Immunity, Pathogenesis, and Prevention of Poxvirus Infections: A Dissertation

Vaccinia virus (VAC) is the prototypical member of the orthopoxvirus genus of the poxvirus family and the virus used for smallpox vaccinations. The following describes the testing of VAC variants designed to have similar immuno-protective profiles with decreased pathogenicity, examines the immune response to VAC after lethal infection in wild type and lupus-prone mice, and describes a method that allows for the enumeration of VAC-specific CD8+ T in naïve and VAC-immune mice. The first part describes work examining VAC Wyeth (VAC-Wy) variants engineered to be less pathogenic in vivo. VAC-Wy variants included genes that code for three immunomodulatory proteins, an interferon-γ (IFNγ) binding protein (B8R), an interleukin 18 (IL-18) binding protein (C12L), and a complement binding protein (C3L, or C21L) or various combinations of the three knockouts, and a triple knockout (VAC-Wy -/-/-) in which all three genes were knocked out of a variant virus. The immunomodulatory effects of other IFNγ binding proteins on VAC-Wy pathogenesis in the mouse were also examined. Virus recombinants where the B8R gene was replaced with a truncated mouse IFNγ receptor gene or a gene that encodes a B8R/IFNγ fusion that allows for dimerization of the secreted IFNγ receptor were studied. As the knockouts and variants were made in the current vaccine VAC-Wy strain, only high dose (1x107 PFU’s) intra nasal (I.N.) infection of mice reliably resulted in detectable virus in the lungs. Further testing revealed that all knockout and variant viruses grew to similar levels after high dose I.N. infections. Protection induced by vaccination with the VAC-Wy variants was studied in comparison to immunizations with the VAC-Wy parental strain. Mice were immunized by tail skin scarification to mimic human immunizations, and this was followed months later by I.N. challenge with 20 LD50’s of VAC-WR. All VAC-Wy recombinants tested, including the VAC-Wy -/-/-, provided similar levels of protection as the parental VAC-Wy strain from a lethal VAC-WR I.N. infection. Mice immunized with the VAC-Wy -/-/- induced similar amounts of neutralizing antibody and similar numbers of CD8+ T cells specific to a subdominant determinant as VAC-Wy. While examining high dose, normally lethal, VAC-WR I.N. infections, a profound splenic CD8+ T cell immune suppression was noted that might have been caused by Fas dependent activation induced cell death (AICD). Using high dose intra-peritoneal (I.P.) and I.N. models of VAC-WR infection, decreased weight loss, decreased virus titers, and increased T cell numbers were found in Fas mutant (B6.MRL-Faslpr/J) mice in comparison to B6 wild type mice on day 6. It would be expected that Fas-deficient CD8+ T cells from B6.MRL-Faslpr/J mice (B6-lpr) would survive a high dose VAC-WR infection better than CD8+ T cells that could express Fas if T cells were being eliminated by Fas-dependent AICD, but co-adoptive transfer experiments using splenocytes from B6-lpr and B6.Cg- IgHaThy-1aGPi-1a/J (IgHa) wild type counterparts found no difference in the numbers or proliferation of donor CD8+ T cells at day 6. As the B6-lpr mice were better protected from VAC-induced weight loss early after lethal VAC-WR infections, it was possible that B6-lpr mice might be protected early in infection. In fact, Fas mutant mice had decreased virus loads in the fat pads, livers, and spleens in comparison to B6 wild type mice at days 2 and 3. In addition to the decreased virus titers, the severe splenic lymphocyte deficiency noted in B6 wild type mice as early as day 2 after high dose I.P. infection was ameliorated in B6-lpr mice. Further experiments demonstrated that uninfected B6-lpr mice had increased numbers of memory phenotype (CD44+) CD4+, CD8+ and γδ+ T cells, with an increased number of γδ+ T cells and NK cells in splenic lymphocytes in comparison to wild type B6 mice. Uninfected B6-lpr mice also had increased numbers of IFNγ+ CD8+ T cells after polyclonal stimulation with an antibody against CD3ε. In lymphocyte depletion experiments performed at day 3, antibody depletion of CD4, CD8, or NK or treatment with an antibody that was specific to the γδ+ TCR did not significantly alter virus loads in B6-lpr mice. In co-adoptive transfer experiments, splenocytes from wild type or B6-lpr mice survived high dose VAC-WR challenge similarly suggesting that B6- lpr splenocytes were not intrinsically better protected from lymphocyte depletion by lack of the Fas protein. On day 2 after high dose I.P. VAC-WR infection, B6- lpr mice had increased numbers of IFNγ+ NK cells, IFNγ+ CD8+ T cells, and IFNγ+ CD4+ T cells. B6-lpr and B6 mice treated with an antibody against IFNγ had significantly increased virus titers in the spleens and livers. Interestingly, there was no significant difference in liver or spleen virus titers when comparing anti- IFNγ antibody treated B6 mice or anti-IFNγ antibody treated B6-lpr mice. These results suggest that multiple leukocyte populations co-operatively or redundantly provide B6-lpr mice with increased protection from high dose VAC-WR infections through increased production of IFNγ. The third part of this work describes the enumeration of total numbers of pathogen-specific CD8+ T cells in a mouse through use of an in vivo limiting dilution assay (LDA). The extensive proliferation of virus-specific CD8+ T cells that occurs after virus infection was used to enumerate numbers of virus-specific CD8+ T cells in a naïve mouse. By transferring limiting amounts of carboxyfluorescein succinimidyl ester (CFSE)-labeled Thy1.1+Ly5.2+ heterogeneous CD8+ T cells into Thy1.2+Ly5.1+ hosts, CD8+ T cell precursor frequencies to whole viruses can be calculated. The calculations are based on finding the number of donor CD8+ T cells that results in CFSElo (i.e. proliferated) donor CD8 T cells in 50% of the hosts. Using probit or Reed and Muench 50% endpoint calculations, CD8+ T cell precursor determinations were made for naïve and immune states to a virus challenge. It was found that in naïve B6 mice, 1 in 1444 CD8+ T cells proliferated in response to VAC-WR (~13,852 VAC-WR-specific CD8+ T cells per mouse) and 1 in 2956 proliferated in response to lymphocytic choriomeningitis virus (LCMV) (~6,761 LCMV-specific CD8+ T cells per mouse). In mice immune to VAC-WR, the number of VAC-WR-specific LDA precursors, not surprisingly, dramatically increased to 1 in 13 (~1,538,462 VAC-WR- specific CD8+ T cells per mouse) consistent with estimates of VAC-WR-specific memory T cells. In contrast, precursor numbers to LCMV did not increase in VAC-WR-immune mice (1 in 4562, ~4384 LCMV-specific CD8+ T cells in a VAC-WR-immune mouse) consistent with the fact that VAC-WR provides no heterologous immunity to LCMV. Using H-2Db-restricted LCMV GP33-specific P14 transgenic T cells it was found that, after accounting for take of donor T cells, approximately every T cell transferred underwent a full proliferative expansion in response to an LCMV infection and a high efficiency was also seen in memory populations. This suggests that most antigen-specific T cells will proliferate in response to infections at limiting dilution. These results, which are discussed in comparison to other methods, show that naïve and memory CD8+ T cell precursor frequencies to whole viruses can be remarkably high. In total this work further advances knowledge of the immunity, pathogenesis, and prevention of poxvirus infections. This was accomplished by studying VAC-Wy recombinants as improved vaccines, by examining the mechanisms and cell types important in early protection from high dose poxvirus infections in B6 and B6-lpr mice, and by describing a method to enumerate total numbers of virus-specific CD8+ T cells in a mouse

Predictors of Delay in Seeking Health Care among Myocardial Infarction Patients, Minia District, Egypt

Objectives. To determine the barriers that hinder early seeking of medical care among Minia's myocardial infarction patients. Methods. The study was based on individual interviews with 207 men and women with a first confirmed myocardial infarction (MI), admitted to the coronary care units of hospitals in Minia city in the period from April 1 to August 30, 2014. Data was collected via structured questionnaire and patient medical charts. The delay was evaluated by assisting patients to triangulate time of symptom onset and time of professional health care by placing both times in context of daily activities that participants could easily remember. Results. The median (25th, 75th percentiles) delay time was 4 (2, 10) h. Only 32.8% of patients arrived within 2 hours of symptoms onset. Variables that significantly predicted prehospital delay time were patient's misinterpretation of nature of pain with OR 8.98 (95% CI) (3.97-20.32), illiteracy 7.98 (2.77-22.95), age (>65) 5.07 (1.57-16.29), and pain resistance behavior 4.61 (2.04-10.41). Conclusions. Interventions to decrease prehospital delay must focus on improving public awareness of acute myocardial infarction symptoms and increasing their knowledge on early treatment benefits

Myc controls a distinct transcriptional program in fetal thymic epithelial cells that determines thymus growth

Interactions between thymic epithelial cells (TEC) and developing thymocytes are essential for T cell development, but molecular insights on TEC and thymus homeostasis are still lacking. Here we identify distinct transcriptional programs of TEC that account for their age-specific properties, including proliferation rates, engraftability and function. Further analyses identify Myc as a regulator of fetal thymus development to support the rapid increase of thymus size during fetal life. Enforced Myc expression in TEC induces the prolonged maintenance of a fetal-specific transcriptional program, which in turn extends the growth phase of the thymus and enhances thymic output; meanwhile, inducible expression of Myc in adult TEC similarly promotes thymic growth. Mechanistically, this Myc function is associated with enhanced ribosomal biogenesis in TEC. Our study thus identifies age-specific transcriptional programs in TEC, and establishes that Myc controls thymus size

Real-time Visualisation of Actin Filaments within Native and Tissue Engineered Cartilage using SiR-Actin

Click on the DOI link to access the article (may not be free).Astrocytes, the most abundant glial cells in the central nervous system, play a critical role in supporting the normal physiological function of neurons. Recent studies have revealed that astrocyte transplantation can promote axonal regeneration and functional recovery after spinal cord injury. Biomaterial can be designed as a growth-permissive substrate and serve as a carrier for astrocyte transplantation into injured spinal cord. In this study, we developed a method to generate collagen microspheres encapsulating astrocytes by injecting the mixture of collagen and astrocytes into the cell culture medium with a syringe controlled by a syringe pump

In-vitro engineering of high modulus cartilage-like constructs.

To date, the outcomes of cartilage repair have been inconsistent and have frequently yielded mechanically inferior fibro-cartilage, thereby increasing the chances of damage recurrence. Implantation of constructs with biochemical composition and mechanical properties comparable to natural cartilage could be advantageous for long term repair. This study attempted to create such constructs, in-vitro, using tissue engineering principles. Bovine synoviocytes were seeded on non-woven polyethylene terephthalate fibre scaffolds and cultured in chondrogenic medium for 4 weeks, after which uniaxial compressive loading was applied using an in-house bioreactor for 1 hour per day, at a frequency of 1 Hz, for a further 84 days. The initial loading conditions, determined from the mechanical properties of the immature constructs after 4 weeks in chondrogenic culture, were strains ranging between 13 and 23 %. After 56 days (sustained at 84 days) of loading, the constructs were stained homogenously with Alcian blue and for type-II collagen. Dynamic compressive moduli were comparable to the high end values for native cartilage and proportional to Alcian blue staining intensity. We suggest that these high moduli values were attributable to the bioreactor setup, which caused the loading regime to change as the constructs developed i.e. the applied stress and strain increased with construct thickness and stiffness, providing continued sufficient cell stimulation as further matrix was deposited. Constructs containing cartilage-like matrix with response to load similar to that of native cartilage could produce long-term effective cartilage repair when implanted

Molecular subtype analysis determines the association of advanced breast cancer in Egypt with favorable biology

<p>Abstract</p> <p>Background</p> <p>Prognostic markers and molecular breast cancer subtypes reflect underlying biological tumor behavior and are important for patient management. Compared to Western countries, women in North Africa are less likely to be prognosticated and treated based on well-characterized markers such as the estrogen receptor (ER), progesterone receptor (PR) and Her2. We conducted this study to determine the prevalence of breast cancer molecular subtypes in the North African country of Egypt as a measure of underlying biological characteristics driving tumor manifestations.</p> <p>Methods</p> <p>To determine molecular subtypes we characterized over 200 tumor specimens obtained from Egypt by performing ER, PR, Her2, CK5/6, EGFR and Ki67 immunohistochemistry.</p> <p>Results</p> <p>Our study demonstrated that the Luminal A subtype, associated with favorable prognosis, was found in nearly 45% of cases examined. However, the basal-like subtype, associated with poor prognosis, was found in 11% of cases. These findings are in sharp contrast to other parts of Africa in which the basal-like subtype is over-represented.</p> <p>Conclusions</p> <p>Egyptians appear to have favorable underlying biology, albeit having advanced disease at diagnosis. These data suggest that Egyptians would largely profit from early detection of their disease. Intervention at the public health level, including education on the benefits of early detection is necessary and would likely have tremendous impact on breast cancer outcome in Egypt.</p

Biomechanical considerations in the pathogenesis of osteoarthritis of the knee

Osteoarthritis is the most common joint disease and a major cause of disability. The knee is the large joint most affected. While chronological age is the single most important risk factor of osteoarthritis, the pathogenesis of knee osteoarthritis in the young patient is predominantly related to an unfavorable biomechanical environment at the joint. This results in mechanical demand that exceeds the ability of a joint to repair and maintain itself, predisposing the articular cartilage to premature degeneration. This review examines the available basic science, preclinical and clinical evidence regarding several such unfavorable biomechanical conditions about the knee: malalignment, loss of meniscal tissue, cartilage defects and joint instability or laxity