ALBERT

Description: Transcriptional profiling has defined pancreatic ductal adenocarcinoma (PDAC) into distinct subtypes with the majority being classical epithelial (E) or quasi-mesenchymal (QM). Despite clear differences in clinical behavior, growing evidence indicates these subtypes exist on a continuum with features of both subtypes present and suggestive of interconverting cell states. Here, we investigated the impact of different therapies being evaluated in PDAC on the phenotypic spectrum of the E/QM state. We demonstrate using RNA-sequencing and RNA-in situ hybridization (RNA-ISH) that FOLFIRINOX combination chemotherapy induces a common shift of both E and QM PDAC toward a more QM state in cell lines and patient tumors. In contrast, Vitamin D, another drug under clinical investigation in PDAC, induces distinct transcriptional responses in each PDAC subtype, with augmentation of the baseline E and QM state. Importantly, this translates to functional changes that increase metastatic propensity in QM PDAC, but decrease dissemination in E PDAC in vivo models. These data exemplify the importance of both the initial E/QM subtype and the plasticity of E/QM states in PDAC in influencing response to therapy, which highlights their relevance in guiding clinical trials.

Description: Stretch activation is important in the mechanical properties of vertebrate cardiac muscle and essential to the flight muscles of most insects. Despite decades of investigation, the underlying molecular mechanism of stretch activation is unknown. We investigated the role of recently observed connections between myosin and troponin, called “troponin bridges,” by analyzing real-time X-ray diffraction “movies” from sinusoidally stretch-activated Lethocerus muscles. Observed changes in X-ray reflections arising from myosin heads, actin filaments, troponin, and tropomyosin were consistent with the hypothesis that troponin bridges are the key agent of mechanical signal transduction. The time-resolved sequence of molecular changes suggests a mechanism for stretch activation, in which troponin bridges mechanically tug tropomyosin aside to relieve tropomyosin’s steric blocking of myosin–actin binding. This enables subsequent force production, with cross-bridge targeting further enhanced by stretch-induced lattice compression and thick-filament twisting. Similar linkages may operate in other muscle systems, such as mammalian cardiac muscle, where stretch activation is thought to aid in cardiac ejection.

Description: Hematopoietic stem cell (HSC) regulation is highly dependent on interactions with the marrow microenvironment. Controversy exists on N-cadherin's role in support of HSCs. Specifically, it is unknown whether microenvironmental N-cadherin is required for normal marrow microarchitecture and for hematopoiesis. To determine whether osteoblastic N-cadherin is required for HSC regulation, we used a genetic murine model in which deletion of Cdh2, the gene encoding N-cadherin, has been targeted to cells of the osteoblastic lineage. Targeted deletion of N-cadherin resulted in an age-dependent bone phenotype, ultimately characterized by decreased mineralized bone, but no difference in steady-state HSC numbers or function at any time tested, and normal recovery from myeloablative injury. Intermittent parathyroid hormone (PTH) treatment is well established as anabolic to bone and to increase marrow HSCs through microenvironmental interactions. Lack of osteoblastic N-cadherin did not block the bone anabolic or the HSC effects of PTH treatment. This report demonstrates that osteoblastic N-cadherin is not required for regulation of steady-state hematopoiesis, HSC response to myeloablation, or for rapid expansion of HSCs through intermittent treatment with PTH.

Description: Abstract 407 Since the hematopoietic system is exquisitely sensitive to environmental and iatrogenic injury, the bone marrow microenvironment likely provides protective mechanisms during times of injury or stress. We have previously demonstrated that prostaglandin E2 (PGE2), which can be produced by many cell types in the bone marrow, targets both the bone marrow microarchitecture and primitive hematopoietic cells when administered systemically to mice (Porter, Frisch et. al., Blood, 2009). Since PGE2 is a local mediator of injury and is known to play a protective role in other cell types, we hypothesized that it could be an important microenvironmental regulator of HSPCs during times of injury. To test this hypothesis, we injured mice with a sub-lethal dose of gamma radiation, 6.5 Gy TBI, and sacrificed mice at varying time points from 1 hour to 6 days post-radiation. Bone marrow supernatant was collected and used for quantification of local PGE2 levels by ELISA. We found that, compared to non-irradiated mice, the PGE2 levels were increased greater than two-fold by 4 hours after irradiation (p=0.0030; n=3–6 mice/group), and these levels remain elevated until at least 6 days after injury (p

Description: Abstract 642 HSCs are rare immature cells capable of reconstituting all blood cell lineages throughout the life of an individual. We have previously shown that intermittent treatment with PTH is sufficient to increase the number of HSCs in the marrow of mice. This PTH effect is blocked in vitro with inhibition of gamma-secretase, the mediator of a required step in Notch signaling. Osteoblastic cells are a critical component of the HSC niche and are likely mediators of the PTH-induced increase in HSCs. Specifically the Notch ligand Jag 1 is expressed on osteoblasts and is therefore implicated as a mechanism through which PTH acts on HSCs. Therefore we investigated in vivo the role of osteoblastic Jag 1 in the PTH-dependent increase in HSCs. We utilized the 2.3kb collagen 1 promoter driven cre recombinase to specifically excise Jag 1 from osteoblastic cells in mice (OBJag1 mice). As we previously reported treatment of wild type (WT) controls with PTH 3 times daily for 10 days resulted in a significant increase in phenotypic HSC populations including Lin-Sca1+cKit+CD48-CD150- short-term HSCs (ST-HSCs) (VEH/PTH 0.0405±0.001 vs 0.0650±0.0038, p≤0.0001) and Lin-Sca1+cKit+CD48-CD150+ long-term HSCs (LT-HSCs) (VEH/PTH 0.0077±0.0008 vs 0.0125±0.00096, p≤0.01) as determined by flow cytometric analysis. In contrast treatment of OBJag1 mice did not result in a phenotypic increase in these populations. Despite the lack of a phenotypic increase in HSCs in OBJag1 mice, when HSC function was assessed by competitive repopulation assay, OBJag1 marrow cells demonstrated the same increased repopulating ability as WT mice (WT: VEH/PTH 12.16±2.7 vs 22.32±2.4, p≤0.01, OBJag1: VEH/PTH 13.6±1.8 vs 31.6±5.9, p≤0.01). Upon secondary transplantation however, HSCs from OBJag1 donors treated with PTH resulted in a lower engraftment rate than VEH treated controls (VEH/PTH 14.61±3.8 vs 4.38±0.9, p≤0.05). This result suggests that osteoblastic Jag 1 is necessary for the increase in phenotypic HSCs resulting from PTH treatment and is required to maintain LT-HSC self-renewal. However these data also suggest an osteoblastic Jag 1 independent mechanism that mediates a transient increase in repopulating ability. Decreased apoptosis is a potential mechanism by which PTH may functionally increase HSCs in the absence of increased self-renewal. To determine if PTH treatment decreases the apoptosis rate of HSCs, WT mice were treated intermittently with PTH once a day for 7 days. Despite a lack of increased HSCs by phenotypic analysis at 7 days, marrow from PTH treated mice displayed an increase in LT-HSC function as measured by competitive transplantation. We determined to measure the effect of PTH on apoptotic rates of HSCs using Annexin V membrane expression. By the 7th day of PTH treatment, LT-HSC apoptotic rates were decreased in the PTH treated group (VEH/PTH 10.482±2.25 vs. 6.27±1.93, p≤0.01) suggesting that changes in apoptotic rate of LT-HSCs precedes the HSC increase. These results were confirmed by flow cytometric measurement of activated caspase 3. PTH treatment decreased the percentage of LT-HSCs that were positive for activated caspase 3 (VEH/PTH 4.3±0.5 vs. 2.4±0.3, p≤0.01). PTH induced micro-architectural changes in trabecular bone at day 7 of treatment suggesting bone involvement despite the lack of an increase in bone volume. These results suggest for the first time that PTH may exert its beneficial effect on bone marrow reconstitution through both Jag 1 dependent and independent effects. Additionally, HSCs demonstrate decreased apoptotic rates and increased reconstitution ability prior to a demonstrable phenotypic increase, mimicking the effect seen in the absence of osteoblastic Jag 1. Together these results suggest that the decreased apoptotic rate may be mediated by an osteoblastic Jag 1 independent mechanism. Whether osteoblasts are required for the observed osteoblastic Jag 1 independent effects remains to be seen as these effects could be mediated by a Jag 1 independent osteoblastic mechanism or by an altogether different cellular component of the HSC niche. Further, since stressful manipulation of HSCs ex vivo is essential for their use in transplantation, defining factors regulating and decreasing their apoptosis may improve their engraftment efficiency, expanding their clinical use when their numbers are limited. Disclosures: No relevant conflicts of interest to declare.

Description: Enucleation is the hallmark of erythropoiesis in mammals. Previously, we determined that yolk sac-derived primitive erythroblasts mature in the bloodstream and enucleate between E14.5–16.5 of mouse gestation (Kingsley, et al. Blood104:19, 2004), however, it is unclear by what mechanism or even where primitive erythroblasts enucleate. Definitive erythroblasts in the fetal liver and adult marrow enucleate by nuclear extrusion, generating reticulocytes and small, nucleated cells with a thin rim of cytoplasm referred to as “extruded nuclei”, that rapidly lose cell membrane phosphatidylserine asymmetry and are engulfed by macrophages. Since the cell membrane plays an important role in the biology of these cells, “extruded nucleus” is a misleading term. We propose that these cells be termed “pyrenocytes”, derived from the Greek “pyren” (the pit of a stone fruit) and “cyte” (cell), reflecting their highly condensed nucleus and high nuclear to cytoplasmic ratio. Careful examination of murine fetal blood by immunohistochemistry and multispectral imaging flow cytometry (Amnis ImageStream) revealed a transient population of εy-globin-positive pyrenocytes temporally coincident with the enucleation of primitive erythroblasts (E14.5–15.5). A high percentage (40%) of these circulating primitive pyrenocytes were annexin V-positive, suggesting that they, like their definitive counterparts, generate a signal that can facilitate engulfment by macrophage cells. At E14.5–15.5, the highest frequency of macrophage cells within the conceptus is found in the liver. Immunohistochemical studies with anti-εy-globin and F4/80 antibodies revealed that many primitive erythroblasts in the liver, but not the spleen, are in close proximity to macrophages. Furthermore, the frequency of nucleated primitive erythroblasts was higher in the liver than in the bloodstream at E15.5. These results, taken together, suggest that late-stage primitive erythroblasts do not passively flow through the liver but may, rather, interact with macrophage cells there. Surprisingly, primitive erythroblasts, but not co-circulating fetal definitive erythrocytes, can reconstitute erythroblast islands by attaching to fetal liver-derived macrophages in vitro. α4 integrin blocking antibodies, but not isotype control antibodies, reduce erythroblast island reconstitution, indicating that these primitive erythroid-macrophage interactions are mediated in part by α4 integrin. Finally, we found that unlike definitive erythroblasts, late-stage primitive erythroblasts fail to autonomously enucleate in vitro unless co-cultured with macrophage cells. We conclude that primitive erythroblasts in the mammalian fetus enucleate by nuclear extrusion as a semi-synchronous cohort and generate a transient population of pyrenocytes. Furthermore, our studies suggest that this process occurs in the fetal liver in association with macrophage cells. Continued investigation of the differences and similarities between primitive and definitive erythropoiesis will lead to an improved understanding of the terminal steps of erythroid maturation.

Description: HSCs are pluripotent cells responsible for the establishment and renewal of the entire hematopoietic system. Our group and others have established that osteoblastic cells in the bone marrow microenvironment regulate HSC cell fate decisions. Specifically, Parathyroid hormone (PTH) expands HSCs by activating osteoblasts in the HSC niche. However, the molecular mechanisms for this increase are unknown. PTH increases local production of prostaglandin E2 (PGE2) in osteoblasts by stimulating cyclo-oxygenase 2 (Cox-2). We also recently found that treatment of osteoblastic MC3T3 cells with PTH (10−7 M) rapidly induces PGE2 Synthase expression. Therefore, we hypothesized that PGE2 may act as a mediator of the PTH effect on HSCs. We have shown that in vivo PGE2 treatment caused a 2.75-fold increase in lineage− Sca-1+ c-kit+ (LSK) cells within the bone marrow compared with vehicle treated mice (p=0.0061, n=8/group). Bone marrow mononuclear cells (BMMC) from mice treated with PGE2 also demonstrated superior lymphomyeloid reconstitution in competitive repopulation analyses, suggesting that HSCs are being expanded or modulated to more efficiently reconstitute the hematopoietic system in the recipients. It is known that HSCs that reside in the G0 phase of the cell cycle have increased ability to reconstitute myeloablated recipient mice. Since PGE2 treatment resulted in superior reconstitution, we hypothesized that PGE2 may increase the percentage of HSCs residing in G0. To test this hypothesis, we treated BMMC from male C57b/6 mice with 10−6 M PGE2 or vehicle for 90 minutes. The percentage of cells in G0 vs. G1 was determined by flow-cytometric analysis using the RNA and DNA dyes, Pyronin-Y and Hoechst 33342 respectively. As we predicted, PGE2 treatment increased the percentage of wild-type LSK cells in G0 1.85 fold over vehicle-treated LSK cells (23.63% in vehicle-treated, n=4 vs. 43.7% in PGE2-treated, n=6). Since the PTH-dependent increase in HSCs is Protein Kinase A (PKA) mediated and the PGE2 receptors EP2 and EP4 signal via PKA, we assayed the effect of PGE2 on the percentage of cells in G0 in mice lacking the EP2 receptor (EP2−/− mice). Interestingly, there was no enrichment for HSC in G0 when BMMC from EP2−/− mice were treated with PGE2 (55.25% in vehicle-treated, n=4 vs. 56.06% in PGE2-treated, n=5). These findings suggest that PGE2-dependent regulation of HSC activity may involve increasing the percentage of HSCs that reside in G0 by activation of EP2, thereby augmenting their ability to reconstitute the hematopoietic system of a myeloablated recipient. 5-bromo-2-deoxyuridine (BrdU) incorporation was also used to investigate the effect of PGE2 on cell cycling of HSCs. Male 6–8 week old C57b/6 mice were injected intraperitoneally with 1 mg BrdU and PGE2 (6 mg/kg) or vehicle. After 30, 60, 90 or 120 minutes, mice were sacrificed and BMMC were subjected to flow cytometric analysis for incorporation of BrdU and DNA content in HSCs. As expected for the highly quiescent HSC population, only a small fraction of HSCs incorporated BrdU. After 30 and 60 minutes of treatment, there was no difference in the percentage of cells that incorporated BrdU between vehicle and PGE2-treated mice. However, at the 90 and 120 minute time points, there were significantly less HSCs cycling in the bone marrow from the PGE2 treated mice (12.1% vs. 5.3% at 90 min, n=2 per group; 11.1% vs. 1.8% at 120 min, n=5 per group, p=0.0060), suggesting that fewer PGE2-treated cells were synthesizing DNA. Taken together, the increase in the percentage of HSCs in G0 and the decrease in cycling HSCs after PGE2 treatment indicate that PGE2 could improve engraftment and reconstitution of the hematopoietic system by enriching for HSCs in G0. These results suggest that PGE2 may exert its beneficial effect on bone marrow reconstitution by altering cell cycle dynamics in HSCs. Identification of the molecular events mediating this novel PGE2 action on HSC could provide additional targets for HSC manipulation in clinical situations requiring rapid and efficient bone marrow reconstitution, such as recovery from iatrogenic or pathologic myeloablative injury.

Description: Abstract 3401 Hematopoietic stem and progenitor cells (HSPCs) are responsible for the continual production of all mature blood cells during homeostasis and times of stress. These cells are known to be regulated in part by the bone marrow microenvironment in which they reside. We have previously reported that the microenvironmentally-produced factor Prostaglandin E2 (PGE2) expands HSPCs when administered systemically in naïve mice (Porter, Frisch et. al., Blood, 2009). However, the mechanism mediating this expansion remains unclear. Here, we demonstrate that in vivo PGE2 treatment inhibits apoptosis of HSPCs in naïve mice, as measured by Annexin V staining (p=0.0083, n=6–7 mice/group) and detection of active-Caspase 3 (p=0.01, n=6–7 mice/group). These data suggest that inhibition of apoptosis is at least one mechanism by which PGE2 expands HSPCs. Since PGE2 is a local mediator of injury and is known to play a protective role in other cell types, we hypothesized that it could be an important microenvironmental regulator of HSPCs during times of injury. Thus, these studies explored the role of PGE2 signaling in the bone marrow following myelosuppressive injury using a radiation injury model. Endogenous PGE2 levels in the bone marrow increased 2.9-fold in response to a sub-lethal dose of 6.5 Gy total body irradiation (TBI)(p=0.0004, n=3–11 mice/group). This increase in PGE2 correlated with up-regulation of microenvironmental Cyclooxygenase-2 (Cox-2) mRNA (p=0.0048) and protein levels at 24 and 72 hr post-TBI, respectively. Further augmentation of prostaglandin signaling following 6.5 Gy TBI by administration of exogenous 16,16-dimethyl-PGE2 (dmPGE2) enhanced the survival of functional HSPCs acutely after injury. At 24 hr post-TBI, the bone marrow of dmPGE2-treated animals contained significantly more LSK cells (p=0.0037, n=13 mice/group) and colony forming unit-spleen cells (p=0.037, n=5 mice/group). Competitive transplantation assays at 72 hr post-TBI demonstrated that bone marrow cells from irradiated dmPGE2-treated mice exhibited increased repopulating activity compared with cells from vehicle-treated mice. Taken together, these results indicate that dmPGE2 treatment post-TBI increases survival of functional HSPCs. Since PGE2 can inhibit apoptosis of HSPCs in naïve mice, the effect of dmPGE2 post-TBI on apoptosis was also investigated. HSPCs isolated from mice 24 hr post-TBI demonstrated statistically significant down-regulation of several pro-apoptotic genes and up-regulation of anti-apoptotic genes in dmPGE2-treated animals (3 separate experiments with n=4–8 mice/group in each), suggesting that dmPGE2 initiates an anti-apoptotic program in HSPCs following injury. Notably, there was no significant change in expression of the anti-apoptotic gene Survivin, which has previously been reported to increase in response to ex vivo dmPGE2 treatment of bone marrow cells (Hoggatt et. al., Blood, 2009), suggesting differential effects of dmPGE2 in vivo and/or in an injury setting. Additionally, to ensure that this inhibition of apoptosis was not merely increasing survival of damaged and non-functional HSPCs, the effect of early treatment with dmPGE2 post-TBI on hematopoietic recovery was assayed by monitoring peripheral blood counts. Interestingly, dmPGE2 treatment in the first 72 hr post-TBI significantly accelerated recovery of platelet levels and hematocrit compared with injured vehicle-treated mice (n=12 mice/group). Immunohistochemical analysis of the bone marrow of dmPGE2-treated mice also exhibited a dramatic activation of Cox-2 in the bone marrow microenvironment. This suggests that the beneficial effect of dmPGE2 treatment following injury may occur, both through direct stimulation of hematopoietic cells and also via activation of the HSC niche. In summary, these data indicate that PGE2 is a critical microenvironmental regulator of hematopoietic cells in response to injury. Exploitation of the dmPGE2-induced initiation of an anti-apoptotic program in HSPCs may represent a useful method to increase survival of these cells after sub-lethal radiation injury. Further, amplification of prostaglandin signaling by treatment with PGE2 agonists may also represent a novel approach to meaningfully accelerate recovery of peripheral blood counts in patients with hematopoietic system injury during a vulnerable time when few therapeutic options are currently available. Disclosures: No relevant conflicts of interest to declare.

Description: Parathyroid Hormone (PTH) expands hematopoietic stem cells (HSC) through activated osteoblasts in the bone marrow (BM). Since PTH stimulates osteoblastic production of Prostaglandin E2 (PGE2), we hypothesized that PGE2 could also regulate HSC. In vivo PGE2 treatment demonstrated a time and dose dependent increase in BM lineage− Sca-1+ c-kit+ (LSK) BM mononuclear cells (BMMC) from PGE2 vs. vehicle treated mice (0.11 vs. 0.04% BMMC, P=0.0061, n=8 mice per treatment group), an effect superior to PTH (350 vs. 100% increase in LSK). There were no significant PGE2 effects on CFU-Cs or peripheral Hct, Plts or WBC counts compared to vehicle. Therefore PGE2-dependent cell expansion was not global across differentiated subsets, but was restricted to primitive hematopoietic cells, similar to the effects of PTH treatment. Consistent with a PGE2-dependent HSC increase, cells from PGE2 vs vehicle-treated mice had superior lymphomyeloid reconstitution by competitive repopulation analysis. However, this increase was short-lived: specifically, PGE2-dependent myeloid (CD11b+) reconstitution was no longer superior at 6 weeks, while the PGE2-dependent increase in lymphoid (CD3e+ and B220+) reconstitution ceased by 16 weeks. This surprising result suggests that in vivo PGE2 treatment selectively expands short-term HSC (or ST-HSC), which have highly proliferative properties, but limited self-renewal. To further confirm this targeted PGE2 effect, LSK subset analysis based on Flt3 and Thy1.1 expression was performed. Consistent with the competitive repopulation data, PGE2 treatment significantly increased Flt3+Thy1.1int LSK ST-HSC (0.0273 vs 0.0140% n=4 in each group, p=0.0307) as well as Flt3+Thy1.1− LSK Multipotent Progenitors (0.0305 vs 0.0195% n=4 in each group, p=0.0070), while Flt3−Thy1.1int LSK Long-Term HSC or LT-HSC (0.0126 vs 0.0078% n=4 in each group, p=0.1069) were unchanged compared to vehicle treatment. ST vs LT-HSC activity can also be quantified by the in vivo clonogenic Colony Forming Unit-Spleen (CFU-S) assay, where day 8 CFU-S represent ST-HSC, while day 10–12 CFU-S represent LT-HSC. Consistent with a PGE2-dependent specific ST-HSC increase, BMMC from PGE2 treated mice gave rise to a significantly higher number of CFU-Sd8 compared to cells from vehicle treated mice (10.5 vs 4.75 CFU-S per 60,000 BMMC, n=4 in each group, p=0.0053), while CFU-Sd10 were unchanged (12.5 vs 11.5 CFU-S per 60,000 BMMC, n=6, p=0.4950). Finally, since ST-HSC confer radioprotection, PGE2-dependent ST-HSC expansion would be expected to improve survival of lethally irradiated recipients receiving limiting numbers of BMMC from PGE2 vs vehicle-treated mice. As predicted, recipients of BMMC from PGE2 treated mice had increased survival 30 days after transplantation compared to animals receiving BMMC from vehicle treated donors (150,000 donor cells: 80% vs 0% survival, p=0.0018; 75,000 donor cells: 53% vs 0% survival, p=0.0173). Taken together, these data demonstrate specific PGE2-dependent regulation of ST-HSC, and provide a unique and novel model to define control of HSC subsets. This finding implicates for the first time specialized regulation of HSC subsets. Moreover, these data indicate that selective therapeutic manipulation of ST-HSC could be exploited in clinical situations requiring rapid bone marrow reconstitution, such as in recovery from iatrogenic or pathologic myeloablative injury.

Description: Abstract 1449 Poster Board I-472 Osteoblastic cells have been identified as a component of the hematopoietic stem cell (HSC) niche. This identification provides a novel strategy for in vivo expansion of HSCs by stimulating their regulatory microenvironment. This therapeutic approach could potentially expand the clinical use of HSCs in conditions where their number limits use. Activation of osteoblastic cells by Parathyroid Hormone (PTH) increases bone, osteoblastic and osteoclastic cell number and expands the HSC pool. Since HSCs do not express the PTH receptor, and genetic activation of PTH receptors in osteoblastic cells is sufficient to expand HSCs, the HSC increase must be initiated by PTH-dependent activation of osteoblastic cells. The HSC-enriched lineage-, sca-1+, and c-kit+ (LSK) compartment is heterogeneous and contains at least three subpopulations of cells with multilineage potential, but with progressively decreased quiescence and more limited self-renewal. In the initial evaluation of PTH effects on the HSC niche, the effects of osteoblastic activation on HSC subsets were not characterized in detail. The effect of PTH and osteoblastic activation on individual HSC subsets could suggest additional therapeutic uses if PTH increases not only Long Term-HSC (LT-HSC) but also the more proliferative Short-Term HSCs/ Multi Potent Progenitors (ST-HSCs/MPPs). In this study, our goal was to identify changes in the proportion of LT-HSC vs ST-HSC and MPPs after PTH treatment by utilizing flow cytometric analysis and competitive repopulation assays (primary, secondary and tertiary reconstitution). We developed an accelerated PTH treatment regimen in mice (40μg/kg body weight ip three times daily for 10 days), which minimizes the effects of normal aging on bone and hematopoiesis. We first analyzed the effect of this PTH regimen on bone. PTH-treated mice had increased trabecular bone volume (% BV/TV VEH 25±2 vs PTH 43±4 p