Nutrition and stem cell integrity in aging.

Adult stem cells (SCs) represent the regenerative capacity of organisms throughout their lifespan. The maintenance of robust SC populations capable of renewing organs and physiological systems is one hallmark of healthy aging. The local environment of SCs, referred to as the niche, includes the nutritional milieu, which is essential to maintain the quantity and quality of SCs available for renewal and regeneration. There is increased recognition that SCs have unique metabolism and conditional nutrient needs compared to fully differentiated cells. However, the contribution of SC nutrition to overall human nutritional requirements is an understudied and underappreciated area of investigation. Nutrient needs vary across the lifespan and are modified by many factors including individual health, disease, physiological states including pregnancy, age, sex, and during recovery from injury. Although current nutrition guidance is generally derived for apparently healthy populations and to prevent nutritional deficiency diseases, there are increased efforts to establish nutrient-based and food-based recommendations based on reducing chronic disease. Understanding the dynamics of SC nutritional needs throughout the life span, including the role of nutrition in extending biological age by blunting biological systems decay, is fundamental to establishing food and nutrient guidance for chronic disease reduction and health maintenance. This review summarizes a 3-day symposium of the Marabou Foundation (www.marabousymposium.org) held to examine the metabolic properties and unique nutritional needs of adult SCs and their role in healthy aging and age-related chronic disease.

Nutrition and its role in human evolution.

Our understanding of human evolution has improved rapidly over recent decades, facilitated by large-scale cataloguing of genomic variability amongst both modern and archaic humans. It seems clear that the evolution of the ancestors of chimpanzees and hominins separated 7-9 million years ago with some migration out of Africa by the earlier hominins; Homo sapiens slowly emerged as climate change resulted in drier, less forested African conditions. The African populations expanded and evolved in many different conditions with slow mutation and selection rates in the human genome, but with much more rapid mutation occurring in mitochondrial DNA. We now have evidence stretching back 300 000 years of humans in their current form, but there are clearly four very different large African language groups that correlate with population DNA differences. Then, about 50 000-100 000 years ago a small subset of modern humans also migrated out of Africa resulting in a persistent signature of more limited genetic diversity amongst non-African populations. Hybridization with archaic hominins occurred around this time such that all non-African modern humans possess some Neanderthal ancestry and Melanesian populations additionally possess some Denisovan ancestry. Human populations both within and outside Africa also adapted to diverse aspects of their local environment including altitude, climate, UV exposure, diet and pathogens, in some cases leaving clear signatures of patterns of genetic variation. Notable examples include haemoglobin changes conferring resistance to malaria, other immune changes and the skin adaptations favouring the synthesis of vitamin D. As humans migrated across Eurasia, further major mitochondrial changes occurred with some interbreeding with ancient hominins and the development of alcohol intolerance. More recently, an ability to retain lactase persistence into adulthood has evolved rapidly under the environmental stimulus of pastoralism with the ability to husband lactating ruminants. Increased amylase copy numbers seem to relate to the availability of starchy foods, whereas the capacity to desaturase and elongate monounsaturated fatty acids in different societies seems to be influenced by whether there is a lack of supply of readily available dietary sources of long-chain polyunsaturated fatty acids. The process of human evolution includes genetic drift and adaptation to local environments, in part through changes in mitochondrial and nuclear DNA. These genetic changes may underlie susceptibilities to some modern human pathologies including folate-responsive neural tube defects, diabetes, other age-related pathologies and mental health disorders.

Management goals for type 1 Gaucher disease: An expert consensus document from the European working group on Gaucher disease.

Gaucher Disease type 1 (GD1) is a lysosomal disorder that affects many systems. Therapy improves the principal manifestations of the condition and, as a consequence, many patients show a modified phenotype which reflects manifestations of their disease that are refractory to treatment. More generally, it is increasingly recognised that information as to how a patient feels and functions [obtained by patient- reported outcome measurements (PROMs)] is critical to any comprehensive evaluation of treatment. A new set of management goals for GD1 in which both trends are reflected is needed. To this end, a modified Delphi procedure among 25 experts was performed. Based on a literature review and with input from patients, 65 potential goals were formulated as statements. Consensus was considered to be reached when ≥75% of the participants agreed to include that specific statement in the management goals. There was agreement on 42 statements. In addition to the traditional goals concerning haematological, visceral and bone manifestations, improvement in quality of life, fatigue and social participation, as well as early detection of long-term complications or associated diseases were included. When applying this set of goals in medical practice, the clinical status of the individual patient should be taken into account.

Differences in bone mineral density, markers of bone turnover and extracellular matrix and daily life muscular activity among patients with recent motor-incomplete versus motor-complete spinal cord injury.

Spinal cord injury (SCI) leads to severe bone loss, but the associated mechanisms are poorly described in incomplete SCI individuals. The purpose of the study is to compare alterations in bone mineral density (BMD) and serum biomarkers of bone turnover in recent motor-incomplete to -complete SCI men, as well as to describe their physical activity and spasticity. We studied 31 men with acute SCI. Whole-body DXA scans, serum biomarkers and self-reported activity and spasticity were examined 1 and/or 3 and 12 months after the injury. We observed a decrease in proximal femur BMD (p < 0.02) in both the groups. Serum phosphate and carboxy-terminal-collagen crosslinks were significantly lower in motor-incomplete versus complete SCI men, whereas albumin-corrected Ca(2+) (p = 0.02) were lower only 3 months after injury. When data from all 31 SCI participants were pooled, we observed increased serum matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of MMP-2 (TIMP-2) (p < 0.02) whereas TIMP-1 decreased (p = 0.03). BMD correlated positively with self-reported activity (r = 0.59, p = 0.04) and negatively with spasticity (r = 0.74, p = 0.02) 12 months after injury. As a summary, men with motor-incomplete SCI developed significant proximal femur bone loss 12 months after injury and exhibited increased bone resorption throughout the first year after the injury. Compared with complete SCI men, incomplete SCI men show attenuated bone resorption. Our pooled data show increased turnover of extracellular matrix after injury and that increased exercise before and after injury correlated with reduced bone loss.

Modified Atkins diet may reduce serum concentrations of antiepileptic drugs.

BACKGROUND: Modified Atkins diet is a treatment option for patients with pharmacoresistant epilepsy that is not suitable for surgery. In the last few years, we have tried dietary treatment added to antiepileptic drugs (AEDs) in adult patients with severe epilepsy. AIM OF THE STUDY: To examine a possible pharmacokinetic interaction between the modified Atkins diet and AEDs. METHODS: In four patients, AED serum concentrations were measured before onset and after 4 and 12 weeks on the diet. The patients used combinations of two or three AEDs, including carbamazepine, clobazam, lamotrigine, nitrazepam, oxcarbazepine, valproate, zonisamide, and topiramate. The patients did not change the type or dose of their AEDs during the diet period. RESULTS: After 12 weeks on the diet, the average serum concentrations of the respective AEDs were reduced by 35% (range 6-46%) compared to prediet values. CONCLUSIONS: Modified Atkins diet used as add-on therapy to AEDs in four patients with drug resistant seizures caused a considerable decrease in AED serum concentrations. In individual patients, this could be of clinical relevance, and we recommend that AED serum concentrations should be closely monitored when offering this diet to adults with epilepsy.

Circadian rhythms of hemostatic factors in tetraplegia: a double-blind, randomized, placebo-controlled cross-over study of melatonin.

STUDY DESIGN: This is a double-blind, randomized, placebo-controlled cross-over study of melatonin in complete tetraplegia. OBJECTIVES: Tetraplegic patients have an increased risk of venous thrombosis despite prophylaxis, blunted variations in melatonin and altered circadian variation of several hemostatic markers. To examine whether melatonin could modify the regulation of hemostasis, we measured plasma melatonin and several markers of hemostasis in tetraplegic subjects with or without melatonin supplement. SETTING: The study was conducted in the Section for Spinal Cord Injury, Sunnaas Hospital, Nesoddtangen, Norway. METHODS: Six subjects with long-standing complete tetraplegia were included in this cross-over study with 2 mg of melatonin or placebo given 4 days before sampling. We also included six able-bodied men without any intervention. Plasma samples were then collected frequently during a 24-h awake/sleep cycle. The plasma concentrations of melatonin and the various markers were analyzed using linear mixed models. RESULTS: The 24-h profiles of prothrombin fragment 1+2 and von Willebrand factor, but not D-dimer, activated FVII, tissue factor pathway inhibitor and plasminogen activator inhibitor type 1, differed (P<0.05) between tetraplegic patients and able-bodied subjects. The absolute plasma concentration of activated FVII was higher (P<0.05) among the able-bodied compared with the tetraplegic groups. Supplementation of melatonin had no impact on these findings. CONCLUSIONS: We found differences in circadian variation of several hemostatic markers between able-bodied and tetraplegics. These differences were apparently unrelated to fluctuations in the melatonin concentrations, suggesting little or no role of melatonin in the regulation of hemostasis in tetraplegia. SPONSORSHIP: Financial support was provided from the Throne Holst Foundation.

Nausea and vomiting in pregnancy: associations with maternal gestational diet and lifestyle factors in the Norwegian Mother and Child Cohort Study.

OBJECTIVE: To investigate primarily the dietary intake, as well as demographics and selected lifestyle factors, of women experiencing nausea and vomiting in pregnancy, nausea only, or women who are symptom free. DESIGN: Prospective cohort study. SETTING: The Norwegian Mother and Child Cohort Study, a population-based pregnancy cohort. SAMPLE: Analyses were based on 51 675 Norwegian pregnancies. METHODS: Dietary intake was assessed by a self-reported food frequency questionnaire answered in the first trimester of pregnancy, as were data regarding nausea and vomiting. Chi-squared tests, one-way analysis of variance, and multiple linear regression were used. MAIN OUTCOME MEASURES: Nausea and vomiting in pregnancy (NVP), gestational weight gain (GWG), and dietary intake. RESULTS: We found that 17 070 (33%) women experienced NVP, 20 371 (39%) experienced only nausea, and 14 234 (28%) were symptom free. Women with NVP were younger and heavier at pregnancy onset, with the lowest GWG and highest energy intake during pregnancy, primarily from carbohydrates and added sugars, compared with the other groups (P < 0.001). In multiple linear regression analysis of GWG and group adjusted for body mass index (BMI), gestational length, smoking during pregnancy, and energy intake, a significant interaction was found between BMI and group (P < 0.001). A significant effect of group (P < 0.001) was found in all BMI strata, except among underweight women (P = 0.65). CONCLUSIONS: Our study suggests that women with NVP are characterised by high intakes of carbohydrates and added sugar, primarily from sugar-containing soft drinks. Whether higher intakes of carbohydrates are a response aimed to alleviate symptoms, or are actually provoking the condition, is not known.

No effect of energy supply during childbirth on delivery outcomes in nulliparous women: a randomised, double-blind, placebo-controlled trial.

The effect of providing oral energy supplements of energy on duration of labour and labour outcomes remains to be clarified. The purpose of this study was to examine whether extra energy supply beyond a self-regulated dietary intake during labour would shorten duration of labour in nulliparous women. A total of 213 healthy women at gestational age >36 weeks received either 1 litre of isotonic energy-drink (n = 111) or placebo-drink (n = 102) at the start of labour. A total of 61% in the intervention group gave birth within the hospital median of 9 hours, compared with 58% in the placebo group (p = 0.68). The mean (SD) durations of labour were 528 (240) minutes and 506 (233) minutes in the intervention and placebo group (p = 0.50), respectively. Extra oral supply of 1 litre energy drink beyond self-regulated intake of food and drink to healthy nulliparous women in birth does not affect the duration of labour.

Body weight, body composition and survival after 1 year: follow-up of a nutritional intervention trial in allo-HSCT recipients.

The role of body weight change in survival among recipients of hematopoietic stem-cell transplantation is controversial. We assessed the effect of optimizing energy and protein intake on 1-year survival, body weight and body composition, and the effect of body weight and body composition on 1-year survival in 117 patients (57 intervention, 60 control) in a randomized controlled trial. Cox regression was used to study effects of the intervention, weight and body composition on death, relapse, and nonrelapse mortality (NRM). We found no significant effect of intervention versus control on death hazard ratio (HR) 1.05, 95% confidence interval (CI) 0.54-2.04, p = 0.88), relapse (HR 1.15, 95% CI 0.48-2.27, p = 0.75), and NRM (HR 0.95, 95% CI 0.39-2.28, p = 0.90). Body weight, fat-free mass index, body fat mass index and total body water changed over time (p < 0.001), similarly in both groups (0.17 ≤ p ≤ 0.98). In multivariable analyses adjusted for group, gender and age, HRs and 95% CIs per one kilo increase in weight were 1.03 (1.01-1.06) and 1.04 (1.01-1.08) for death and NRM after 1 year (p ≤ 0.02), respectively, and 1.08 (1.01-1.15) for relapse after 3 months (p = 0.02). In conclusion, weight gain is possibly due to fluid retention and is an indicator of a complication in HSCT, rather than a marker of improved nutritional status.

Protein translation, proteolysis and autophagy in human skeletal muscle atrophy after spinal cord injury.

AIM: Spinal cord injury-induced loss of skeletal muscle mass does not progress linearly. In humans, peak muscle loss occurs during the first 6 weeks postinjury, and gradually continues thereafter. The aim of this study was to delineate the regulatory events underlying skeletal muscle atrophy during the first year following spinal cord injury. METHODS: Key translational, autophagic and proteolytic proteins were analysed by immunoblotting of human vastus lateralis muscle obtained 1, 3 and 12 months following spinal cord injury. Age-matched able-bodied control subjects were also studied. RESULTS: Several downstream targets of Akt signalling decreased after spinal cord injury in skeletal muscle, without changes in resting Akt Ser473 and Akt Thr308 phosphorylation or total Akt protein. Abundance of mTOR protein and mTOR Ser2448 phosphorylation, as well as FOXO1 Ser256 phosphorylation and FOXO3 protein, decreased in response to spinal cord injury, coincident with attenuated protein abundance of E3 ubiquitin ligases, MuRF1 and MAFbx. S6 protein and Ser235/236 phosphorylation, as well as 4E-BP1 Thr37/46 phosphorylation, increased transiently after spinal cord injury, indicating higher levels of protein translation early after injury. Protein abundance of LC3-I and LC3-II decreased 3 months postinjury as compared with 1 month postinjury, but not compared to able-bodied control subjects, indicating lower levels of autophagy. Proteins regulating proteasomal degradation were stably increased in response to spinal cord injury. CONCLUSION: Together, these data provide indirect evidence suggesting that protein translation and autophagy transiently increase, while whole proteolysis remains stably higher in skeletal muscle within the first year after spinal cord injury.

Deoxyribonucleic acid triplex formation inhibits granulocyte macrophage colony-stimulating factor gene expression and suppresses growth in juvenile myelomonocytic leukemic cells.

Juvenile myelomonocytic leukemia (JMML) is a severe childhood malignancy. The autocrine production of GMCSF is believed to be responsible for the spontaneous proliferation of JMML cells. A nuclear factor-kappaB (NF-kappaB)/Rel binding site within the GM-CSF gene promoter, termed the kappaB element, plays an important role in controlling transcription from the GM-CSF gene. We investigated the effect of an oligonucleotide GM3, directed to form a DNA triple helix across this kappaB element, on growth and GM-CSF production by JMML cells. Treatment of these cells, either unstimulated or induced by TNFalpha, with GM3 led to a significant and specific inhibition of both GM-CSF production and spontaneous colony formation. This constitutes the first report linking specific triplex-mediated inhibition of gene transcription with a functional outcome; i.e., cell growth. We observed the constitutive presence of NF-kappaB/Rel proteins in the nucleus of JMML cells. The constitutive and TNFalpha-induced NF-kappaB/Rel complexes were identical and were composed mainly of p50 and c-Rel proteins. Treatment of the cells with a neutralizing anti-TNFalpha monoclonal antibody completely abrogated constitutive nuclear expression of NF-kappaB/Rel proteins. These results indicate that the aberrant, constitutive GM-CSF gene activation in JMML is maintained by TNFalpha-mediated activation of NF-kappaB/Rel proteins. Our findings identify the molecular basis for the autocrine TNFalpha activation of the GM-CSF gene in JMML and suggest potential novel and specific approaches for the treatment of this aggressive childhood leukemia.

Intake of a Protein-Enriched Milk and Effects on Muscle Mass and Strength. A 12-Week Randomized Placebo Controlled Trial among Community-Dwelling Older Adults.

OBJECTIVES: To investigate the effect of 20 g protein with breakfast and evening meal on muscle mass, muscle strength and functional performance in older adults. DESIGN: A double-blinded randomized controlled study. SETTING: Oslo and Akershus University College of Applied Sciences, Norway. PARTICIPANTS: Healthy community-dwelling men and women (≥ 70 years) with reduced physical strength and/or performance. INTERVENTION: Subjects were randomly assigned to receive either protein-enriched milk (2 x 0.4 L/d; protein group) or an isocaloric carbohydrate drink (2 x 0.4 L/d; control group) with breakfast and evening meal for 12 weeks. MEASUREMENTS: The primary endpoints were muscle mass measured by dual X-ray absorptiometry, and tests of muscle strength (one repetition maximum test of chest press and leg press) and functional performance (handgrip strength, stair calimb and repeated chair rise). RESULTS: In total, 438 subjects were screened, 50 subjects were randomized and 36 completed the study. Chest press improved significantly in the protein (1.3 kg (0.1-2.5), p=0.03) and the control group (1.5 kg (0.0-3.0), p=0.048), but with no difference between the groups (p=0.85). No significant change in leg press (p=0.93) or muscle mass (p=0.54) were observed between the protein and the control group. Nor did we observe any significant differences in the functional performance tests (p>0.05 for all tests) between the groups. CONCLUSION: Increased protein intake (2 x 20 g/d) did not significantly improve muscle mass, muscle strength or functional performance in healthy older weight stable adults. Whether intake of > 20 g protein to each meal is necessary for preservation of muscle mass and strength in older adults should be further investigated in a larger study. This underscores the need for well-designed studies that can differentiate between the effect of protein intake and increased energy. This trial was registered at Clinicaltrials.gov (ID no. NCT02218333).

Fat-soluble vitamins in breast-fed preterm and term infants.

OBJECTIVE: To examine the supply and status of fat-soluble vitamins in very low birth weight (VLBW) infants compared to a reference group of normal birth weight (NBW) infants. DESIGN: A longitudinal study of VLBW infants in the early neonatal period. Blood samples were drawn at 1 week of age and at discharge from hospital. Plasma was analyzed for the fat-soluble vitamins: retinol, 25-OH-vitamin D, alpha-tocopherol and phylloquinone (vitamin K(1)) using high-performance liquid chromatography. SUBJECTS: A total of 40 VLBW infants were included in the study. A reference group of 33 NBW infants was randomly selected from one of our previous studies. RESULTS: The VLBW infants received fortified human milk, and daily oral vitamin supplement (Multibionta). In VLBW infants, plasma retinol concentrations decreased and plasma 25-OH-vitamin D increased during the study period. VLBW infants had significantly lower plasma retinol (0.3 vs 0.7 mu M) and higher plasma 25-OH-vitamin D (166 vs 25 nM) at discharge compared to NBW infants. Plasma phylloquinone concentration in VLBW infants was very high (53 ng/ml) at one week of age, especially in the youngest infants (192 ng/ml), but decreased rapidly during the study period resulting in low/normal plasma concentrations (0.9 ng/ml) at discharge. CONCLUSIONS: We observed alterations in plasma concentration of retinol and 25-OH-vitamin D in VLBW infants in the early neonatal period, resulting in marked differences between VLBW at discharge and NBW. Further trials are needed to evaluate whether changes in vitamin supplementation may improve clinical outcome in VLBW infants.

Interleukin (IL)-10, but not IL-4 or IL-13, inhibits cytokine production and growth in juvenile myelomonocytic leukemia cells.

Juvenile myelomonocytic leukemia (JMML) carries a poor prognosis. The endogenous production of cytokines by the JMML cells contributes to their growth and therapeutic resistance. Interleukin (IL)-4, IL-10, and IL-13 inhibit cytokine production in monocytes. We have now studied whether these cytokines can inhibit JMML cell cytokine production, thereby potentially reducing the malignant cell load in this disorder. We found that IL-10, but not IL-4 or IL-13, dose dependently inhibited JMML cell production of the hemopoietic growth factors granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha, and IL-1beta. Similarly, IL-10, but not IL-4 or IL-13, suppressed JMML colony formation and cell viability. This was not due to the absence of receptors because we could detect mRNAs for the IL-4 and the IL-13 receptor alpha subunits and the IL-2 common gamma subunit in JMML cells. Furthermore, the receptors were active since both IL-4 and IL-13 up-regulated surface expression of MHC class II and down-regulated CD14 antigens on JMML cells and monocytes. Unlike activated monocytes, the JMML cells did not produce IL-10. It is suggested that the loss of cytokine inhibitory effects of IL-4 and IL-13 could play a role in the pathogenesis of this disorder. On the other hand, the inhibition of cytokine production, growth, and viability of JMML cells by IL-10 suggests that this cytokine may have a therapeutic potential in JMML.

Biomarkers for nutrient intake with focus on alternative sampling techniques.

Biomarkers of nutrient intake or nutrient status are important objective measures of foods/nutrients as one of the most important environmental factors people are exposed to. It is very difficult to obtain accurate data on individual food intake, and there is a large variation of nutrient composition of foods consumed in a population. Thus, it is difficult to obtain precise measures of exposure to different nutrients and thereby be able to understand the relationship between diet, health, and disease. This is the background for investing considerable resources in studying biomarkers of nutrients believed to be important in our foods. Modern technology with high sensitivity and specificity concerning many nutrient biomarkers has allowed an interesting development with analyses of very small amounts of blood or tissue material. In combination with non-professional collection of blood by finger-pricking and collection on filters or sticks, this may make collection of samples and analyses of biomarkers much more available for scientists as well as health professionals and even lay people in particular in relation to the marked trend of self-monitoring of body functions linked to mobile phone technology. Assuming standard operating procedures are used for collection, drying, transport, extraction, and analysis of samples, it turns out that many analytes of nutritional interest can be measured like metabolites, drugs, lipids, vitamins, minerals, and many types of peptides and proteins. The advantage of this alternative sampling technology is that non-professionals can collect, dry, and mail the samples; the samples can often be stored under room temperature in a dry atmosphere, requiring small amounts of blood. Another promising area is the potential relation between the microbiome and biomarkers that may be measured in feces as well as in blood.

Angiogenesis and hemostasis in hematological neoplasias.

Angiogenesis is essential for tumor growth and metastasis. This is firmly established in solid tumors, but accumulating evidence suggests that this is also an important event in hematological neoplasias. Angiogenesis is therefore a putative target for therapy. The potential application of different angiogenesis inhibitors is currently under intense clinical investigation, and we will here review a number of these trials. The association between cancer and thromboembolic disease is even better documented, and again, this is not limited to solid tumors. It appears that many patients with hematological malignancies have a dysfunctional hemostatic system, with increased risk of thromboembolism. Furthermore, effective antithrombotic therapy seems to reduce the risk of cancer progression and even prolongs overall survival. In this review we will thus discuss the mechanisms involved in the regulation of angiogenesis and hemostasis and present evidence for a shared biology. A number of factors regulating the hemostatic system also have pro- or anti-angiogenic properties. Tissue factor (TF) and TF pathway inhibitor (TFPI) seem to play a central role, and there are several lines of evidence suggesting a close cooperation between TF/TFPI and pro-angiogenic factors like members of the vascular endothelial growth factor family. A better understanding of this shared biology may reveal new targets, and will probably increase the safety of targeting the blood supply.

Inhibitors of angiogenesis selectively reduce the malignant cell load in rodent models of human myeloid leukemias.

Angiogenesis is essential for growth and metastasis of solid tumors and probably also for hematological malignancies. Angiogenic inhibitors, like endostatin (ES) and PI-88, retard cancer growth. We tested these in mice with juvenile myelomonocytic leukemia (JMML), and in rats with acute myeloid leukemia (BNML). Eight weeks after transplantation and with a continuous drug treatment for the last 4 weeks, the leukemic cell mass decreased from almost 90% of all bone marrow cells to about 15 and 45% with ES, to about 35 and 55% with PI-88, and to about 10 and 25% with ES + PI-88 in the leukemic mice and rats, respectively. The numbers of normal human bone marrow cells transplanted into mice were unchanged by the treatments. The microvessel density in leukemic animals given ES or PI-88 was 10-50% of that in untreated animals. Notably, simultaneous treatment with ES and PI-88 led to a reduction of about 95% in JMML mice and 85% in BNML rats. In vitro proliferation of either JMML or BNML cells was not significantly altered by either drug, demonstrating the selectivity of ES and PI-88 as angiogenic inhibitors. In conclusion, anti-angiogenic therapy may be a valuable adjunct to conventional treatment of leukemia.

The leukopoietic cytokine granulocyte colony-stimulating factor increases blood flow to rat bone marrow.

The current knowledge concerning the blood supply to hematopoietic bone marrow during increased marrow metabolism is scanty. We have previously shown that an accelerated erythropoiesis in the awake rat is accompanied by a rapid increase in perfusion of the tibial marrow and its bony encasement. We have now measured blood flow to tibial marrow and bone in rats with stimulated granulopoiesis, caused by injection of recombinant human granulocyte colony-stimulating factor (rhG-CSF). In awake, adult rats, blood flow was measured with the microsphere method before and at intervals during a 48-hour period after subcutaneous (s.c.) injection of rhG-CSF (10 micrograms/kg). Administration of rhG-CSF caused a marked leukocytosis, mostly due to an increase in blood granulocytes, amounting to 4 times the control value at 8 hours. Concomitantly, the perfusion of tibial marrow rose to about 200% of control by 8 hours before it declined toward baseline. Denervation of the marrow had no effect on this hyperemic response. The perfusion of tibial bone was apparently unaffected by rhG-CSF injection. We conclude that rhG-CSF injection increases blood flow to hematopoietic marrow, but not to bone. This may have important implications for marrow transplantation and drug therapy for patients with marrow failure.

Endogenous nitric oxide causes vasodilation in rat bone marrow, bone, and spleen during accelerated hematopoiesis.

There is a marked increase in blood flow to rat bone marrow during increased erythro- or granulopoiesis. Furthermore, stimulated erythropoiesis increases bone and splenic perfusion, whereas granulopoietic hyperactivity does not. The mechanism behind this hyperemia is unknown. Endogenous nitric oxide (NO) has been shown to be a potent vasodilator in many vascular beds, but its possible role in the regulation of bone marrow, bone, and spleen vascular resistance and perfusion has not been explored. With the radioactive microsphere method, we determined blood flow to bone marrow, bone, and spleen in awake rats. Eight rats were bled heavily (1.5% of body weight), eight others received 10 micrograms/kg recombinant human granulocyte colony-stimulating factor (rhG-CSF) subcutaneously, and eight other untreated rats served as controls. We used 300 micrograms/kg, intraaortal, of the potent NO synthase blocker N-monomethyl-L-arginine (L-NMMA) (Calbiochem, La Jolla, CA). The inhibition of NO formation was subsequently reversed with 1000 mg/kg intraaortal arginine. Marrow vascular resistance was reduced to approximately 30% of control baseline in the experimental rats 10 hours after hematopoietic stimulation with either bleeding or rhG-CSF. Concomitantly, marrow blood flow increased to about 260% of control baseline in the bled rats, while it almost tripled after rhG-CSF injection. Inhibition of NO formation increased marrow vascular resistance in all three groups. After L-NMMA treatment, marrow perfusion was reduced to about 50% of baseline in the bled and 75% in the rhG-CSF-treated rats, while perfusion in the controls remained apparently unaltered. These changes were completely reversed with arginine. The increases in vascular resistance after NO blockade could not be explained by a concomitant change in arterial blood pressure. L-NMMA increased the vascular resistance in the bone and spleen both in controls and in stimulated rats, but since arterial blood pressure rose proportionally, perfusion remained unchanged. We conclude that NO plays an important role in the regulation of both the normal bone marrow vascular tone and the vasodilation that occurs during accelerated hematopoiesis. NO apparently also regulates bone and splenic vascular tone, but less conspicuously than in the stimulated bone marrow.

Increased cellular hypoxia and reduced proliferation of both normal and leukaemic cells during progression of acute myeloid leukaemia in rats.

The microenvironmental changes in the bone marrow, spleen and liver during progression of the transplantable promyelocytic leukaemia in the Brown Norwegian rat (BNML) have been studied. We used flow cytometry to estimate cellular hypoxia and proliferation based on in vivo pulse-labelling with a mixture of 2-nitroimidazole linked to theophylline (NITP) and bromodeoxyuridine (BrdUrd). The leukaemic cells were identified with the RM124 antibody. In rats inoculated with leukaemic cells the fraction of RM124+ cells was significantly increased from day 20 onwards in the spleen and from day 27 in the bone marrow and liver, reaching a level of 65-87% in these organs at day 32. At day 32, the NITP+ fraction of RM124+ cells had increased significantly in the bone marrow and spleen to 88% and 90%, respectively. The corresponding fractions of NITP+ normal cells reached 63% and 65%, respectively. From day 13 to day 32, the DNA-synthesizing (BrdUrd+) fraction of RM124+ cells in the bone marrow decreased significantly from 52% to 25%, and of normal cells from about 20% to 6%. In the bone marrow and spleen at day 27 and 32, the S-phase and G2/M-phase fractions according to DNA content were higher for the NITP+ than for the NITP- cells. This could partly be explained by an impaired cell cycle progression due to hypoxia. Nevertheless, we found indications of leukaemic cells that were simultaneously labelled with NITP and BrdUrd, in the bone marrow and spleen. These latter findings suggest that in contrast to normal cells some of the leukaemic cells can proliferate even during hypoxia, and this subpopulation may consequently renew and expand the leukaemic cell load.