Antidiuretic effect of desmopressin chimera agglomerates by nasal administration in rats

In the present study, a nasal powder of the antidiuretic peptide desmopressin (DDAVP) formulated as chimera agglomerates was studied to improve drug bioavailability and provide a flexible drug product. Firstly, DDAVP was spray-dried along with mannitol and lecithin to produce primary microparticles capable of instantaneous dissolution in water. The chimera agglomerates were spontaneously formed by mechanically vibrating the microparticles on two stacked sieves. Agglomerate formation and strength were favored by the presence of lecithin. Drug content and dissolution rate remained unmodified after agglomeration. However, owing to the agglomerate larger size, powder flowability was greatly improved in comparison with the original microparticles, allowing accurate powder dosing into the nasal delivery device. DDAVP in vitro permeation across excised rabbit nasal mucosa from the agglomerates was significantly higher than that obtained from a commercial liquid nasal spray.     

Enhancing the utility of a prM/E-expressing chimeric vaccine for Japanese encephalitis by addition of the JEV NS1 gene

Recently, we demonstrated that a single-cycle West Nile virus (WNV) named RepliVAX WN could be used to produce a chimeric Japanese encephalitis (JE) vaccine (RepliVAX JE) by replacing the WNV prM/E genes with those of JEV. Here, we tested if replacement of WNV NS1 gene in RepliVAX JE with that of JEV (producing TripliVAX JE) could produce a superior vaccine. TripliVAX JE elicited higher anti-E immunity and displayed better efficacy in mice than RepliVAX JE. Furthermore, TripliVAX JE displayed reduced immune interference caused by pre-existing anti-NS1 immunity. Thus, we propose prM/E/NS1 chimerization as a new strategy for flavivirus vaccine development.     

Immunogenicity of candidate chimeric DNA vaccine against tuberculosis and leishmaniasis

Mycobacterium tuberculosis and Leishmania donovani are important intracellular pathogens, especially in Indian context. In India and other South East Asian countries, both these infections are highly endemic and in about 20% cases co-infection of these pathogens is reported. For both these pathogens cell mediated immunity plays most important role. The available treatment of these infections is either prolonged or cumbersome or it is ineffective in controlling the outbreaks and spread. Therefore, potentiation of a common host defense mechanism can be used to prevent both the infections simultaneously. In this study we have developed a novel chimeric DNA vaccine candidate comprising the esat-6 gene of M. tuberculosis and kinesin motor domain gene of L. donovani. After developing this novel chimera, its immunogenicity was studied in mouse model. The immune response was compared with individual constructs of esat-6 and kinesin motor domain. The results showed that immunization with chimeric DNA vaccine construct resulted in stronger IFN-γ and IL-2 response against kinesin (3012 ± 102 and 367.5 ± 8.92 pg/ml) and ESAT-6 (1334 ± 46.5 and 245.1 ± 7.72 pg/ml) in comparison to the individual vaccine constructs. The reciprocal immune response (IFN-γ and IL-2) against individual construct was lower (kinesin motor domain: 1788 ± 36.48 and 341.8 ± 9.801 pg/ml and ESAT-6: 867.0 ± 47.23 and 170.8 ± 4.578 pg/ml, respectively). The results also suggest that using the chimeric construct both proteins yielded a reciprocal adjuvant affect over each other as the IFN-γ production against chimera vaccination is statistically significant (p < 0.0001) than individual construct vaccination. From this pilot study we could envisage that the chimeric DNA vaccine construct may offer an attractive strategy in controlling co-infection of leishmaniasis and tuberculosis and have important implication in future vaccine design.     

Creation of chimeric mutant axolotls: a model to study early embryonic heart development in Mexican axolotls

The Mexican axolotl (Ambystoma mexicanum) provides an excellent model for studying heart development since it carries a cardiac lethal mutation in gene c that results in failure of contraction of mutant embryonic myocardium. In cardiac mutant axolotls (c/c) the hearts do not beat, apparently because of an absence of organized myofibrils. To date, there has been no way to analyze the genotypes of embryos from heterozygous spawnings (+/c×+/c) until stage 35 when the normal (+/c or +/+) embryos first begin to have beating hearts; mutant (c/c) embryos fail to develop normal heartbeats. In the present study, we created chimeric axolotls by using microsurgical techniques. The general approach was to transect tailbud embryos and join the anterior and posterior halves of two different individuals. The chimeric axolotl is composed of a normal head and heart region (+/+), permitting survival and a mutant body containing mutant gonads (c/c) that permits the production of c/c mutant offspring: 100% c/c offspring were obtained by mating c/c chimeras (c/c×c/c). The mutant phenotypes were confirmed by the absence of beating hearts and death at stage 41 in 100% of the embryos. Examination of the mutant hearts with electron microscopy and comfocal microscopy after immunofluorescent staining for tropomyosin showed identical images to those described previously in naturally-occurring c/c mutant axolotls (i.e., lacking organized sarcomeric myofibrils). These ”c/c chimeric” axolotls provide a useful and unique way to investigate early embryonic heart development in cardiac mutant Mexican axolotls. Accepted: 5 December 2000     

供体脾灌注对高度致敏肾移植受者嵌合体形成的影响

目的探讨供体脾灌注对高度致敏肾移植受者稳定期嵌合体形成及移植肾功能的影响。方法对16例高度致敏患者进行配对分组,实验组肾移植术中先予供体脾灌注40min,前瞻性观察脾灌注对患者术后6个月内嵌合体形成、移植肾排斥反应发生及移植肾功能的变化。结果脾灌注后受者外周血中供者来源的有核细胞数量显著增加,受者形成稳定嵌合体的时间较早,例数比对照组更多;肾移植术后脾灌注组排斥反应发生时间较对照组延迟,排斥反应严重程度明显较对照组轻微;术后6个月时,脾灌注组患者血肌酐值低于对照组。结论供体脾灌注可以显著提高高敏肾移植受者外周血中供者来源的有核细胞数量,减轻排斥反应强度,从而促进受者嵌合体的形成,有利于改善稳定期移植肾功能。     

人／猪造血嵌合体构建及特异性免疫耐受形成的初步研究

目的初步探析人/猪造血嵌合体的构建及所诱导形成特异性免疫耐受的条件,为实现嵌合体人源性造血提供实验支持。方法实验分为3组,新生猪5只/组,实验1组:将人脐血来源CD34+细胞移植入正常新生小型猪体内后,持续注射人源SCF、EPO;实验2组:新生猪移植细胞后不注射人源因子;实验3组(正常对照组):新生猪注射生理盐水。移植后10、20、30、40、50、60d时取嵌合体猪外周血,检测人源CD71+细胞比例,同时检测猪外周血抗人种属抗体。结果2组嵌合体猪外周血中均有一定比例的人源CD71+细胞,但实验1组外周血比例显著高于同期实验2组(P<0.01);正常小型猪外周血于16d出现抗人种属抗体,嵌合体猪外周血于移植后50d出现,且抗体效价低于同龄正常猪。结论利用新生乳猪可建立人/猪造血嵌合体,可诱导形成供者型免疫耐受;在人源造血因子作用下,可实现人造血干细胞在嵌合体内分化。     

Immunoprophylaxis using polypeptide chimera vaccines plus adjuvant system promote Th1 response controlling the spleen parasitism in hamster model of visceral leishmaniasis

In recent years, several advances have been observed in vaccinology especially for neglected tropical diseases (NTDs). One of the tools employed is epitope prediction by immunoinformatic approaches that reduce the time and cost to develop a vaccine. In this scenario, immunoinformatics is being more often used to develop vaccines for NTDs, in particular visceral leishmaniasis (VL) which is proven not to have an effective vaccine yet. Based on that, in a previous study, two predicted T-cell multi-epitope chimera vaccines were experimentally validated in BALB/c mice to evaluate the immunogenicity, central and effector memory and protection against VL. Considering the results obtained in the mouse model, we assessed the immune response of these chimeras in Mesocricetus auratus hamster, which displays, experimentally, similar pathological status to human and dog VL disease. Our findings indicate that both chimeras lead to a dominant Th1 response profile, inducing a strong cellular response by increasing the production of IFN-γ and TNF-α cytokines associated with a decrease in IL-10. Also, the chimeras reduced the spleen parasite load and the weight a correlation between protector immunological mechanisms and consistent reduction of the parasitic load was observed. Our results demonstrate that both chimeras were immunogenic and corroborate with findings in the mouse model. Therefore, we reinforce the use of the hamster as a pre-clinical model in vaccination trials for canine and human VL and the importance of immunoinformatic to identify epitopes to design vaccines for this important neglected disease.     

A multivariate morphometric analysis of hippocampal anatomical variation in C57BL/6 ↔ BALB/c chimeric mice

We investigated hippocampal anatomy in artificially-produced chimeras derived by the aggregation of embryos from two widely-studied inbred mouse strains, C57BL/6J and BALB/cJ. Contrary to expectations, the chimeras were not always intermediate between the parental strains. For a number of characters, the chimeras exceeded qualitatively as well as quantitatively the phenotypical range displayed by both inbred parental strains. These findings imply that if only one parent is available for comparison, for instance, in studies involving a normally inviable genotype, separating effects of this genotype from idiosyncratic effects inherent to the chimeric model will be very difficult, if not impossible.     

小鼠类胚胎干细胞分离培养及用于制作嵌合体小鼠的实验研究

目的探讨分离小鼠囊胚内细胞群类胚胎干细胞及用于制作嵌合体小鼠的方法及应用价值。方法分离3.5d小鼠囊胚内细胞群的类胚胎干细胞作为供体细胞,通过显微注射方法将分离的类胚胎干细胞注射到供体小鼠的囊胚腔中,再将注射后的囊胚移植到假孕雌鼠的子宫中制作嵌合体小鼠。结果分离36枚囊胚的内细胞群类胚胎干细胞,注射256只昆明小鼠囊胚中,移植32只假孕雌鼠子宫中,获产崽2窝,共12只,其中2只获毛色嵌合体小鼠。结论采用该技术分离所获得的类胚胎干细胞作为供体细胞制作嵌合体小鼠获得成功,该方法为ES细胞介导的转基因动物制作增添了一条新的途径,在同种不同品系的动物改良及遗传病基因治疗中有一定的应用价值,尤其是对未能建立ES细胞系的大动物的遗传工程操作具有一定意义。     

Age-dependent loss of naïve T cells in TCR transgenic bone marrow chimeras

Study of immune senescence is complicated by low numbers of antigen-specific lymphocytes, particularly na?ve T (Tn)cells which disappear with aging. Although T cell receptor (TCR) transgenic mice facilitate aging research, their large number of Ag-specific T cells renders their T cell pool abnormal, precluding normal in vivo immunity. To create a physiologically relevant model with measurable numbers of TCR transgenic CD4+ T cells in the context of normal lymphocytes, mixed (DO11.10+BALB/c) bone marrow (BM) chimeras were constructed. As found in normal mice, the total number of transgenic T cells and the Tn:memory T cell ratio declined with the aging of the BM chimeras. Although these shifts in T cell subsets were evident in both the lymph nodes and the spleen (SP), they were more pronounced in the SP. Unlike DO11.10 mice, transgenic T cells in the chimera acquired an effector/memory phenotype upon antigen challenge. These results reveal a pliable model to study the impact of the constriction of the Tn cell repertoire upon optimal vaccine responses in the elderly.