Production of Aujeszky's disease (pseudorabies) virus envelope glycoproteins gB and gC as recombinant polyhedra in baculovirus-infected silkworm larvae

The expression of viral antigens in baculovirus-infected insect cells is often ineffective. As an alternative approach, therefore, we developed the recombinant polyhedra technology, which is an efficient strategy for the production of viral subunit vaccine. Here, we report a strategy for the large-scale production of a pseudorabies virus (PRV) gB or gC in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the epitope regions of PRV gB or heparin-binding domains of PRV gC. Recombinant BmNPV-PRV-gB or BmNPV-PRV-gC-infected silkworm larvae expressed native polyhedrin and fusion protein that was detected using both anti-polyhedrin and anti-PRV gB or anti-PRV-gC antibodies. Electron and confocal microscopy demonstrated that the recombinant polyhedra contained both the fusion protein and native polyhedrin with a normal morphology and that the recombinant polyhedra contained PRV gB or gC. The yield of gB or gC antigen produced in BmNPV-PRV-gB or BmNPV-PRV-gC-infected silkworm larvae reached 0.69 or 0.46 mg per larva, respectively, at 6 days post-infection. These results demonstrate that the recombinant polyhedra strategy can be used for the large-scale production of PRV gB or gC antigen.     

Abnormal Calcium Handling and Exaggerated Cardiac Dysfunction in Mice with Defective Vitamin D Signaling

Aim

Altered vitamin D signaling is associated with cardiac dysfunction, but the pathogenic mechanism is not clearly understood. We examine the mechanism and the role of vitamin D signaling in the development of cardiac dysfunction.

Methods and Results

We analyzed 1α-hydroxylase (1α-OHase) knockout (1α-OHase^−/−) mice, which lack 1α-OH enzymes that convert the inactive form to hormonally active form of vitamin D. 1α-OHase^−/− mice showed modest cardiac hypertrophy at baseline. Induction of pressure overload by transverse aortic constriction (TAC) demonstrated exaggerated cardiac dysfunction in 1α-OHase^−/− mice compared to their WT littermates with a significant increase in fibrosis and expression of inflammatory cytokines. Analysis of calcium (Ca²⁺) transient demonstrated profound Ca²⁺ handling abnormalities in 1α-OHase^−/− mouse cardiomyocytes (CMs), and treatment with paricalcitol (PC), an activated vitamin D₃ analog, significantly attenuated defective Ca²⁺ handling in 1α-OHase^−/− CMs. We further delineated the effect of vitamin D deficiency condition to TAC by first correcting the vitamin D deficiency in 1α-OHase^−/− mice, followed then by either a daily maintenance dose of vitamin D or vehicle (to achieve vitamin D deficiency) at the time of sham or TAC. In mice treated with vitamin D, there was a significant attenuation of TAC-induced cardiac hypertrophy, interstitial fibrosis, inflammatory markers, Ca²⁺ handling abnormalities and cardiac function compared to the vehicle treated animals.

Conclusions

Our results provide insight into the mechanism of cardiac dysfunction, which is associated with severely defective Ca²⁺ handling and defective vitamin D signaling in 1α-OHase^−/− mice.     

Gliosis in the Mice Hippocampus Without Neuronal Death After Systemic Administration of High Dosage of Tetanus Toxin

Tetanus toxin (TeT), an exotoxin, has been studied to cause tetanus in mammalian brains, and it can block the release of some neurotransmitters and affect seizure propagation. In the present study, we investigated neuronal damage/death and glial changes in the mouse hippocampus after systemic administration (intraperitoneal injection) of TeT 10 and 100 ng/kg. In both the 10 and 100 ng/kg TeT-treated groups, no neuronal death occurred in any subregions of the mouse hippocampus until 24 h post-treatment; however, there were changes in glia in the hippocampus depending on time course and dosage. The morphology of GFAP-immunoreactive astrocytes and Iba-1-immunoreactive microglia was apparently changed in the 100 ng/kg TeT treated-group compared to the 10 ng/kg TeT treated-group. In the 100 ng/kg TeT treated-group, they were increased in size and their immunoreactivity was distinctively increased from 12 h post-treatment. We also found that their protein levels were increased in the hippocampus at 12 h post-treatment of 100 ng/kg TeT. In conclusion, these results indicate that the systemic administration of 100 ng/kg TeT induced a distinctive microglia changes in the mouse hippocampus without any neuronal death/damage.     

Additive Effect between IL-13 Polymorphism and Cesarean Section Delivery/Prenatal Antibiotics Use on Atopic Dermatitis: A Birth Cohort Study (COCOA)

Background

Although cesarean delivery and prenatal exposure to antibiotics are likely to affect the gut microbiome in infancy, their effect on the development of atopic dermatitis (AD) in infancy is unclear. The influence of individual genotypes on these relationships is also unclear. To evaluate with a prospective birth cohort study whether cesarean section, prenatal exposure to antibiotics, and susceptible genotypes act additively to promote the development of AD in infancy.

Methods

The Cohort for Childhood of Asthma and Allergic Diseases (COCOA) was selected from the general Korean population. A pediatric allergist assessed 412 infants for the presence of AD at 1 year of age. Their cord blood DNA was subjected to interleukin (IL)-13 (rs20541) and cluster-of-differentiation (CD)14 (rs2569190) genotype analysis.

Results

The combination of cesarean delivery and prenatal exposure to antibiotics associated significantly and positively with AD (adjusted odds ratio, 5.70; 95% CI, 1.19–27.3). The association between cesarean delivery and AD was significantly modified by parental history of allergic diseases or risk-associated IL-13 (rs20541) and CD14 (rs2569190) genotypes. There was a trend of interaction between IL-13 (rs20541) and delivery mode with respect to the subsequent risk of AD. (P for interaction = 0.039) Infants who were exposed prenatally to antibiotics and were born by cesarean delivery had a lower total microbiota diversity in stool samples at 6 months of age than the control group. As the number of these risk factors increased, the AD risk rose (trend p<0.05).

Conclusion

Cesarean delivery and prenatal antibiotic exposure may affect the gut microbiota, which may in turn influence the risk of AD in infants. These relationships may be shaped by the genetic predisposition.     

Prognostic factors in primary diffuse large B-cell lymphoma of adrenal gland treated with rituximab- CHOP chemotherapy from the Consortium for Improving Survival of Lymphoma (CISL)

ABSTRACT: BACKGROUND: The objective of this study was to identify prognostic factors for survival in patients with primary diffuse large B-cell lymphoma (DLBCL) of the adrenal gland. METHODS: Thirty one patients diagnosed with primary adrenal DLBCL from 14 Korean institutions and treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) were analyzed. RESULTS: Complete remission (CR) and overall response rate after R-CHOP chemotherapy were 54.8% and 87.0%. The 2-year estimates of overall survival (OS) and progression-free survival (PFS) were 68.3% and 51.1%. In patients achieving CR, significant prolongations of OS (P = 0.029) and PFS (P = 0.005) were observed. Ann Arbor stage had no influence on OS. There was no significant difference in OS between patients with unilateral involvement of adrenal gland and those with bilateral involvement. When staging was modified to include bilateral adrenal involvement as one extranodal site, early stage (I or II) significantly correlated with longer OS (P = 0.021) and PFS (P <0.001). CONCLUSIONS: Contrary to prior reports, our data suggests that outcomes of primary adrenal DLBCL are encouraging using a regimen of R-CHOP, and that achieving CR after R-CHOP is predictive of survival. Likewise, our modified staging system may have prognostic value.     

Enhancement of centelloside production from cultured plants of Centella asiatica by combination of thidiazuron and methyl jasmonate

In order to produce centellosides from whole plant cultures of Centella asiatica (L.) Urban, we evaluated the synergistic effects of thidiazuron (TDZ) and methyl jasmonate (MJ) on whole plant growth and centelloside production. After 4 weeks of treatment with 0.025 mg/L of TDZ coupled with 0.1 mM MJ, the production of madecassoside and asiaticoside from whole plant cultures was estimated to be 2.40- and 2.44-fold, respectively, above that of MJ elicitation alone. When whole plants were treated with a growth regulator and an elicitor, the growth of whole plants, as compared to the controls, did not differ. Additionally, total phytosyterol content in the leaves of whole plants co-treated with MJ and TDZ was 1.08-fold greater than those of MJ alone. These results demonstrate that combined treatments not only stimulate the accumulation of centellosides in the leaves but also inhibit the reduction of phytosterol levels caused by MJ elicitation.     

Alternative chaperone machinery may compensate for calreticulin/calnexin deficiency in Caenorhabditis elegans

Proper folding and maintenance of the native structure are central to protein function and are assisted by a family of proteins called chaperones. Calreticulin and calnexin are ER resident chaperones well conserved from worm to human. Calreticulin/calnexin knock-out mice exhibit a severe phenotype, whereas in Caenorhabditis elegans, calreticulin [crt-1(jh101)]- and calnexin [cnx-1(nr2009)]-null mutant worms exhibit only a mild phenotype, suggesting the possible existence of alternative chaperone machinery that can compensate for the deficiency of calreticulin and/or calnexin. In order to rapidly identify the compensatory chaperone components involved in this process, we analyzed the proteome of crt-1(jh101) mutants and [crt-1(jh101);cnx-1(nr2009)] double mutants. When grown at 20 degrees C, we found that five proteins were up-regulated and two proteins were down-regulated in crt-1(jh101) mutants; nine proteins were up-regulated and five proteins were down-regulated in [crt-1(jh101);cnx-1(nr2009)] double mutants. In addition, elevation of the cultivation temperature to 25 degrees C, which is still permissive to growth but causes specific defects in mutants, led to the identification of several additional proteins. Interestingly, the consistent increment of heat shock protein-70 family members (hsp70) together with protein disulfide isomerase (PDI) at all the examined conditions suggests the possible compensatory function imparted by hsp70 and PDI family members in the absence of calreticulin and/or calnexin.     

Redox treatment ameliorates diabetes mellitus-induced skin flap necrosis via inhibiting apoptosis and promoting neoangiogenesis

Intractable wound healing is the habitual problem of diabetes mellitus. High blood glucose limits wound healing by interrupting inflammatory responses and inhibiting neoangiogenesis. Oxidative stress is commonly thought to be a major pathogenic cause of diabetic complications. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one, EDV) is a free radical scavenger which suppress oxidative stress. This study investigates whether EDV can reduce oxidative stress in wound healing HaCaT/human dermal fibroblasts cells (HDFs) in vitro and in vivo animal model. Cell viability and wound healing assays, FACS flow cytometry, and Hoechst 33342 staining were performed to confirm apoptosis and cytotoxicity in H₂O₂ and EDV-treated HaCaT and HDFs. A streptozotocin-induced hyperglycemic animal model was made in adult C57BL6 mice. Full-thickness skin flap was made on dorsomedial back and re-sutured to evaluate the wound healing process. EDV was delivered slowly in the skin flap with degradable fibrin glue. The flap was monitored and analyzed on postoperative days 1, 3, and 5. CD31/DAPI staining was done to detect newly formed blood vessels. The expression levels of NF-κB, bcl-2, NOX3, and STAT3 proteins in C57BL6 mouse tissues were also examined. The wound healing process in hyper- and normoglycemic mice showed a difference in protein expression, especially in oxidative stress management and angiogenesis. Exogenous H₂O₂ reduced cell viability in a proportion to the concentration via apoptosis. EDV protected HaCaT cells and HDFs from H₂O₂ induced reactive oxygen species cell damage and apoptosis. In the mouse model, EDV with fibrin resulted in less necrotic areas and increased angiogenesis on postoperative day 5, compared to sham-treated mice. Our results indicate that EDV could protect H₂O₂-induced cellular injury via inhibiting early apoptosis and inflammation and also increasing angiogenesis. EDV might be valuable in the treatment of diabetic wounds that oxidative stress has been implicated.