Vitrification of in vitro-derived bovine embryos: targeting enhancement of quality by refining technology and standardising procedures.

Bovine invitro fertilisation technology has been widely exploited in commercial settings. The majority of invitro-derived cattle embryos are transferred into recipient cows as recently collected (i.e. 'fresh') embryos due to the lack of a reliable cryopreservation method that results in favourable pregnancy rates following transfer of thawed embryos. This is a primary reason for the poor industry uptake of this extreme temperature freezing process. Numerous investigations into vitrification have revealed the importance of rapid cooling and warming rates, enhancing embryo viability after cryopreservation compared with conventional slow freezing. Those studies spawned a considerable assortment of cryovessels and diversity of procedures, delivering variable rates of success, which makes performing vitrification consistently a practical challenge. Hence, further research is required in order to both optimise and standardise vitrification methodology and to design a cryovessel that enables direct transfer of vitrified embryos to recipients after warming. In parallel with improvements in vitrification, it is important to continue to raise the quality of invitro-derived cattle embryos through modifications in laboratory culture techniques. The twin goals of methodology refinement and standardisation, leading to embryo quality enhancement, are each imperative if invitro fertilisation technology is to be adopted in the field.

Does the Addition of Salubrinal to in vitro Maturation Medium Enhance Bovine Blastocyst Yields and Embryo Cryotolerance?

BACKGROUND: Modifications to in vitro maturation (IVM) media are made to improve rates of blastocyst formation and quality of mammalian embryos. Embryo quality is an important factor in the viability of embryos following cryopreservation. Salubrinal is a specific inhibitor of endoplasmic reticulum stress-induced apoptosis in eukaryotic cells. Here, it was added to IVM medium in an attempt to increase blastocyst formation and to enhance embryo quality in cattle. OBJECTIVE: To assess the effect on blastocyst formation and cryotolerance of the supplementation of salubrinal to bovine IVM medium. MATERIALS AND METHODS: Cumulus-oocyte complexes (COCs) collected from slaughterhouse ovaries were assigned randomly to two groups, either cultured in IVM medium that was supplemented with 400 mM salubrinal (treated, 262 COCs) or that was not supplemented (control, 263 COCs). All oocytes of the matured COCs were fertilized in vitro with sperm from the same proven bull and cultured for 6-7 d. At the time of blastocyst collection, expanded blastocysts were chosen for cryopreservation, while early, hatching and hatched blastocysts and those of poor quality were not used. There were 83 expanded blastocysts classified to be of good quality in both the control and salubrinal-treated groups that were subjected to vitrification. After 5 to 10 months of cold storage, the embryos were warmed and cultured in vitro for 24 h to assess the survival rate and for 48 h to assess the hatching rate. RESULTS: The blastocyst developmental rate in the salubrinal-treated group was similar to that in the control group, 61.5% compared with 62.7% (P > 0.05). The survival rate of blastocysts after vitrification was also similar, at or very close to 100%. In addition, there was no statistically significant difference in the hatching rate of expanded blastocysts derived from the COCs cultured with (treated) and without (control) addition of salubrinal to the IVM medium (91.6% compared with 85.5%; P > 0.05). CONCLUSION: Supplementation of salubrinal to the IVM medium neither improved nor reduced rates of bovine blastocyst formation and of embryo cryotolerance as determined by post-warming viability.

An in vitro assessment of the value of sucrose supplementation to warming media for the vitrification of bovine embryos.

　　BACKGROUND: In order to thaw slow-cooled bovine embryos it is standard practice to draw out permeating cryoprotectants by passing embryos through successively decreasing osmotic solutions. However, recently it has been suggested that sucrose may not be needed in the warming media. OBJECTIVE: The aim of this experiment was to compare the effect of warming media prepared with or without the inclusion of sucrose on the survival and hatching capacity of vitrified in vitro-derived bovine embryos. MATERIALS AND METHODS: Expanded blastocysts were produced in vitro and vitrified. Vitrified embryos were warmed either successively through 0.5, 0.3 and 0.2 M sucrose solutions ('stepwise'), or by placing directly into the blastocyst solution without the addition of sucrose ('direct'). A total of 93 expanded blastocysts were assigned randomly to two treatment groups, respectively. RESULTS: The re-expansion rates of vitrified embryos warmed after 24h in vitro culture were similar between the two groups (46/46, 100%; 46/47, 97.9%). From those vitrified embryos that expanded at 24 h there was also no significant difference in hatching rates after 48 h in vitro culture (42/46, 91.3%; 40/46, 87.0%). CONCLUSION: The findings indicate that stepwise warming through sucrose solutions is not required for continued embryo development. Hence, a more time-efficient warming method for vitrified embryos may be followed when conducting cattle embryo transfers.

Evaluation of liquid culture media to support growth of Mobiluncus species.

Mobiluncus curtisii and M. mulieris are anaerobic, gram-negative, motile curved rods isolated commonly from the vagina of women with bacterial vaginosis. Hitherto, there has been difficulty in isolating and growing these bacteria and little attention has been paid to growth in liquid media. Reasons for establishing the means of attaining optimal growth in such media include production of antigens for diagnostic and immunological studies and production of the soluble cytotoxin. In this study the efficacy of 12 liquid culture media in supporting growth was examined. M. mulieris (strain A198) multiplied > or =10-fold in only five media - Schaedler broth, Columbia blood broth (CBB), peptone-starch-dextrose (PSD) broth, brain-heart infusion plus arginine and spent tissue-culture medium. Similarly, M. curtisii (strain A98) multiplied > or =10-fold in only three media -Schaedler broth, CBB and PSD. Some strains of both bacterial species grew very poorly or not at all, in all the media tested. With an inoculum of > or =10(5)/ml, CBB, or PSD plus 10% horse serum, supported the growth of some strains of both bacterial species to 10(9) organisms/ml within 48 h, and viable bacteria persisted longer in some media (e.g., CBB) than in others. While variation in growth of Mobiluncus spp. may occur between one laboratory and another, these observations provide the basis for optimisation of a universal liquid culture medium that should facilitate production of antigens and cytotoxin.

A model of development of acquired immunity to malaria in humans living under endemic conditions.

Malaria remains a significant global health problem. Most morbidity and mortality in an endemic setting is in children less than 5 years old, and increasing resistance to infection and disease with age is thought to reflect a slow, gradual acquisition of protective immunity. It is not clear if the semi-immune status of adults, in which parasites are present at below clinical threshold, is the result of cumulative exposure to Plasmodium falciparum or reflects an underlying difference between adult and infant immunity. Immuno-epidemiological studies of people living in malaria-endemic areas have not produced consistent examples of surrogate markers of protection. This gulf in our understanding of immunity to malaria may be addressed by novel application of an established murine model of immune regulation of blood stage infection. This exploits two examples of loss of immunity, selective immunosuppression in pregnancy, and waning of maternally transferred protection in neonates, to distinguish the immunological determinants involved in the radical transition between susceptible and resistant immune status. It is suggested that application of this unique model should significantly advance knowledge of how acquired immunity to malaria develops and is highly relevant to the pathogenesis of malaria in human pregnancy and the design of antimalarial vaccines for use in children.

Potentiation by a novel alkaloid glycoside adjuvant of a protective cytotoxic T cell immune response specific for a preerythrocytic malaria vaccine candidate antigen.

We have recently demonstrated that the novel glycoalkaloid tomatine, derived from leaves of the wild tomato Lycopersicon pimpinellifolium, can act as a powerful adjuvant for the elicitation of antigen-specific CD8+ T cell responses. Here, we have extended our previous investigation with the model antigen ovalbumin to an established malaria infection system in mice and evaluated the cellular immune response to a major preerythrocytic stage malaria vaccine candidate antigen when administered with tomatine. The defined MHC H-2kd class I-binding 9-mer peptide (amino acids 252-260) from Plasmodium berghei circumsporozoite (CS) protein was prepared with tomatine to form a molecular aggregate formulation and this used to immunise BALB/c (H-2kd) mice. Antigen-specific IFN-gamma secretion and cytotoxic T lymphocyte activity in vitro were both significantly enhanced compared to responses detected from similarly stimulated splenocytes from naive and tomatine-saline-immunised control mice. Moreover, when challenged with P. berghei sporozoites, mice immunised with the CS 9-mer-tomatine preparation had a significantly delayed onset of erythrocytic infection compared to controls. The data presented validate the use of tomatine to potentiate a cellular immune response to antigenic stimulus by testing in an important biologically relevant system. Specifically, the processing of the P. berghei CS 9-mer as an exogenous antigen and its presentation via MHC class I molecules to CD8+ T cells led to an immune response that is an in vitro correlate of protection against preerythrocytic malaria. This was confirmed by the protective capacity of the 9-mer-tomatine combination upon in vivo immunisation. These findings merit further work to optimise the use of tomatine as an adjuvant in malaria vaccine development.

Heterologous gene expression in bacterial systems under reduced oxygen tensions. Small-scale optimization precedes industrial fermentation.

The expression of heterologous fusion proteins from the anaerobically inducible Escherichia coli nitrite reductase nirB promoter has been described using a number of different industrial regimes, but which have proved impractical for scaling down to suit primary research purposes. This paper describes the novel application of microbiological gas sachets generating anaerobic and microaerophilic environments to evaluate the inducible expression under the influence of nirB of heterologous proteins by attenuated vaccine strains of Salmonella typhimurium. The conditions of reduced oxygen tension model those found in lymphoid organs colonized by Salmonella in vivo and so can be used to optimize the vaccine dose prior to administration. Modeling in vivo promoter inducibility to monitor the stability of a plasmid within attenuated vaccine strains of bacteria offers an attractive alternative to antibiotic resistance, which is not permitted for clinical use in humans. This technological advance may be utilized to optimize heterologous gene expression in any microaerophilic bacterial system as a pilot, prior to production-scale applications.

The sequestration hypothesis: an explanation for the sensitivity of malaria parasites to nitric oxide-mediated immune effector function in vivo.

While the effect of reactive nitrogen intermediates (RNI) against macrophage-dwelling protozoa, such as Leishmania and Toxoplasma has become established, the possible antiparasitic function of nitric oxide (NO) and RNI against the intracellular blood stages of malaria and babesia has, until recently, been less well accepted. This was, at least in part, due to the long-standing notion that haemoglobin (Hb) universally scavenges NO and thus that erythrocytes act as a permanent sink for this molecule. It is now known that NO can be released as well as scavenged by Hb, and that the less oxygenated the blood the lower the affinity of Hb for NO. As a consequence, NO is preferentially released by venous erythrocytes. Based on the increased sensitivity of Plasmodium falciparum -infected erythrocytes to RNI in venous blood that was recently demonstrated, it is proposed that the noted greater susceptibility of mature intra-erythrocytic forms of malaria, late-stage schizonts, is coincidental with their peripheral blood withdrawal by sequestration to deep-tissue capillaries. This environment is non NO-scavenging in nature and one which would bring schizonts and macrophages into intimate proximity, providing diffusion distances sufficiently short for RNI to be effective. Given its short half-life, this hypothesis explains the potential for NO to be toxic for malaria parasites in vivo, and suggests that sequestration, a mechanism adopted by the parasite to supposedly avoid immune surveillance, may in fact have a partially counteractive effect.

Vaccination against malaria: targets, strategies and potentiation of immunity to blood stage parasites.

Malaria is the world's major parasitic disease, for which effective control measures are urgently needed. One of the difficulties hindering successful vaccine design against Plasmodium is an incomplete knowledge of antigens eliciting protective immunity, the precise types of immune response for which to aim, and how these can be induced. A greater appreciation of the mechanisms of protective immunity, on the one hand, and of immunopathology, on the other, should provide critical clues to how manipulation of the immune system may best be achieved. This review discusses the current state of malaria vaccine development and research to understand the factors involved in the modulation of vaccine-potentiated immunity to the pathogenic blood-borne stages of the parasite.

Increased production of acute-phase proteins corresponds to the peak parasitaemia of primary malaria infection.

Recent studies have implicated non-specific mediators associated with CD4+ T cells of the T helper 1 subset in resistance to experimental malarias. As part of continuing studies into the multifactorial role of nitric oxide and other contributors to the innate immune response in control of acute-phase malaria infection, the production of the acute-phase proteins, caeruloplasmin and serum amyloid P, following infection of naive mice with blood stages of the rodent malaria parasite Plasmodium chabaudi was investigated. Levels of both acute-phase proteins in the serum of infected mice were significantly elevated on days 7-12 post-infection compared both to other times of infection, and to background levels detected in uninfected control mice. These times corresponded to the ascending and peak primary parasitaemia, when production of interferon-gamma, tumour necrosis factor-alpha and nitric oxide is known to be raised. Although it is not apparent whether the production of caeruloplasmin and serum amyloid P has a causal effect in reducing parasitaemia or is simply a by-product of innate immunity, the detection of increased levels of circulating acute-phase proteins may act as a useful surrogate marker of high level parasitaemia, and therefore, of blood-borne malaria pathology.

A role for cytokines in potentiation of malaria vaccines through immunological modulation of blood stage infection.

Malaria is the world's major parasitic disease, for which effective control measures are urgently needed. One of the difficulties hindering successful vaccine design against Plasmodium is an incomplete knowledge of antigens eliciting protective immunity, the precise types of immune response for which to aim, and how these can be induced. A greater appreciation of the mechanisms of protective immunity, on the one hand, and of immunopathology, on the other, should provide critical clues to how manipulation of the immune system may best be achieved. We are studying the regulation of the balance between T helper 1 (Th1) and T helper 2 (Th2) CD4+ T lymphocytes in immunity to asexual blood stages of malaria responsible for the pathogenicity of the disease. Protective immunity to the experimental murine malarias Plasmodium chabaudi and Plasmodium yoelii involves both Th1 and Th2 cells, which provide protection by different mechanisms at different times of infection characterised by higher and lower parasite densities, respectively. This model therefore facilitates a clearer understanding of the Th1/Th2 equilibrium that appears central to immunoregulation of all host/pathogen relationships. It also permits a detailed dissection in vivo of the mechanisms of antimalarial immunity. Here, we discuss the present state of malaria vaccine development and our current research to understand the factors involved in the modulation of vaccine-potentiated immunity.

Expression and immunogenicity of a liver stage malaria epitope presented as a foreign peptide on the surface of RNA-free MS2 bacteriophage capsids.

We have designed a novel vaccine strategy which enables display of short peptides expressed from chimeras of the gene encoding the coat protein of the RNA bacteriophage MS2 and inserted foreign DNA. MS2 coat protein has a beta-hairpin loop at the N-terminus which forms the most radially distinct feature of the mature capsid. The coat protein gene was modified to enable insertion of DNA at the central part of the beta-hairpin loop. Upon expression of the recombinant gene in E. coli, the MS2 coat protein subunits self-assemble into capsids, each comprising 180 copies of the monomer. This system was used to produce chimeras containing a putatively protective epitope, T1, from the immunodominant liver stage antigen-1 (LSA-1) of the malaria parasite Plasmodium falciparum. The immunogenicity of the native MS2 capsid and the recombinant construct was investigated in BALB/c (H-2(d)) mice. The native protein appeared to elicit both humoral and cellular immune responses, observed as a predominance of type 2 cytokines but with a mixed profile of immunoglobulin isotypes. In contrast, the LSA-1 chimera stimulated a type 1-polarised response, with significant upregulation of interferon-gamma, a finding which corroborates naturally acquired resistance to liver stage malaria. These results validate RNA phage capsid display of immunogenic determinants as a basis for the development of novel peptide vaccines and indicate that further evaluation of MS2 coat protein as a vector for malaria epitopes is merited.

Modulation of experimental blood stage malaria through blockade of the B7/CD28 T-cell costimulatory pathway.

Recent studies have implicated cytokines associated with CD4+ T lymphocytes of both T helper (Th)1 and Th2 subsets in resistance to experimental blood stage malaria. As the B7/CD28 costimulatory pathway has been shown to influence the differentiation of Th cell subsets, we investigated the contribution of the B7 molecules CD80 and CD86 to Th1/Th2 cytokine and immunoglobulin isotype profiles and to the development of a protective immune response to malaria in NIH mice infected with Plasmodium chabaudi. Effective blockade of CD86/CD28 interaction was demonstrated by elimination of interleukin (IL)-4 and up-regulation of interferon (IFN)-gamma responses by P. chabaudi-specific T cells and by reduction of P. chabaudi-specific immunoglobulin G1 (IgG1). The shift towards a Th1 cytokine pattern corresponded with efficient control of acute parasitaemia but an inability to resolve chronic infection. Moreover, combined CD80/CD86 blockade by using anti-CD80 and anti-CD86 monoclonal antibodies raised IFN-gamma production over that seen with CD86 blockade alone, with augmentation of this Th1-associated cytokine reducing levels of peak primary parasitaemia. These results demonstrate that IL-4 production by T cells in P. chabaudi-infected NIH mice is dependent upon CD86/CD28 interaction and that IL-4 and IFN-gamma contribute significantly, at different times of infection, to host resistance to blood stage malaria. In addition, combined CD80/CD86 blockade resulted in preferential expansion of IFN-gamma-producing T cells during P. chabaudi infection, suggesting that costimulatory pathways other than B7/CD28 may contribute to T-cell activation during continuous antigen stimulation. This study indicates a role for B7/CD28 costimulation in modulating the CD4+ T-cell response during malaria, and further suggests involvement of this pathway in other infectious and autoimmune diseases in which the Th cell immune response is also skewed.

A dichotomous role for nitric oxide in protection against blood stage malaria infection.

Nitric oxide (NO) is cytotoxic and cytostatic to blood stage malaria parasites in vitro, but the precise mechanism(s) by which it mediates an effect in vivo is not known. In particular, whether or not control of acute parasitemia depends on the presence of NO is unclear. We have shown previously that blocking NO synthesis at the time of its induction may cause an increase in peak primary parasitemia during infection of mice with Plasmodium chabaudi, suggesting that NO may be parasiticidal in vivo. However, as recent data indicate that NO suppresses Th1 cell proliferation in vitro by downregulating IL-2 production, we have investigated whether this immunoregulatory function of NO affects its capacity for anti-malarial activity. Treatment of P. chabaudi-infected mice with the iNOS inhibitor aminoguanidine hemisulfate (AG) starting just prior to the peak of primary parasitemia caused a significant elevation and extension of the acute infection and led to a partial but significant abrogation of the suppression of spleen cell proliferation to both mitogen and specific antigen observed when NO synthesis was not blocked. In the absence of NO, levels of IL-2, but not of IFN-gamma, TNF-alpha, or of any Th2-regulated cytokines examined, increased significantly. However, when AG treatment was brought forward to the early ascending phase of primary parasitemia, significantly increased levels of IFN-gamma and TNF-alpha, as well as of IL-2, were observed over those for infected control mice similarly treated with phosphate-buffered saline. Moreover, despite the absence of NO, parasitemias of AG-treated mice were not significantly elevated. The effect of AG therefore appeared to be dependent upon the timing of its administration in vivo. We propose that during malaria infections, there is a dynamic balance between the regulatory and anti-parasitic roles of NO. While the immunosuppressive function of NO leads to a downregulation in vivo of production of IL-2, and indirectly of IFN-gamma and TNF-alpha, this perceived weakening of the host cell-mediated immune response is in part masked by the protective anti-malarial effects of NO itself.