Aichi virus (AiV) is a small, nonenveloped RNA virus categorized to Picornaviridae. AiV infection causes mild gastroenteritis, but in neonates, AiV usually causes the risk of certain enterovirus-related clinical syndromes, such as fever, nausea, vomiting and diarrhea. The first case of AiV infection in Taiwan was diagnosed from a young patient with diarrhea in Kaohsiung Veterans General Hospital, and the AiV was successfully isolated. Antiviral innate immune system of our body plays the major role to defense virus invasion. Because AiV is an emerging picornavirus, the knowledge about its pathogenesis and the interaction with host innate immunity were totally absent. This study aims to investigate the mechanism of AiV regulating innate immune response. We first demonstrated that AiV is a type I IFN sensitive virus. IFN-£\2 treatment potently inhibited AiV replication. Real-time quantitative PCR data indicated that AiV induced only small amout of type I IFN gene expression, and the similar result was observed using IFN-£] luciferase reporter assay. In addition, the AiV triggered IFN-£] luciferase activity was progressively decreased in the late phase of infection. Immunoblotting assay showed that AiV evidently activated IRF-3 and IRF-7, the transcription factors of type I IFN induction. However, the retinoic acid inducible gene I (RIG-I) protein was cleavaged and its activity was downregulated by AiV. This data suggested that AiV triggered low level of type I IFN response may due to the negative feedback regulation of RIG-I activity. This immune evasion might be important for AiV replication in cells. Our study first reveals the status of innate immune response of AiV infection, and provides the basic virological theory for the development of anti-AiV drugs and vaccines in the future.

Blood was obtained from 5 adult horses and 16 juvenile horses (foals) at the time of birth and subsequently at 2-, 4-, and 8-weeks of age. Neutrophils from adult horses were purified and incubated for 2 h and 4 h with media, avirulent R. equi, virulent R. equi, or recombinant-human granulocyte-macrophage colony stimulating factor (rhGM-CSF). Neutrophils from foals were purified and incubated for 2 h and 4 h with media or virulent R. equi. Total RNA was extracted from both adult and foal neutrophils immediately after purification to measure baseline expression levels (0 h), and immediately after each of the prescribed incubation times. For each sample, 1 µg of total RNA was reverse-transcribed and analyzed for differential gene expression using real-time PCR. After 2 h and 4 h incubation with virulent or avirulent R. equi, neutrophils from adult horses expressed significantly (P< 0.05) greater TNFα, IL-12p40, IL-6, IL-8, and IL-23p19 mRNA relative to expression by unstimulated neutrophils, but not IFNγ or IL-12p35 mRNA. Furthermore, virulent R. equi induced significantly greater IL-23p19 mRNA expression than avirulent R. equi. Stimulation with rhGM-CSF of adult equine neutrophils failed to induce significant changes in cytokine expression. In foal neutrophils, stimulation with virulent R. equi induced significantly greater expression of IFNγ, TNFα, IL-6, IL-8, IL-12p40, and IL-12p35 mRNA relative to expression by unstimulated neutrophils. Furthermore, there were significant effects of age on expression of IL-6, IL-8 and IL-12p40 mRNA. Neutrophil mRNA expression of IL-6 and IL-8 in newborn foals was significantly greater than expression at 2-, 4-, and 8-weeks of age. There was no significant difference between unstimulated and R. equi-stimulated neutrophils from newborn and 2-week-old foals in expression of IL-12p40; however, expression of IL-12p40 by R. equi-stimulated neutrophils from 4- and 8-week-old foals was significantly greater than expression by unstimulated neutrophils. These results demonstrate that R. equi-stimulated neutrophils are a source of many pro-inflammatory cytokines, and that the magnitude of this expression with respect to IL-6, IL-8, and IL-12p40 mRNA expression was influenced by age. Collectively, the data presented indicate a non-phagocytic role for neutrophils that may influence the type of adaptive immune response to R. equi.

Abstract At present, diagnostic reagent is the most fruitful and important realm in internal biotech product commercialization. Compared to other biotech medicine with the industrial characteristics of complications such as: high investment, high risk, and long-term research and development; diagnostic reagent, relatively, acquires a lower threshold for technique, a short-term research and development, less investment and a short time for feedback. In addition, people gradually acquire the notions of prophylaxis and health care, as a result, the potentiality of diagnostic reagent comes to the market¡¦s notice. The process of diagnostic reagent development can be divided by the mainstream techniques into four phases including: the technology of chemical test, the technology of enzyme test, the technology of immunoassay test, the technology of nuclease molecule test and biochip. From this, we know that the technology of immunity test is still the mainstream in the market, but there is a trend toward the development of the technology of nuclease molecule immunoassay. General Biologicals Corp. is the first built diagnostic reagent manufacture in our country, and, so far, it is the only diagnostic reagent manufacture that meets with the Department of Health¡¦s CGMP standard. The General Biologicals Corp. is the only internal manufacture that produces ELISA and EIA microplate. However, the General Biologicals Corps has less than 1% of six hundred million, and foreign manufactures have it all. The General Biologicals Corps went public in recent years, and the company has a microplate immunoassay diagnostic reagent factory for over twenty years. Those doctors, examiners and sales clerks who used the immunoassay reagent produced by the General Biologicals Corps are all in high positions in this industry. They know this company and also their reagent kits which have a low sensitivity. More important is that the reagent kits are not improved for all these years. The bad impression of the company makes the customers and collaborators have doubts about the quality, the attitude of the personnel and the sincerity of the company. It results in a trust crisis. In light of the five competitiveness analyses in the executive strategies: dynamic competition of the company in enterprise; S.W.O.T competitiveness analysis; smiling curve and bitter smiling curve; red ocean and blue ocean strategy and, finally, the administration of registering examined medical instrument, to see the integral competitiveness of the company in the industrial competitive environment. Can the General Biologicals Corp. seek league and collaboration in the competitors to seize the internal market, and then the overseas market. By these analyses and advises, to reengeneering the General Biologicals Corp. has a new management on the visible foundation. Key Word¡GImmunity Test, Nuclear Molecular Immuno assay. Five Force, Reengeneering

The present study used Escherichia coli¡]BL21¡^that contained pET-23a-bpGH plasmids, to express black porgy growth hormone ¡]bpGH¡^. The bpGH was refolded at pH 11.3 in the presence of catalytic amounts of cysteine and purified by ion exchanger chromatography and gel filtration chromatography. The purified bpGH is a monomer and has a molecular weight of 22 kDa. Using the bpGH, the effects of the growth hormone on growth, essential amino acid deposition and nonspecific immunity in black porgy were studied. The experiment was a 4*3 ¡]diet*GH¡^ factorial design. Four experimental diets were formulated in that fish meal was replaced by the mixture of soya protein and gelatin so that fish meal / soya mixture = 100 / 0, 75 / 25, 50 / 50 and 25 / 75, respectively. GH treatments included non-injection, once per 3 days and once per 12 days. GH was injected intramuscularly at a dosage of 0.05 &#x00B5;g / g wet body weight. The growth trial lasted for 72 days. The results showed that GH administration significantly enhanced weight gain, feed efficiency, protein efficiency ratio and muscle methionine concentration of the fish. GH injection improved the growth performance of the fish fed low protein quality diets to a level equals to the groups fed high protein quality diets. These results indicate that GH injections enhanced the perferential absorption and deposition of the first limiting amino acid methionine from the diets. In addition, GH administration enhanced alternative complement activity and increased serum lysozyme concentration, implicating the enhancement of the immunity.

B rucella lipopolysaccharide is an important component of virulence in brucellosis. Recent research in macrophage models has shown that Brucella LPS does not behave like classical LPS by stimulating potent inflammatory responses. The central hypothesis of this work is that O-antigen is dynamic signaling molecular and participates in complex interactions with the host to promote productive infection. A corollary to this is that the host environment is dynamic, and Brucella has evolved mechanisms to cope with changing environments. In an effort to understand the contribution of Brucella LPS to virulence and pathogenesis, the function of a metabolic locus important in the synthesis of LPS has been demonstrated and complemented. The spontaneous loss of LPS expression has been characterized. Contribution of LPS to acquisition of the host environment in tissue culture and mouse models has been explored. This work demonstrated that genes outside the O-antigen biosynthesis ( manBA) cluster contribute to LPS biosynthesis. Further high frequency mutation involving manBA is partly responsible for observed dissociation of Brucella strains. Finally, work herein attempts to look at the role of LPS in acquisition of the host environment and shows that LPS is important for recruiting particular cell populations within a host model of brucellosis.

Thesis (Ph. D.)--University of Chicago, Committy on immunology, June 2001. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph.D.)--Ohio University, November, 2009. / Release of full electronic text on OhioLINK has been delayed until December 1, 2010. Title from PDF t.p. Includes bibliographical references.

Thesis (Ph. D.)--University of Chicago, Committee on Immunology, August 2001. / Includes bibliographical references. Also available on the Internet.

Nearly every species on the planet has at least one parasite, which, by definition, incurs a cost in the host. Therefore, organisms must resist parasites - preventing or reducing infections - or tolerate parasites - reducing the costs of infection - in order to maintain their fitness despite the presence of parasites. Here, I investigated: 1) whether parasitic, larval trematodes (cercariae) can detect the least resistant tadpole host species, 2) a hypothetical framework for how host life history impacts the utilization of inflammation and thus, resistance and tolerance, 3) whether a common anesthesia technique used in experimental infections immunocompromises tadpoles, 4) the relationship between tadpole host life history, tolerance, and behavioral resistance to cercarial infection, 5) how tadpole behavior affects trematode infection location, and 6) how host life history impacts trematode infection location and the implications of this for host tolerance. In the first chapter, I investigated whether trematode cercariae could discriminate among several tadpole host species to identify the most susceptible hosts. Cercariae were consistently more attracted to Bufo terrestris tadpoles, which was the most susceptible host species. Other tadpole species varied in attractiveness in an order similar to their susceptibility to infection. Furthermore, there was consistent and significant variation among individual attractiveness and susceptibility within host species. If susceptibility to infection is heritable, chemical cues used by cercariae to identify susceptible hosts could represent a substrate on which natural selection acts, setting up a "Red Queen" arms race between host cues and parasite detection of those cues. In the second chapter, I proposed a framework which outlined the cost-benefit relationship between host life history and immune responses. Because inflammatory immune responses are known to cause self-damage to hosts, anti-inflammatory immune responses should vi be used for long-lived, slowly-developing ("slow-paced") hosts, those infected with relatively less virulent parasites, and/or ongoing but ineffective inflammatory responses. Conversely, the cost of inflammation might be less expensive than the cost of infection among shortlived, rapidly-developing ("fast-paced") hosts, those infected with virulent parasites, and/or those undergoing protracted but ineffective anti-inflammatory immune responses. In the third research chapter, I investigated whether two common anesthesia agents, benzocaine and tricaine mesylate (MS-222), immunocompromise tadpoles. These chemicals are used extensively to study the behavioral resistance of tadpoles to cercariae; if treatment increases infections not only by removing these behaviors but also by suppressing the immune system, behavior might appear artificially effective at preventing trematode infection. I found that neither benzocaine nor MS-222 affected the abundance of circulating white blood cells relative to waterexposed control tadpoles. Furthermore, there was no difference in trematode infection success when tadpoles were anesthetized, allowed to recover from anesthesia, and subsequently experimentally infected. The results of this experiment indicate that benzocaine and MS-222 are both practical, non-immunosuppressive anesthesia agents to use when studying trematode infections in amphibians. In the fourth research chapter, I quantified tadpole hosts' use of behavioral resistance (parasite-induced behaviors) and tolerance of exposure to cercariae. Across seven host species, parasite-induced behaviors were negatively correlated with pace-of-life, with rapidly-developing ("fast-paced") tadpoles exhibiting significantly more behavior than slowly-developing ("slowpaced") tadpoles. The opposite pattern was true of tolerance, where fast-paced species had poorer tolerance of cercarial exposure than slow-paced species. Given that slow-paced species are more likely to be exposed to cercariae because they 1) occur in water more likely to harbor cercariae and 2) have longer developmental times, tolerance to trematode exposure might be an vii evolutionary adaptation that circumvents the costs of behavioral - and, possibly, immunological - resistance to infection. In the fifth research chapter, I investigated whether parasite-induced behaviors were capable of affecting encystment location of trematode cercariae in Hyla femoralis tadpoles and whether the resulting encystment location affected tolerance of infection. Benzocaineanesthetized and control tadpoles had similar infection intensities. However, among benzocaineanesthetized tadpoles, the majority of cercarial infections occurred in tadpoles' heads, but unanesthetized control tadpoles were predominantly tail-infected. Furthermore, the number of head infections were negatively associated with mass change (poor tolerance), whereas the number of tail infections was positively associated with mass change (good tolerance). These results suggest that parasite-induced behavior is not only an important mechanism of resistance to trematodes, as other researchers have described, but also a mechanism of tolerance, whereby tadpoles can prevent the deleterious effects of trematode infection by controlling infection location. The sixth research chapter extends the work of the fifth and investigates whether host life history predicts encystment location of cercariae and whether encystment location affects tolerance of infection. Among seven host species, fast-paced species had significantly more infections in the head and body, whereas slow-paced species had the majority of infections in their tails. Slow-paced species were also less resistant to trematode infections in any body location than fast-paced species. These patterns were partially explained by surface area, with a slow-paced species having more surface area (making them more likely to be contacted and infected by cercariae) and a larger proportion of slow-paced species' surface area was comprised by tail than heady and body. Tail infections were less expensive than head and body infections; slow-paced species were more tolerant of infection and across species, tail infections had no effect on tolerance (mass change) whereas head infections were negatively associated with tolerance. These results suggest that slow-paced tadpoles, which are relatively more likely to viii become infected by cercariae and probably have more evolutionary history with these parasites, have invested in a morphology that improves their tolerance to parasites. The body of work that I have produced demonstrates that variation in resistance and tolerance to trematode parasites is ubiquitous among tadpole hosts. Furthermore, this variation is predictable based on host life history. Because tadpole life history can dramatically impact the likelihood of exposure to cercariae and encounters are necessary for host-parasite selective pressure to occur, life history can predict the adaptations of hosts and parasites. Given amphibians' status as the most rapidly declining taxon on the planet and the ubiquity of emerging infectious diseases for amphibians and other organisms, these findings should inform future research on host- and parasite-mediated mechanisms of disease.

Mucosal delivery of recombinant adenovirus serotype 5 (rAd5)-based vaccine preparations are appealing for vaccine development in terms of lowering toxicity induced by high viral loads and substantial liver accumulation following systemic injection of the vaccine. However, this mode of delivery is currently under-developed due to the relatively low T-cell mediated immune responses generated against the encoded transgene. The first study described in this thesis demonstrated that sublingual immunization induces rapid migration of MHCII+, CD11C+ antigen presenting cells to the delivery site and elicit antigen-specific T and B cell-mediated immune responses in naïve mice and those with pre-existing immunity (PEI) to Ad5 at a level higher than that achieved after oral immunization. More importantly, this strategy improved protection of animals with PEI to Ad in contrast to poor protection after IM injection. The second study was designed to establish a method for inducing PEI that most accurately reflects natural infection in rodents and identifies the immunologic parameters elicited by rAd5-based Ebola vaccine necessary for protection against lethal infection. When immunization occurred by the same route in which PEI was induced, the antigen-specific multifunctional CD8+ T cell and antibody responses were significantly reduced. This correlated with poor survival after challenge with a lethal dose of Ebola Zaire in rodents. The data suggests that 1) establishment of PEI by the same route used for immunization is the most stringent test for a novel formulation designed to be effective in those with PEI to Ad5, and 2) for a formulation to be effective in those with PEI, it must be capable of restoring antigen-specific multifunctional CD8+ T cell and antibody responses, compromised by PEI. The third study screened novel formulations for their ability to improve in vitro transduction efficiency and immunogenicity and efficacy in vivo in the presence of anti-Ad5 neutralizing antibodies. Formulations consisting of pharmaceutically acceptable, non-immunogenic excipients that can prime the arms of immune response compromised by PEI improved survival after lethal challenge with Ebola Zaire challenge for rAd5-based Ebola vaccine in rodents with PEI. Taken together, these studies provide insight on how to reconstitute necessary immune responses in vaccine protocols by establishing a reliable PEI model in rodents, testing routes of administration, and formulations of the rAd5-based Ebola vaccine. / text