The effect of continual antigenic stimulation on the immune system of mice was studied using two different experimental approaches. A GVHR was induced in Fi mice by the injection of parental spleen cells at weekly intervals. Several weeks later the spleen cells of mice undergoing a GVHR were shown to be immunosuppressed as their in vitro responses to the mitogens Con A and LPS were substancially lower than control animals. The serum from these treated mice was also immunosuppressive to normal spleen cells. The proliferative response to Con A and allogeneic cells of normal: syngeneic, allogeneic, and parental spleen cells was 90% suppressed when GVH serum was added in comparison to the addition of normal serum. Similarly, the in vitro antibody response to a T dependant antigen was impaired; however, the antibody response to a T independant antigen was not impaired. These results indicate that T cell functions are more sensitive than are B cell function to immunosuppressive factors in the serum of mice undergoing a GVHR. The serum was fractionated by gel filtration on a Bio-Gel P-200 column. The inhibitory material in GVH serum eluted in the immunoglobulin fraction of serum which indicate that it has a molecular weight of 150,000 or greater. The second approach studied involved continual allogeneic stimulation. Parental type mice were injected at five day intervals with Fi spleen cells in order to induce a HVG reaction. After several injections the spleen cells from these mice were tested in vitro. The spleen cells from HVG mice responded the same as normal spleen cells to the mitogens Con A and LPS. The spleen cells from HVG mice showed an enhanced in vitro antibody response as compared to normal spleen cells. This enhancement was attributed to the allogeneic effect. This series of experiments have shown that the induction of a GVHR in mice can later lead to immunosuppression and production of immunosuppressive factors in the serum of these mice. The induction of a short term HVG reaction has no adverse effects on the immune system except for enhancing an antibody response. It is possible that a more prolonged HVG reaction would parallel the immunosuppression observed in mice undergoing a GVHR. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Recent work has shown that in vitro, the cytotoxic immune response to cell surface antigens is enhanced if the antigen to which the immune response is directed, is on cells bearing major histocompatibility antigens identical to those of the responding cells. This 'H-2 restriction' has been demonstrated in the mouse using virally infected cells, haptenated cells, cells bearing the male Y antigen, and cells differing at the minor histocompatibility loci. Other investigations have shown that antigenic determinants coupled to tolerated antigens or isologous serum proteins, elicit a humoral response which is weaker than that to the same determinant coupled to a heterologous carrier. This and other evidence suggest an inverse relationship between humoral and cell mediated immunity. The purpose of this investigation was to explore the humoral response to antigens on cells which are syngeneic or allogeneic to the recipient, in order to determine the influence of a tolerated as opposed to allogeneic background. The approach used in this study was as follows: Mice were immunized with antigen (rabbit Fab' fragments) attached to syngeneic, allogeneic, or F₁ (semi syngeneic), irradiated spleen cells. Specific anti-rabbit Fab' plaque forming cell numbers were determined five days after the third, weekly injection of Fab' coated spleen cells. Some of the spleen cells taken from the responding animals, on the day of sacrifice, were incubated in vitro with soluble antigen (rabbit Fab' fragments not specific for mouse cells) for four days. The results showed that the humoral response to antigens attached to cells bearing 'self histocompatibility antigens (i.e. syngeneic or F₁ semi syngeneic cells) was significantly weaker than the humoral response to the same antigen on allogeneic cells. The effect of in vitro incubation of responder spleen cells for four days with soluble antigen was to reverse this difference. Those spleen cells exhibiting lowered plaque forming cell numbers initially (i.e. those cells from mice immunized with antigen on syngeneic or F₁ cell surfaces) showed, after incubation, a response equal to or greater than those cells which intially (before in vitro incubation) demonstrated a larger response (i.e. cells from those mice immunized with antigen on allogeneic cell surfaces). / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

This work attempts to establish some phenomenon of a rise in titer of C'3 and C'4 due to antigenic stimulation. Normal level of complement is established and compared to other workers as well as against guinea pig levels. Young rats were bled to establish normal levels of complement. The animals were then injected with an antigenic substance and after a period of seven days were bled again to determine the level of complement. Various antigenic and non-antigenic substances were used as well as normal saline injections for control.

Francisella tularensis is the causative agent of tularemia and is categorized by the CDC as a Tier 1 select agent. This gram-negative, facultative-intracellular bacterium infects macrophages by escaping the phagosome and replicating with high efficacy in the cytosol. Multiple virulence factors, including capsule and lipopolysaccharide (LPS), are expressed by F. tularensis. Biosynthesis of capsule and LPS O-antigen requires the same O-antigen biosynthesis gene cluster and, together, expression of capsule and O-antigen confer serum resistance. Mutations in the O-antigen biosynthesis gene cluster not only result in serum sensitivity, but also attenuate the ability to cause disease in vivo. In addition to changes in F. tularensis virulence, individual capsule and O-antigen mutants appear to have distinct intracellular phenotypes in macrophages. As previously shown by Lindemann et al. (2011), the capsule and O-antigen mutants FTT1236, FTT1237, and FTT1238 all replicated in human monocyte derived macrophages (MDMs) up to 16 hr and then ceased to replicate after that. This is hypothesized to be due to MDM cytotoxicity. In contrast, Raynaud et al. (2007) showed that the capsule and O-antigen mutant wbtA completely lacked replication in J774 macrophages, the reason for which has not been identified. A potential explanation for the loss of F. tularensis capsule and O-antigen mutant replication is capture and degradation by the host cell's autophagy pathway. Capture and degradation by autophagy is an accepted innate immune response to many intracellular pathogens. When small subpopulations of bacteria that normally replicate in membrane-bound vacuoles become cytosolic, such as Mycobacterium tuberculosis and Salmonella enterica serovar Typhimurium, they are targeted to forming autophagosomes through ubiquitination and binding of autophagy receptors. Pathogens have also developed methods to circumvent recognition and degradation by autophagy. Since F. tularensis replicates in the cytosol, it stands to reason that it has a means of evading detection by autophagy. We propose that expression of capsule and O-antigen acts as a mechanism used by F. tularensis to protect itself in an extracellular environment, as well as during intracellular infection. In this thesis we characterized nine different capsule and O-antigen mutants, and found different replication phenotypes in MDMs and varying degrees of MDM cytotoxicity. Also, only a subset of the mutants was detected by the autophagy marker, ubiquitin, supporting our hypothesis that different capsule and O-antigen mutants have diverse fates in MDMs. We also show that LVS and Schu S4 wbtA mutants had similar phenotypes. Upon further evaluation, we found that LVS wbtA more readily colocalized with ubiquitin, autophagy receptors, and the autophagy membrane protein LC3B, but not Beclin-1 or LAMP-1. This supports our hypothesis that capsule and O-antigen mutants are more susceptible to recognition by autophagy. Yet, because we did not observe LAMP-1 colocalization, there may be defects in the maturation of autophagosomes to degradative autolysosomes. Finally, we found that the fate of LVS wbtA in MDMs is dissimilar from J774 macrophages, suggesting macrophage species affect mutant fate. This thesis shows that different capsule and O-antigen mutants have multiple fates in MDMs, and suggest that F. tularensis capsule and O-antigen act as protective virulence factors that limit detection by autophagy.

Acute myeloid leukemia (AML) is a hematological disorder characterized by blockage of differentiation of myeloblasts. To date, the main therapy for AML is chemotherapy. Yet, studies are seeking a better treatment to enhance the survival rate of patients and minimize the relapsing of the disease. Since the major problem in these cells is that they are arrested in cellular differentiation, drugs that could induce their differentiation have proven to be efficient and of major interest for AML therapy. CD44 triggering appeared as a promising target for AML therapy as it has been shown that specific monoclonal antibodies, such as A3D8 and H90, reversed the blockage of differentiation, inhibited the proliferation of all AML subtypes, and in some cases, induced cell apoptosis. Studies conducted in our laboratory have added strength to these antibodies as potential treatment for AML. Indeed, our laboratory found that treating HL60 cells with A3D8 shows a decrease in the phosphorylation of the mammalian target of Rapamycin (mTOR) kinase correlated with the inhibition of proliferation/induction of differentiation of AML cells.The relationship between the induction of differentiation and the inhibition of proliferation and the decrease of mTOR phosphorylation remains to be clarified. To study the importance of the de-phosphorylation of mTOR and the observed effect of CD44 triggering on differentiation and/or proliferation, we sought to prepare phospho-mimic mutants of the mTOR kinase that will code for a constitutively phosphorylated form of mTOR and used two main methods to express this mutant in HL60 cells: lentiviral and simple transfection (cationic-liposomal transfection).

Diese Arbeit beschäftigt sich mit den Biomarkern Osteopontin und CD44 Standard, sowie CD44 Isovariante 6 beim Rektumkarzinom. Wir konzentrierten uns auf die prognostische Bedeutung von Osteopontin und CD44 Standard, sowie CD44 Isovariante 6. In einigen Vorgängerarbeiten zeigten sich Zusammenhänge vor allem bei der Tumorinduktion, Metastasierung und Überleben. In unserer Arbeit konnten wir bestätigen, dass sich hohe Serumkonzentrationen von OPN bei Patienten mit Rektumkarzinom hochsignifikant negativ auf das Gesamtüberleben auswirken. Niedrigere Serumkonzentrationen sind daher mit einer günstigeren Prognose assoziiert. Dies zeigte sich auch in der durchgeführten multivariaten Analyse. Wir kommen daher zu dem Schluss, dass sich OPN als prognostischer Marker eignet. In der Literatur zeigte sich CD44v6 mit verstärkter Metastasierung assoziiert. Dies konnten wir nicht bestätigen. Wir sahen CD44std und auch CD44v6 weder mit Gesamtüberleben, noch mit Tumorstadium und Metastasierung assoziiert. Auch wenn wir CD44 mit OPN gemeinsam auf das Gesamtüberleben untersuchten, fanden wir keinen signifikanten Einfluss. Als mögliche Schlussfolgerung dieser Arbeit könnte man die aktuelle Therapie des Rektumkarzinoms bei hohen OPN Werten reevaluieren. Bei hohen Osteopontin Werten wären dann ggfs. aggressivere Therapieprotokolle vorstellbar. / This thesis deals with the biomarkers osteopontin and CD44 standard, as well as CD44 isovariant 6 in rectal carcinoma. We focused on the prognostic importance of osteopontin and CD44 standard, as well as CD44 isovariant 6. In some previous studies, correlations were found, especially with tumor induction, metastasis and survival. In this thesis, we were able to confirm that high serum concentrations of osteopontin have a highly significant negative effect on overall survival in patients with rectal cancer. Lower serum concentrations are therefore associated with a better prognosis. This was also reflected in the multivariate analysis that was carried out. We therefore conclude that osteopontin is useful as a prognostic marker. In the literature, CD44v6 is shown to be associated with increased metastasis. We could not confirm this. We saw CD44std and CD44v6 associated neither with overall survival nor with tumor stage and metastasis. Even when we tested CD44 with OPN together on overall survival, we found no significant impact. As a possible conclusion of this thesis, therapy for rectal carcinoma could be re-evaluated with high OPN values. In the case of high OPN values, more aggressive therapy protocols might be conceivable.

Chlamydia trachomatis is an important cause of immune-mediated damage to the reproductive tract of infected patients. Certain chlamydial antigens and host genetic factors have been identified as contributing to immunopathological events, but a comprehensive understanding of specific components involved in destructive vs. protective immune responses to chlamydial infections is far from clear. In this study, it is shown that C. trachomatis-infected patients generate antibodies against an iron-responsive chlamydial protein, YtgA. The identity of YtgA was confirmed by mass spectrometry following two-dimensional polyacrylamide gel electrophoresis and Western blot analysis. This finding underscores a necessity to examine patient sera samples to identify chlamydial antigens that are likely encountered and important to the immune response during human infections.

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the causative agents of botulism and represents a family of seven structurally similar but antigenically different serotypes (A to G). The BoNTs are expressed in C. botulinum as a single polypeptide chain and then posttranslationally nicked, forming a di-chain polypeptide chain consisting of a 100-kDa heavy chain and a 50-kDa light chain held together by a disulfide bond. Topologically, the neurotoxins are composed of three domains, a binding domain (HC), a translocation domain (HN) and a catalytic domain. The BoNTs act preferentially on cholinergic nerve endings in both humans and animals and thus produce a flaccid paralysis that may result in death. In southern Africa, BoNT types C and D have been associated with botulism in cattle. To combat the disease, a bivalent vaccine consisting of formalin-inactivated type C and D holotoxins is currently available, and although it is efficacious, several concerns regarding its production has been raised, most notably its cost. The development of efficacious recombinant subunit vaccines may provide a means whereby many of the production problems may be eliminated or minimized. Consequently, the aim of this investigation was to produce a recombinant botulinum neurotoxin serotype D binding domain [BoNT/D(HC)] vaccine candidate for preventing BoNT/D intoxication. Towards this end, the gene fragment for the heavy chain (HC) of the BoNT produced by the C. botulinum type D vaccine strain D-50 was amplified, cloned in Escherichia coli and characterized by nucleotide sequence analyses. An alignment of the deduced amino acid sequence with that of characterized clostridial type C and D neurotoxins demonstrated that the heavy chains are composed of highly conserved domains interceded with tracts of amino acids exhibiting little overall relatedness, although considerable identity between the components ofa specific pair is apparent in certain of the regions. The deduced amino acid sequence exhibited 99, 66 and 73% identity with the reported amino acid sequences of BoNT/D-SA, BoNT/D and BoNT/C1, respectively. Attempts at expressing the native gene sequence for the HC from BoNT/D-50 in Brevibacillus brevis 47-5Q were unsuccessful. This may have been due to differences in codon bias between the heterologous gene and B. brevis. Consequently, a completely synthetic codonoptimized gene encoding the HC of BoNT/D-SA was constructed and expressed using a B. brevis 47-5Q mutant as expression host, obtained on mutagenesis with N-methyl-N’-nitro-Nnitrosoguanidine (NTG). Extracellular expression of the 48-kDa recombinant protein was verified by Western blot analyses with anti-BoNT/D antibodies. The recombinant BoNT/DSA(HC) protein was purified from the culture supernatant and used to vaccinate mice, after which their survival against challenge with active toxin was evaluated. Mice given two subcutaneous vaccinations were protected against intraperitoneal administration of 4 X 102 mouse lethal dosages (MLDs) of 16S BoNT/D-50 toxin. Antibody levels in mice surviving challenge were determined by enzyme-linked immunosorbent assays and confirmed that BoNT/D-SA(HC) was successful in evoking a protective immune response, whilst Western blot analyses indicated the presence of anti-16S BoNT/D-50 toxin antibodies in the serum. From these results it could be concluded that the recombinant BoNT/D-SA(HC) protein is an effective immunogen, able to protect against a high challenge dose of BoNT/D-50 neurotoxin. / Dissertation (MSc)--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

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Indiana University-Purdue University Indianapolis (IUPUI) / Triple-negative breast cancer (TNBC), which accounts for 15-20% of all breast cancers, is highly aggressive and metastatic with the poorest overall rates. While surgery, radiation, and chemotherapy remain the main treatment options, TNBC represents an unmet medical need for better treatment strategies. Tremendous efforts have been made to develop effective therapies over the past years. However, TNBC treatment options are still very limited due to the lack of good drug targets and the low response rate of current therapies. In this study, we developed two different strategies to treat TNBC based on its cancer genomic features: 1) heterozygous loss of chromosome 17p (17p loss) and 2) high mutation load. 17p loss is one of the most frequent genomic events in breast cancer including TNBC, rendering cancer cells vulnerable to the inhibition of POLR2A via α-amanitin (POLR2A-specific inhibitor). Here, we developed a new drug T-Ama (α-amanitin-conjugated trastuzumab) targeting HER2-low TNBC with 17p loss by combining the effects of α-amanitin and trastuzumab (HER2+ breast cancer therapy). Our results showed that T-Ama exhibited superior efficacy in treating HER2-low TNBC with 17p loss in vitro and in vivo, and surprisingly induced immunogenic cell death (ICD) which further enhanced T cell infiltration and cytotoxicity levels and delivered greater efficacy in combination with immune checkpoint blockade therapy. Collectively, the therapeutic window created by 17p loss and HER2 expression will make HER2-low TNBC clinically feasible targets of T-Ama. As another genetic feature of TNBC, the higher genomic instability and mutational burden results in more neoantigens presented on MHC-I, along with the higher level of tumor-infiltrating T cells, making TNBC a perfect model for immunotherapy compared to the other breast cancer subtypes. Here, we designed a deconvolution-algorithm-derived library screening to find new therapeutic targets and identified PIK3C2α as a key player that determines MHC-I turnover and reduces the MHC-I-restricted antigen presentation on tumor cells. In preclinical models, inhibition of PIK3C2α profoundly suppressed breast tumor growth, increased tumor-infiltrating CD8+ T cells, and showed high potential enhancing the efficacy of anti-PD-1 therapy, suggesting that PIK3C2α is a potential therapeutic target for TNBC immunotherapy. / 2025-05-22