Comparison of in vitro and in vivo methods to study stability of PLGA microencapsulated tetanus toxoid vaccines.

The purpose of this study was to investigate the utility of various in vitro and in vivo methods to assess the stability of experimental vaccines containing tetanus toxoid (TT) within PLGA microspheres. In vitro, the breakdown of the encapsulating polymers into their acid components led to changes in the structure of TT, as determined by the physico-chemical methods, rendering it undetectable by capture ELISA and altering its structural integrity. The changes in TT were directly related to increasing acidity of the vaccine supernate. Purified toxoid (not encapsulated) exposed to low pH (2.5) underwent similar changes but re-neutralisation of buffer containing free toxoid, even after one week at pH 2.5 led to some re-folding of protein as determined by fluorescence spectroscopy and gel filtration chromatography. The microencapsulated vaccines were still able to generate an antibody response in mice even after prolonged pre-incubation at 37 degrees C and the apparent absence of detectable toxoid in the vaccine supernate. Electron microscopy demonstrated differences in the amount of degradation between different formulations of microspheres. Vaccines that had retained their spherical morphology after incubation in vitro for up to 28 days were able to induce protective antibodies response equal to that of freshly prepared vaccines, which indicates that the toxoid within intact microspheres remained immunogenic. Immunochemical and physico-chemical detection methods, performed on antigen released from PLGA vaccines in vitro, are valuable in providing information on product characteristics but may not be able to predict effectiveness and should be used with in vivo methods to evaluate the stability of such formulations.

Temporal changes in the granulocytic responses to experimental infection of the skin of mice and sheep with Dermatophilus congolensis.

The patterns of dermal inflammatory cell response to infection with Dermatophilosis congolensis were determined in mice and sheep from histological samples taken before and at intervals after topical application of infective zoospores to ether-swabbed skin. Neutrophils, eosinophils, basophils and mast cells were identified by histochemical staining. Temporal changes in the B cell, T cell, and MHC Class II+ dendritic cell populations form part of a separate report. The filamentous stages of the bacterium were observed in the stratum corneum of both species; in the sheep they were also found in the outer layers of the living epidermis. In both species, large numbers of neutrophils and some lymphocytes penetrated the epidermis and entered the infected surface region. Within the underlying dermis there was an accumulation of dendritic cells immediately below the infected epidermis and evidence of mast cell degranulation; the basophils and eosinophils did not appear to be actively involved. The striking difference between the two species was the duration of the infection and the associated response which, in the mouse, lasted about five days in comparison with over 21 days in the sheep. Neutrophil numbers in the mouse for example were elevated by 12 h and had peaked at 60 h after infection, while in the sheep they did not peak until about 120 h.

Experimental dermatophilosis in murine models of immunodeficiency.

Gnotobiotic mice with congenital immune deficiencies were infected with the skin pathogen Dermatophilus congolensis. Athymic (nude) mice with T cell deficiency were less susceptible than nude mice which also carried the beige mutation (beige-nude) with NK cell and granulocyte defects, as part of the murine equivalent of Chediak-Higashi syndrome. The additional presence of the x-linked immunodeficiency gene in other beige mutant mice, giving reduced B cell responsiveness, did not increase their susceptibility. BALB/c mice with the nude mutation and evidence of macrophage insufficiency, had a moderate level of susceptibility, greater than that of outbred nude mice but less than that of beige, nude mice. The appearance of the lesions on the haired mice was different from that on those with hairless skin (nude and beige-nude). On the haired mice thin crusts developed and healed rapidly, while on the hairless mice the lesions started as nodules and later progressed to crusts. The nude BALB/c mice developed atypical lesions, which resembled ulcers. Germ-free nude and beige-nude mice showed the same types and time course of infection as the gnotobiotic animals, suggesting that bacterial interference, by a limited skin flora, did not play a major role in defence against D. congolensis. However, bacteriological analysis indicated that D. congolensis could survive in the gut of germ-free mice. This work emphasizes the importance of non-specific immune mechanisms, such as epidermal hyperproliferation and the neutrophil, in resistance to D. congolensis.

Factors regulating the steroid 11-hydroxylation by non-germinating spores of Cunninghamella elegans (Lendner).

In the presence of malate or citrate sporangiospores of C. elegans were able to hydroxylate cortexolone with a rate twofold exceeding that of the control, water suspended spores. Analysis of the intracellular nicotinamide coenzyme pools revealed an increased NADPH:(NADP+ + NADPH) ratio, indicating more effective NADPH-generating systems in malate- or citrate-stimulating spores. Swollen spores remaining in the pregermination state, retained higher cortexolone-hydroxylating activity in the absence of malate and citrate. In these spores degradation of endogenous alanine and glutamic acid was observed. Possible NADPH-generating systems in C. elegans sporangiospores were discussed.

Adenylate energy charge and the steroid 11 alfa-hydroxylase activity in Monosporium olivaceum during growth and starvation.

During growth of Monosporium olivaceum its energy charge, E.C., (i.e. the adenylates ratio ATP + 0.5 ADP/ATP + ADP + AMP) increased from an initial value of 0.59 up to 0.85 after 25 hr of growth and then decreased to 0.51. The increase of energy charge was followed by the decrease of the activity of the 11 alpha-hydroxylase of cortexolone. This occured very clearly in the starved mycelium. Highest hydroxylation activity was observed when the lowest E.C. level (0.39-0.33) was reached.