Human hematopoietic microenvironments, in vivo, in vitro and on chip

Cells are directed by signals in their microenvironment. Knowledge about this communication is necessary to understand both normal processes and cancer. This PhD thesis focuses on the human bone marrow microenvironment that governs the generation of B-cells (early B-lymphopoiesis). Multiple environmental elements affecting the maturation process were explored by use of both classical and new technologies. Immunohistochemical investigation of bone marrow biopsies revealed that all stages of differentiating B-cells harbor a niche formed by slender CD10+ stromal cells, suggesting an essential supporting role. A cDNA cloning strategy (signal sequence trap, SST-REX) identified several B-cell surface proteins that may contribute in the crosstalk with the microenvironment. One unknown protein was characterised (TMEM-9), but turned out to have no obvious role in cell communication. Next, classical and new cultivation platforms were developed for assaying the impact of microenvironmental factors on early B-lymphopoiesis. Conventional cultivation based on a feeder layer of stromal cells showed that the morphogen Wnt3a reduced both proliferation of B-cell progenitors and the production of B-cells from hematopoietic stem cells. The existence of an operational Wnt-pathway in B-cell progenitors was verified by detection of central pathway molecules both at protein level (Western blot) and at expression level (RT-PCR). For the purpose of obtaining more advanced models of hematopoietic environments, a microfluidic system (chip) was fabricated by soft lithography. The system was compatible with long-term cultivation and stem cell differentiation, as shown by extensive adipogenic and osteogenic differentiation of mesenchymal stem cells (MSCs), which are reported to provide essential hematopoietic support. Exploitation of the microscale properties of this system can be used for housing hematopoietic cells in more realistic environments than those enabled by existing methods

Interstitial fluid drainage from rat apical area takes place via vessels in the mandibular canal.

We sought to investigate the transport route for protein‐rich fluid from the apical area towards the draining lymph nodes. The first mandibular molar root canals in 24 female Wistar rats were instrumented and filled with radioactive‐labelled human serum albumin. The rats were sacrificed at different intervals beginning after 10 min (time 0) and continuing up to 72 h. Three jaw segments, gingiva around the first molar, blood samples, submandibular and cervical lymph nodes were collected and analyzed for radioactivity. The starting volume of tracer (control) for all experiments was calculated from measurements at time 0. At time 0, radioactivity was only detected in the jaw segments. Within lymph nodes and serum, the tracer was found after 4 h, with the highest amount recorded in serum up to 24 h. Lymphatics were found within the mandibular canal along blood vessels and nerves and exiting via foramen mandibularis, after immunohistochemical staining in four untreated rats. Our results show tracer distribution from the apical area towards the mandibular canal in a posterior direction. The tracer washout rate was low, and the fluid was mainly absorbed into blood vessels. The lymphatics in the mandibular canal may be more important for immune cell transport than for fluid drainage.publishedVersio

Wnt expression and canonical Wnt signaling in human bone marrow B lymphopoiesis

BACKGROUND: The early B lymphopoiesis in mammals is regulated through close interactions with stromal cells and components of the intracellular matrix in the bone marrow (BM) microenvironment. Although B lymphopoiesis has been studied for decades, the factors that are implicated in this process, both autocrine and paracrine, are inadequately explored. Wnt signaling is known to be involved in embryonic development and growth regulation of tissues and cancer. Wnt molecules are produced in the BM, and we here ask whether canonical Wnt signaling has a role in regulating human BM B lymphopoiesis. RESULTS: Examination of the mRNA expression pattern of Wnt ligands, Fzd receptors and Wnt antagonists revealed that BM B progenitor cells and stromal cells express a set of ligands and receptors available for induction of Wnt signaling as well as antagonists for fine tuning of this signaling. Furthermore, different B progenitor maturation stages showed differential expression of Wnt receptors and co-receptors, β-catenin, plakoglobin, LEF-1 and TCF-4 mRNAs, suggesting canonical Wnt signaling as a regulator of early B lymphopoiesis. Exogenous Wnt3A induced stabilization and nuclear accumulation of β-catenin in primary lineage restricted B progenitor cells. Also, Wnt3A inhibited B lymphopoiesis of CD133(+)CD10(- )hematopoietic progenitor cells and CD10(+ )B progenitor cells in coculture assays using a supportive layer of stromal cells. This effect was blocked by the Wnt antagonists sFRP1 or Dkk1. Examination of early events in the coculture showed that Wnt3A inhibits cell division of B progenitor cells. CONCLUSION: These results indicate that canonical Wnt signaling is involved in human BM B lymphopoiesis where it acts as a negative regulator of cell proliferation in a direct or stroma dependent manner

Maritim militærtrening i en usikker verden: En studie av sammenheng mellom personlig hardførhet, opplevd mestringstro, kognitiv fleksibilitet og beslutningsstil i en maritim kontekst

Den stadig endrende sikkerhetspolitiske situasjonen Norge står overfor, stiller krav til en økt operativ evne, noe som kan oppnås gjennom militærtrening. Inneværende studie tok utgangspunkt i en maritim operasjon som Sjøkrigsskolen i Bergen gjennomfører årlig. Målet var å undersøke hvorvidt det finnes en gruppeforskjell mellom høy og lav personlig hardførhet i nivåer av opplevd mestringstro, kognitiv fleksibilitet og preferanse for beslutningsstil. Ved bruk av et pre-postdesign, ble det videre mulig å undersøke effekten av den maritime operasjonen på kadettenes nivåer av opplevde mestringstro, kognitive fleksibilitet og preferanse for beslutningsstil, samt identifisere om effekten var ulik for de to gruppene (høy og lav personlig hardførhet). Studien benyttet Banduras sosial-kognitive teori som teoretisk rammeverk. Dataene ble samlet inn ved bruk av spørreskjema distribuert til kadettene før og etter den maritime operasjonen i desember 2021, og ble videre analysert ved bruk av Statistica versjon 64. Resultatene viste at den maritime operasjonen medførte en økning i opplevd mestringstro for begge gruppene. Det ble også funnet en gruppeforskjell i kognitiv fleksibilitet, der gruppen høy personlig hardførhet hadde et høyere nivå av kognitiv fleksibilitet. Det ble avdekket en samlet preferansetendens for bruk av rasjonell beslutningsstil, etterfulgt av intuitiv, avhengig og spontan beslutningsstil. Preferansen var lavest for unnvikende beslutningsstil. Studien gir et unikt innblikk i effekten maritim militærtrening har på psykologiske faktorer i en usikker verden, noe som kan være av betydning for revidering av eksisterende treningsprogram og seleksjonsprosesser i Forsvaret.The shifting political and safety situation in Norway demands the need for more military operational capacity, which can be achieved through military training. The present study was based on a maritime operation that the Norwegian Naval Academy in Bergen conducts annually. The aim was to examine whether there was a group difference between high and low personal hardiness in levels of self-efficacy, cognitive flexibility, and preference for decision-making style. Furthermore, the objective was to examine the effect of the maritime operation on the cadets' self-efficacy, cognitive flexibility, and preference for decision-making style, and if the maritime operation had different effects on the two groups (high and low personal hardiness). Bandura's social-cognitive theory was used as a theoretical framework. The study used a pre-post design and data were therefore collected through a questionnaire distributed to the cadets before and after the maritime operation in December 2021, and further analyzed using Statistica version 64. The results showed that the maritime operation led to an increase in self-efficacy for both groups. A group difference in cognitive flexibility was also discovered, where the high personal hardiness group had a higher level. An overall preference for rational decision-making style was uncovered, followed by intuitive, dependent, and spontaneous decision-making style. The preference was lowest for the avoidant decision-making style. The study provides a unique insight into the effect of maritime military training on psychological factors in an uncertain world, which may be beneficial when revising training programs and selection in the Norwegian Armed Forces.Masteroppgave i psykologiMAPSYK360INTL-KMDINTL-JUSINTL-MNINTL-PSYKINTL-SVINTL-HFMAPS-PSYKINTL-ME

High-salt diet causes osmotic gradients and hyperosmolality in skin without affecting interstitial fluid and lymph

The common notion is that the body Na+ is maintained within narrow limits for fluid and blood pressure homeostasis. Several studies have, however, shown that considerable amounts of Na+ can be retained or removed from the body without commensurate water loss and that the skin can serve as a major salt reservoir. Our own data from rats have suggested that the skin is hypertonic compared with plasma on salt storage and that this also applies to skin interstitial fluid. Even small electrolyte gradients between plasma and interstitial fluid would represent strong edema-generating forces. Because the water accumulation has been shown to be modest, we decided to reexamine with alternative methods in rats whether interstitial fluid is hypertonic during salt accumulation induced by high-salt diet (8% NaCl and 1% saline to drink) or deoxycorticosterone pellet implantation. These treatments resulted both in increased systemic blood pressure, skin salt, and water accumulation and in skin hyperosmolality. Interstitial fluid isolated from implanted wicks and lymph draining the skin was, however, isosmotic, and Na+ concentration in fluid isolated by centrifugation and in lymph was not different from plasma. Interestingly, by eluting layers of the skin, we could show that there was an osmolality and urea gradient from epidermis to dermis. Collectively, our data suggest that fluid leaving the skin as lymph is isosmotic to plasma but also that the skin can differentially control its own electrolyte microenvironment by creating local gradients that may be functionally important.acceptedVersio

Abnormal neonatal sodium handling in skin precedes hypertension in the SAME rat

We discovered high Na(+) and water content in the skin of newborn Sprague-Dawley rats, which reduced ~ 2.5-fold by 7 days of age, indicating rapid changes in extracellular volume (ECV). Equivalent changes in ECV post birth were also observed in C57Bl/6 J mice, with a fourfold reduction over 7 days, to approximately adult levels. This established the generality of increased ECV at birth. We investigated early sodium and water handling in neonates from a second rat strain, Fischer, and an Hsd11b2-knockout rat modelling the syndrome of apparent mineralocorticoid excess (SAME). Despite Hsd11b2(-/-) animals exhibiting lower skin Na(+) and water levels than controls at birth, they retained ~ 30% higher Na(+) content in their pelts at the expense of K(+) thereafter. Hsd11b2(-/-) neonates exhibited incipient hypokalaemia from 15 days of age and became increasingly polydipsic and polyuric from weaning. As with adults, they excreted a high proportion of ingested Na(+) through the kidney, (56.15 ± 8.21% versus control 34.15 ± 8.23%; n = 4; P < 0.0001), suggesting that changes in nephron electrolyte transporters identified in adults, by RNA-seq analysis, occur by 4 weeks of age. Our data reveal that Na(+) imbalance in the Hsd11b2(-/-) neonate leads to excess Na(+) storage in skin and incipient hypokalaemia, which, together with increased, glucocorticoid-induced Na(+) uptake in the kidney, then contribute to progressive, volume contracted, salt-sensitive hypertension. Skin Na(+) plays an important role in the development of SAME but, equally, may play a key physiological role at birth, supporting post-natal growth, as an innate barrier to infection or as a rudimentary kidney

CD24-targeted intraoperative fluorescence image-guided surgery leads to improved cytoreduction of ovarian cancer in a preclinical orthotopic surgical model

Background: The completeness of resection is a key prognostic indicator in patients with ovarian cancer, and the application of tumour-targeted fluorescence image-guided surgery (FIGS) has led to improved detection of peritoneal metastases during cytoreductive surgery. CD24 is highly expressed in ovarian cancer and has been shown to be a suit

Proteoglycans contribute to the functional integrity of the glomerular endothelial cell surface layer and are regulated in diabetic kidney disease

All capillary endothelia, including those of the glomeruli, have a luminal cell surface layer (ESL) consisting of glycoproteins, glycolipids, proteoglycans (PGs) and glycosaminoglycans. Previous results have demonstrated that an intact ESL is necessary for a normal filtration barrier and damage to the ESL coupled to proteinuria is seen for example in diabetic kidney disease (DKD). We used the principles of ion exchange chromatography in vivo to elute the highly negatively charged components of the ESL with a 1 M NaCl solution in rats. Ultrastructural morphology and renal function were analyzed and 17 PGs and hyaluronan were identified in the ESL. The high salt solution reduced the glomerular ESL thickness, led to albuminuria and reduced GFR. To assess the relevance of ESL in renal disease the expression of PGs in glomeruli from DKD patients in a next generation sequencing cohort was investigated. We found that seven of the homologues of the PGs identified in the ESL from rats were differently regulated in patients with DKD compared to healthy subjects. The results show that proteoglycans and glycosaminoglycans are essential components of the ESL, maintaining the permselective properties of the glomerular barrier and thus preventing proteinuria.publishedVersio

Novel clearance of muscle proteins by muscle cells

Blood levels of cardiac troponins (cTn) and myoglobin are analysed when myocardial infarction (MI) is suspected. Here we describe a novel clearance mechanism for muscle proteins by muscle cells. The complete plasma clearance profile of cTn and myoglobin was followed in rats after intravenous or intermuscular injections and analysed by PET and fluorescence microscopy of muscle biopsies and muscle cells. Compared with intravenous injections, only 5 % of cTnT, 0.6 % of cTnI and 8 % of myoglobin were recovered in the circulation following intramuscular injection. In contrast, 47 % of the renal filtration marker FITC-sinistrin and 81 % of cTn fragments from MI-patients were recovered after intramuscular injection. In addition, PET and biopsy analysis revealed that cTn was taken up by the quadriceps muscle and both cTn and myoglobin were endocytosed by cultured muscle cells. This local clearance mechanism could possibly be the dominant clearance mechanism for cTn, myoglobin and other muscle damage biomarkers released by muscle cells