A disponibilidade de hemoderivados é um parâmetro importante para medir a qualidade da saúde em um país. Dentre os produtos hemoderivados, imunoglobulinas tem alto valor agregado. O Instituto Butantan tem por objetivo o estabelecimento de uma planta industrial para fracionamento de plasma, com um processo produtivo baseado principalmente em cromatografias. Eventos tromboembólicos a partir de infusões de imunoglobulinas por via intravenosa (IgIV) foram relacionados com presença de Fator XI de coagulação (FXI) como contaminantes nas preparações de IgIVs. Com objetivo de detectar o FXI nas frações, o processo cromatográfico foi testado em escala piloto, bancada, e em cromatografia direta e o FXI foi dosado nas frações iniciais e no produto final IgIV. Foram estabelecidos os métodos de dosagem de atividade de FXI por tempo de coagulação e ensaio cromogênico. Concluímos que o FXI acompanha a IgG nas etapas iniciais dos processos cromatográficos e verificamos que ocorre a presença de FXI nos produtos finais. Este trabalho contribui para o desenvolvimento do conjunto de testes de controle de qualidade de biofármacos derivados de plasma humano. / The availability of hemoderivatives is an important parameter to measure a countrys health quality. Among hemoderivatives products, immunoglobulin have high value.. Instituto Butantan, aims the establishment of an industrial plant for plasma fractionation with a process drawn, based mainly in chromatographies. Thromboembolic events from infusions of intravenous immunoglobulins (IVIg) have been related with the presence of coagulation Factor XI (FXI) as contaminant in the IVIG preparations. With an objective of tracking FXI in the chromatographic fractions, the process was tested in pilot and bench scales and in direct chromatography and the FXI was measured in the initial fractions and in the final product IVIg. The methods of measurement of FXI activity thru coagulation time and chromogenic assay were established. We have concluded that FXI accompanies IgG in the early stages of the chromatographic processes and verified that the presence occurs in the final products, This work contributes to the development of the set of quality control tests of biopharmaceuticals derivatives from human plasma.

Orientador: Claudio Lucio Rossi / Tese (doutorado) - Universidade Estadual de Campinas. Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T09:51:57Z (GMT). No. of bitstreams: 1 Suzuki_LisandraAkemi_D.pdf: 1217880 bytes, checksum: 4ceb1f773f112c88145bc69e02976ef2 (MD5) Previous issue date: 2010 / Resumo: A neurocisticercose (NC) e uma importante causa de doença neurológica em muitos paises em desenvolvimento, incluindo o Brasil. O diagnostico clinico da NC e dificultado pelo polimorfismo e pela não especificidade dos sintomas. As tecnicas de neuroimagem e pesquisa de anticorpos específicos tem contribuído para o diagnostico da NC e uma melhor compreensão dos processos fisiopatológicos dessa infecção. O presente trabalho teve como objetivo avaliar, por meio de técnicas imunoenzimaticas (ELISA), a resposta imune humoral na NC, utilizando como preparações antigênicas o liquido vesicular (LV) e uma fração glicoproteica obtida do extrato bruto de cisticercos de Taenia solium (T. solium) com afinidade por lentil-lectina (fração Gp). Cinquenta e seis amostras de liquido cefalorraquidiano (LCR), 22 de pacientes com NC e 34 de pacientes com outros problemas neurológicos, foram utilizadas para a pesquisa de IgG e suas subclasses, com os seguintes resultados: IgG-LV: 100% de sensibilidade e especificidade; IgG1 -LV: 72,73% de sensibilidade e 100% de especificidade; IgG2-LV: 81,81% de sensibilidade e 100% de especificidade; IgG3-LV: 59,09% de sensibilidade e 97,06% de especificidade; IgG4-LV: 90,91% de sensibilidade e 97,06% de especificidade; IgG-fração Gp: 90,91% de sensibilidade e 97,06% de especificidade; IgG1-fração Gp: 59,09% de sensibilidade e 91,18% de especificidade; IgG2-fração Gp: 68,18% de sensibilidade e 94,12% de especificidade; IgG3-fração Gp: 36,36% de sensibilidade e 100% de especificidade; IgG4-fração Gp: 86,36% de sensibilidade e 100% de especificidade. Quarenta e sete amostras de LCR, 16 de pacientes com NC e 31 de pacientes com outros problemas neurológicos foram utilizadas para a pesquisa de IgE, com os seguintes resultados: IgE-LV e IgE-fração Gp: 93,75% de sensibilidade e 100% de especificidade. Cinquenta e sete amostras de soros, 22 de pacientes com NC, 18 de pacientes com outras infecções e 17 de pessoas presumivelmente sadias, foram utilizadas para a pesquisa da IgG e suas subclasses, IgE, IgA e IgM, com os seguintes resultados: IgG-LV: 100% de sensibilidade e especificidade; IgG1-LV: 86,36% de sensibilidade e 94,28% de especificidade; IgG2-LV: 90,91% de sensibilidade e 97,14% de especificidade; IgG3-LV: 86,36% de sensibilidade e 97,14% de especificidade; IgG4-LV: 100% de sensibilidade e de especificidade; IgG-fração Gp: 95,45% de sensibilidade e 100% de especificidade; IgG1-fração Gp: 63,64% de sensibilidade e 94,28% de especificidade; IgG2-fração Gp: 68,18% de sensibilidade e 97,14% de especificidade; IgG3-fração Gp: 54,54% de sensibilidade e 88,57% de especificidade; IgG4-fração Gp: 90,91% de sensibilidade e 100% de especificidade; IgELV: 90,91% de sensibilidade e 97,14% de especificidade; IgE-fração Gp: 86,36% de sensibilidade e 100% de especificidade; IgA-LV: 54,54% de sensibilidade e 94,28% de especificidade; IgA-fração Gp: 13,63% de sensibilidade e 100% de especificidade. Anticorpos IgM não foram detectados com as preparações de LV e fração Gp. Nossos resultados mostraram que, com ambas as preparações antigênicas, tanto em amostras de LCR quanto em amostras de soros, a maior positividade foi obtida na detecção de anticorpos das classes IgG e IgE, seguida da positividade da IgA. Anticorpos IgM não foram detectados em amostras de soros com reações de ELISA realizadas com LV e fração Gp. Com relação as subclasses da IgG, a IgG4 apresentou, tanto em amostras de LCR como em amostras de soros, valores de positividade e concentração iguais ou superiores as outras subclasses. As reações ELISA realizadas com LV mostraram sensibilidades iguais ou superiores aquelas obtidas com a fração Gp. Considerando a complexidade e o custo final da obtenção da fração Gp, o LV pode ser considerado mais adequado para a pesquisa de anticorpos em amostras de LCR e soros de pacientes com NC. / Abstract: Neurocysticercosis (NC) is an important cause of neurological disease in many developing countries, including Brazil. The clinical diagnosis of NC is hindered by the polymorphism and non-specificity of the symptoms. Neuroimaging techniques and detection of specific antibodies have contributed to the diagnosis of NC and a better understanding of the physiopathological processes of this infection. The purpose of this study was to evaluate the humoral immune response in NC by using immunoenzymatic techniques (ELISA) in which vesicular fluid (VF) and a glycoprotein fraction purified from a crude extract of Taenia solium cysticerci with affinity for lentil-lectin (fraction Gp) were used as antigenic preparations. Fifty-six cerebrospinal fluid (CSF) samples, 22 from patients with NC and 34 from patients with other neurological disorders, were assayed for IgG and IgG subclasses, with the following results: IgG-VF: 100% sensitivity and specificity, IgG1 - VF: 72.73% sensitivity and 100% specificity, IgG2 -VF: 81.81% sensitivity and 100% specificity, IgG3 -VF: 59.09% sensitivity and 97.06% specificity, IgG4 -VF: 90.91% sensitivity and 97.06% specificity, IgG-fraction Gp: 90.91% sensitivity and 97.06% specificity, IgG1- fraction Gp: 59.09% sensitivity and 91.18% specificity, IgG2-fraction Gp: 68.18% sensitivity and 94.12% specificity, IgG3 -fraction Gp: 36.36% sensitivity and 100% specificity, IgG4 - fraction Gp: 86.36% sensitivity and 100% specificity. Forty-seven CSF samples, 16 from patients with NC and 31 from patients with other neurological disorders, were assayed for IgE, with the following results: IgE-VF and IgE-fraction Gp: 93.75% sensitivity and 100% specificity. Fifty-seven serum samples, 22 from patients with NC, 18 from patients with other infections and 17 from presumably healthy individuals, were assayed for IgG, IgG subclasses, IgE, IgA and IgM, with the following results: IgG-VF: 100% sensitivity and specificity, IgG1-VF: 86.36% sensitivity and 94.28% specificity, IgG2 -VF: 90.91% sensitivity and 97.14% specificity, IgG3 -VF: 86.36% sensitivity and 97.14% specificity, IgG4 -VF:100% sensitivity and specificity, IgG-fraction Gp: 95.45% sensitivity and 100% specificity, IgG1- fraction Gp: 63.64% sensitivity and 94.28% specificity, IgG2 -fraction Gp: 68.18% sensitivity and 97.14% specificity, IgG3 -fraction Gp: 54.54% sensitivity and 88.57% specificity, IgG4 - fraction Gp: 90.91% sensitivity and 100% specificity, IgE-VF: 90.91% sensitivity and 97.14% specificity, IgE-fraction Gp: 86.36% sensitivity and 100% specificity, IgA-VF: 54.54% sensitivity and 94.28% specificity, IgA-fraction Gp: 13.63% sensitivity and 100% specificity. No specific IgM antibodies were detected with VF and fraction Gp antigenic preparations. These results show that with the two antigenic preparations the highest positivity in CSF and serum samples was obtained for IgG and IgE antibodies, followed by positivity for IgA. No IgM antibodies were detected in serum samples assayed with VF and fraction Gp. With regard to IgG subclasses, IgG4 positivity and concentration in CSF and serum samples were higher than or equal to the other subclasses. ELISA reactions done with VF showed equal or higher sensitivities than those obtained with fraction Gp. Considering the complexity and high cost of obtaining fraction Gp, VF could be more suitable for detecting specific antibodies in CSF and serum samples from patients with NC. / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

Made available in DSpace on 2017-07-10T17:47:58Z (GMT). No. of bitstreams: 1 Juliandro_Ostapachen.pdf: 456635 bytes, checksum: 34bc017b7ec1271a02e2edb2dac65a1e (MD5) Previous issue date: 2015-02-20 / The challenge of keeping the properly development in the Holstein breeders of reposition (HPB) in the initial stages of creation, depends on both the feed management as well the health condition. The management of heifer s creation, during suckling in collective stalls, with automatic feeders enables the search for new nutritional protocols associated to the transfer of the passive immunity (TIP) which are both determining factors to the success of the livestock. Aiming the evaluation of the influence of the IgG serum in the weight gain of heifers during suckling and after their first year of life, it was conducted an experiment with 45 Holstein heifers distributed in three collective stalls equipped with automatic feeders where the heifers were undergone to three different suckling protocols. These protocols were denominated as: treatment one, with the daily provision of 4 liters of milk replacer at ease; treatment two, with the daily provision of 6 liters of milk replacer at ease; and treatment three with the daily provision of milk replacer at ease. All the heifers began the experiment on their fifth day of life and the concentration of serum immunoglobulin G (IgG), was at least 10 mg/dL, with a variation around 13,4 and 43,7 mg/dL. The experimental period of suckling, in all the treatments was fixed in 55 days and all the animals were weaned on their sixth day of life. The heifers had their weight gain checked weekly. After weaned, all the heifers were conducted to a single collective lot and handled under the same feeding conditions until their first year of life. The feeding management in the post-weaning had a daily ingestion of 2kg of supplement divided in 2 meals during the period of 10 months. During this period, they also had Tifton straw at ease and free access to pickets with Tifton grass. After the weaning, the measurement of the body weight of the heifers was taken in monthly intermissions. The experimental design used was totally randomized (DIC). All the data were evaluated by statistics analysis of the linear correlation of Pearson, analysis of variance ANOVA and submitted to the Tukey's average test at 5% and a paired t-test at 5% of significance. Regarding to the performance in the weight gain, there was no influence of the IgG concentration in the weight gain to the weaning and neither in the weight gain after the heifers were one year old. The evaluation of the weight gain occurred between the treatments and disregarding treatments. When dealing with the performance and taking only the suckling protocol, it has been found that the treatments 2 and 3 have shown a higher weight gain at weaning , but the efficiency in weight gain after the heifer's first year was higher in treatments 1 and 3. It was concluded that since the minimum serum concentration of IgG is 10mg/dL, there is no interference of this parameter about the weight gain at weaning and during the first year of life of the heifer. The milk consumption at ease ensures a better weight gain of animals compared to the animals with controlled milk consumption / O manejo de criação de bezerras na fase de aleitamento em baias coletivas equipadas com alimentadores automáticos possibilita a busca por novos protocolos nutricionais que, associados à transferência da imunidade passiva (TIP), representam fatores determinantes para o sucesso da criação. Com o objetivo de se avaliar a influência da concentração sérica de IgG no desempenho ponderal de bezerras em fase de aleitamento e após um ano de vida, 45 bezerras da raça Holandesa foram distribuídas em três baias coletivas equipadas com alimentadores automáticos e submetidas a três diferentes protocolos de aleitamento (Tratamentos experimentais) denominados de 4L, tratamento que teve o fornecimento de quatro litros diários de sucedâneo lácteo; 6L, tratamento com o fornecimento de seis litros diários de sucedâneo lácteo; e AL, tratamento com fornecimento ad libitum de sucedâneo lácteo. O fornecimento de concentrado para todos os animais foi a vontade. As bezerras entraram no experimento com cinco dias de idade e concentração de imunoglobulina G sérica (IgG), variou entre 13,4 e 43,7 mg/dL. O período experimental de aleitamento em todos os tratamentos foi fixado em 55 dias sendo que os animais foram desmamados aos 60 dias de idade. As bezerras tiveram seu ganho de peso aferido semanalmente. Após o desmame todas as bezerras foram conduzidas para um único lote coletivo e manejadas sob as mesmas condições alimentares até completarem um ano de vida. O manejo alimentar dos 65 dias aos 12 meses de vida contou com a ingestão de 2 Kg de concentrado diário divido em duas refeições, feno de Tifton à vontade, e, acesso contínuo à piquetes de pastoreio com grama Tifton. Após o desmame, a mensuração do peso corporal das bezerras foi feita em intervalos mensais. Utilizou-se o delineamento inteiramente casualizado (DIC), os dados foram avaliados através da análise estatística de correlação linear de Pearson, análise de variância ANOVA e submetidas ao teste de média de Tukey a 5%, e teste t pareado a 5% de significância. Com relação ao desempenho em ganho de peso, não houve influência da concentração de IgG no ganho de peso ao desmame e também no ganho de peso após um ano de vida. As avaliações de ganho de peso aconteceram entre os tratamentos e desconsiderando-se os tratamentos, com o objetivo de encontrar o efeito apenas da concentração de IgG sobre o ganho de peso, porém, o resultado foi o mesmo para ambas as avaliações. Quanto ao desempenho e o protocolo de aleitamento, verificou-se que os tratamentos 6L e AL foram superiores no ganho de peso ao desmame, porém a eficiência em ganho de peso após um ano de vida foi maior nos tratamentos 4L e AL. Conclui-se que desde que a concentração sérica mínima de IgG seja de 10 mg/dL, não há interferência deste parâmetro sobre o ganho de peso no desmame e durante o primeiro ano de vida da novilha. O fornecimento de leite a vontade garante melhor ganho de peso aos animais em relação ao fornecimento controlado

No description available.

The number of cases of Clostridium difficile infection (CDI) has been increasing globally. CDI is the main cause of nosocomial diarrhoea, which may be life-threatening in complicated cases, and also costs the health care societies millions of pounds annually. The predominant types and their resistance to antibiotics have been changing and one of the major selective pressures which causes this is antimicrobial use. Although much is known about the role of the toxins in pathogenesis of CDI, the role of immunogenic cell wall components is unclear. They may play a role in colonisation and pathology and a study of these could clarify the infection process. It is therefore important to study the immune responses against these bacterial wall components from different strains and their effects on stimulation of leukocytes to produce cytokines and chemokines. This study was divided into four parts: 1. An epidemiological study to determine frequencies of the predominant types of C. difficile, thus 140 C. difficile isolates from surgical patients and their environment during 2009 were investigated to define their PCR ribotype. This utilised capillary sequencing gel electrophoresis for their analysis. 2. The determination of antimicrobial susceptibility to six antibiotics (ampicillin, erythromycin, tetracycline, metronidazole, moxifloxacin and vancomycin) was assessed and MIC determination by agar dilutions. 3. Investigation of host immunity to molecules with conserved molecular patterns. Surface-layer proteins (SLPs), lipocarbohydrate (LC) and flagellar proteins were separated and purified from five ribotypes of C. difficile (001, 002, 027, 078 and106) predominant in Scotland. a) The immune responses to these molecules were assessed by ELISA by exposing serum of patients and healthy donors and measuring specific IgG levels. b) Innate immunity was investigated by distinguishing responses of a macrophage cell line (THP1) to the above molecules. Induction of interleukins (IL)-1β, IL-6, IL- 8, IL-10 and IL-12 interleukins and TNF-α was detected by ELISA. In this study 15 different ribotypes were identified. The most frequent were 001, 020, 106 ribotypes (52.8%, 7.4% and 5.7%), respectively, while 13 isolates could not be assigned a ribotype. However, all isolates were sensitive to vancomycin, metronidazole and moxifloxacin, but 74.28% of isolates were resistant to erythromycin. The IgG level against bacterial antigens (SLPs, LC and flagella proteins) in donors’ serum showed almost normal distribution to all antigens from the different ribotypes and the sensitivity of the assays was increased by raising the concentration of antigens. Levels to SLPs were generally the highest, but the flagellar protein exceeded the SLPs of the 027 ribotype. The donors, controls, patients and carrier sera gave similar results. The greatest induction of interleukins was obtained using 50μg of antigen with the THP-1 cells activated with 50ng of PMA. The highest induction of all antigens was for IL-10. The highest values for the control LPS was with IL-12. But the best effect for SLPs of 027 was for IL-10 (109.1ng/ml), while the weakest for TNF for SLPs of 027 (4.7ng/ml). In general the IL-1β, IL-6, IL-8 and TNF concentrations ranged from 4.7-60ng/ml for all antigens and in contrast IL-12 and IL-10 average ranged 11- 109.1ng/ml. To conclude, the prevalence of C. difficile and their antibiotic susceptibility are constantly changing. IgG antibodies to SLPs and flagellar proteins from the hypervirulent ribotype 027 were highest in the community and hospitalized individuals. The molecules of conserved molecular patterns are immunogenic with various levels of response in the monocytic THP1 cells. SLPs were best in inducing interleukins. Flagellar proteins from 027 ribotypes accompanied SLPs in IL-10 induction levels. Consequently SLPs and flagellar proteins from 027 ribotypes appeared the best immunogenic bacterial molecules.

<p>Coeliac disease (CD) is a multifactorial disease of the small intestine. In genetically predisposed individuals the, ingestion of cereals leads to a remodulation of the mucosal architecture, and the production of autoantibodies against tissue transglutaminase (tTG). The treatment is a lifelong gluten-free diet.</p><p>The diagnostic procedure relies on the examination of a small-bowel biopsy that displays villous atrophy. A spectrum of clinical manifestations is associated with CD, ranging from overt enteropathy to atypical and silent symptoms. Approximately 1% of the general population has CD, and the majority is undiagnosed. Although most patients with active CD can be detected by the assessment of elevated IgA-tTG, some patients lack these antibodies. Moreover, individuals with IgA-deficiency cannot be identified by means of IgA serology. </p><p>The aim of this thesis was to investigate the clinical utility of IgG-tTG for the detection and follow-up of subjects with active CD. The included studies showed that IgG-tTG was highly prevalent in IgA-deficient and IgA-competent patients with CD, whereas non-CD patients rarely had these antibodies. During a gluten-free diet, IgG-tTG decreased, demonstrating that IgG-tTG can be used to follow the patient’s adherence such a diet. Furthermore, 10% of healthy IgA deficient blood donors had elevated IgG-tTG, indicating that they had silent CD. </p><p>In IgA-competent subjects, high IgG-tTG levels correlated with a severe mode of CD and profound mucosal deterioration, suggesting that IgG-tTG might be involved in the disease progression. Moreover, we found that although a considerable percentage of IgA-competent patients lack IgG-tTG, the presence of these antibodies in conjunction with high levels of IgA-tTG was highly predictive of a severe small-intestine villous atrophy. It was also demonstrated that IgG-tTG normalisation coincided with clinical remission in IgA-competent CD patients on a gluten-free diet. </p>

Quenched Molecular Dynamics (QMD) used to explore molecular conformations was developed to operate in Insight II platform for two simulation engines: CHARMm and Discover. Two scripts and procedures were written for molecular minimization, dynamics, minimization of each of several hundred conformers, and cut off. Experience with Insight II/Discover versus Quanta/CHARMm, and between Insight II/CHARMm versus Quanta/CHARMm has taught that the forcefield is the key factor in QMD studies. Protein A has been used for the purification of commercial antibodies, but it is expensive. Seven peptidomimetics of protein A were designed based on the hot-spots located at the helix-loop-helix region of protein A, and synthesized via solid phase using the Fmoc approach. These peptidomimetics were characterized by MS and NMR. The conformations of four peptidomimetics were studied by NMR and CD in water/hexafluoroisopropanol (pH 4). The CD and NMR data show that addition of hexafluoroisopropanol stabilizes their a-helical conformations. The structures of these peptidomimetics in solution were generated with Quanta/CHARMm using NMR data as limits for the QMD technique. Protein G has also been used to purify antibodies, but it is expensive too. A number of protein G mimics were designed as trivalent molecules. An efficient preparation of trivalent molecules having a useful primary amine arm has been developed through solid phase synthesis. The cheap, commercially available poly(propylene imine) dendrimers were used as scaffolds which allow multimerization of functionalized compounds. A small library of trivalent compounds were synthesized using this approach. A portion of compounds in this library were tested by Amersham Biosciences. The seven amino acid modified DAB-Am-4 exhibits strong binding to the IgG/Fab, and is a potential ligand for IgG purification. The interactions between neurotrophins (ie NGF and NT-3) and their receptors are typical drug targets. Fourteen second-generation peptidomimetics showing NGF-like or NT3-like activities in a preliminary bioassay, were resynthesized and tested again. Preliminary and retested data were compared. To access a direct binding assay, five fluorescently labeled peptidomimetics 41a-e were synthesized for a fluorescence activated cell sorting (FACScan) assay. Six monomeric precursors 42 and 43 were prepared on large scales for the library of bivalent turn analogs

Coeliac disease (CD) is a multifactorial disease of the small intestine. In genetically predisposed individuals the, ingestion of cereals leads to a remodulation of the mucosal architecture, and the production of autoantibodies against tissue transglutaminase (tTG). The treatment is a lifelong gluten-free diet. The diagnostic procedure relies on the examination of a small-bowel biopsy that displays villous atrophy. A spectrum of clinical manifestations is associated with CD, ranging from overt enteropathy to atypical and silent symptoms. Approximately 1% of the general population has CD, and the majority is undiagnosed. Although most patients with active CD can be detected by the assessment of elevated IgA-tTG, some patients lack these antibodies. Moreover, individuals with IgA-deficiency cannot be identified by means of IgA serology. The aim of this thesis was to investigate the clinical utility of IgG-tTG for the detection and follow-up of subjects with active CD. The included studies showed that IgG-tTG was highly prevalent in IgA-deficient and IgA-competent patients with CD, whereas non-CD patients rarely had these antibodies. During a gluten-free diet, IgG-tTG decreased, demonstrating that IgG-tTG can be used to follow the patient’s adherence such a diet. Furthermore, 10% of healthy IgA deficient blood donors had elevated IgG-tTG, indicating that they had silent CD. In IgA-competent subjects, high IgG-tTG levels correlated with a severe mode of CD and profound mucosal deterioration, suggesting that IgG-tTG might be involved in the disease progression. Moreover, we found that although a considerable percentage of IgA-competent patients lack IgG-tTG, the presence of these antibodies in conjunction with high levels of IgA-tTG was highly predictive of a severe small-intestine villous atrophy. It was also demonstrated that IgG-tTG normalisation coincided with clinical remission in IgA-competent CD patients on a gluten-free diet.

Rhesus macaques represent a valuable model in biomedical research and in development of vaccines and therapeutics. Due to the lack of reagents, the general properties of IgG and corresponding cellular receptors (FcγR) in this species are poorly characterized. We engineered recombinant IgGs containing each of the four rhesus macaque heavy constant region (CH) subclasses. To define FcγRs that mediate IgGs, we identified and characterized three FcγR classes, and generated recombinant cDNA constructs. cDNA IgH constructs were created by fusing – by sequence overlap extension PCRs – a gene segment encoding the murine variable heavy domain specific for the hapten NIP, an established specificity system for assessing antibody effector functions, with rhesus macaque CH fragments. The complete IgH constructs were transfected into J558L cells, a murine IgH-lost myeloma cell line expressing anti-NIP light chain. Secretion of engineered IgGs was determined by ELISAs using NIP-BSA and anti-monkey IgG-specific antibodies. Molecular cloning methods were applied to identify and clone FcγR genes, and recombinant FcγR cDNA constructs were created by the recombinant DNA method. Four engineered IgH cDNA constructs were successfully created. Recombinant IgGs, in the intact Ig form and retaining the original anti-NIP specificity, were successfully produced. Compared to those in humans, FcγRs in rhesus macaques share high homology, yet also feature a relatively high level of intra-species polymorphism and possess different N-linked glycosylation patterns. FcγR constructs and expression vectors were successfully generated. The chimeric recombinant IgGs are powerful tools for defining IgG functional properties and studying CH structure/function relationship. These molecules can also be used as immunogens for generation of antibodies capable of unequivocally detecting individual IgG subclasses. The findings on FcγRs validate rhesus macaques as a model for studying antibody responses, and underscore the need to take into account of the genetic heterogeneity. The FcγR constructs and vectors serve as a tool for further studies of IgG/FcγR interactions. We also reported here our findings from a separate study that the main female hormone, 17β-estradiol, is capable of restoring antibody responses to an influenza vaccine in a postmenopausal mouse model, suggesting that immunogenicity and efficacy of influenza vaccines should be evaluated in postmenopausal women.

Quenched Molecular Dynamics (QMD) used to explore molecular conformations was developed to operate in Insight II platform for two simulation engines: CHARMm and Discover. Two scripts and procedures were written for molecular minimization, dynamics, minimization of each of several hundred conformers, and cut off. Experience with Insight II/Discover versus Quanta/CHARMm, and between Insight II/CHARMm versus Quanta/CHARMm has taught that the forcefield is the key factor in QMD studies. Protein A has been used for the purification of commercial antibodies, but it is expensive. Seven peptidomimetics of protein A were designed based on the hot-spots located at the helix-loop-helix region of protein A, and synthesized via solid phase using the Fmoc approach. These peptidomimetics were characterized by MS and NMR. The conformations of four peptidomimetics were studied by NMR and CD in water/hexafluoroisopropanol (pH 4). The CD and NMR data show that addition of hexafluoroisopropanol stabilizes their a-helical conformations. The structures of these peptidomimetics in solution were generated with Quanta/CHARMm using NMR data as limits for the QMD technique. Protein G has also been used to purify antibodies, but it is expensive too. A number of protein G mimics were designed as trivalent molecules. An efficient preparation of trivalent molecules having a useful primary amine arm has been developed through solid phase synthesis. The cheap, commercially available poly(propylene imine) dendrimers were used as scaffolds which allow multimerization of functionalized compounds. A small library of trivalent compounds were synthesized using this approach. A portion of compounds in this library were tested by Amersham Biosciences. The seven amino acid modified DAB-Am-4 exhibits strong binding to the IgG/Fab, and is a potential ligand for IgG purification. The interactions between neurotrophins (ie NGF and NT-3) and their receptors are typical drug targets. Fourteen second-generation peptidomimetics showing NGF-like or NT3-like activities in a preliminary bioassay, were resynthesized and tested again. Preliminary and retested data were compared. To access a direct binding assay, five fluorescently labeled peptidomimetics 41a-e were synthesized for a fluorescence activated cell sorting (FACScan) assay. Six monomeric precursors 42 and 43 were prepared on large scales for the library of bivalent turn analogs