The immune system produces antibodies to recognize and provide protection against infection. The immunoglobulin G (IgG) antibody isotype is present at high serum concentrations and has a longer half-life than other isotypes due to the interaction between its fragment crystallizable (Fc) region with the neonatal Fc receptor (FcRn). This Fc-FcRn interaction, which takes place in many cell types throughout the cardiovascular system, mediates pH-dependent formation of the IgG-FcRn complex and leads to the rescue of IgG from eventual degradation via transport from the low-pH early endosome back to the cell surface for release into serum at pH 7.4. Because this process is the primary determinant of IgG antibody half-life, and because the Fc region is common to all antibodies of the same subtype, the Fc-FcRn system has been a target of numerous antibody design and engineering studies. Indeed, several engineered Fcs have been reported with extended serum half-lives. These novel Fc variants, however, have generally been the result of extensive experimental screening and combinations of individual Fc mutations with known biophysical properties; there are few reports of predominantly structure-based rational Fc design. Notably, simply increasing Fc binding affinity for FcRn at low pH does not appear to be sufficient to achieve the largest increases in half-life (and in some cases, very high affinity results in reduced serum half-life). Most of these engineered Fcs have increased affinity not only at low pH (~6.0), but also at pH 7.4. The longest-lived Fc variant known to date, however, with mutations L309D/Q311H/N434S (“DHS”), has only a modest 5-fold increase in binding affinity compared to wt Fc at low pH, but also exhibits negligible binding to the receptor at pH 7.4 (Lee et al., 2019). This is consistent with previous reports that identify efficient release at physiologic serum pH to be critical to FcRn-mediated half-life extension. Thus, while engineering for affinity at low pH, it is also important to optimize the pH dependence of binding for optimal release at serum pH. The rational design process requires a detailed understanding of the structural and functional details of the interaction, which for a pH-dependent complex like Fc-FcRn must also include an accurate model of the pH-sensing mechanism. Unfortunately, the only publicly available crystal structure of a human Fc-FcRn complex is of the M252Y/S254T/T256E (“YTE”) variant, and was determined only to a relatively low 3.8 Å resolution, leaving the atomic positions of many sidechains, and even regions of the protein backbone, subject to substantial uncertainty. Furthermore, the widely accepted conventional mechanism of pH sensing, involving protonation of key histidine residues on Fc at low pH due to the assumed histidine pKa of 6.5 being within the range of interest (pH 6.0-7.4), is thermodynamically impossible. In this thesis I present an extensive analysis of the Fc-FcRn system, including the generation of all-atom models of human wild-type (wt) and variant complexes and the rat wt complex, and assignment of dominant protonation states at pH 6.0, at which most binding experiments are performed. I validate these models using retrospective molecular dynamics (MD)-based free-energy perturbation (FEP) calculations to compare to a large dataset of wt and mutant binding affinities. During this validation process I identify a residue on FcRn, glutamic acid 133, which adopts a highly unusual configuration in the complex and, due to quantum mechanical electronic polarization effects, is not described well by the fixed-charge molecular mechanics force field used by the FEP calculations, resulting in systematic errors for mutations that affect its hydrogen bonding network. I also identify a new variant, with a V308P mutation in a YTE background (“YTEP”), which induces a previously unreported conformational change that accounts for its high binding affinity compared to YTE and wt. To address the problem of the pH-sensing mechanism, I describe a general method for calculating the pH-sensing free energy of binding for any complex, based on a study of the pH dependence of protein unfolding free energies (Yang and Honig, 1993). The key observation underlying this method is that pH-dependent complex formation must be accompanied by a change in the pKa of one or more titratable groups between the unbound and bound states. Furthermore, the change in binding energy between two pHs can be directly calculated based on those pKas alone. As there are no experimental pKa measurements available for the Fc-FcRn interface residues, I perform these pH-sensing free energy calculations using FEP-based calculated pKas to quantitatively assess which residues at the interface are involved in sensing pH over the physiologically relevant pH range, and present a residue-level model for pH sensing in the Fc-FcRn system. Finally, I present some preliminary work toward the rational design of modified Fc regions with both increased affinity at low pH, and increased pH dependence of binding, using FEP calculations to guide experiment. This type of approach, of computational screening of a large number of different variants, followed by more limited experimental testing of promising leads, has the potential to streamline Fc design efforts and provide further insight into the structural basis of function for the Fc-FcRn system.

Emerging infectious diseases remain persistent threats that are challenging to predict. Humanity has faced many terrible pandemics and will face more, but to pinpoint the specific time and place of an outbreak, the type of pathogen, and the consequences is effectively impossible. This point was recently highlighted by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) viral pandemic, which led to global clinical and socioeconomic damage. When confronted by such a viral threat, the biomedical research community fervently responded with unprecedented haste to reveal SARS-CoV-2 clinical information, genome sequences, spike fusion protein structures, antigenic properties, antiviral therapeutics, and new vaccine platforms all within a year. As a small part of the tremendous collaborative research response, I used structural methods to study the SARS-CoV-2 spike fusion protein, specifically mechanisms of antibody-mediated viral neutralization. Viral fusion proteins are key components of virus particles that enable a virus to enter an animal host cell. Fusion proteins are the most common targets for neutralizing antibodies and serve a vital role as vaccine immunogens to elicit a protective immune response. To develop an understanding of SARS-CoV-2 antibody-mediated neutralization, one of my primary research interests was solving antibody structures in complex with the spike fusion protein using cryo-EM (cryogenic electron microscopy). With antibody structures I helped characterize spike epitopes, rationalize antigenic properties of emerging variants, and hypothesize viral neutralization mechanisms. I discovered antibody structures with multiple neutralization mechanisms including receptor blocking, conformational “locking” of the RBD (receptor binding domain), and spike disassembly. Viruses are evolving pathogens, and the Omicron sub-lineages are some of the most antibody-resistant SARS-CoV-2 variants to date. I studied mechanisms of Omicron antibody neutralization, which included traditional mechanism such as receptor blocking, as well as new mechanisms involving spike disassembly and conformational locking at SD1 (subdomain 1) epitopes. I also investigated broad antibody recognition at a conserved RBD epitope which neutralized not only SARS-CoV-2 but also SARS-CoV and other sarbecoviruses. Lastly, I had the opportunity to study other classes of viral fusion proteins, including those of alphaviruses and rabies virus, which serve as representative class members of the other varieties of viral fusion proteins, broadening my research for any type of known viral pathogen. Structural studies of antibodies highlight vulnerabilities of the spike protein when targeting SARS-CoV-2 and other fusion proteins in future vaccine design. The trials and tribulations of SARS-CoV-2 and the wealth of new research on coronaviruses offer hope of future pandemic preparedness. Understanding the structural mechanisms of viral fusion proteins and antibody neutralization gives hope of developing further therapeutic interventions. The work described in this thesis on fusion proteins SARS-CoV-2 spike (S), alphavirus envelope (E), and rabies virus glycoprotein (G) have prepared me to combat other infectious viral agents, including those already infecting humans and those at risk of spilling over into humans. When posed with such unpredictable emerging threats, we can learn from the past and position ourselves to be ready for the future.

Probiotic bacteria are increasingly prevalent in food and nutritional products today. These remarkable microorganisms are capable of imparting exceptional health benefits on their host, including prevention of infection by pathogens and stimulation of immune system function. Their most common mode of delivery is through dairy products (e.g. yogurt), which are also one of their preferred habitats. The interactions between probiotic bacteria and dairy systems have been studied, but are still not well discerned. There is a need for better understanding of these associations, as well as those surrounding the mode of bacterial transfer from the food product to the human gastrointestinal tract. Discoveries into the optimal means of probiotic transport to the body may lead to great advancements in both the design of probiotic foods and their exploitation in the support of human health. Much of the previous research on probiotic bacteria has explored their possible means of adherence in the intestine, as well their strengths in the promotion of human health. Studies relating to their interaction with dairy products are lacking, however, thus this work aims to elucidate some of these aspects. The primary endeavor of this thesis was to develop a technique to quantify the binding affinity of probiotic lactic acid bacteria for milk phospholipids. An additional objective was to exploit these bacteria, as well as dairy ingredients rich in bioactive molecules, in the creation of a highly nutritious food product. In these experiments, a collection of methods were used in progression in order to arrive at a novel protocol to assess binding with excellent reproducibility and simplicity. These included various membrane blotting techniques, as well as thin-layer chromatography. Essentially, phospholipids from both animal-derived standards and milk extracts were applied to a surface (e.g. PVDF membrane), and bacteria were incubated with them to allow binding reactions. The lactic acid bacteria selected for the final assays consisted of four strains of Lactobacillus, including L. reuteri (SD2112 and T-1), L. acidophilus, and L. casei (LC-10). Their adhesion to phospholipids was detected by either colorimetric or fluorescent labeling systems. To illustrate this, the final method developed was a procedure in which bacteria fluorescently stained with acridine orange were allowed to bind to dots of PVDF membrane coated with phospholipids. The results of this study showed that lactic acid bacteria undeniably exhibit selective binding affinity for phospholipids as opposed to other lipids such as triglycerides. The bacteria demonstrated significantly greater binding for a phospholipid extract from milk as opposed to individual phospholipid standards from other sources (p<0.05). Nonetheless, adhesion to all phospholipids was substantially greater than that to triglycerides. These findings, as well as the development of this method, should prove valuable in future research regarding the associations of probiotics with dairy systems. An additional purpose of this thesis was to design a dairy-based food product containing ingredient sources rich in milk bioactives. A gel-type product was created using primarily colostrum, buttermilk powder, and whey protein isolate, as well as selected strains of Lactobacillus. With the inclusion of immunoglobulin-rich colostrum, the product was analyzed alongside fluid milk and colostrum in order to quantify and compare these bioactive molecules. An enzyme-linked immunosorbent assay (ELISA) was used to complete this, and the results revealed concentrations that would be expected by the literature. Specifically, immunoglobulin G (IgG) was quantified by interpolation from a bovine IgG standard regression curve. The results showed that the concentration of IgG in the gel was nearly twice that of colostrum, and almost eight-fold higher than that of milk. This indicates that use of bioactive-rich substances, such as colostrum, in a food product may serve as a means of delivering more concentrated doses of bioactives than their respective ingredients. The research completed in this thesis is significant in that it contributes a valuable method to the elucidation of bacterial binding interactions with milk components, and also demonstrates the successful application of dairy ingredients to an innovative food product high in beneficial compounds. The insight provided by these studies could encourage further work in improving the understanding of probiotic delivery and advancing the development of bioactive-rich food products.

An immunoglobulin-fortified milk replacer and a subcutaneous (SC) injectable solution of immunoglobulins (Ig) were examined as methods of achieving passive immunity in neonatal calves. Bovine Ig, from abattoir blood, were purified by polyphosphate fractionation and ion-exchange chromatography. In experiment 1, carried out at Agriculture Canada Research Station, Agassiz, 37 colostrum-deprived Holstein-Friesian bull calves were allotted to one of four treatments. Col/WM calves were fed colostrum on day 1 and whole milk, days 2 - 42. MR-Nolg calves (control) were fed milk replacer with no Ig, days 1-42. MR-Hi/Lo calves were fed milk replacer with Ig at 50 mg/ml on day 1, and at 10 mg/ml, days 2 -21. MR-Hi/No calves were fed milk replacer with Ig at 50 mg/ml, day 1,'and with no Ig, days 2 - 21. From days 2 2 - 42, MR-Hi/Lo and MR-Hi/No treatment calves received milk replacer with no Ig. In experiment 2, carried out at the University Research Farm at Oyster River, 24 colostrum-deprived Holstein-Friesian bull calves were allotted to one of three treatments. The first two treatments were the same as for experiment 1, Col/WM and MR-Nolg fed for days 1-21. MR-Lo Inj calves were fed milk replacer with Ig at 10 mg/ml, days 1-21, and were also given a SC injection of Ig solution within the first 6 hours of life. For days 2-42, calves were fed WM or MR-Nolg, as per experiment 1. For both experiments, blood samples and calf weights were taken at birth, 24 & 48 hours of age, day 7 and weekly thereafter for six weeks. Diarrhea (scours) levels, rectal temperatures and general health of calves were recorded daily for the first three weeks as well. Experiment 1 survival at 6 weeks of age was 11 out of 11 calves for Col/WM treatment, 8 out of 8 calves for MR-Hi/Lo treatment, 7 out of 8 calves for MR-Hi/No and a significantly lower (P>0.05) 7 out of 9 calves for MR-Nolg. In experiment 2, survival was 7 out of 8 calves for both Col/WM and MR-Lo-Inj treatments and a significantly lower (P>0.05) 4 out of 8 calves for MR-Nolg treatment. Calves on MR-Hi/No had significantly higher diarrhea levels than the other three treatments over weeks one and four in experiment 1. In experiment 2, calves which did not receive any Ig had significantly higher diarrhea levels over weeks three and four than calves which received Ig. Experiment 1 average daily gains (ADG) were significantly higher for calves on Col/WM, MR-Hi/Lo and MR-Hi/No treatments than for calves on MR-Nolg at six weeks of age. In experiment 2, six week ADG were significantly higher for calves on Col/WM and MR-Lo Inj treatments than for MR-Nolg. For both experiments, serum Ig levels of calves on Col/WM were significantly higher than calves on the other treatments at 24 and 48 hours of age. MR-Hi/Lo, MR-Hi/No and MR-Lo Inj calves trended to higher serum Ig levels than MR-Nolg calves but were not significantly different. Calves which received Ig, from colostrum, the Ig-fortified milk replacer or a subcutaneous Ig injection, had higher survival rates, lower diarrhea levels, less antibiotic treatment and higher average daily gains than calves hot receiving any Ig. It was concluded that immunoglobulins, administered either orally or parenterally, are an effective, alternative method, for providing passive immunity in neonatal calves. / Land and Food Systems, Faculty of / Graduate

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: The African penguin, which occurs along the south-eastern and south-western shores of South-Africa and Namibia, has experienced a severe reduction in population numbers due to guano and egg collection in the first half of the 19th century, and oil pollution in the second half of the 19th century as a result of oil tankers rounding the Cape of Good Hope. The population would have been reduced by a further 19% had it not been for the rehabilitation of penguins at the South African National Council for the Conservation of Coastal Birds (SANCCOB) facility. Although this has been very successful, mortalities as a result of avian malaria infection have considerably reduced the efficiency of rehabilitation. In an effort to assess the role of immunity against malaria in combating the disease, an enzyme-linked immunosorbent assay (ELISA) for the detection of antibody levels to avian malaria was developed. The ELISA was used to detect antibody levels to avian malaria of penguins on entry and during rehabilitation from October 2001 to January 2003. The aim of this study was to continue the determination of antibody levels to avian malaria of penguins entering the SANCCOB facility, in order to allow an evaluation of the antibody levels to avian malaria for two full calendar years. This investigation was combined with a polymerase chain reaction (PCR)-based method, capable of detecting any Plasmodium species in penguin serum. These two methods were also used to investigate avian malaria in several breeding colonies in order to assess the role avian malaria may play in the survival of the African penguin in the wild. Results indicated that the ability of penguins to produce anti-Plasmodium antibodies was not influenced by oiling and that infection with malaria was not due to recrudescence but rather due to infection via mosquitoes. This indicated a possible role of the SANCCOB facility in exposing the penguins to avian malaria. However a large number of penguins arrived at the facility previously infected with malaria, indicating that malaria was present in the breeding colonies. Investigations in the breeding colonies revealed extremely high avian malaria prevalence even though no sick birds or mortalities were observed. This raised the question whether different types of malaria are responsible for infection in the SANCCOB facility and breeding colonies. / AFRIKAANSE OPSOMMING: Die Afrika Pikkewyn kom langs die suid-oostelike en suid-westelike kus van Suid Afrika en Namibië voor. In die afgelope eeu het hierdie spesie ‘n geweldige afname in populasie getalle ondervind. Dit was hoofsaaklik die gevolg van die versameling van guano en pikkewyneiers in die eerste helfte van die 19de eeu en oliebesoedeling in die tweede helfde van die 19de eeu. Die “South African Foundation for Conservation of Coastal Birds” (SANCCOB) is ‘n seevoëlreddings- en rehabilitasiesentrum vir siek, beseerde en ge-oliede pikkewyne. Dit word geskat dat die Afrika Pikkewyn populasie met ‘n verdere 19% sou afgeneem het as dit nie vir die rehabilitasie by die SANCCOB sentrum was nie. Hierdie sentrum het egter aansienlike vrektes in die somer as gevolg van voëlmalaria, wat sodoende die effektiwiteit van die rehabilitasie verlaag. In ‘n poging om die rol van immuniteit teen malaria te bepaal is ‘n “enzyme-linked immunosorbent assay” (ELISA) ontwikkel vir die bepaling van antiliggaam vlakke teen malaria. Hierdie ELISA is gebruik vir die bepaling van die anti-Plasmodium antiliggaam vlakke van die pikkewyne by aankoms en ten tye van rehabilitasie by SANCCOB vanaf Oktober 2001 to Januarie 2003. Die doel van hierdie studie was eerstens om hierdie ELISA bepalings voort te sit om sodoende antiliggaam vlakke teen malaria oor twee kalender jare te kan evalueer. Hierdie ondersoek was gekombineer met ‘n polimerase ketting reaksie (PCR) metode, wat enige Plasmodium spesie in pikkewynserum sou kon opspoor. Hierdie twee metodes is ook gebruik vir ondersoeke in sommige broeikolonies, met die doel om te bepaal watter rol voëlmalaria in die oorlewing van die Afrika pikkewyn in die natuur speel. Resultate het getoon dat olie nie die vermoë van die pikkewyn beïnvloed om anti- Plasmodium antiliggame te vervaardig nie en dat malaria infeksie hoofsaaklik deur muskiete veroosaak word en nie deur heruitbraak van ‘n bestaande infeksie nie. Dit dui egter daarop dat pikkewyne blootgestel word aan voëlmalaria by die SANCCOB sentrum. Daar is ook gevind dat ‘n groot aantal pikkewyne met malaria infeksies by die sentrum opgedaag het wat dui op die voorkoms van malaria in die broeikolonies. Ondersoeke in die broeikolonies het ‘n besonder hoë voorkoms van malaria onthul. Geen vrektes of siek pikkewyne is in die broeikolonies waargeneem nie, wat moontlik kan beteken dat pikkewyne by SANCCOB met ‘n ander tipe malaria geïnfekteer word as in die broeikolonies.

Sixty-two newborn Holstein-Friesian calves were used to study the role of colostrokinin, serum cortisol, and serum thyroxine in the absorption of maternal immunoglobulin. Calves were removed from their dams prior to suckling and assigned one of four rations: colostrum, whole milk, milk plus colostral immunoglobulin, and milk plus immunoglobulin plus colostrokinin. Calves were fed their assigned ration either at birth or after twelve hours inanition. All calves were fed pooled colostrum at 24 hours postpartum. Blood samples were collected at seventeen times during the first 32 hours postpartum. Calves were born with high cortisol concentrations (88 ng/ml) which decreased (P < .05) within two hours postfeeding. Serum cortisol levels increased (P < .05) between two and three hours after calves ingested a colostral source of immunoglobulin. Time of initial feeding had no effect on the cortisol surge. No such increase was observed in neonates consuming an immunoglobulin-free milk ration. These results demonstrate that the immunoglobulin fraction of colostrum is responsible for initiating an increase in cortisol secretion by the adrenal cortex. Within four hours postpartum, serum thyroxine concentrations increased (P < .05) at least 50% in all treatment groups regardless of whether the calves were fed or fasted. After peaking at 18 μg/dL, the serum thyroxine concentrations fell gradually throughout the duration of the collection period. Colostrokinin exhibited a biphasic effect on serum immunoglobulin concentrations which was dependent on the initial time of feeding. Calves exposed to colostrokinin in 0 hour feedings had serum immunoglobulin G concentrations significantly higher (P < .05) after 16 hours postpartum than animals not fed colostrokinin. Fasted calves, exposed to colostrokinin at 12 hours postpartum, had no increase in serum immunoglobulin G concentrations following a colostrum feeding at 24 hours postpartum. Fasted calves fed a ration not containing colostrokinin exhibited a two-fold increase in serum immunoglobulin G concentrations after the 24 hour colostrum feeding. Colostrokinin did not have an immediate effect on serum immunoglobulin G concentrations, but required an approximate twelve hour period to manifest its regulatory function. The presence or absence of colostrokinin in the experimental rations did not have any effect on the cortisol or thyroxine profiles. The variable serum immunoglobulin G profiles suggest that colostrokinin is involved in the acquisition of passive immunity by the calf, but colostrokinin may have more than one physiological role.

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

The purpose of the present study was to determine if psychological stress, defined as academic examination stress, would systematically produce changes in immune parameters (immunoglobulin concentration) and psychological functioning. It was hypothesized that as examination stress occurred there would be an effect on immunological function consistent with heightened psychological activity/stress. Subjects were 23 master's and doctoral students in psychology who volunteered for the research project. All subjects were administered a series of psychological tests to measure stress, personality factors, emotional states, and anxiety levels. All tests were administered and.blood samples drawn over a period of 15 months across two lowstress and two high-stress periods. Immunological tests included white blood cell (WBC) differential count and radial immunodiffusion (RID) for the determination of concentration of different immunoglobulin classes (IgA, IgG, IgM) in serum. Data were treated to a one-way analysis of variance (ANOVA) with repeated measures, t /test for correlated samples correlational matrix between variables across assessments and discriminant function analysis. Results showed (1) increased immunoglobulin levels during periods of stress; (2) immunoglobulin G most consistently related to stress and probably most indicative of the stressed condition and biological resistance to stress; (3) anxiety related to external events; (4) increase in anxiety under stress; and (5) anxiety inversely correlated with emotional stability and coping skills while positively related to tension, increased number of somatic complaints, and obsessive-compulsive trends. Firm support was provided for the hypothesis that as stress occurred, there would be consistent changes in immunological functioning associated with heightened psychological activity/stress. It was concluded that a response pattern to stress was adaptive along both psychological- and immunological dimensions and that the concept of bodymind interaction was the most realistic approach to understanding the total response patterns.

O presente estudo avaliou o efeito da suplementação mineral injetável de cobre (Cu) e zinco (Zn) sobre a resposta imunológica e o desempenho de bovinos de corte a pasto, foram avaliadas vacas no período pré-parto (experimento I) e seus bezerros até pós desmama (experimento II). O experimento foi realizado na Faculdade de Zootecnia e Engenharia de Alimentos da USP, Campus de Pirassununga-SP. No experimento I, foram avaliadas sessenta vacas prenhas por inseminação, distribuídas aleatoriamente em delineamento inteiramente casualizado com dois tratamentos (T e S). No tratamento testemunha (T), as vacas receberam soro fisiológico como placebo, e no tratamento suplementado (S) receberam mineral injetável via subcutânea (75 mg de cobre e 250 mg de zinco), com aproximadamente 75 dias antes do parto. Foram avaliados os teores de Cu e Zn séricos, IgG e IgM, ceruloplasmina e a atividade fagocitária. Os dados experimentais foram analisados utilizando-se o PROC MIXED do SAS (SAS, 2009), com o nível de significância de 5% para interpretação estatística de todos os testes. Os teores de cobre, zinco, IgM, IgG, ceruloplasmina e a atividade fagocitária das vacas não apresentaram diferença (p&gt;0,05) entre os tratamentos. Da mesma forma, não houve influencia (p&gt;0,05) das vacas nos teores de Cu e Zn séricos, na resposta imunológica e no desempenho dos bezerros aos 70 dias de idade (primeira coleta de sangue). O fornecimento de Cu e Zn injetável aos 75 dias antes do parto não alterou os teores de zinco, cobre e ceruloplasmina sérica, bem como a resposta imunológica até 30 dias após o parto. Como não houve diferença significativa entre as mães, foi considerado que os bezerros eram provenientes de vacas homogêneas. Assim, no experimento II, cinquenta e quatro bezerros filhos destas vacas, foram distribuídos aleatoriamente em delineamento inteiramente casualizado em 2 tratamentos: (T) - bezerros não suplementados, que receberam soro fisiológico como placebo; (S) - bezerros suplementados com cobre (15 mg aos 70 dias, 45 mg aos 140 dias e 60 mg aos 210 dias) e zinco (50 mg aos 70 dias, 150 mg aos 140 dias e 200 mg aos 210 dias). Foram realizadas colheitas de sangue com aproximadamente 70, 140, 170, 210 e 240 dias de idade, para avaliação dos teores de Cu e Zn sérico, ceruloplasmina, IgG e IgM e a atividade fagocitária. A suplementação parenteral de Cu e Zn aos 70, 140 e 210 dias não alteraram (p&gt;0,05) os teores de Cu e Zn séricos, a resposta imunológica, o desempenho, a atividade da enzima superóxido dismutase e a atividade fagocitária de bezerros Nelore. A falta de resposta ao suplemento mineral injetável deveu-se possivelmente ao fato dos bezerros já estarem recebendo as quantidades adequadas de cobre e de zinco nas suas dietas. / This study evaluated the effect of injectable Cu and Zn on the immune response and performance of beef cattle on pasture, cows were evaluated in the antepartum period (experiment I) and their calves to post weaning (experiment II). The experiment was perfomed at the Faculty of Animal Science and Food Engineering, University of São Paulo, Campus Pirassununga. In experiment I, sixty pregnant Nelore cows by insemination were divided in a completely randomized design in two treatments. In the control treatment (T), the 30 cows received saline as placebo, the supplemented (S) received mineral injection subcutaneously (75 mg of copper and 250 mg of zinc), 75 days prior to parturition, when also received clostridiosis vaccine. It were evaluated the serum levels of Cu and Zn, IgG, and IgM, ceruloplasmin, SOD and phagocytic activity. Experimental data were analyzed using PROC MIXED of SAS (SAS, 2009), with 5% significance level for statistical interpretation of all tests. The levels of copper, zinc, IgM, IgG, ceruloplasmin and the phagocytic activity of the cows had no statistical difference (p&gt;0.05) between treatments. Similarly, there wasn\'t influence (p&gt;0.05) of cows for Cu and Zn serum, immune response and performance of calves at 70 days of age (first blood sample). The supply of Cu and Zn injection at 75 days before delivery did not change the levels of zinc, copper and serum ceruloplasmin and the immune response 30 days after delivery. Since there was no significant difference between the mothers, it was considered that the calves were from homogeneous cows. In experiment II, fifty four calves born from these cows were distributed completely randomized design with 2 treatments: (T) - calves not supplemented that receive solution saline as placebo and (S) - calves supplemented with injectable mineral applied subcutaneously containing copper (15 mg at 70 days, 45 mg at 140 days and 60 mg at 210 days) and zinc (50 mg at 70 days, 150 mg at 140 days and 200 mg at 210 days. Blood samples were realized with approximately 70, 140, 170, 210 and 240 days of age. Were assessed levels of serum Cu and Zn, ceruloplasmin, IgG and IgM and the phagocytic activity. Parenteral supplementation of Cu and Zn at 70, 140 and 210 days didn\'t alter (p&gt;0.05) the contents of Cu and Zn serum, the immune response, performance, activity of superoxide dismutase and the phagocytic activity of Nelore calves. The lack of response to injectable mineral supplement possibly was due to the fact that the calves were already taking adequate amounts of copper and zinc in their diets.

Sepse é a primeira causa de morte em Unidades de Terapia Intensiva. A gravidade dessa doença é considerada conseqüência de um desequilíbrio da resposta inflamatória e, apesar dos avanços em diagnóstico e tratamento, os índices de mortalidade se mantêm inalterados. O papel dos receptores Fc de immunoglobulinas nesta situação é pouco esclarecido. Tais receptores deflagram respostas imunes opostas, que dependem do receptor envolvido e podem ser tanto ativatórias, quanto inibitórias. As respostas ativatórias são atribuídas a um motivo chamado ITAM, enquanto as inibitórias são relacionadas ao motivo ITIM. Camundongos apresentam dois receptores de IgG ativatórios (Fc&#947;RI e Fc&#947;RIII), que portam motivos ITAM, associados a uma sub-unidade conhecida como cadeia gamma, assim como um receptor de IgG que apresenta um motivo ITIM na sua porção intra-citoplasmática (Fc&#947;RII). Este trabalho teve como objetivo o estudo do papel destes receptores em bacteremias e sepse. Para isso, utilizamos um modelo de peritonite induzida por ligadura e punção cecal. Este projeto descreve pela primeira vez, um papel importante do FcR&#947;II na indução de apoptose em linfócitos B, durante infecção bacteriana severa. Nossos resultados colocaram em evidência, ainda, o fato de que animais deficientes em cadeia gamma apresentam mortalidade diminuída, quando submetidos a esse modelo de peritonite, e que essa diminuição é associada a menores valores de TNF&#945; no soro e nos fluidos peritoneais, menor recrutamento peritoneal de células inflamatórias, assim como a um surpreendente aumento na fagocitose de E. coli. Hemocultura e cultura do lavado peritoneal desses animais revelaram uma flora multimicrobiana, enquanto camundongos selvagens apresentaram uma forte predominância de E. coli e um número total bastante superior de bactérias. Esse papel inibitório da cadeia gamma pode estar relacionado a mecanismos de auto-tolerância. Lisado total de células peritoneais de camundongos deficientes em cadeia gamma apresentam fosforilação aumentada de diversas proteínas, quando comparados a lisados obtidos, a partir de camundongos selvagens. Estudos semelhantes realizados com camundongos transgênicos para o receptor de IgA (Fc&#945;RI), entretanto, não demonstraram um papel crucial desse receptor nesta doença. Este trabalho abre, portanto, novas perspectivas para o tratamento de doenças infecciosas, através de intervenção sobre a cadeia gamma e coloca em rediscussão os conceitos atuais de ITAM e ITIM. / Sepsis is the first cause of death in Critical Care Units and despite the development in its diagnosis and treatment, mortality remains unaffected. The role of immunoglobulin Fc receptors in sepsis is not clearly understood. These receptors initiate opposing responses, depending on their aggregation by the ligand and can induce activating or inhibitory responses. The activating responses are attributed to a motif known as ITAM, and the inhibitory responses to another one called ITIM. Mice express two activating IgG receptors (Fc&#947;RI et Fc&#947;RIII) which have ITAM motifs in the intracytoplasmic domain of an associated subunit, called the FcR&#947; chain, as well as an inhibitory IgG receptor which possesses an ITIM motif in its intracytoplasmic domain. The objective of this work is to study the importance of these receptors in bacteremia and in sepsis. To this aim, we have used a peritonitis model, induced by cecal ligation and puncture (CLP). This project describes for the first time, an important role of Fc&#947;RII in B lymphocytes apoptosis. Moreover, our results show that FcR&#947; chain knockout mice have a decreased mortality in this model, which is associated to diminished TNF&#945; serum and peritoneal fluids levels, to a reduced recruitment of peritoneal inflammatory cells and to a surprising increase in E. coli phagocytosis. Blood and peritoneal fluid cultures have shown a polymicrobial flora 24 hours post-CLP for FcR&#947;-chain deficient mice, whereas wild-type mice present a strong predominance of E. coli in the same cultures and an increased bacteria total count. Lysates from FcR&#947;-chain deficient peritoneal cells revealed augmented phosphorylation of many proteins, as compared to wild-type cells. This FcR&#947; chain inhibitory role could be related to self-tolerance mechanisms. This work opens new perspectives for the treatment of bacterial diseases, proposing FcR&#947; chain targeting and the reexamination of the actual concepts of ITAM and ITIM.