Objetivo: Determinar a influência da presença de cafeína no sangue de cordão umbilical na ocorrência de apneia. Métodos: Estudo de coorte prospectivo de recém-nascidos pretermos com peso de nascimento menor de 2.000 g. Os critérios de exclusão foram: mães que receberam opióides , ventilação mecânica durante os primeiros 4 dias de vida, malformação congênita cerebral e cardíaca maiores, asfixia perinatal, hemorragia peri-intraventricular severa, exsanguíneotransfusão antes do quarto dia de vida e uso de metilxantina antes da extubação. Os recém-nascidos foram divididos em: com e sem cafeína detectável no sangue de cordão umbilical e acompanhados nos primeiros quatro dias de vida para a ocorrência de apneia. Resultados: 87 com e 40 sem cafeína detectável no sangue de cordão umbilical foram estudados. A mediana da concentração de cafeína dos 87 pacientes com cafeína detectável no sangue de cordão umbilical foi 2,3 µg/ml (0,2-9,4 µg/ml). Não houve associação entre a ocorrência de apneia e a presença de cafeína no sangue de cordão umbilical. Recém-nascidos com cafeína detectável no cordão umbilical tiveram apnéia mais tarde (66.3 horas) do que aqueles com níveis indetectáveis (54.2 horas). Conclusão: a detecção de níveis de cafeína no sangue de cordão umbilical não diminuiu a ocorrência de apneia da prematuridade. Nós sugerimos que novos estudos com a administração de altas doses de cafeína para mães antes do parto prematuro, como estratégia para prevenir a apneia da prematuridade, devam ser realizados. / Objective: To determine the influence of presence of caffeine in umbilical cord blood on apnea occurrence. Methods: A prospective cohort study with preterm newborns with birth weight less than 2,000 g was undertaken. Exclusion criteria were: mothers that received opioids, mechanical ventilation during the first 4 days of life, cerebral and major cardiac malformations, perinatal asphyxia, severe periintraventricular hemorrhage, exchange transfusion before the fourth day of life, and those that received methylxantine prior to extubation. Neonates were divided in: with detectable and undetectable caffeine in umbilical cord blood. Newborns were followed for the first 4 days for occurrence of apnea spells. Results: 87 with and 40 without detectable caffeine in umbilical cord blood were studied. The median caffeine concentration of the 87 patients with detectable caffeine in umbilical blood was 2.3 µg/ml (0.2-9.4 µg/ml). There was no association between occurrence of apnea spells and presence of caffeine in umbilical cord blood. Neonates with detectable caffeine in umbilical blood had apnea later (66.3 ± 4.14 hours) than those with undetectable levels (54.2 ± 6.26 hours). Conclusion: The detected levels of caffeine in umbilical cord blood did not decrease the occurrence of apnea of prematurity. We suggest that further studies on administration of high dose of caffeine to mothers prior to a preterm delivery as a preventive measure for apnea of prematurity deserve to be conducted.

abstract: The unique anatomical and functional properties of vasculature determine the susceptibility of the spinal cord to ischemia. The spinal cord vascular architecture is designed to withstand major ischemic events by compensating blood supply via important anastomotic channels. One of the important compensatory channels of the arterial basket of the conus medullaris (ABCM). ABCM consists of one or two arteries arising from the anterior spinal artery (ASA) and circumferentially connecting the ASA and the posterior spinal arteries. In addition to compensatory function, the arterial basket can be involved in arteriovenous fistulae and malformations of the conus. The morphometric anatomical analysis of the ABCM was performed with emphasis on vessel diameters and branching patterns. A significant ischemic event that overcomes vascular compensatory capacity causes spinal cord injury (SCI). For example, SCI complicating thoracoabdominal aortic aneurysm repair is associated with ischemic injury. The rate of this devastating complication has been decreased significantly by instituting physiological methods of protection. Traumatic spinal cord injury causes complex changes in spinal cord blood flow (SCBF), which are closely related to a severity of injury. Manipulating physiological parameters such as mean arterial pressure (MAP) and intrathecal pressure (ITP) may be beneficial for patients with a spinal cord injury. It was discovered in a pig model of SCI that the combination of MAP elevation and cerebrospinal fluid drainage (CSFD) significantly and sustainably improved SCBF and spinal cord perfusion pressure. In animal models of SCI, regeneration is usually evaluated histologically, requiring animal sacrifice. Thus, there is a need for a technique to detect changes in SCI noninvasively over time. The study was performed comparing manganese-enhanced magnetic resonance imaging (MEMRI) in hemisection and transection SCI rat models with diffusion tensor imaging (DTI) and histology. MEMERI ratio differed among transection and hemisection groups, correlating to a severity of SCI measured by fraction anisotropy and myelin load. MEMRI is a useful noninvasive tool to assess a degree of neuronal damage after SCI. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2016

Introdução: A célula NK possui uma importante função no sistema imune inato de defesa primária contra vírus e patógenos e também realiza a imunovigilâcia tumoral. Muitos estudos clínicos tem avaliado o uso dessas células na imunoterapia adotiva. A expansão e a ativação da célula NK requer sinais e estímulos para manter a sua sobrevivência. Atualmente existem muitos protocolos para a expansão e ativação da célula NK, porém não existe uma definição do melhor método para uso clínico. Objetivo: O estudo tem como objetivo avaliar a melhor forma para expansão das células NK isoladas de células mononucleares do sangue de cordão umbilical e placentário.Método: Foram avaliadas cinco diferentes condições para expansão de células NK de mononucleares isoladas do sangue do cordão umbilical e placentário. Foram testados protocolos utilizando as interleucinas (IL), IL-2, IL-3, IL-15; com ou sem a presença do co-cultivo com células-tronco mesenquimais do cordão umbilical (CTM-CU) e, também o co-cultivo com células apresentadoras de antígeno artificiais ligadas a IL-21 à membrana (mbIL21 APC). Resultados: Os protocolos utilizando co-cultivo com APC mbIL21 foram superiores aos demais quanto à capacidade de expansão de células NK (CD3-, CD56+, CD16+). O protocolo de co-cultura de APC, CTM-CU e estímulo com IL-2 apresentaram um aumento significativo de NK (CD3-, CD56+, CD16+) quando comparado ao protocolo de APC/IL-2 sem CTM-CU (p<0,05). Conclusão: A expansão ex vivo de células NK na presença das APC e CTM-CU apresentaram uma proporção estatisticamente superior de célula NK CD16+ quando comparada com condições de cultivo com apenas a APC, tendo essas células NK potencial para utilização na imunoterapia adotiva associada com anticorpos monoclonais ou anticorpos bi-específicos. / Background: Natural killer (NK) cells play a major role in innate immunity, especially against viral pathogens, and are also a part of the immune surveillance of tumors. Several clinical trials have evaluated the use of these cells for adoptive cell immunotherapy. Ex vivo expansion of NK cells, however, is a complex process which requires multiple cell signals to ensure cell survival, proliferation, and activation. There are many protocols used for NK cell expansion and activation, however, there is a lack of evidence regarding which method is the most effective for clinical grade NK cells expansion. Objective: The main purpose of this study is to evaluate an optimal protocol for the ex vivo expansion of NK cells isolated from umbilical cord blood mononuclear cells (CB-MNC). Methods: Five different conditions for the expansion of umbilical cord-derived NK cells were evaluated. Each protocol was a different combination of interleukins (IL-2, IL-3, and IL-15) with or without the presence of feeder cells or artificial antigen presenting cells (aAPCs). Feeder cells utilized were umbilical cord-derived mesenchymal stem cells (UC-MSC), and aAPCs were membrane-bound IL-21 artificial APCs (mbIL21 aAPCs). Results: Protocols employing mbIL21 aAPCs demonstrated greater expansion of natural killer cells (CD3- CD56+) than the other protocols. The protocol employing aAPCs, IL-2 and UC-MSC feeder cells had a statistically significant higher proportion of CD16+ NK cells when compared to the protocol without the MSC feeder cells, but there was no significant difference in the expansion of total natural killer cells concerning these two protocols. Conclusion: Ex vivo expansion of NK cells in the presence of aAPCs and UC-MSC feeder cells yielded a significant higher proportion of CD16+ NK when compared to the aAPCs only culture condition, and could be a better product for NK adoptive immunotherapy in conjunction with monoclonal or bi-specific antibodies.

Objetivo: Determinar a influência da presença de cafeína no sangue de cordão umbilical na ocorrência de apneia. Métodos: Estudo de coorte prospectivo de recém-nascidos pretermos com peso de nascimento menor de 2.000 g. Os critérios de exclusão foram: mães que receberam opióides , ventilação mecânica durante os primeiros 4 dias de vida, malformação congênita cerebral e cardíaca maiores, asfixia perinatal, hemorragia peri-intraventricular severa, exsanguíneotransfusão antes do quarto dia de vida e uso de metilxantina antes da extubação. Os recém-nascidos foram divididos em: com e sem cafeína detectável no sangue de cordão umbilical e acompanhados nos primeiros quatro dias de vida para a ocorrência de apneia. Resultados: 87 com e 40 sem cafeína detectável no sangue de cordão umbilical foram estudados. A mediana da concentração de cafeína dos 87 pacientes com cafeína detectável no sangue de cordão umbilical foi 2,3 µg/ml (0,2-9,4 µg/ml). Não houve associação entre a ocorrência de apneia e a presença de cafeína no sangue de cordão umbilical. Recém-nascidos com cafeína detectável no cordão umbilical tiveram apnéia mais tarde (66.3 horas) do que aqueles com níveis indetectáveis (54.2 horas). Conclusão: a detecção de níveis de cafeína no sangue de cordão umbilical não diminuiu a ocorrência de apneia da prematuridade. Nós sugerimos que novos estudos com a administração de altas doses de cafeína para mães antes do parto prematuro, como estratégia para prevenir a apneia da prematuridade, devam ser realizados. / Objective: To determine the influence of presence of caffeine in umbilical cord blood on apnea occurrence. Methods: A prospective cohort study with preterm newborns with birth weight less than 2,000 g was undertaken. Exclusion criteria were: mothers that received opioids, mechanical ventilation during the first 4 days of life, cerebral and major cardiac malformations, perinatal asphyxia, severe periintraventricular hemorrhage, exchange transfusion before the fourth day of life, and those that received methylxantine prior to extubation. Neonates were divided in: with detectable and undetectable caffeine in umbilical cord blood. Newborns were followed for the first 4 days for occurrence of apnea spells. Results: 87 with and 40 without detectable caffeine in umbilical cord blood were studied. The median caffeine concentration of the 87 patients with detectable caffeine in umbilical blood was 2.3 µg/ml (0.2-9.4 µg/ml). There was no association between occurrence of apnea spells and presence of caffeine in umbilical cord blood. Neonates with detectable caffeine in umbilical blood had apnea later (66.3 ± 4.14 hours) than those with undetectable levels (54.2 ± 6.26 hours). Conclusion: The detected levels of caffeine in umbilical cord blood did not decrease the occurrence of apnea of prematurity. We suggest that further studies on administration of high dose of caffeine to mothers prior to a preterm delivery as a preventive measure for apnea of prematurity deserve to be conducted.

South Africa is in dire need of a public umbilical cord blood stem cell bank (UCB SCB). A severe shortage of genetically compatible samples for BM transplantation precludes the majority of South Africans from receiving the relevant medical care. UCB is a viable alternative to BM but is currently disposed of post-delivery. UCB could furthermore serve as a resource of genetically compatible haematopoietic progenitor cells (HPCs) that could be used in gene therapy approaches directed towards a cure for HIV-1. Knowing whether HIV-1 affects or infects primitive HPCs is vital to determine the course of action for transplantation of UCB-derived genetically resistant HPCs. Collecting and storing UCB in a public UCB bank could thus serve as a vital resource of genetically compatible samples for BM transplantation. It was thought that the high incidence of HIV-1 in South African patients and the persistent stigma surrounding HIV-1 would be problematic for collecting sustainable numbers of UCB units and subjecting units to compulsory screening for infectious diseases. This was however, not the case. In the South African context, we are faced with unique and rich challenges relating to cultural and religious differences that are further augmented by linguistic constraints and educational insufficiencies. Nevertheless, the majority of patients within the interviewed patient cohort were supportive of the idea of establishing a public UCB SCB in SA and were willing to undergo additional HIV-1 screening. The Ultrio-Plus® assay was verified in this study for screening UCB units for HIV-1 and could be used in routine analyses of UCB units prior to banking. Conflicting results in the literature exist with regard to HIV-1’s ability to infect or affect haematopoietic progenitor cells. Results from this study revealed that HIV-1 was not only able to affect HPCs’ ability to form colonies in vitro, but was also capable of infecting CD34+ HPCs in some individuals. These results substantiate the theory that some CD34+ HPCs serve as viral reservoirs which could account for residual viraemia in patients on antiretroviral therapy. Results suggest that allogeneic transplantation of HIV-1 infected individuals with UCB-derived, genetically modified HPCs, should be pursued. / Thesis (PhD)--University of Pretoria, 2012. / Immunology / unrestricted

Type 1 Diabetes (T1D) is organ-specific autoimmune disease which causes pancreatic beta cells to be irreversibly destroyed. The only possible treatment represents life-lasting insulin administration. The real trigger of destructive insulitis isn't known. T1D is a multi- factorial disease involving both external and internal factors in the disease pathogenesis. The presence of autoreactive T lymphocytes in pancreas is necessary for development of diabetes. T regulatory cells have protective function in the destructive insulitis. The aim of this diploma thesis was to study cord blood T regulatory cells and their connection to type 1 diabetes development. We tried to find the difference among T regulatory cells in mononuclear cord blood cells (CBMC) in different study groups. Samples were collected from mothers suffering from T1D, gestational diabetes. Healthy controls were tested as well. Sixty-eight samples of cord blood were included in the study among the years 2009 - 2011. Samples were divided into 3 groups (CBMC from children born to T1D mothers, mothers with gestational diabetes and healthy mothers without T1D). CBMC were ana- lysed by flow cytometry. T regulatory cells (defined as CD4+CD25+) were isolated by magnetic separation (MACS). The functional capacity of these cells was studied as well by...

Osteoblasts (OST) provide strong intrinsic growth modulatory activities on hematopoietic stem and progenitor cells via different mechanisms that include secretion of growth factors, and cellular interaction. Previously we showed that medium conditioned by mesenchymal stromal cell (MSC)-derived osteoblasts (M-OST) improve the expansion of cord blood (CB) CD34+ cells. I hypothesize that the hematopoietic supporting activity of M-OST would vary as a function of their maturation. This was tested by producing osteoblast conditioned media (OCM) from M-OST at distinct stages of maturation, and testing their growth regulatory activities in CB CD34+ cell cultures. My results showed that some of the growth promoting activity of OCM on CB cells are not dependent on the maturation status, while others are and those are largely independent of Notch signalling. In conclusion, these results provide further evidence that osteoblasts release factors that can promote the growth of immature CB progenitors in a Notch-independent way.

Cellular mechanisms involved in neurodegeneration and neuroprotection are continuing to be explored, and this paper focuses on some novel discoveries that give further insight into these processes. Oligodendrocytes and activated astroglia are likely generators of the pro-inflammatory cytokines, such as the tumor necrosis factor family and interleukin family, and these glial support cells express adhesion receptors (e.g., VCAM) and release intercellular adhesion molecules (ICAM) that have a major role in neuronal apoptosis. Even brief exposure to some substances, in ontogeny and sometimes in adulthood, can have lasting effects on behaviors because of their prominent toxicity (e.g., NMDA receptor antagonists) or because they sensitize receptors (e.g., dopamine D2 agonists), possibly permanently, and thereby alter behavior for the lifespan. Cell cycle genes which may be derived from microglia, are the most-recent entry into the neuroprotection schema. Neuroprotection afforded by some common substances (e.g., melatonin) and uncommon substances [e.g., nicotine, green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG), trolox], ordinarily thought to be simple radical scavengers, now are thought to invoke previously unsuspected cellular mechanisms in the process of neuroprotection. Although Alzheimer's disease (AD) has features of a continuous spectrum of neural and functional decline, in vivo PET imaging and and functional magnetic resonance imaging, indicate that AD can be staged into an early phase treatable by inhibitors of β and γ secretase; and a late phase which may be more amenable to treatment by drugs that prevent or reverse tau phosphorylation. Neural transplantation, thought to be the last hope for neurally injured patients (e.g., Parkinsonians), may be displaced by non-neural tissue transplants (e.g., human umbilical cord blood; Sertoli cells) which seem to provide similar neurotrophic support and improved behavior-without posing the major ethical dilemma of removing tissue from aborted fetuses. The objective of this paper is to invite added research into the newly discovered (or postulated) novel mechanisms; and to stimulate discovery of additional mechanisms attending neurodegeneration and neuroprotection.

Due to high incidence, medical and socioeconomic burden and impact on individual quality of life and productivity, allergic disorders are a crucial issue for 21st century immunology. Much still remains to be elucidated, particularly regarding the very early processes in allergy development. In order to introduce timely, effective preventive measures, novel, more reliable predictive factors of allergy risk also need to be established. Dysregulation of proper balance between the branches of immune response, particularly unwarranted dominance of Th2, is the underlying cause of allergy. After birth, new immune balance needs to be established to prepare the neonate for adequate reactivity towards newly encountered environmental stimuli. Regulatory T cells (Treg) play a central role in finely setting this balance and inducing tolerance towards harmless environmental antigens, including allergens. Interactions with external factors, most importantly microbiota, modulate this process during the early postnatal "window of opportunity." Analysis of cord blood Treg of children of allergic mothers uncovered decreased presence of function-associated surface markers and lower production of IL-10. Furthermore, decreased proportion of Helios- induced Treg was observed in children with higher risk of allergy....

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