Immunity to nematode infections requires a host T helper 2 (Th2) response promoted by epithelial cell driven IL-33 induction of cytokine secretion of Interleukin (IL)-4, 5 and 13 by a range of immune cells including innate lymphoid cells type 2 (ILC2s) and CD4+ T cells. This induces effector responses such as goblet cell mucus secretion and mast cell activation driving disease resolution. Finding candidate molecules and discrete cell populations that enhance these responses would provide new targets for treating infection via specific host immune-modulation and would contribute to the development of effective vaccines against nematode infections. In this study we addressed how novel components of host adaptive and innate immunity can contribute to pulmonary control of Nippostrongylus brasiliensis infections. Murine reinfection studies with the parasitic nematode N. brasiliensis have shown development of a Th2 CD4+ T cell responses in the lung to be essential for immunity to secondary N. brasiliensis infection. To test if T cell recruitment from secondary lymphoid tissue contributed to this immunity, we used the drug Fingolimod (FTY720) to block T cell egress from lymph nodes (LN) to peripheral tissue. T cell egress from the LN was required for resolution of a primary infection but not for secondary infection. The presence of tissue-resident IL-4Rα responsive CD4+ T cells in the lung was sufficient for protective immunity to N. brasiliensis reinfection. These results demonstrated that effective CD4+ T cell Th2 immunity can be generated at peripheral sites by pre-existing T cell populations, independently of T cell recruitment from secondary lymphoid organs (SLO). Additionally, we identify that the pulmonary epithelial cell-secreted collectin, surfactant protein D (SP-D), is an important component of host immunity to N. brasiliensis infection. We demonstrate here that SP-D production is induced following N. brasiliensis infection in a Th2 dependent manner, it bound preferentially to lung stage L4 parasites and enhanced macrophage and ILC2 protective responses essential for controlling infection. vi Taken together the data presented in this thesis provides two new important insights into pulmonary host immunity to parasitic helminth infections.

Immunity to nematode infections requires a host T helper 2 (Th2) response promoted by epithelial cell driven IL-33 induction of cytokine secretion of Interleukin (IL)-4, 5 and 13 by a range of immune cells including innate lymphoid cells type 2 (ILC2s) and CD4+ T cells. This induces effector responses such as goblet cell mucus secretion and mast cell activation driving disease resolution. Finding candidate molecules and discrete cell populations that enhance these responses would provide new targets for treating infection via specific host immune-modulation and would contribute to the development of effective vaccines against nematode infections. In this study we addressed how novel components of host adaptive and innate immunity can contribute to pulmonary control of Nippostrongylus brasiliensis infections. Murine reinfection studies with the parasitic nematode N. brasiliensis have shown development of a Th2 CD4+ T cell responses in the lung to be essential for immunity to secondary N. brasiliensis infection. To test if T cell recruitment from secondary lymphoid tissue contributed to this immunity, we used the drug Fingolimod (FTY720) to block T cell egress from lymph nodes (LN) to peripheral tissue. T cell egress from the LN was required for resolution of a primary infection but not for secondary infection. The presence of tissue-resident IL-4Rα responsive CD4+ T cells in the lung was sufficient for protective immunity to N. brasiliensis reinfection. These results demonstrated that effective CD4+ T cell Th2 immunity can be generated at peripheral sites by pre-existing T cell populations, independently of T cell recruitment from secondary lymphoid organs (SLO). Additionally, we identify that the pulmonary epithelial cell-secreted collectin, surfactant protein D (SP-D), is an important component of host immunity to N. brasiliensis infection. We demonstrate here that SP-D production is induced following N. brasiliensis infection in a Th2 dependent manner, it bound preferentially to lung stage L4 parasites and enhanced macrophage and ILC2 protective responses essential for controlling infection. vi Taken together the data presented in this thesis provides two new important insights into pulmonary host immunity to parasitic helminth infections.

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Orientador: Iguatemy Lourenço Brunetti / Banca: João Olimpio Tognoli / Banca: Regina Célia Vendramini / Resumo: O uso de amostras-controle para acompanhamento do desempenho analítico é necessário para reproduzir a veracidade dos resultados das análises, pois monitora as variações que podem ocorrer no sistema analítico. O ideal para amostras-controle é ter a mesma matriz que as amostras dos pacientes, assim elas comportar-se-ão da mesma forma. Entretanto, para alcançar a estabilidade necessária, as amostras-controle passam por manipulações durante sua produção que podem alterar as propriedades da matriz. Dentre estas manipulações estão a adição de aditivos e mudanças físicas do meio como o congelamento ou liofilização. Neste estudo, nós produzimos amostras-controle com pool de soro humano trabalhados na forma líquida, congelados, conservados a ≤ -18°C e ≤ -80°C e sólida a 2 - 8°C liofilizados, sem preservante e com preservante e analisamos 23 analitos da rotina de laboratório clínico: ácido úrico, albumina, fosfatase alcalina, alanina aminotransferase, amilase, aspartato aminotransferase, bilirrubina direta, bilirrubina total, cálcio, capacidade latente de ligação do ferro, colesterol, creatinina, ferro, fósforo, gama-glutamil transferase, glicose, colesterol- HDL, desidrogenase lática, potássio, proteínas totais, sódio, triglicérides e uréia. As amostras foram analisadas por 300 dias, e comparado a estabilidade entre os soros sem preservante e com preservante nos diferentes processos de conservação. Os coeficientes de variação encontrados foram bastante satisfatórios, mas a estabilidade no decorrer do tempo foi melhor observada no soro com preservante, especialmente a 2 - 8°C - liofilizado, onde todos os analitos estudados permaneceram estáveis por 300 dias, com exceção do ácido úrico que foi de 240 dias / Abstract: Using control-samples for analytical performance monitoring is required to reproduce the veracity of the results of biochemical analyses, since them monitors changes that may occur in analytical system. The ideal for control-samples is to have the same matrix that samples of the patients, so they will behave the same way. However, to achieve the necessary stability control-samples undergo operations such as sum additives and/or physical changes of middle (freezing or lyophilization) during its production which can change the properties of the matrix. In this study, we produced control-samples with pool of human serum worked in frozen liquid form kept at ≤ -18°C and ≤ -80°C and freeze-dried solid kept between 2 - 8°C, without or with a preservative for 23 analytes of clinical laboratory routine: uric acid, serum albumin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, amylase, direct bilirubin, total bilirubin, calcium, latent iron-binding capacity, cholesterol, creatinine, iron, phosphorus, gamma glutamyl transferase, HDL-cholesterol, glucose, lactic dehydrogenase, total proteins, potassium, sodium, triglycerides and urea. The samples were analyzed for 300 days, and the stability compared between the without a preservative and with a preservative sera in different processes of conservation. The coefficient of variation found were quite satisfactory, but stability over time was better observed in with a preservative serum, mainly in serum stored between 2 - 8°C - freeze-dried, in which all analytes studied remained stable for 300 days, with the exception of uric acid that was for 240 days / Mestre

Allergies are a common chronic disease and considerably decrease the quality of life for affected individuals. Understanding the immune responses during allergic diseases is essential for both diagnosis and the development of effective therapies. The route of sensitisation to allergens is one factor that influences the immune response and the outcome of allergic diseases and both human and animal studies have highlighted IL-4Ra as an important component in the induction of allergy. The aim of this study was to investigate the contributions of the route of sensitisation to allergens with focus on the significance of cell specific expression of IL-4Ra in the onset of allergy. The route of sensitization to Anisakis pegreffii influences the outcome of experimental allergic asthma: Worldwide, increasing numbers of allergies to the fish parasite Anisakis pegreffii are reported. Anisakis can cause allergies after accidental infection of humans and in the occupational environment. Currently it is not clear if different exposure routes to Anisakis affect the development of allergic asthma and if they have an influence on the immune response. To address these questions, the present study investigated immune responses and disease development after Anisakis live infection and after nasal sensitisation in a mouse model of allergic airway disease. We showed that the route of sensitisation influences the outcome of Anisakis pegreffii induced allergic asthma and demonstrated important contributions of IL-4Ra to the underlying immune response. Alternatively activated macrophages are not necessary for the development of experimental allergic lung inflammation: Development of alternatively activated macrophages (AAM) is induced by signals of IL-4Ra. Alternatively activated macrophages (AAM) are a feature of allergic asthma in clinical and experimental investigations but their role in the development of allergy is not defined. To address this, a model of acute allergic asthma was used to compare mice deficient in AAM (LysMcrelL-4Ra-110x mice) with control mice. We found that the presence of AAM at early stages of allergic airway inflammation these cells was not required for the onset of the disease. Smooth muscle IL-4Ra is not required for experimental allergic asthma: In vitro studies have suggested that IL-4Ra signalling on airway smooth muscle cells (ASMC) is critical for airway irrflammation and airway hyperresponsiveness. Using mice deficient for IL-4Ra in ASMC, the in vivo effects of impaired IL-4Ra signalling in ASMC on the outcome of asthmatic disease were investigated. The impairment of IL-4Ro: on SMC had no effect on major aetiological markers of allergic asthma. These findings suggest that IL-4Ra responsiveness in airway SMC during the acute phase of allergic asthma is not critical for the outcome of the disease. Conclusions: The present study showed the importance of the route of sensitisation and IL4Ra in the development of allergy to Anisakis pegreffii. The use of in vivo models of experimental allergic asthma revealed that the route of sensitisation can influence the underlying immune response of the disease. Furthermore, by using mice with cell specific deficiencies in IL-4Ra it was demonstrated that expression of this receptor on smooth muscle cells and macrophages is not essential for the development of acute experimental allergic airway disease, as it has been previously suggested.

The field of clinical laboratory science is experiencing a critical shortage of qualified professionals. Because health care practitioners depend on the results of laboratory tests to help diagnosis and treat patients, it is important to address the current and future shortage in the laboratory workforce. There is limited research on factors affecting the turnover intentions of clinical laboratory employees. Accordingly, the research questions for this study examined the effect of burnout (BO) and organizational commitment (OC) on the turnover intention of laboratory employees in Florida. A cross-sectional survey design was used to examine the relationship between BO and OC on turnover intentions. Data were collected from licensed clinical laboratory directors, supervisors, technologists, and technicians using the following scales: demographic questionnaire, Maslach Burnout Inventory - General Survey, and Organizational Commitment Questionnaire. Linear regression and ANOVA were used to examine the relationships between these variables. The response rate was 18.4% (N = 184). Among clinical laboratory employees in Florida, the findings revealed significant predictive relationships between BO and turnover intention, OC and turnover intention, age and BO, and work shift and OC among clinical laboratory employees in Florida. Potential implications for positive social change from this study include reducing turnover among laboratory employees by allowing laboratory managers to create strategies that will reduce BO and increase OC, and thus decrease turnover intention.

The purpose of this study was to determine the impact of Regulation 1 of CLIA '88 on personnel needs in clinical laboratories of acute care hospitals in Virginia, resulting from proficiency testing, complexity level of testing and personnel standards. Because the legislation was enacted and passed with the intent of improving the quality of laboratory testing in every setting, the problem of the study was to determine the effects Regulation 1 of CLIA ’88 had on personnel needs for the delivery of quality clinical laboratory services in acute care hospitals of Virginia. A survey was sent to 140 acute care hospital laboratories in Virginia. There were 107 respondents, with 75 respondents providing usable data for this study. The remainder did not provide full laboratory services. Demographic information was obtained regarding bed capacity, educational levels of current personnel serving in different capacities, test volume, and percent of tests performed by different complexity levels. Comparisons were made among small, medium, and large facilities. The majority of respondents were representative of a facility with a bed capacity of 200 or less. The level of test complexity performed was similar regardless of the facility size. The majority of facilities did not anticipate an increase in personnel needs as a result of CLIA personnel standards. All facilities had personnel at all capacities that met required educational levels at this time. The majority of facilities did not anticipate an increase in personnel as a result of increased proficiency testing; however, when a projected need was indicated, there was a greater need indicated for AS degree level personnel followed by BS level personnel with a decline indicated in non degreed personnel. Staffing pattern changes related to increased proficiency testing indicated differences in the projected needs of small, medium, and large facilities. Barriers for implementing CLIA ’88 personnel standards were identified from the literature review and the pilot study. Respondents were asked to identify the barriers that were most Significant; they are in order as ranked: cost, availability of qualified personnel, and CLIA not reflecting the depth of knowledge and judgment needed to make independent competent judgment. The barriers were also reviewed by hospital bed size. It was concluded that as the number of tests being sent out increased, the number of tests being performed in-house have increased at the moderate complexity level, a level which requires less qualified personnel. The intent of the law to improve the quality of laboratory testing has not occurred in every setting. If the intent of the law is implemented, a need exists to provide educational opportunities at the AS and BS level for experienced personnel. Respondents did not perceive criteria as established by CLIA ‘88 as being adequate to determine the qualifications of personnel, who are responsible for quality patient test results in all settings. / Ed. D.

Retiring baby boomers and the lack of interest and awareness among college students to enroll in an accredited Clinical Laboratory Science (CLS) program have created a shortage of CLS professionals in the 21st century. The U.S. Bureau of Labor Statistics predicts 18,000 CLS vacancies by 2018. However, only about 5,000 students graduate from accredited CLS programs each year. The purpose of this study was to explore students' perceptions of allied health professions and factors that influenced students and CLS professionals to select CLS as a profession. Bandura's social cognitive career theory served as the theoretical framework for this phenomenological study. Convenient purposeful sampling was used to select the 7 CLS professionals, 5 high school students, and 5 college students in the Chicago area. Participants took part in either a 30- to 60-minute group session or a 45- to 90-minute semi structured interview. Qualitative analysis included open axial coding to identify emerging patterns and themes from the transcripts. Findings revealed that the perceptions of both high school and college students' knew little about the CLS profession, and factors influencing CLS as a career choice included interests in science, health care, and family. CLS professionals indicated their interests in science and a high demand for CLS services in the workforce led them to pursue careers in the field. Implications for social change include improving professional-development programs for student awareness of allied health professions and mitigating the shortage of clinical laboratory scientists.

Darbo tikslas. Palyginti darbo sąlygas privačiose ir viešųjų įstaigų klinikinėse laboratorijose. Tyrimo metodika. Tyrime dalyvavo 192 respondentai. 96 respondentai buvo vienos privačios X laboratorijos darbuotojai (penki padaliniai), kiti 96 viešųj�� įstaigų laboratorijų darbuotojai. Atsako dažnis 76,8 proc. Tyrimas atliktas 2007 m. spalio – 2008 m. sausio mėnesiais. Duomenys surinkti anoniminės anketinės apklausos būdu. Statistinė duomenų analizė atlikta naudojant kompiuterinį SPSS 13.0 statistinį paketą ir MS Excel. Rezultatai. Darbo aplinkoje su kenksmingomis cheminėmis medžiagomis, susiduria 97,9 proc. viešųjų ir 94,8 proc. privačios laboratorijos darbuotojai. Nepatenkinti darbo apsaugos priemonėmis buvo 55( 57,3 proc.) viešosios laboratorijos darbuotojų ir 25 (26 proc.) privačios laboratorijos darbuotojų. Geresniu užmokesčiu pasižymi privačių laboratorijų darbuotojai (p<0,05), o tai įtakoja ir tai, kad jie rečiau, nei viešųjų laboratorijų darbuotojai galvoja apie darbo keitimą. Didžiausias stresorius darbo aplinkoje buvo atsakomybė, už aliekamą darbą. (viešoje 82,3proc., privačioje 57,3 proc.) Statistiškai patikimiau (p<0,05) didesnę atsakomybę už atliekamą darbą jautė viešųjų laboratorijų darbuotojai. Labiausiai motyvaciją darbui didina atlygis, už padaryta darbą. Dažnesnį nuovargį darbo metu dažniau jaučia privataus sektoriaus darbuotojai, kurį įtakoja, didesni darbo krūviai, pamaininis darbas. Išvados. Privataus sektoriaus laboratorijų darbuotojai uždirba daugiau... [toliau žr. visą tekstą] / Final Paper Aim: Compare the work conditions in the public and private clinical laboratories. Work Methods: We have taken for the research work 192 respondents: 96 of them were employed in the private X laboratory (five departments) and the other 96 respondents were working in the public laboratories. Answer period 76,8 percent. This research was being run in the duration from the October, 2007 until the January, 2008. The questionnaire data have been gathered anonym. The statistic data analysis were put up by the help of statistical package SPSS and MS Excel. Results. In the environment with the toxic sanitary conditions work 97,9 percent of employees of public laboratories, and 94,8 percent of workers are engaged for the private laboratories. 55 (57,3 percent) of the public laboratories workers were not satisfied with the work protection conditions, and 25 (26 percent) of the private laboratory workers weren’t quite happy about the mentioned matter. A better income is assessable for the private laboratories employees (p<0,05), - this can admit them the more constant post of work than that of the public laboratories workers, who have to change their work therefore. The most ordinary factor of hectic in the environment was set the stressor of responsibility for the accomplished work as follows: 82,3 percent of the public sector respondents, and 57,3 percent of the private laboratories workers. The statistic reliable (p<0,05) responsibility for the work was shown by the... [to full text]

Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal alloantibodies against fetal platelet antigens inherited from the father and which are absent from maternal platelets. In Caucasians, antibodies against the Leu33 (HPA-1a) polymorphism of integrin β3 (part of the platelet αIIbβ3 complex) account for >70% of cases. Antenatal screening for these antibodies does not currently take place in the UK, partly because of the absence of sensitive, predictive tests. We hypothesized that the poor sensitivity and predictive abilities of current assays are due to the use of β3 in an inappropriate conformation, resulting in sub-optimal binding of HPA-1a antibodies. We hypothesized firstly that in vitro induced changes to αIIbβ3 might alter accessibility of the HPA-1a epitopes to alloantibodies, thus reducing assay sensitivity. Secondly, we hypothesized that HPA-1a antibodies are stimulated by, and preferentially recognise, β3 in association with αv, a molecule present on placental syncytiotrophoblasts, and that reactivity against platelet αIIbβ3 reflects only cross-reactivity with αvβ3. Our first hypothesis was proven by demonstrating that use of the cation chelating compound EDTA, used by many diagnostic laboratories as a component of assay reagents or present in blood samples as anticoagulant, resulted in significantly reduced assay sensitivity. These findings were confirmed in an international workshop. Support for our second hypothesis was provided by demonstrating enhanced reactivity of a small panel of examples of anti-HPA-1a against αvβ3 compared to αIIbβ3 and by molecular modelling data. We also showed that HPA-1a antibodies can inhibit platelet function by using a novel application of the ROTEM® delta thromboelastograph and an immunofluorescence assay in which we demonstrated blocking of platelet function using a monoclonal antibody, PAC-1, that binds only to activated αIIbβ3. These studies provide possible explanations for the poor sensitivity and predictive abilities of current assays and suggest further areas for research.