Neste trabalho realizamos a análise macroscópica das estruturas do funículo umbilical e da placenta, a análise macroscópica da vascularização funicular e placentária, e a análise à microscopia de luz dos componentes funiculares da paca, procurando melhor conhecer a morfologia reprodutiva desta espécie. Para tanto, utilizamos nove pacas fêmeas, provenientes do Setor de Animais Silvestres do Departamento de Zootecnia da Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal - UNESP, nos estágios inicial, intermediário e final de prenhez, os animais tiveram o corno uterino gestante retirado cirurgicamente. Com o útero aberto identificamos as membranas fetais e fizemos a mensuração do funículo umbilical e da distância céfalo-coccígea fetal, em seguida identificamos os vasos funiculares que foram injetados com látex do tipo Neoprene corado com pigmento específico; após injeção, esses vasos foram dissecados para a visualização da disposição das artérias e veias na placenta. Três animais foram direcionados à análise microscópica do funículo umbilical que depois de colhido foi seccionado em porções proximal, intermediária e distal que foram fixadas e sofreram processamento histológico usual com posterior colorações em hematoxilina- eosina e tricromico de Masson. Constatamos nestes animais uma placenta de formato discoidal globoso e as membranas fetais constavam de cório-alantóide e âmnio. As médias das medidas dos comprimentos dos funículos umbilicais e das distâncias cefalococcígeas foram de 7,31cm e 14,5 cm respectivamente. Macroscopicamente observamos no funículo umbilical a presença de duas artérias umbilicais, uma veia umbilical, o alantóide, vasos onfalomesentéricos; microscopicamente, observamos que as artérias e veias umbilicais eram musculares, o ducto alantóide era constituído por epitélio bi-estratificado cúbico e, essas estruturas, juntamente com os vasos onfalomesentéricos estavam englobadas pela gelatina de Wharton, também se observou a presença de vasa vasorum. A dissecação dos vasos umbilicais injetados indicou que as veias se localizavam basicamente na periferia da placenta enquanto as artérias ocupavam posição centralizada. / On this research we peform the macroscopic analysis of the umbilical cord and placenta structure, the macroscopic analysis of umbilical cord and placenta valcularization, and analysis with light microscopy of umbilical cords constituents of paca, aiming to increase the knowledge about the reproductive morphology in this species. To achieve that we used nine female pacas, which came from the Wild Animal Sector of the Zootechnology Departament at the Faculty of Agrarian and Veterenary Sciences of Jaboticabal - UNESP -, in early, entermediary and late phases of pregnancy. The animals had their pregnanty uterine horn taken out on surgery. After opening the uterus, we identified the fetal membranes and measured the umbilical cord and the cephalococcyga distance. Than we identified the umbilical cords vessels with were injected with latex (Neoprene®) dyed by specific pigment; after the injections, the vessels were dissected for visualization of the disposition of arteries and veins in the placenta. Three animals were carried out to microscopic analysis of the umbilical cord that , after the collect, was sectioned in proximal, intermediary and distal portions wich were set and than went to an usual histological processing with furthering colorations with hematoxilin-eosin and Masson tricome method. In these animals we noticed a discoid globular placenta, and the fetal membranes were constituted by chorion-alantois and amnion. The averages of the umbilical cords length and the crown-rump distance were 7,31 cm and 14,5 cm respectively. Macroscopically, we noticed the presence of two umbilical cord arteries, one umbilical cord vein, the allantois and onfalomesenteric vessels; microscopically, we noticed that the umbilical cord arteries and veins were muscly, the alantoic duct was formed by a cubic double layer epthelium and this sctructures, together with the onfalomezenteric vessels, were surrounded by the Wharton´s gelatin. The presence of vasa vasorum was also observed. The dissection of the injected umbilical cord vessels pointed that veins were basically placed that the placenta periphery and arteries presented a central position.

Estudos envolvendo a utilização do sangue de cordão umbilical foram intensificados na última década, devido ao grande potencial que estas possuem nas pesquisas de transplantes e ontogenia celular. A investigação dos métodos para a purificação e caracterização dessas células em diferentes animais pode aumentar a utilização destes como modelos experimentais para uma variedade de propostas científicas e terapêuticas. Para desenvolver a caracterização das células de sangue de cordão umbilical foram utilizadas 20 amostras oriundas de fetos de com idades compatíveis ao segundo e terceiro terço de gestação. Estas amostras foram processadas de duas formas distintas. A primeira forma visou às células presentes na camada leucocitária total, a segunda visou as células com densidade menor que 1077 separadas pelo Ficoll Paque. Os “pellets" resultantes foram processados para posterior observação por microscopia eletrônica de transmissão. Foram constatadas a presença de células granulares (basófilos, eosinófilos e neutrófilos) e agranulares (células bláticas, precursores eosinofílicos e linfócitos) em diferentes estágios de maturação em ambos os grupos e também células com características morfológicas compatíveis a apoptose. / Studies involving the use of umbilical cord blood were intensified in the last decade, due to its huge potential for transplant research and the ontogeny of leucocytes cells. The investigation of the methods to purify and characterize these cells in different animals are a base for experimental models for a variety of scientific and therapeutic proposals. To develop the characterization of the umbilical cord cells, twenty samples of fetal were collected. The fetal were in the second and third thirds of the gestation. These samples were processed in two distinct forms. The first form, studied the cells in the buffet coat. The second, studied the cells with density less than, 1,077 separated by the Ficoll Paque. The resulting pellets were processed for later observation by electronic microscopy transmission. The presence of granular and non granular cells were detected in different maturation stages in both groups and also morphological compatible cells with apoptosis.

Alzheimer's disease (AD) is the most prevalent form of progressive dementia and is characterized by the accumulation of amyloid beta (Aß) peptide in the brain and in the cerebral vessels forming cerebral amyloid angiopathy (CAA). As previously reported, an active immunization strategy of mice with Aß1-42 peptide results in decreased Th1 and increased Th2 cytokine responses as well as an effectively clearance of CNS Aß. This approach has also yielded favorable results for many patients, unfortunately, a small percentage of these study participants developed severe aseptic meningoencephalitis likely secondary to CNS invasion of activated T-cells. We have previously demonstrated that disruption of CD40-40L pathway reduces Aß plaque load, promotes Th2 response, and rescues from cognitive impairments. However, direct blockage of the CD40 pathway by passive vaccination with anti-CD40L antibody leads to immunosupression. Therefore, in its current form this therapeutic strategy poses an unacceptable risk to the recipient of treatment, aged individual. For those reasons, the identification and characterization of alternative modulators/inhibitors of CD40 signaling may be necessary for the development of safe and effective AD immunotherapy. This proposal introduces novel immunomodulatory therapies that are based on previous vaccination strategies or cell based therapies across medial field. We showed that transcutaneous vaccination can both be efficacious and safe, thus clearly demonstrating that the right combination of the antigens, adjuvants, and the routes of administration are crucial for the right vaccine. Furthermore, we demonstrated that the effects of current Aß vaccine strategies could be enhanced by a simultaneous blockade of CD40-40L signaling. As an alternative approach, we explored the possibility of cell-based therapies and showed that human umbilical cord blood cells, which are currently used as a treatment for systemic lupus erythematosus and leukemia, and currently investigated against stroke, amyotropic lateral sclerosis, age-related macular degeneration, multiple sclerosis, and Parkinson's disease, and showed that not just they improved the AD like pathology in transgenic animals but altered both the brain and peripheral inflammation levels. Lastly, we discussed the involvement of microglia, one of the key players in both AD pathogenesis and Aß clearance and suggesed that microglia in actuality has a continuum of physiological activation states that contribute to proinflammation, antiinflammation, and phagocytosis.

Eine Erhöhung des Bilirubins über 2 mg/dl betrifft 90 % aller Neugeborenen. Sie ist meist physiologisch und tritt optisch sichtbar bei 60-70 % dieses Kollektivs auf. In der pathologischen, exzessiv erhöhten Form ist sie der häufigste Grund für eine stationäre Wiederaufnahme während der ersten sieben Lebenstage. Ihre schwerste Komplikation, der Kernikterus, scheint - trotz allgemein verfügbarer, preiswerter und sicherer Therapiemöglichkeiten - wieder vermehrt aufzutreten. Die Gründe liegen im Überwachungsdefizit bei früher Entlassung von schlecht aufgeklärten Eltern, Nichtbeachtung der Besonderheiten der Neugeborenen ≤ 38 Schwangerschaftswochen und der zunehmenden Tendenz zum Stillen bei häufig unzureichender Anleitung. Ferner werden ikterische Kinder nur zu oft lediglich visuell bezüglich des Grades der Bilirubinämie eingeschätzt und die Therapie somit erheblich verzögert. Gegenstand dieser Arbeit ist die Frage, ob aus der Dynamik des Serumbilirubinspiegels von der Geburt bis zum 3. Lebenstag die Wahrscheinlichkeit des Auftretens einer phototherapiepflichtigen Hyperbilirubinämie abgeschätzt werden kann. Dazu wurde der Serumbilirubinspiegel direkt postnatal aus dem Nabelschnurblut, bzw. am 3. Lebenstag gleichzeitig mit dem Stoffwechselscreening ermittelt und der Phototherapiebedarf im Verlauf festgehalten. Um die Aussage zu präzisieren, wurde die Studienpopulation aus 2573 Kindern weiter unterteilt in 2180 reife tAGA- (hier Eu- und Hypertrophe), 267 reife tSGA-Kinder (Hypotrophe) und 126 FG (Frühgeborene). In allen 3 Gruppen korrelierten das Nabelschnurbilirubin und der Serumbilirubinwert vom 3. Lebenstag positiv mit der Entwicklung einer Hyperbilirubinämie. Anhand dieser Ausgangswerte konnten Grenzen für Hoch-, Mittelhoch-, Mittelniedrig- und Niedrigrisikogruppen definiert werden, welche die Entwicklung einer Hyperbilirubinämie mit einer Wahrscheinlichkeit von ≥ 20 %, 5-20 %, 0 < x <5 % und 0 % voraussagen. Damit kann man bereits früh eine Vorabselektion entsprechend dem Gefährdungspotential treffen und die Verlaufskontrollen entsprechend terminieren. Als Risikofaktoren einer therapiepflichtigen Hyperbilirubinämie wurden außerdem Frühgeburtlichkeit, seltener tSGA, geringes Geburtsgewicht und niedriges Gestationsalter (in der vorliegenden FG-Gruppe nicht signifikant) gefunden. Im Falle einer Sectiogeburt und bei Zuhilfenahme von Hilfsmitteln im Rahmen einer vaginalen Entbindung nahm der Bedarf an Phototherapie in der tAGA- und tSGA-Gruppe zu.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年1月31日 匠原龍太郎氏の博士論文として提出された

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This work examines the osteogenic potential and immune-privileged properties of human umbilical cord perivascular cells (HUCPVCs) in normal Wistar rats and athymic rnu/rnu rats for up to 60 days. HUCPVCs demonstrated a mesenchymal stromal cell phenotype, assayed through flow cytometry, and RT-PCR analysis detected their expression of osteogenic genes. A bone tissue engineering construct was developed through centrifugal seeding of HUCPVCs onto calcium phosphate-coated PLGA scaffolds. These cell-scaffold constructs were transplanted into bilateral femoral defects. HUCPVCs did not induce any systemic biological response in normal rats; however, they did not engraft and impaired bone healing up to 60 days. When transplanted into athymic rats, HUCPVCs were detected up to 30 days in the femoral defects, improved bone regeneration at 15 and 30 days, as measured by micro computed tomography, and expressed osteogenic proteins. These findings demonstrate that HUCPVCs are suitable for bone tissue engineering studies in larger animals.

This work examines the osteogenic potential and immune-privileged properties of human umbilical cord perivascular cells (HUCPVCs) in normal Wistar rats and athymic rnu/rnu rats for up to 60 days. HUCPVCs demonstrated a mesenchymal stromal cell phenotype, assayed through flow cytometry, and RT-PCR analysis detected their expression of osteogenic genes. A bone tissue engineering construct was developed through centrifugal seeding of HUCPVCs onto calcium phosphate-coated PLGA scaffolds. These cell-scaffold constructs were transplanted into bilateral femoral defects. HUCPVCs did not induce any systemic biological response in normal rats; however, they did not engraft and impaired bone healing up to 60 days. When transplanted into athymic rats, HUCPVCs were detected up to 30 days in the femoral defects, improved bone regeneration at 15 and 30 days, as measured by micro computed tomography, and expressed osteogenic proteins. These findings demonstrate that HUCPVCs are suitable for bone tissue engineering studies in larger animals.

The objective of these experiments is to explore the effect of LIPUS on the ultraexpansion and osteogenic differentiation of harvested passage-4 HUCPV-SCs. HUCPV-SCs were divided into two groups: a treatment group that received LIPUS for 10 minutes for 1, 7, and 14 days and a control group that received a sham treatment utilizing both basic and osteogenic media. The results in basic media and osteogenic media demonstrated nonsignificant differences in cell count, ALP, DNA content, and CD90. Statistically significant expression of OSP and PCNA was observed on day 14 in LIPUS treated group. Nucleostemin expression in the LIPUS-treated group was insignificant on days 1 and 7. However, a selective increase in osteogenic markers was obtained on day 7 for ALP and OCN and on day 14 for OPN. Future experiments are required to explore the effects of different application times and/or techniques of LIPUS on the behaviour of HUCPV-SCs. / Medical Science

Umbilical cord blood (UCB) is a source of hematopoietic stem and progenitor cells (HSPC) used for allogeneic transplantation. Ex vivo expansion of HSPC can improve the slow platelet and neutrophil engraftment associated with UCB transplants. HSPCs reside in niches, some of which are near the endosteal bone surface, where they can associate with immature osteoblasts. Interestingly, osteoblasts can enhance the growth of HSPC in culture and their platelet engraftment activity. Using a proteomics approach, I identified 47 differentially expressed proteins between mesenchymal stem cells and immature osteoblasts. Several of these were previously implicated in HSPC maintenance such as IGF2, IGFBP2, DCN, GAS6 and VCAM1. Moreover, several other proteins belong to the alternative and classical complement pathways. Finally, I discovered that microvesicles found in osteoblast conditioned medium may also modulate the growth of HSPC, at least in ex vivo cultures.