A meningite meningocócica permanece como importante causa de morbidade e mortalidade em todo o mundo. Extratos purificados de LOS de Neisseria meningitidis foram utilizados em um ensaio de ELISA a fim de detectar a especificidade dos anticorpos anti-LOS produzidos por 41 pacientes com doença meningocócica. Durante o período de convalescença, 100% dos pacientes apresentaram aumento da concentração sérica de IgG anti-LOS (L1, L2, L3,7, L6, L8 e L10) em no mínimo três vezes a concentração verificada nos soros coletados durante a fase aguda. O maior aumento foi de oito vezes para IgG anti-L3,7 adicionada de resíduo de ácido siálico. Portadores de Neisseria meningitidis e Neisseria lactâmica na nasofaringe não produziram anticorpos anti-LOS. Através de um ensaio ELISA de Inibição, foi possível verificar a especificidade dos anticorpos anti-LOS, exceto para os anticorpos IgG anti-L10, que apresentaram reação cruzada com outros tipos antigênicos de LOS como L2, L3,7 e L6, revelando-se um importante candidato a antígeno vacinal. / Meningococcal meningitis remains as an important cause of morbidity and mortality all over the world. Purified LOS extracts were used in an ELISA assay to verify the specificity of the antibodies produced due to meningococcal disease. All 41 convalescent sera collected from patients had at lowest a three-folder increase of anti-LOS IgG concentrations when compared with acute sera. A higher increase of eight fold was seen to anti-L3,7 IgG specific antibodies. Safe carries of Neisseria meningitidis and Neisseria lactamica had no detectable specific anti-LOS antibodies. In an Inhibition ELISA assay, except for anti-L10 IgG, antibodies against L1, L2, L3,7, L6 and L8 immunotypes were specific. Anti-L10 antibodies cross-reacted with L2, L3,7 and L6 epitopes.

The interaction between molecules is essential in a wide range of biological processes. A detailed knowledge of these interactions is necessary for understanding these processes. Among the systems that involve important interactions is the immune system. NMR spectroscopy has a large number of spectral parameters that were used in this work to study antibody-antigen interactions. These same parameters were also used to begin a structural analysis of a medium-sized protein, neurophysin, that has important interactions with neurohormones, and served here as a model antigen. A set of ligands differing in size and charge was designed and used to probe the binding site of anti-phosphocholine antibodies. These ligands ranged from small organic species to medium sized proteins. Amino acids, peptides and proteins were homogeneously linked to phenyl phosphocholine and analyzed by NMR techniques. Transferred nuclear Overhauser effect measurements were used to determine the conformation of bound ligands. The conformational change observed in some ligands was explained as either due to the antibody selecting one conformation that already exists, or the antibody binding inducing a conformational change. Titration data and detailed NMR analysis showed a more rigid M3C65 antibody fragment upon binding. In summary, with eight examples of ligands and four examples of antibodies studied by NMR, a spectrum of effects was seen, including a lock-and-key model and limited local induced fit. The contribution of the carrier molecule to antibody binding was in restricting the conformation of the ligand. Bigger ligands that are expected to be more immunogenic, showed less binding avidity as determined by immunological assays. Fluorinated ligands were synthesized to determine the kinetics of binding using 19F NMR spectra. Higher concentration of a fragment of the antibody M3C65 was analyzed to determine assignments of some residues in the combining site of the antibody. High resolution NMR techniques were used to assign resonances in neurophysin. The physiological role of neurophysin includes hormone storage and stabilization of oxytocin and vasopressin against proteolytic degradation within the posterior pituitary. Neurophysin is a 10 KD protein that dimerizes at high concentrations needed for NMR studies. An organic cosolvent was used to lower the dimerization constant, and hence inrease the spectral resolution. This permitted sequence-specific assignments that were then used to identify residues in the neurophysin-hormone binding site. Chemical shift differences and conformational changes were observed for the residues glutamate 47 and leucine 50. The 3₁₀ helix was further stabilized towards a more ideal helix upon hormone-analog peptide binding. Some of the residues contributing to the monomer-monomer interface were also assigned. Dimerization ill1duced chemical shift differences and conformational changes were observed for phenylalanine 35, threonine 38, and alanine 69. Tyrosine: 49 and phenylalanine 22 were affected but to a lesser extent. One characteristic of neurophysin in all studied cases was dynamic equilibrium between different folding states.

Profound vascular leakage in conjunction with elevated viremia is the hallmark of Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS). Antibody (Ab)-dependent enhancement (ADE), in which pre-existing, cross-reactive Abs enhance virus infectivity, is thought to be responsible for increased viremia, while loss of endothelial cell (EC) barrier integrity is the precursor to plasma leakage. However, the relationship between viremia and vascular leak has not been established. The objective of this dissertation project was to determine the involvement of antibodies in the pathogenesis of vascular leak syndrome associated with DHF/DSS by establishing a relationship between Ab-mediated increase in viremia and changes in vascular permeability, the hallmark of DHF/DSS. Our approach focused on characterization of human monoclonal antibodies (hMAbs) from a previously dengue virus (DENV)-infected patient for their ability to both neutralize and enhance infection and increase vascular permeability in vitro. Our results revealed that the human antibody response to DENV E protein elicited by natural infection is predominantly comprised of broadly cross-reactive antibodies targeting domain II epitopes. Using a multiplex cytokine immunoassay, qRT-PCR, and plaque assay, we demonstrated an association between viral load and cytokine production in DENV-infected FcγR-bearing K562 cells, and determined that DENV infection of K562 cells in the presence of hMAb resulted in a modulated inflammatory cytokine response with an overall pro-inflammatory profile. Using human microvascular ECs (HMEC-1), we further demonstrated an association between viral load, cytokine production, and the onset of permeability changes via an indirect mechanism in which inflammatory mediators released by DENV-infected K562 cells altered HMEC-1 barrier function and observed a synergistic effect between active DENV infection and release of inflammatory mediators by both K562 and HMEC-1 that increased permeability. Collectively, our results support the multifactorial nature of the pathogenesis underlying vascular leak, involving a complex interaction between ECs and FcγR-bearing cells, and a synergistic relationship between enhanced viremia and inflammatory mediators leading to increased permeability. Our use of hMAbs provided a novel approach to understanding how Abs impact the vasculature during DENV infection and enable identification of Ab characteristics that may trigger vascular leak, a crucial concern for DENV vaccine design. / acase@tulane.edu

Monoclonal antibodies (mAbs) have become an important part of therapy for a number of cancers. The first mAb to be approved for clinical use is rituximab, which is currently used for the treatment of various B cell malignancies. Despite its clinical value, the mechanisms in which rituximab induces tumor regression are unclear. Growing evidence suggests that multiple mechanisms involving complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) are involved. However, the direct interactions between CDC and ADCC have yet to be investigated. My studies examine the relationship between complement fixation and the activation of NK cells by utilizing in vitro assays, a syngeneic murine lymphoma model, and clinical samples from patients. Using these systems, I demonstrate that the initiation of the complement cascade inhibits NK cell activation and ADCC induced by rituximab in vitro. I also show that depletion of complement enhances the activation of NK cells and improves the efficacy of mAb therapy in a murine model. Lastly, I demonstrate that NK cell activation correlates with decreased complement activity in patients after rituximab treatment. The studies described in this dissertation have furthered the understanding of the mechanisms involved in antibody therapy. These results have described a novel inhibitory role for complement activity in the anti-tumor responses of mAbs. Furthermore, these findings suggest that strategies to circumvent the inhibitory effect of complement may improve how current mAbs are used and the how mAbs are designed in the future.

This thesis reports the characterisation of a novel outer membrane protein (OMP) from M. catarrhalis, designated M35, with a molecular mass of 36.1 kDa. This protein is structurally homologous to classic Gram-negative porins, such as OMP C from E. coli and OMP K36 from K. pneumoniae, with a predicted structure of 8 surface loops connecting 16 antiparallel -sheets. Comparison of the DNA sequences of the M35 genes from 18 diverse clinical isolates showed that the gene was highly conserved (99.6-100 % of nucleotides) with only one isolate (ID78LN266) having base variations that resulted in amino acid substitutions. A single amino acid mutation in the 3rd external loop of M35 in isolate ID78LN266 significantly affected antibody recognition, indicating that loop 3 contains an immunodominant B-cell epitope. The reduction in antibody-binding to M35 from ID78LN266 was similar to that caused by complete removal of loop 3. Since loop 3 folds into the porin channel in the classic structure, the antibody specificity to loop 3 was hypothesised to be a potential mechanism for evasion of host immune responses targeted to M35, potentially explaining the high degree of conservation across isolates. A series of recombinant proteins were constructed to analyse the binding to M35 of antibodies specificity for loop 3 or the remainder of the protein. It was found that loop 3- specific antibodies were not able to bind to M35 on the surface of M. catarrhalis and that this corresponds both with a lack of ability to enhance opsonophagocytosis in vitro and bacterial clearance in vivo. Additionally, antibodies raised against a version of M35 lacking loop 3 and M35 from the variant isolate ID78LN266 were both no less effective than the full consensus M35 by both these measures. It therefore appears that while the majority of antibodies raised against M35 are specific for loop 3 these antibodies do not mediate anti-M. catarrhalis actions. Two deletion mutant strains of M. catarrhalis that do not contain the outer membrane protein M35 were created by insertional inactivation of the M35 gene. Growth comparisons between these mutant strains and their wildtype parent strains initially led to the hypothesis that M35 is necessary for efficient glutamic acid uptake by M. catarrhalis, however this hypothesis was later shown to be incorrect. Efficient uptake of glutamic acid seemed to be mediated by a novel 40 kDa protein that was up-regulated in the deletion mutant strains, presumably to compensate for the lack of M35. M35 was also found to be essential for in vivo survival of M. catarrhalis in the nasal cavities of mice, indicating that it is an essential functional protein for colonisation of the mucosal surface.

Xenotransplantation is the final frontier of using vascularised organs or cellular grafts to treat end-organ disease and offers a potential solution to the worldwide shortage of human tissue available for transplantation. The main immunological barrier to xenografting from pig-to-primate is the antigen, Galactose-α1,3-Galactose (Gal) which is found in all species except humans and other higher primates. Even with the major advancement of deleting Gal from the potential pig donor species with the aid of cloning technology, complete elimination may be elusive as alternative genes yet to be fully characterised, may still produce Gal at low levels. Thus, the human immune response against Gal may continue to be a barrier to successful xenotransplantation. The aim of this project was to develop small animal models of the important components of xenograft rejection that largely relate to the anti-Gal immune response. These include models of hyperacute, acute vascular and chronic xenograft-like rejection that in turn, provide new insights in the immune mechanisms of the rejection processes. The role of antibody and both innate and cognate cellular immunity are explored. Both vascularised heart grafts and non-vascularised skin graft models are examined as rejection of solid organs may differ from cellular transplantation. The project also provides a platform for future studies in testing genetic and pharmacotherapeutic strategies to overcome the rejection processes uncovered.

<p>This study aims at introducing and describing a novel multi parameter analysis method to identify potential acquisition targets and to qualitatively and quantitatively evaluate the overall match between a target company and its acquirer. The method was tested with two recent real cases involving each an antibody based biotech company and a large fully integrated pharmaceutical company. The model was validated by comparing two independent antibody companies against the real cases, testing if they would have made better targets. It was found out that the in reality acquired companies scored highest, thus proving the validity of the method. One of the four potential targets got the highest scores for both acquirers. Consequently one of the acquired targets was only the second best match. The still independent companies would not have been better targets. The lowest scoring target company did get identical scores for both acquiring companies. Despite the proper prediction of targets, the scoring did not reveal the true underlying motives for the acquisitions, nor could significant parameters be identified to discriminate between target and non-target. This study adds a novel, valuable tool to the still limited arsenal of methods to qualitatively and quantitatively measure a match between target and acquirer solely based on publicly available data.</p>

<p>Background. Immunohistochemistry is a useful method in the differential diagnosis between pleural mesotheliomas and metastatic adenocarcinomas in the pleura. Cytokeratin 20 and 7 have been used successfully as markers in studies determining primary location of adenocarcinomas from metastases. The current study is a complementary research of archived paraffininbedded material of cases with cancer origin. This study contributes a bigger statistical material that may facilitate the search for unknown primary site of adenocarcinoma by identification of metastatic cells in the pleura.</p><p>Methods. Cells from the pleura taken from fifteen patients with diagnosed cancer of different types and eleven patients with cancer of unknown origin, were stained with antibodies against the tumour markers: Ber EP 4, calretinin, cytokeratin 20 and 7, estrogen receptor α, thyroid transcription factor, prostate-specific antigen and Cdx2.The staining was conducted in an automated immunohistochemical system. The staining of each kind of antibody was confirmed by a control section staining.</p><p>Results. All control staining ended perfect The whole panel of antibodies used on mammary cancer showed the same pattern for every antibody. Of the patients with cancer of unknown origin there were four that gave the same pattern, two men and two women. The women are deceased. To make a more careful evaluation more information and clinic background is needed. The number of samples is too small to draw any statistical conclusions.</p><p>Comment. Although the control staining was perfect the negative result of CK20 in the cases of diagnosed colon cancer was unexpected. This staining should be performed again to confirm the result. In some cases the number of cells were to few for a certain evaluation. The slides and the results of this work will be archived for further research.</p>

B lymphopoiesis in aged mice is characterized by reduced B cell precursors and an altered antibody repertoire. Aged mice maintain an ordinarily minor pool of early c-kit+ pre-B cells, indicative of poor preBCR expression, even as preBCR competent early pre-B cells are significantly reduced. Therefore, in aged mice, preBCR-mediated B2 B lymphopoiesis is significantly diminished; likely as a consequence of poor surrogate light chain expression. Notably, the remnant B1 B cell lineage present in adult bone marrow is retained in aged mice as evidenced by normal numbers (~0.3%) of Lin-CD19+B220low/- B1 B cell precursors. Of interest, B1 progenitors express substantially less lambda 5 surrogate light chain protein than do B2 pro-B cells and the surrogate light chain levels are further reduced in aged mice. B cells derived from putatively preBCR-deficient precursors, either B2 c-kit+ B cell precursors or B1 B cell progenitors, from either young or aged mice, generate new B cells in vitro that are biased to larger size, higher levels of CD43/S7, and decreased kappa light chain expression. Notably, immature B cells in aged bone marrow exhibit a similar phenotype in vivo, consistent with the changes seen in B cell precursor subpopulations. In aged mice, the B2 pathway is partially blocked with limited preBCR expression and signaling; however, continued B cell development via preBCR-deficient pathways, including B1 pathways, is observed. Increased generation of new B cells by these alternative pathways may contribute to altered phenotype, repertoire, and function in senescence.

This study aims at introducing and describing a novel multi parameter analysis method to identify potential acquisition targets and to qualitatively and quantitatively evaluate the overall match between a target company and its acquirer. The method was tested with two recent real cases involving each an antibody based biotech company and a large fully integrated pharmaceutical company. The model was validated by comparing two independent antibody companies against the real cases, testing if they would have made better targets. It was found out that the in reality acquired companies scored highest, thus proving the validity of the method. One of the four potential targets got the highest scores for both acquirers. Consequently one of the acquired targets was only the second best match. The still independent companies would not have been better targets. The lowest scoring target company did get identical scores for both acquiring companies. Despite the proper prediction of targets, the scoring did not reveal the true underlying motives for the acquisitions, nor could significant parameters be identified to discriminate between target and non-target. This study adds a novel, valuable tool to the still limited arsenal of methods to qualitatively and quantitatively measure a match between target and acquirer solely based on publicly available data.