expressional and mutational analysis of pro-apoptotic Bcl-2 member PUMA in hepatocellular carcinomas.

Deregulation of apoptosis is involved in mechanisms of cancer development. PUMA is a pro-apoptotic member of the Bcl-2 family and mediates p53-dependent and -independent apoptosis. The aim of this study was to investigate whether alterations of PUMA protein expression and somatic mutations of PUMA gene are characteristics of human hepatocellular carcinoma (HCC). We analyzed expression of PUMA protein in 20 HCCs using immunohistochemistry. Also, we analyzed mutation of the Bcl-2 homology 3 (BH3) domain of PUMA gene, which is an important domain in apoptosis function of PUMA by single-strand conformation polymorphism (SSCP) in 69 HCCs. PUMA protein expression was detected in both HCC cells and non-tumor hepatocytes in all of the 20 HCCs. In 10 of these HCCs, cancer cells showed higher PUMA expression than non-tumor (cirrhotic) hepatocytes of the same patients; whereas in the remaining 10, cancer cells and non-tumor hepatocytes showed similar levels. Mutational analysis revealed no PUMA BH3 domain mutation in the 69 HCCs, suggesting that PUMA BH3 domain mutation is not a direct target of inactivation in hepatocellular cancer development. The increased expression of PUMA in malignant hepatocellular cells relative to that in non-tumor hepatocytes suggests that PUMA expression may play a role in HCC development.

Transport of leukotriene C4 by a cysteine-less multidrug resistance protein 1 (MRP1).

Overexpression of multidrug resistance protein 1 (MRP1), an ATP-binding cassette protein, causes multidrug resistance. We developed a functional cysteine-less version of MRP1 that provides a framework for detailed biochemical and biophysical studies. The 18 Cys residues of a truncated MRP1 (tMRP1; lacking the first multispanning transmembrane domain) were replaced with Ala to generate Cys-less tMRP1 (CL tMRP1). CL tMRP1 expressed in Saccharomyces cerevisiae membranes displayed high-affinity ATP-dependent transport of the MRP1 substrate leukotriene C4. Compared with full-length MRP1, the K m for leukotriene C4 transport by CL tMRP1 was increased approximately 3-fold, while V max was not affected. Thus a functional CL tMRP1 can be expressed using a low-cost and rapid-generation yeast expression system. This Cys-less protein can be used for biochemical, spectroscopic and structural studies to elucidate the mechanism of drug transport by MRP1.

Sodium dodecyl sulfate-capillary gel electrophoresis of polyethylene glycolylated interferon alpha.

Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using a hydrophilic replaceable polymer network matrix was applied to characterize the polyethylene glycol(PEG)ylated interferon alpha (PEG-IFN). The SDS-CGE method resulted in a clearer resolution in both the PEG-IFN species and the native IFN species. The distribution profile of PEGylation determined by SDS-CGE was consistent with that obtained by SDS-polyacrylamide gel electrophoresis (PAGE) with Coomassie blue or barium iodide staining. The result was also compared using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. SDS-CGE was also useful for monitoring the PEGylation reaction to optimize the reaction conditions, such as reaction molar ratio. This study shows the potential of SDS-CGE as a new method for characterizing the PEGylated proteins with advantages of speed, minimal sample consumption and high resolution.