One strain of Bacillus subtilis and three antibiotic-resistant strains of Staphylococcus aureus were employed to determine the biological effects of everninomicin B (EvB), a naturally occurring antibiotic produced by Micromonospora carbonacea (NRRL 2972) and M. carbonacea var. aurantiaca. Minimum inhibitory concentration (MIC) of EvB for B. subtilis was 1.2 x 10-3 µmole/ml of EvB in glucose minimal broth and 2.6 x 10-3 µmole/ml of EvB in nutrient broth. MIC values of EvB for S. aureus were 3.25 x 10-4 µmole/ml of EvB for strains resistant to penicillin or tetracycline and 6.5 x 10-4 µmole/ml of EvB for the aminoglycoside-resistant strain. The inhibitory effect of EvB was found to be reversible for all concentrations of EvB and all bacterial strains tested. The inhibitory effect of EvB for B. subtilis was not dependent upon the initial concentration of cells nor the stage in the growth cycle at the time the compound was introduced. The inhibitory effect was dependent upon the initial concentration of cells for S. aureus. Electron microscopy studies showed distinct morphological changes in treated cells of S. aureus. Cellular lysis in these cells was also detected.

The drug of choice for the treatment of Stieptococcal pharyngitis is penicillin G. However, a common home remedy prescribes the use of salt-water solutions for gargling. Members of Beta -hemolytic streptococcal groups A, B, and C were isolated from the upper -respiratory tracts of patients diagnosed as having streptococcal pharyngitis. These cultures we:e obtained from HCA Greenview Hospital (Bowling Green, Kentucky) and used to study the effects of sodium chloride on the isolates. The minimum inhibitory concentration of sodium chloride was determined for each of eight hospital isolates. Croup A streptococci were inhibited at a concentration of 7.2% sodium chloride while Group C streptococci were inhibited at a 7.0% concentration. Group P streptococci were more resistant, and inhibition of growth occurred at 12.0% sodium chloride concentrations. Scanning electron microscopic studies showed no significant differences in the external structure of cells treated with sodium chloride when compared to non-treated cells. Despite the lack of changes in the external structure of treated cells, fine structural alterations were observed with transmission electron microscopic studies. Treatment of the cells with sodium chloride resulted in a condensation of nucleoid deoxyribonucleic acid (DNA) and some loss of ribosomes. These changes were followed by a dissolution of the cytoplasmic cell contents resulting in an intact cell wall with capsule. Other parameters such as the rate of growth, minimum bactericidal concentrations, DNA content and protein content of cells treated with sodium chloride were examined and compared to control cells.

No description available.

The study of antibody responses in prominent aquaculture species such as the rainbow trout, Oncorhynchus mykiss, can facilitate vaccine development and contribute to producing useful paradigms of adaptive immunity in lower vertebrates. Thus, it is essential to identify genes responsible for antibody responses. In the mouse model, hybridoma technology allows for the association of monoclonal antibodies possessing various affinities for antigen with specific VH sequences, gene family utilization, and other molecular events (i.e. somatic hypermutation) that occur during the specific immune response. The absence of a comparable hybridoma technology in piscine systems has limited similar studies of fish immunogenetics to date. Molecular and serological experiments were performed in an attempt to obtain information regarding somatic mutation and VH gene utilization for trout antibodies without reliance on hybridoma technology. PCR primers recognizing consensus sequences of FR1 and FR3 were used to amplify antibody VH sequences from panned, antigen-specific B cells. to follow the development of the expressed VH repertoire, lymphocytes were obtained at weeks 0, 5, 10, and 20 post primary immunization with trinitrophenylated-keyhole limpet hemocyanin (TNP-KLH) or infectious hematopoietic necrosis virus (IHNV). Lymphocytes were also collected 10 weeks post secondary immunization (week 35). These studies were conducted in parallel with serological analyses of plasma antibodies obtained from the same sample in order to correlate molecular data with serological data from individual trout. Antigen-specific lymphocytes were processed to isolate RNA templates to produce cDNA which was cloned and sequenced. This sequence analysis allowed us to report, for the first time, the temporal accumulation of potential somatic variants that correlate to the development of new, high affinity antibody subpopulations during the immune response, some with the emergence of new antibody heavy chain isoelectropherotypes as identified by 2D-IEF/SDS-PAGE. Southern analysis and gene titration using various antigen-specific cDNA probes allowed us to correlate trout antibodies possessing various affinities for antigen with specific VH sequence and gene family utilization. Thus, trout Ig VH gene family utilization appears to follow the mouse model of differential use for specific immune response. These results reveal a capability for fine-tuning the piscine immune response previously not recognized.

Leishmania infantum chagasi (Lic) is a pathogenic protozoan parasite and one of the etiological agents of human visceral leishmaniasis (VL). VL is a potentially deadly disease characterized by variable fevers, cachexia, hepatosplenomegaly, and global immune suppression. Many questions regarding the pathogenesis of VL and the mechanisms of host defense during Lic infection remain to be elucidated. The primary focus of this thesis is the relationship between Lic and the mammalian immune system. We studied parasite-host interactions during Lic infection at the molecular, cellular, and organismal level. We generated transgenic parasites that expressed firefly luciferase and/or fluorescent proteins to expand our capacity to detect, observe, and quantify the parasites in a variety of experimental settings with modern analytical methodologies. Using luciferase-expressing Leishmania, we developed an experimental infection model in which parasites were detected and the relative parasite burden in specific anatomical locations could be quantified in a live animal host using bioluminescence imaging. This method allowed the parasite burden to be assessed in the same host throughout the course of infection. Utilizing this model we have made some intriguing observations relating to the kinetics and distribution of the parasite burden over time. The parasite burden was observed primarily in the liver and bone marrow over the first few weeks and then shifts to the spleen and bone marrow. To gain a better understanding of the initial parasite-host immune interactions in vivo, we studied the early inflammatory response after intradermal (i.d.) inoculation. We observed a rapid and abundant influx of neutrophils into the inoculated ears. The neutrophil influx was transient, dose dependent and specific for the local inoculation site. While there was not a significant neutrophil influx into the draining lymph nodes (dLN), there was an increase in the total cellularity and a striking increase in the relative proportion of B cells to T cells over the first week after intradermal parasite challenge. By inoculating transgenic mCherry-Lic we found that neutrophils were the primary parasite-laden host cell in the dermal tissue during the first day, but macrophages harbored most of the parasites by 2 days. Neutrophil depletion using low-dose antibody treatment resulted in a reduced rate of parasite uptake initially at the site of inoculation, but no significant change in the dLN dynamics. We further examined the parasite-host relationship by studying molecular signaling and cellular interactions between Leishmania and human neutrophils. We investigated the nature of the chemotactic activity of Leishmania-conditioned growth medium for human neutrophils by testing physical properties of the activity and ruled out some of the major Leishmania surface molecules as potential candidates. We aim to identify the agent(s) responsible for the activity in on-going studies. To this end, we are collaborating with a group at the NIH and testing biochemical purification/separation samples. We conclude that intradermal Lic challenge induces a rapid innate immune response at the local site of infection, that neutrophils sense Leishmania-derived factors leading to directed migration, and that neutrophils function as a primary site for Leishmania entry into the mammalian host.

The research desires to evaluate the preventing policies on emerging infectious diseases by system dynamics, and takes the SARS situation in Taiwan for example. According to epidemiology and everything about SARS, we build the model of SARS transmission and prevention. Therefore we can simulate the situation and policies, and find the effective policies. After the simulation and the evaluation, we find that most SARS patients at later stage are affected in hospital. For the reason, the most effective policies are the ¡uPolicy about enhancing protection abilities in hospital¡v and ¡uPolicy about reducing the interaction with people in hospital¡v. Furthermore, the effectiveness of ¡uQuarantine policies¡v is not stronger than the above policies. The most important thing is that we discover Taiwan is very lucky, because the infectivity is very low (about 3.7%). If the infectivity of SARS were as high as 10% and we still took the same policies as we took in 2003, the situation would be terrible. Anyway, when we confront this kind of emerging infectious diseases, the better way is taking policies in hospital intently.

Interventions for influenza virus infections are essential to minimize the worldwide annual morbidity, mortality, and economic loss caused by this highly contagious respiratory pathogen. Establishment of universal, long-lasting protection against epidemic and pandemic strains of the virus can potentially eradicate the necessity of annual reformulation and readministration of low-efficacious seasonal vaccines, increasing pandemic preparedness. The protective potential of Type 1 T helper (TH1)-polarized memory CD4+ T cells against Influenza A virus (IAV) infection and generation of secondary memory populations following viral clearance are well-characterized. To assess the potential of CD4+ T memory cells as a candidate for adoptive immunotherapy, here we validated and optimized cryopreserved IAV-specific memory CD4+ T cell-mediated protection against infection and evaluated their potential for subsequent memory formation. Donor-derived in vitro-generated memory CD4+ T cells were transferred into IAV-infected naïve mice following cryopreservation of these cells for 6-12 months and overnight activation with gamma-chain cytokines, interleukin (IL)-7 and IL-7+IL-2. Results showed that cytokine-cultured cryopreserved memory CD4+ T cells, compared to their non-cultured counterparts, controlled viral titer in the lung at the peak infection phase, decreased morbidity, expedited recovery, and formed increased secondary memory cells in the lung, the primary site of infection, including lung tissue-resident memory (TRM) CD4+ T cells. Phenotypic and functional analysis confirmed that donor-derived secondary memory CD4+ T cells retain a TH1-phenotype and produce cytokines associated with protection against IAV. These observations support that the protectiveness and memory-forming potential of host- and/or donor-derived memory CD4? T cells can be preserved and harnessed for future use. This T-cell based adoptive immunotherapy addresses some of the current challenges of available preventative and therapeutic options, such as low vaccine efficacy, availability of only early treatment drugs, lack of immunity against pandemic strains and effective memory cell generation.

No description available.

The consistent seasonal signal that characterizes annual influenza epidemics has long suggested a causal link between the physical environment and the transmission of influenza. Yet, despite considerable interest--dating as far back as Hippocrates--the environmental factors that facilitate the seasonal spread of influenza remain unclear. Historically, significant study of influenza seasonality was based almost exclusively on temperate regions,.due to a lack of high-quality influenza data in low-latitudes. In turn, although numerous hypotheses have been forwarded to explain the seasonal nature of influenza in temperate regions, few acknowledge the seasonal patterns in lower latitudes.This dissertation examines the scientific evidence for the seasonal mechanisms that potentially explain the complex seasonal patterns of influenza disease activity across the latitudinal gradient extending from temperate to tropical regions. I identified seasonal climatic variables that are potentially responsible for influenza seasonality from observational, experimental, ecological and anecdotal studies. I then used a global database of influenza seasonality to assess the consistency of relationships between influenza seasonality and the seasonality of relevant climatic variables. I determined that no single climatic variable is consistently correlated with seasonal influenza activity across temperate, subtropical and tropical regions.However, I did find a significant U-shaped relationship between specific humidity and influenza epidemics globally with epidemics becoming increasingly likely as specific humidity increases or decreases from approximately 12 g/kg. Further, I examined the temporal and spatial variation of influenza activity and specific humidity during the 2009 A/H1N1 pandemic across Mexico, which spans temperate, subtropical and tropical regions. I show that specific humidity may have modified the progression of three distinct waves of infection during the pandemic. These patterns are in agreement with the U-shaped relationship between specific humidity and seasonal influenza epidemics observed at a global scale. In all, this is the first time that relationships between climate and influenza (both seasonal and pandemic) activity have been successfully synthesized into a single parsimonious model across temperate, subtropical and tropical regions.

Cholera in Haiti has persisted since its introduction after the 2010 earthquake. The outbreak demonstrates how a combination of socioeconomic factors, mainly a lack of infrastructure, can cause an outbreak to become a much more serious epidemic and the current enduring endemic. Because cholera came to a previously unexposed nation through United Nations peacekeepers, the outbreak in Haiti offers a unique perspective on the impact of globalization on public health. In addition, it provides a deeper look into the disproportionate impact of diarrheal diseases on low-income countries. Several biological and socioeconomic factors have facilitated the outbreak. Biological risk factors include immunologically naïve populations, low gastric acidity, and blood type. In addition, socioeconomic factors include a lack of clean drinking water and sanitation as well as a fragile and over-taxed healthcare system. The persisting struggle surrounding water and sanitation combined with a lack of knowledge on cholera prevention have precipitated the outbreak into an epidemic and further into its current endemic status. Current efforts to battle cholera include water and sanitation improvements, a national vaccination campaign, as well as the mobilization of community health workers. However, without the construction of sustainable water and sanitation infrastructure, it is unlikely that cholera in Haiti can be eliminated.