The development of oral vaccines based on live attenuated Salmonella strains

Abstract Safe, live attenuated Salmonella strains can be produced by introducing defined non-reverting mutations into the chromosome. Such rationally attenuated strains have proved to be excellent oral vaccines in several animal species and can therefore be considered as candidate vaccines against invasive salmonellosis in both animals and man. A panel of attenuating lesions is now available from which it is possible to tailor the level of attenuation and hence produce strains with different immunogenic properties. Because of the spectrum of immune responses produced by such Salmonella vaccine strains they have been utilised extensively as vectors for delivering heterologous antigens to the mammalian immune system. We have focussed on the development of a single dose oral tetanus vaccine based on attenuated Salmonella strains expressing a non-toxic, immunogenic protein derived from tetanus toxin (fragment C). Several different expression systems have been used for fragment C and candidate vaccine strains have been constructed that are capable of protecting orally immunised mice against a lethal challenge with tetanus toxin. An oral tetanus vaccine may help to reduce the mortality rate from tetanus in the developing world by overcoming the problems associated with the implementation of vaccine programmes using the current parenteral vaccine.     

Characterisation of Candida albicans infections of haematogenous and mucosal origin in mice lacking the interferon gamma receptor protein

Mice harbouring a null deletion mutation in the IFNgamma receptor gene were used to study the role of IFNgamma responsiveness during experimental systemic candidiasis of mucosal or haematogenous origin. After intravenous (i.v.) or intranasal (i.n.) challenge with Candida albicans the progression of infection and concomitant cellular and antibody anti-C. albicans immune responses were analysed. During the week following i.v. challenge, the rate of C. albicans multiplication in kidneys, liver and spleen was faster in IFNgammaR (-/-) than IFNgammaR (+/+) mice. As a result, IFNgammaR (-/-) mice perished earlier than IFNgammaR (+/+) mice when challenged with equal numbers of live yeast cells. However, the overall susceptibility of the two mouse strains, in terms of survival against different C. albicans challenge doses over a 60-day period, was similar. No differences were found in the cellular anti-C. albicans response generated by i.v. challenge in both mouse strains. In contrast the kinetics and strength of the serum anti-C. albicans antibody responses were markedly different. Significantly stronger, predominantly IgG2a antibody responses accompanied the eventual control of C. albicans infection in IFNgammaR (-/-) mice. Following intranasal challenge, there was no difference in the rate of C. albicans clearance from the lungs of IFNgammaR (-/-) and IFNgammaR (+/+) mice. However, 48 h after challenge, large, conspicuous abscesses appeared in the lungs, liver, kidneys and spleen of IFNgammaR (-/-) mice. These abscesses were characterised by the presence of C. albicans and abundant neutrophilic infiltrates, but very few macrophages. No such abscesses developed in i.n. challenged IFNgammaR (+/+) mice. In both mouse strains, i.n. challenge induced strong systemic anti-C. albicans cellular responses, but relatively low titre systemic antibody responses. Mucosal anti-C. albicans antibody responses were detected in IFNgammaR (+/+), but not IFNgammaR (-/-) mice. Splenic adherent macrophages obtained from IFNgammaR (-/-) mice exhibited a significantly lower candidacidal activity than those of IFNgammaR (+/+) mice, and as expected, were not responsive to IFNgamma. In summary, these data suggest that IFNgamma has a role in limiting C. albicans multiplication during the early stages of infection, as well as in preventing the development of C. albicans-associated abscesses. Activation of macrophages by IFNgamma might be pivotal in mediating this role.     

Genomewide scan for linkage reveals evidence of several susceptibility loci for alopecia areata

Alopecia areata (AA) is a genetically determined, immune-mediated disorder of the hair follicle that affects 1%-2% of the U.S. population. It is defined by a spectrum of severity that ranges from patchy localized hair loss on the scalp to the complete absence of hair everywhere on the body. In an effort to define the genetic basis of AA, we performed a genomewide search for linkage in 20 families with AA consisting of 102 affected and 118 unaffected individuals from the United States and Israel. Our analysis revealed evidence of at least four susceptibility loci on chromosomes 6, 10, 16 and 18, by use of several different statistical approaches. Fine-mapping analysis with additional families yielded a maximum multipoint LOD score of 3.93 on chromosome 18, a two-point affected sib pair (ASP) LOD score of 3.11 on chromosome 16, several ASP LOD scores >2.00 on chromosome 6q, and a haplotype-based relative risk LOD of 2.00 on chromosome 6p (in the major histocompatibility complex locus). Our findings confirm previous studies of association of the human leukocyte antigen locus with human AA, as well as the C3H-HeJ mouse model for AA. Interestingly, the major loci on chromosomes 16 and 18 coincide with loci for psoriasis reported elsewhere. These results suggest that these regions may harbor gene(s) involved in a number of different skin and hair disorders.     

Metabolism of 35S-labelled Antithyroid Drugs in Man

Differences in the metabolic fate of antithyroid drugs influence the optimal frequency of administration and their therapeutic efficacy. ³⁵S propylthiouracil differed from the ³⁵S imidazoles (carbimazole and methimazole) in the more rapid absorption and excretion and the shorter biological half-life in the plasma of the former. Renal function may have a more important influence on the biological half-life of the drugs than thyroid status. Further work is required to determine the optimal frequency of administration for each compound.     

Glomerular CD34 expression in short- and long-term diabetes.

Aging and diabetes are associated with exacerbated expression of adhesion molecules. Given their importance in endothelial dysfunction and their possible involvement in the alteration of glomerular permeability occurring in diabetes, we have evaluated expression of the sialomucin-type adhesion molecule CD34 in renal glomerular cells of normal and diabetic animals at two different ages by colloidal gold immunocytochemistry and immunoblotting. CD34 labeling was mostly assigned to the plasma membranes of glomerular endothelium and mesangial processes. Podocyte membranes were also labeled, but to a lesser degree. Short- and long-term diabetes triggers a substantial increase in immunogold labeling for CD34 in renal tissues compared with young normoglycemic animals. However, the level of labeling in old diabetic and healthy control rats is similar, suggesting that the effect of diabetes and aging on CD34 expression is similar but not synergistic. Western blotting of isolated glomerular fractions corroborated immunocytochemical results. Increased expression of CD34 may reflect its involvement in the pathogenesis of glomerular alterations related to age and diabetes. Alterations present in early diabetes, resembling those occurring with age, strengthen the concept that diabetes is an accelerated form of aging.     

MicroRNA Signature Characterizes Primary Tumors That Metastasize in an Esophageal Adenocarcinoma Rat Model

Objective

To establish a miRNA signature for metastasis in an animal model of esophageal adenocarcinoma (EAC).

Background

The incidence of esophageal adenocarcinoma (EAC) has dramatically increased and esophageal cancer is now the sixth leading cause of cancer deaths worldwide. Mortality rates remain high among patients with advanced stage disease and esophagectomy is associated with high complication rates. Hence, early identification of potentially metastatic disease would better guide treatment strategies.

Methods

The modified Levrat’s surgery was performed to induce EAC in Sprague-Dawley rats. Primary EAC and distant metastatic sites were confirmed via histology and immunofluorescence. miRNA profiling was performed on primary tumors with or without metastasis. A unique subset of miRNAs expressed in primary tumors and metastases was identified with Ingenuity Pathway Analysis (IPA) along with upstream and downstream targets. miRNA-linked gene expression analysis was performed on a secondary cohort of metastasis positive (n=5) and metastasis negative (n=28) primary tumors.

Results

The epithelial origin of distant metastasis was established by IF using villin (VIL1) and mucin 5AC (MUC5AC) antibodies. miRNome analysis identified four down-regulated miRNAs in metastasis positive primary tumors compared to metastasis negative tumors: miR-92a-3p (p=0.0001), miR-141-3p (p=0.0022), miR-451-1a (p=0.0181) and miR133a-3p (p=0.0304). Six target genes identified in the top scoring networks by IPA were validated as significantly, differentially expressed in metastasis positive primary tumors: Ago2, Akt1, Kras, Bcl2L11, CDKN1B and Zeb2.

Conclusion

In vivo metastasis was confirmed in the modified Levrat’s model. Analysis of the primary tumor identified a distinctive miRNA signature for primary tumors that metastasized.     

The ubiquitin-proteasome system is responsible for cysteine-responsive regulation of cysteine dioxygenase concentration in liver

Hepatic cysteine dioxygenase (CDO) activity is a critical regulator of cellular cysteine concentration and availability of cysteine for anabolic processes and is markedly higher in animals fed diets containing excess sulfur amino acids compared with those fed levels at or below the requirement. Rat hepatocytes responded to a deficiency or excess of cysteine in the culture medium with a decrease or increase in CDO level but no change in CDO mRNA level. The cysteine analog, cysteamine, but not cysteine metabolites or thiol reagents, was also effective in increasing CDO. Inhibitors of the 26S proteasome blocked CDO degradation in cysteine-deficient cells but had little or no effect on CDO concentration in hepatocytes cultured with excess cysteine. High-molecular-mass CDO-ubiquitin conjugates were observed in cells cultured in cysteine-deficient medium, whether or not proteasome inhibitor was present, but these CDO-ubiquitin conjugates were not observed in cells cultured in cysteine-supplemented medium with or without proteasome inhibitor. Similar results were observed for degradation of recombinant CDO expressed in human heptocarcinoma cells cultured in cysteine-deficient or cysteine-supplemented medium. CDO is an example of a mammalian enzyme that is robustly regulated via its substrate, with the presence of substrate blocking the ubiquitination of CDO and, hence, the targeting of CDO for proteasomal degradation. This regulation occurs in primary hepatocytes in a manner that corresponds with changes observed in intact animals.