Comparison of acellular pertussis-based combination vaccines by Japanese control tests for toxicities and laboratory models for local reaction

Two batches each of diphtheria–tetanus–acellular pertussis vaccine (DTaP) and that combined with inactivated polio vaccine purchased from the U.S.A., European and Asian markets were compared with Japanese DTaPs by Japanese control tests for DTaP and laboratory models for local reaction. All the imported vaccines met Japanese criteria for toxicities of acellular pertussis vaccine except for the toxicity to mouse weight gain (body weight decreasing (BWD) toxicity). When injecting into mouse footpad, rabbit back skin and mouse quadriceps muscle, the imported vaccines induced much severer inflammation and tissue injury comparing to Japanese DTaPs irrespective of animal species, injection site and injection volume suggesting that these vaccines may induce stronger local reactogenicity.     

Current prospects for mRNA gene delivery

Replication-deficient viruses have been used most successfully in the field of gene therapy because of their high transfection efficiency. However, the risk of insertional mutagenesis and induction of unwanted immune responses remains still critical for their safe application. On the other hand, nonviral vectors have been intensively investigated for plasmid DNA (pDNA) delivery as a safer alternative although their gene transfer efficiency is still many folds lower than for viral vectors, which has been predominately attributed to the insufficient transport of pDNA into the nucleus. Instead of pDNA, messenger RNA (mRNA) has recently emerged as an attractive and promising alternative in the nonviral gene delivery field. This strategy combines several advantages compared to pDNA: (i) the nuclear membrane, which is a major obstacle for pDNA, can be avoided because mRNA exerts its function in the cytoplasm; (ii) the risk of insertional mutagenesis can be excluded; (iii) the determination and use of an efficient promoter is omitted; (iv) repeated application is possible; (v) mRNA is also effective in non-dividing cells, and (vi) vector-induced immunogenicity may be avoidable. In this review, we summarize recent improvements of mRNA gene delivery and discuss its opportunities for the usage in gene therapy.     

Generation of monoclonal antibody that distinguishes PrP from PrP and neutralizes prion infectivity

To establish PrP^Sc-specific mAbs, we immunized Prnp^-/- mice with PrP^Sc purified from prion-infected mice. Using this approach, we obtained mAb 6H10, which reacted with PrP^Sc treated with proteinase K, but not with PrP^Sc pretreated with more than 3 M GdnHCl. In contrast, reactivity of pan-PrP mAbs increased with increasing concentrations of GdnHCl used for pretreatment of PrP^Sc. In histoblot analysis, mAb 6H10 showed a positive reaction on a non-denatured histoblot but reactivity was lower when the histoblot was pretreated by autoclaving. Epitope analysis suggested that the extreme C-terminus of PrP is likely to be part of the epitope for mAb 6H10. MAb 6H10 immunoprecipitated PrP^Sc from brains of mice, sheep, and cattle infected with prions. Furthermore, pretreatment of purified PrP^Sc with mAb 6H10 reduced the infectious titer more than 1 log. Taken together, these results suggest that mAb 6H10 recognizes a conformational epitope on PrP^Sc that is related to prion infectivity.     

Increased O<Subscript>2</Subscript> consumption in excitation–contraction coupling in hypertrophied rat heart slices related to increased Na<Superscript>+</Superscript>–Ca<Superscript>2+</Superscript> exchange activity

The goal of our study was to evaluate the origin of the increased O₂ consumption in electrically stimulated left ventricular slices of isoproterenol-induced hypertrophied rat hearts with normal left ventricular pressure. O₂ consumption per minute (mVO₂) of mechanically unloaded left ventricular slices was measured in the absence and presence of 1-Hz field stimulation. Basal metabolic mVO₂, i.e., mVO₂ without electrical stimulation, was significantly smaller, but mVO₂ for the total Ca²⁺ handling in excitation–contraction coupling (E–C coupling mVO₂), i.e., delta mVO₂ (=mVO₂ with stimulation − mVO₂ without stimulation), was significantly larger in the hypertrophied heart. Furthermore, the fraction of E–C coupling mVO₂ was markedly altered in the hypertrophied heart. Namely, mVO₂ consumed by sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2) was depressed by 40%; mVO₂ consumed by the Na⁺/K⁺-ATPase (NKA)-Na⁺/Ca²⁺ exchange (NCX) coupling was increased by 100%. The depressed mVO₂ consumption by SERCA2 was supported by lower protein expressions of phosphorylated-Ser¹⁶ phospholamban and SERCA2. The increase in NKA–NCX coupling mVO₂ was supported by marked augmentation of NCX current. However, the increase in NCX current was not due to the increase in NCX1 protein expression, but was attributable to attenuation of the intrinsic inactivation mechanisms. The present results demonstrated that the altered origin of the increased E–C coupling mVO₂ in hypertrophy was derived from decreased SERCA2 activity (1ATP: 2Ca²⁺) and increased NCX activity coupled to NKA activity (1ATP: Ca²⁺). Taken together, we conclude that the energetically less efficient Ca²⁺ extrusion pathway evenly contributes to Ca²⁺ handling in E–C coupling in the present hypertrophy model.     

Expression and function of toll-like receptors in human basophils

We investigated the expression and function of a panel of Toll-like receptors (TLRs) in human basophils. Basophil preparations constitutively expressed TLR2, TLR4, TLR9 and TLR10 mRNAs (TLR4 > TLR2 > TLR9, TLR10). Although TLR mRNA expression in basophils was generally less prominent compared with those in neutrophils and monocytes, basophils expressed significantly higher levels of TLR2 and TLR4 mRNA than eosinophils. Various TLR ligands (Pam3Cys-Ser-Lys4, poly I:C, lipopolysaccharide, R-848, CpG DNA) were tested, but none affected the expression level of adhesion molecule CD11b or the viability of freshly purified basophils. On the other hand, when basophils were pretreated with interferon-gamma before stimulation with TLR ligands, only the TLR4 ligand, lipopolysaccharide, upregulated CD11b expression. However, the surface levels of TLR2 and TLR4 on the interferon-gamma-treated basophils showed no obvious changes. These results suggest that TLR4 on basophils may be involved in the pathogenesis of infection-induced exacerbation of allergic inflammation by modulating basophil functions.     

Advantages for transdermal over oral oxybutynin to treat overactive bladder: Muscarinic receptor binding, plasma drug concentration, and salivary secretion

To clarify pharmacological usefulness of transdermal oxybutynin in the therapy of overactive bladder, we have characterized muscarinic receptor binding in rat tissues with measurement of plasma concentrations of oxybutynin and its metabolite N-desethyl-oxybutynin (DEOB) and salivation after transdermal oxybutynin compared with oral route. At 1 and 3 h after oral administration of oxybutynin, there was a significant increase in apparent dissociation constant (Kd) for specific [N-methyl-3H]scopolamine ([3H]NMS) binding in the rat bladder, submaxillary gland, heart, and colon compared with control values. Concomitantly, submaxillary gland and heart showed a significant decrease in maximal number of binding sites (Bmax) for [3H]NMS binding, which lasted until 24 h. Transdermal application of oxybutynin caused dose-dependent increases in Kd values for specific [3H]NMS binding in rat tissues. The increment of Kd values by transdermal oxybutynin was dependent on the application time. Plasma concentrations of oxybutynin and DEOB peaked at 1 h after oral oxybutynin. In contrast, plasma concentrations of oxybutynin increased slowly, depending on the transdermal application time of this drug until 12 h. Suppression of pilocarpine-induced salivation in rats due to transdermal oxybutynin was significantly weaker and more reversible than that by oral oxybutynin, which abolished salivary secretion. The present study has shown that transdermal oxybutynin binds significantly to rat bladder muscarinic receptors without producing both long-lasting occupation of exocrine receptors and cessation of cholinergic salivation evoked by oral oxybutynin. Thus, the present study provides further pharmacological basis for advantage of transdermal over oral oxybutynin in the therapy of overactive bladder.     

Hemolytic disease of the newborn due to anti-Di: a case study and review of the literature

BACKGROUND: The severity of hemolytic disease of the newborn (HDN) due to Diego(b) (Di(b)) mismatch ranges from no symptoms to severe jaundice that requires exchange transfusion (ET). The clinical significance of anti-Di(b) is incompletely recognized. CASE REPORT: A male newborn, referred with jaundice, was revealed to have HDN due to Di(b) mismatch and was treated successfully with phototherapy and high-dose intravenous gamma globulin (IVGG). STUDY DESIGN AND METHODS: The literature of HDN caused by Di(b) mismatch was reviewed. The cases were classified into three groups according to their severity: the mildest needed no therapy (NO), the moderate group received phototherapy alone (PHOTO), and the most severe was treated with ET and/or high-dose IVGG therapy plus phototherapy (ET/IVGG). RESULTS: Among 27 cases of HDN due to Di(b) reported to date, 10, 6, and 11 cases required NO, PHOTO, and ET/IVGG, respectively. A significant correlation (p < 0.01) was found between the maternal anti-Di(b) titer and the severity of the disease when the ET/IVGG group was compared with the NO group. All mothers of the group that needed ET/IVGG had an anti-Di(b) titer of 64 or greater. CONCLUSION: A maternal high titer (> or =64) of anti-Di(b) is associated with a higher risk of severe hyperbilirubinemia for mismatched newborns.     

Folate antagonist,methotrexate induces neuronal differentiation of human embryonic stem cells transplanted into nude mouse retina

Transplanted embryonic stem (ES) cells can be integrated into the retinas of adult mice as well-differentiated neuroretinal cells. However, the transplanted ES cells also have a tumorigenic activity as they have the ability for multipotent differentiation to various types of tissues. In the present study, human ES (hES) cells were transplanted into adult nude mouse retinas by intravitreal injections 20 h after intravitreal N-methyl-d-aspartate (NMDA) administration. After the transplantation of hES cells, the folate antagonist, methotrexate (MTX) was administrated in order to control the differentiation of the transplanted hES cells. Neuronal differentiation and teratogenic potential of hES cells were examined immunohistochemically 5 weeks after transplantation. The proliferative activity of transplanted cells was determined by both the mitotic index and the Ki-67 proliferative index. Disappearance of Oct-4-positive hES cells showing undifferentiated morphology was observed after intraperitoneal MTX treatment daily, for 15 days. Decreased mitotic and Ki-67 proliferative indices, and increased neuronal differentiation were detected in the surviving hES cells after the MTX treatment. These results suggest two important effects of intraperitoneal MTX treatment for hES cells transplanted into nude mouse retina: (1) MTX treatment following transplantation induces neuronal differentiation, and (2) MTX decreases proliferative activity and tumorigenic potential.     

Involvement of p21waf1/cip1 expression in the cytotoxicity of the potent histone deacetylase inhibitor spiruchostatin B towards susceptible NALM-6 human B cell leukemia cells

Spiruchostatin B (SP-B) is a potent histone deacetylase (HDAC) inhibitor that has potential for the chemotherapy of leukemia. The aim of this study was to study the susceptibility of human leukemia cell lines to SP-B. We found that NALM-6 human B cell leukemia cells are the most susceptible to SP-B. There was a low correlation between the expression of HDAC1 mRNA and HDI susceptibility of leukemia cells. NALM-6 has higher endogenous p21waf1/cip1 mRNA expression than other leukemia cells. SP-B-induced cytotoxicity was mediated by induction of histone acetylation via inhibition of HDACs, and this effect of SP-B was associated with apoptosis, which was mediated by caspase activation in NALM-6 cells. SP-B time-dependently increased the size of the sub-G1 (apoptotic) peak, and this effect correlated with SP-B induction of cellular apoptotic features such as changes in nuclear morphology. SP-B significantly increased p21waf1/cip1 expression prior to induction of apoptosis. In conclusion, NALM-6 cells, which have a higher expression of p21waf1/cip1 mRNA than other leukemia cell lines, were susceptible to SP-B-induced cytotoxicity that resulted in induction of apoptosis. Our findings may be useful when establishing a therapeutic strategy based on SP-B.