Antibodies neutralize and eliminate pathogens, malignancies, and toxins by acting either alone or in association with Fc receptors which, once engaged, activate the elimination mechanisms of phagocytic cells. Based on structural differences, antibodies are divided into functionally distinct classes (IgM, IgD, IgG, IgE and IgA). Structure-function relationships within these classes are not well characterized. In addition, animal models for the assessment of potential therapeutic strategies for the modulation of the interaction between antibodies and Fc receptors are not established. Nonhuman primates are widely used to model human diseases and, represent excellent in vivo systems for this assessment. Therefore, we have studied nonhuman primate IgD as well as IgG and IgA specific Fc receptors in rhesus macaques, cynomolgus macaques, baboons and sooty mangabeys. IgD genes had not been identified in nonhuman primates nor the IgD receptors characterized in any species. We characterized IgD genes of the four monkey species, as well as chimpanzees and dogs. In contrast to other antibody classes, the IgD hinge regions are highly conserved between human and nonhuman primates, thus indicating a role in Fc receptor binding. In humans, Fc receptors CD16a (natural killer cells) and CD16b (neutrophils) bind IgG1 and IgG3, and CD89 (myeloid cells) binds IgA. To assess ligand binding and glycosylation properties of nonhuman primate CD16a, CD16b, and CD89, we sequenced, cloned, and generated recombinant molecules in a mammalian expression system. Our results verify the presence of CD16a, but not CD16b in nonhuman primates. CD16a is expressed on monocytes and a subpopulation of lymphocytes. In sooty mangabeys, CD16 is also expressed on neutrophils. Recombinant sooty mangabey/baboon CD16a binds to human IgG1 and IgG2, but not IgG3 and IgG4. Monkey CD89 has the same peripheral blood leukocyte expression profiles as humans, and binds human and recombinant macaque IgA. Blocking of N-glycans inhibited expression of CD89, but only marginally CD16a expression. Although extensive similarities of antibody/Fc receptor interactions exist between human and nonhuman primates, several differences must be considered when evaluating therapeutic strategies. However, these differences can be exploited to further characterize the structure-function relationships existing within antibody molecules and respective receptors.

Broadly neutralizing antibodies against HIV are rare; most patients never develop them at detectable levels. The discovery of four such antibodies therefore warrants research into their origins and their presumed unique characteristics. Such studies, however, require baseline knowledge about commonalities and biases affecting human immunoglobulin development. Obtaining that knowledge requires large sets of gene sequence data and the appropriate statistical techniques and tools. The Genbank repository provides a free and easily accessible source for such data. Several large datasets cumulatively comprising over 10,000 human Ig heavy chain genes were identified, downloaded, and carefully filtered. We then developed a special software tool called SoDA, which employs a unique dynamic programming algorithm to provide a statistical reconstruction of the events that led to a given antigen receptor gene. Once developed, tested, and peer-reviewed, we used SoDA to provide initial data about each downloaded gene with respect to gene segment usage, n-nucleotide addition, CDR3 length, and mutation frequency, thereby establishing the most precise estimates currently available for human Ig heavy chain gene segment usage frequencies. We compared data from productive non-autoreactive Ig to non-productive Ig and found evidence for gene segment usage biases, D/J segment pairing preferences resulting from multiple sequential D-to-J recombination events, and biases in TdT action between the V-D and D-J. Further analysis of autoreactive Ig genes yielded evidence that n-nucleotide addition comes at a cost: the higher the ratio of n-nucleotides to germline-encoded nucleotides for a given CDR3 length, the greater the probability of autoreactivity. These results suggest that the germline gene segments have been selected for lack of autoreactivity. It has previously been shown that human Ig gene segments have evolved efficient evolvability under somatic hypermutation. We have now extended these results, showing that Ig gene sequences are "tuned" to preferentially produce consequential mutations in the antigen-binding domains, and synonymous mutations in the framework regions. Together, these analyses provide new insights into the genetic and mechanistic biases shaping the human Ig repertoire. / Dissertation

Cellular adhesion research has recently focused on the small scale at the level of individual receptor-ligand bonds. This trend in research is primarily due to experimental advances which allow such individual bond force measurements. Here, one of these techniques, micromanipulation, has been extended to not only determine the bond force of individual receptor-ligand pairs, but also the intrinsic kinetic rates of the interaction. Using transmembrane (TM ) Fc gamma receptor III (CD16a-TM) and human IgG (hIgG), the dependence of adhesion probability on receptor-ligand expression densities, contract duration and contact area was quantitated. A probabilistic based theoretical formulation was developed and validated that relates the intrinsic molecular kinetic rates of the receptorVligand interaction to the experimentally determined adhesion probability. This theoretical formulation describing individual receptor-ligand kinetics has also allowed direct evaluation of existing biophysical bond strength/kinetics paradigms at the extreme condition of single bonds. A force-displacement model was also developed to quantitate the force exerted on the RBC membrane transducer during the micropipette retraction process and found to be in agreement with previous work. In addition to CD16a-TM, the kinetic rates of CD16a anchored via a glycosyl phosphatidylinositol (GPI) moiety (CD16a-GPI) and the two alleles of CD16b (NA1 and NA2) were determined for human, rabbit, and mouse IgG species. The binding affinity of these CD16 interactions to soluble IgG was also measured by traditional bulk chemistry approaches and compared to those measured via the micromanipulation protocol in which the IgG ligand is membrane bound in the solid phase. These data suggest that the membrane anchor itself can alter CD16 binding properties. This represents the first reported effect of the anchor on an intrinsic receptor property, its kinetic rates and binding affinity. This thesis presents two specific aims or goals. These goals were achieved and reported in this thesis. During the course of this research, I also explored other directions and gathered initial data. These directions were further explored by other researchers but the initial data is also presented here.

Utilizing self-assembled monolayer nanotechnology, micro-electro-mechanical systems (MEMS) and IC technologies, a novel flexural plate-wave (FPW) biosensor is developed in this dissertation for detecting the immunoglobulin-E (IgE) concentration of human serum. The acoustic waves of the proposed FPW devices are launched by the 25-pair inter-digital transducer (IDT) input electrodes and propagated through the 4.82 £gm-thick Si/SiO2/Si3N4/Cr/Au/ZnO floating thin-plate. Since the thickness of such floating thin-plate is much smaller than the designed wavelength of FPW device (80 £gm), most of the propagating wave energy will not be dissipated into outside of thin-plate and the mass sensitivity is very high. To further reduce the insertion loss of the proposed FPW devices, two 3 £gm-thick Al reflection grating electrodes (RGE) are designed beside the input and output IDTs. To implement a FPW-based IgE biosensor, a Cr/Au electrode layer has to be deposited on the backside of the floating thin-plate to serve as a substrate for further coating the cystamine SAM/glutaraldehyde/IgE antibody layers. Once the IgE antigens of human serum are bound to the IgE antibody layer, the small change in the mass of floating thin-plate will result in a shift of center frequency of the testing FPW-based biosensor. Compared to the reference FPW biosensors, the shift of center frequency generated by the testing FPW biosensor under different IgE antigen concentration can be detected by commercial network analyzer or the frequency-shift readout system developed by our collaboration laboratory (VLSI Design Lab. of NSYSU). Compared to commercial enzyme linked immunosorbent assay (ELISA) analyzer (sample volume >25 £gl/well, testing time >60 min, dimension>40 cm ¡Ñ30 cm¡Ñ10 cm), the implemented FPW-based IgE biosensor presents a smaller sample volume (<5 £gl), faster response (<10 min) and smaller size (<9 mm¡Ñ6 mm¡Ñ0.5 mm). In addition, a very low insertion loss (-9.2 dB), a very high mass sensitivity (-6.08¡Ñ109 cm2 g-1) and a very high sensing linearity (99.46 %) of the proposed IgE biosensor can be demonstrated at 6.6 MHz center frequency. This study successfully developed a novel FPW-based allergy biosensor by MEMS technology, which has great potential to be further applied into point-of-care testing (POCT) microsystem.

Prevalence of childhood asthma in Taiwan has increased 9 times from 1.3% to 10-14% in the past 4 decades. Many studies worldwide have demonstrated that many genes in different chromosomes are implicated in childhood asthma in different ethnic populations. A growing body of evidence suggests that allergic sensitization could occur in perinatal stage and correlate to the development of childhood asthma. Epidemiological studies, however, indicate that prevalence of childhood asthma is much higher in developed countries than that in developing countries; and prevalence of childhood asthma in metropolitan area is higher than that in country sites. This suggests that certain genes can interact with the environmental factors in developed countries to promote the development of childhood atopic disorders. Interests are now increasing on what is (are) the real pathogenic gene-gene interaction(s) for childhood atopic disorders under influence of age, gender and environmental factors? In a large perinatal cohort study with 1,211 pregnant women and their offspring from the obstetrics and pediatrics of Kaohsiung Chang Gung Memorial Hospital, we analyzed 159 allergy candidate genes with 384 single nucleotide polymorphisms and showed that 14 genes over 22 somatic and X chromosomes risk to or protective from cord blood immunoglobulin E (CBIgE) elevation are different from those genes associated with IgE elevation in children under 1.5, 3 and 6 years of age (12, 15 and 12 genes, respectively). CX3CL1, IL13, PDGFRA and FGF1 polymorphisms were associated with elevated IgE at earlier ages (newborn, 1.5 and 3 years); HLA-DPA1, HLA-DQA1, CCR5 and IL5RA polymorphisms were associated with IgE production at 6 years of age. Further analysis by multifactor dimensionality reduction (MDR) developed from data reduction strategy, we found that there are interactions among innate immunity, adaptive immunity, and response and remodeling genes on IgE production begin in prenatal stage. For example, The gene-gene interaction among IL13, rs1800925, CYFIP2, rs767007 and PDE2A, rs755933 was significantly associated with IgE production at 3 years of age. This suggests that different genotypes of genes interact one another on the IgE production contributing to the development of allergic diseases. Since the concentration of IgE is an important indicator of atopic disorders and allergic sensitization, we believe after clarifying the natural course of the genomic profiles on IgE elevation, certain early predictor(s) and preventive regimens for allergic sensitization or atopic disorders may be made possible.

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2008.

<p>Diss. (sammanfattning) Umeå : Umeå universitet, 1995, härtill 5 uppsatser.</p> / digitalisering@umu

No description available.

Analysis of M-components is an important part of the diagnosis of monoclonal gammopathies and for the evaluation of disease response during treatment. In this project, two widely used electrophoresis methods and their corresponding immunotyping method were compared to evaluate the sensitivity of each method for the detection of small M-components. The project included 30 plasma samples from patients with identified M-components; 10 samples containing each IgG, IgA and IgM, respectively. All samples were diluted with normal EDTA plasma to achieve M-components of 5,00g/L. The samples were then serially diluted to achieve M-component concentrations of; 5,00, 2,50, 1,25, 0,63, 0,31 and 0,16g/L. All 180 samples were analysed with agarose gel electrophoresis and capillary electrophoresis. The dilutions above and below the detection level of each method were then analysed with immunofixation and immunosubtraction. The results showed good agreement between agarose gel electrophoresis and capillary electrophoresis in the highest concentrations of IgG and IgM. With agarose gel electrophoresis, IgA was detected in the same location as transferrin and the lowest concentration detected were therefore 1,25g/L. Besides the samples containing IgG, immunofixation was the most sensitive method.

Focused ultrasound (FUS) can temporarily increase blood-brain barrier (BBB) permeability and locally deliver therapeutic agents to the brain. To date, applications of FUS for treatment of Alzheimer’s disease (AD) have not been explored. Here, I propose that FUS can facilitate a rapid reduction in amyloid-β peptide (Aβ) pathology in a mouse model of AD. Firstly, FUS was used to enhance delivery of an antibody directed against Aβ, which aggregates and forms extracellular plaques. FUS mediated the delivery of antibodies to the targeted right cortex by 4 hours post-treatment and antibodies remained bound to Aβ plaques for 4 days. At 4 days post-treatment, stereological quantification of plaque burden demonstrated a significant reduction of 23%. Secondly, FUS treatment alone resulted in a significant reduction in plaque load (13%). I then investigated effects of FUS that may contribute to Aβ plaque reduction, specifically the delivery of endogenous antibodies to the brain and, activation of microglia and astrocytes. Endogenous immunoglobulin was found bound to plaques within the treated cortex at 4 days post-FUS. Western blot analysis confirmed that immunoglobulin levels were increased significantly. Further, FUS led to a time-dependent increase in glial response. The expression of ionized calcium-binding adaptor molecule 1, a marker of phagocytic microglia, was increased at 4 hours and 4 days, and it was resolved by 15 days. Astrocytes had a slightly delayed response, with an increase in the expression of glial fibrillary acidic protein at 4 days, which declined by 15 days. After 4 days, microglia and astrocytes had significantly greater volumes and surface areas, signifying enhanced activation in the FUS-treated cortex, without an apparent increase in cell count. Co-localization of Aβ within activated glia revealed a significant increase in Aβ internalization following FUS. In conclusion, it was demonstrated that the delivery of exogenous antibodies by FUS, and FUS alone can lead to plaque reduction. Mechanisms by which FUS alone reduces plaque load may include entry of endogenous antibodies to the brain and the induction of a transient glial response. This work details acute effects of FUS that highlight the promise of this delivery method for AD treatment.