pH homeostasis in Leishmania donovani amastigotes and promastigotes.

Intracellular pH and pH gradients of Leishmania donovani amastigotes and promastigotes were determined over a broad range of extracellular pH values. Intracellular pH was determined by 31P NMR and by equilibrium distribution studies with 5,5-dimethyloxazolidine-2,4-dione or methylamine. Promastigotes maintain intracellular pH values close to neutral between extracellular pH values of 5.0 and 7.4. Amastigote intracellular pH is maintained close to neutral at external pH values as low as 4.0. Both life stages maintain a positive pH gradient to an extracellular pH of 7.4, which is important for active transport of substrates. Treatment with ionophores, such as nigericin and carbonyl cyanide m-chlorophenylhydrazone and the ATPase inhibitor dicyclohexylcarbodiimide, reduced pH gradients in both stages. Maintenance of intracellular pH in the physiologic range is especially relevant for the survival of the amastigote in its acidic in vivo environment.     

The Leishmania chagasi proteasome: role in promastigotes growth and amastigotes survival within murine macrophages

Proteasomes are multisubunit proteases that exist universally among eukaryotes. They have multiple proteolytic activities and are believed to have important roles in regulating cell cycle, selective intracellular proteolysis, and antigen presentation. Here we have partially purified Leishmania chagasi proteasome. The L. chagasi proteasome rich fraction displayed the typical features of eukaryotic 20S proteasome complexes, being active towards peptidyl substrates with hydrophobic and acidic residues, and sensitive to the proteasome-specific inhibitor lactacystin. We have shown that lactacystin, or its active form clasto-lactacystin beta-lactone, but not E-64, blocks the in vitro growth of L. chagasi promastigotes, demonstrating that the interference with parasite growth is due to the lack of proteasome activity. Furthermore, pre-treatment of L. chagasi promastigotes with lactacystin did not prevent parasite entry in host cells, but markedly restricted its intracellular survival. These results demonstrate that intact parasite proteasome function is required for replication of L. chagasi and for amastigotes survival inside the vertebrate host cell.     

Insulin-like growth factor I is a comitogen for hepatocyte growth factor in a rat model of hepatocellular carcinoma

Hepatocyte growth factor-scatter factor (HGF-SF) is a potent hepatic mitogen yet inhibits hepatocellular carcinoma (HCC) cell growth in vitro. Insulin-like growth factor I (IGF-I) is a pleiotropic growth factor shown to be important in cell growth and differentiation in other tumors. We hypothesized that IGF-I may play a role in regulating HGF-SF activity and HCC progression. Using an in vivo model of HCC, we showed elevated IGF-I messenger RNA (mRNA) expression in normal liver from tumor-burdened animals in the absence of changes in circulating IGF-I levels. Analysis of IGF-I receptor (IGF-IR) and HGF-SF (c-met) receptor expression showed significantly higher expression of both receptors in normal liver compared with an HCC specimen. Using cultured HCC cells from this model, we next showed that treatment with IGF-I led to significant increases in mitogen-activated protein kinase (MAPK) activity. Furthermore, we observed significant time-dependent increases in the expression of the c-fos and c-jun proto-oncogenes after addition of IGF-I (n = 5 per group, P <.05). Despite activation of a MAPK pathway and increased proto-oncogene expression, IGF-I failed to significantly affect cell mitogenesis. In contrast, HGF significantly inhibited cell mitogenesis in HCC lines (68.4% +/- 9.4% vs. control, n = 4, P <.05). Pretreatment of HCC cells with IGF-I (60 minutes) led to significant HGF-SF stimulation of total cell mitogenesis dependent on both IGF-I and HGF-SF dose (194% +/- 8% increase vs. control, n = 4, P <.05). In conclusion, tumor burden is important in altering intrahepatic growth factor synthesis. Signal cooperation between multiple cytokine pathways is an important factor in the progression of HCC.     

Insulin-like growth factor I is a growth and survival factor in human multiple myeloma cell lines

Human multiple myeloma (MM) represents a highly aneuploid tumor as shown by cytogenetic studies. This may partly explain the heterogeneity with regard to growth factor requirements demonstrated among MM cells. We have previously reported the expression of insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR) mRNA in some MM cell lines. In this study we investigated the role of IGF-I as a growth and/or survival factor in three MM cell lines: LP-1, EJM, and Karpas 707. We report that all cell lines expressed IGF-I and IGF-IR mRNA and protein. LP-1 and Karpas 707, but not EJM, were stimulated to proliferation in a dose-dependent manner by exogenous IGF-I. An IGF-IR blocking antibody inhibited both the IGF-I-induced and spontaneous growth of LP-1, and Karpas 707, while the EJM cell line was unaffected by the addition of the antibody. In conclusion, our results show that IGF-I can act as a growth factor in human MM, and they suggest that an autocrine IGF-I loop may contribute to the growth and survival in some MM cell lines.     

Insulin-like growth factor I is a dual effector of multiple myeloma cell growth

Multiple myeloma (MM) is an invariably fatal disease that accounts for approximately 1% to 2% of all human cancers. Surprisingly little is known about the cellular pathways contributing to growth of these tumors. Although the cytokine interleukin-6 has been suggested to be the major stimulus for myeloma cell growth, the role of a second potential growth factor, insulin-like growth factor I (IGF-I), has been less clearly defined. The IGF-I signaling cascade in 8 MM cell lines was examined. In 7 of these, the IGF-I receptor (IGF-IR) was expressed and autophosphorylated in response to ligand. Downstream of IGF-IR, insulin receptor substrate 1 was phosphorylated, leading to the activation of phosphatidylinositol-3'-kinase (PI-3K). PI-3K, in turn, regulated 2 distinct pathways. The first included Akt and Bad, leading to an inhibition of apoptosis; the second included the mitogen-activated protein kinase (MAPK), resulting in proliferation. Biologic relevance of this pathway was demonstrated because in vitro IGF-I induced both an antiapoptotic and a proliferative effect. Importantly, in vivo administration of IGF-I in SCID mice inoculated with the OPM-2 line led to approximately twice the growth rate of tumor cells as in controls. These results suggest that IGF-I activates at least 2 pathways effecting myeloma cell growth and contributes significantly to expansion of these cells in vivo. (Blood. 2000;96:2856-2861)     

Insulin-like growth factor I is essential for postnatal growth in response to growth hormone.

Insulin-like growth factor I (IGF-I) is essential for cell growth and intrauterine development while both IGF-I and GH are required for postnatal growth. To explore the possibility of direct GH action on body growth, independent of IGF-I production, we have studied the effects of GH in an IGF-I-deficient mouse line created by the Cre/loxP system. The IGF-I null mice are born with 35% growth retardation and show delayed onset of peripubertal growth, grow significantly slower, and do not attain puberty. Their adult body weight was approximately one third and body length about two thirds that of their wild-type litter mates. Injection of recombinant human GH (rhGH, 3 mg/kg, twice daily, sc) between postnatal day 14 (P14) to P56 failed to stimulate their growth as measured as both body weight and length. In contrast, wild-type mice receiving the same doses of rhGH exhibited accelerated growth starting at P21 that continued until P56, when their body weight was increased by 30% and length by 12% compared with control mice treated with diluent. Despite the lack of response in growth, IGF-I null mice have normal levels of GH receptor expression in the liver and increased liver Jun B expression and liver size in response to rhGH treatment. Our results support an essential role for IGF-I in GH-induced postnatal body growth in mice.     

Insulin-like growth factor I is required for vessel remodeling in the adult brain

Although vascular dysfunction is a major suspect in the etiology of several important neurodegenerative diseases, the signals involved in vessel homeostasis in the brain are still poorly understood. We have determined whether insulin-like growth factor I (IGF-I), a wide-spectrum growth factor with angiogenic actions, participates in vascular remodeling in the adult brain. IGF-I induces the growth of cultured brain endothelial cells through hypoxiainducible factor 1 alpha and vascular endothelial growth factor, a canonical angiogenic pathway. Furthermore, the systemic injection of IGF-I in adult mice increases brain vessel density. Physical exercise that stimulates widespread brain vessel growth in normal mice fails to do so in mice with low serum IGF-I. Brain injury that stimulates angiogenesis at the injury site also requires IGF-I to promote perilesion vessel growth, because blockade of IGF-I input by an anti-IGF-I abrogates vascular growth at the injury site. Thus, IGF-I participates in vessel remodeling in the adult brain. Low serum/brain IGF-I levels that are associated with old age and with several neurodegenerative diseases may be related to an increased risk of vascular dysfunction.     

Leishmania major: identification of stage-specific antigens and antigens shared by promastigotes and amastigotes

Summary A battery of antisera to Leishmania major was used to identify stage-specific antigens, or antigens expressed predominantly in amastigotes or promastigotes. At least 30 protein antigens common to amastigotes and promastigotes could be detected in ³⁵S-methionine labelled preparations. They ranged in molecular weight from 25000 to 165000. Two amastigote specific antigens and five antigens expressed predominantly in the amastigote were detected in biosynthetically-labelled preparations. Five promastigote specific antigens were also identified. Antibodies from hyperimmunized mice that were resistant to reinfection with L. major recognized mainly antigens shared by the two life-cycle stages of the parasite. Analysis of parasite antigens on 'western blots' provided a different picture from that obtained by immunoprecipitation and gel electrophoresis of ^3SS-methionine labelled polypeptides. Only 19 antigens were detected and they were all shared by the two parasite forms. However, the abundance and immunogenicity of some of these antigens may be different in the two life-cycle stages of the parasite. Using the various sera, seven shared membrane antigens were identified in radio-iodinated preparations. Antibodies from hyperimmunized resistant mice that recognized shared antigens in ³⁵S-meth-ionine labelled preparations, detected four amastigote membrane antigens not detected by other sera. The function of the stage-specific antigens remains to be established. It is expected that individual antigens produced by recombinant DNA technology will allow these studies to proceed.     

Insulin-like growth factor I and growth hormone in canine starvation

The roles of plasma insulin-like growth factor I (IGF I) and growth hormone (GH) were studied in 7 beagle dogs before and during starvation and during refeeding. IGF I levels significantly decreased from 75.2 +/- 5.9 ng/ml at 7 days prior to the start of starvation to 9 +/- 1.7 ng/ml at 19 days after the commencement of starvation (mean +/- SEM; P less than 0.0001). During refeeding IGF I significantly rose from 9 +/- 1.7 ng/ml to 55.5 +/- 7.5 ng/ml within 9 days (mean +/- SEM; P less than 0.002). During starvation plasma GH levels significantly increased (P less than 0.05) and these elevated levels returned to normal during refeeding. The dogs' GH secretory capacity significantly increased during starvation (P = 0.012) and became normal again during refeeding. The following conclusions can be drawn from this study: 1) starvation in the dog leads to a significant and drastic reduction of the circulating levels of IGF I, and 2) starvation in the dog, as in man, leads to increased circulating GH levels and to an increased GH-secretory capacity possibly brought about by a lack of a negative feedback normally exerted by IGF I.     

Insulin-like growth factor I and anthropometric parameters in a Danish population

During the last decade several studies indicated that low insulin-like growth factor (IGF) I levels are related to higher risk of cardiovascular disease and mortality. Obesity represents one further main cardiovascular risk factor which might also be related to IGF-I. The objective of the present study was to analyse the associations between anthropometric measures and IGF-I levels in a population-based sample. From the Danish cross-sectional Health2006 study 3,328 subjects (1,835 women; 1,493 men) aged 19-72 years were included in the analyses. Serum IGF-I levels were determined by an immunoassay. Body height, weight as well as waist and hip circumferences were measured. Body-mass-index, waist-to-hip ratio and waist-to-height ratio were calculated. Circulating IGF-I levels were inversely associated with all anthropometric markers as evaluated by linear regression adjusting for age, alcohol consumption, smoking and physical activity. Our large cross-sectional study suggests that IGF-I may serve as the link between obesity and mortality although any causal relation cannot be inferred and longitudinal analyses are needed to clarify the causal relation.     

Insulin-like growth factor I: pros and cons of a bioassay

BACKGROUND: Insulin-like growth factor I (IGF-I) immunoassays are primarily used to estimate IGF-I status. Recently an IGF-I-specific kinase receptor activation assay (KIRA) was developed as an alternative method for measuring IGF-I bioactivity. When compared with IGF-I immunoassays, the IGF-I KIRA has the theoretical advantage of measuring the net effects of IGF-binding proteins on IGF-I receptor activation. The IGF-I KIRA is sensitive and specific and has, for a bioassay, a very low intra- and inter-assay coefficient of variation (<7 and <15%, respectively). Several studies have shown that the IGF-I KIRA is more sensitive than IGF-I immunoassays for detecting differences in clinical state. In addition, the IGF-I KIRA seems superior to IGF-I immunoassays for monitoring acute effects of therapeutic interventions. CONCLUSIONS: It is not known if IGF-I KIRA is superior to IGF-I immunoassays for evaluating the efficacy of growth hormone (GH) treatment for GH deficiency and of GH receptor antagonist treatment for acromegaly. However, IGF-I KIRA undoubtedly will provide new insights into the factors that regulate IGF-I bioactivity in the circulation in both healthy and diseased states.     

Insulin-like growth factor I gene expression is induced in astrocytes during experimental demyelination.

To investigate insulin-like growth factor I (IGF-I) and IGF-I receptor gene expression during experimental demyelination and myelin regeneration, young mice were fed cuprizone (( bis(cyclohexanone) oxaldihydrazone )). This copper-chelating agent produces demyelination in the corpus callosum and superior cerebellar peduncles, and when treatment is stopped, there is rapid remyelination. At intervals during cuprizone treatment and recovery, brain sections were hybridized with specific probes and immunostained with antibodies to determine the localization and relative amounts of IGF-I and IGF-I receptor mRNAs and peptides. In untreated littermates, IGF-I and IGF-I receptor mRNAs and peptides were not detected in white matter. In cuprizone-treated mice, high levels of both IGF-I mRNA and peptide were expressed by astrocytes in areas of myelin breakdown. Astrocyte IGF-I expression decreased rapidly during recovery and oligodendroglial expression of myelin-related genes increased. In severely demyelinated areas, immature oligodendroglia exhibited a transient increase in IGF-I receptor mRNA and peptide immunoreactivity during early recovery. This highly specific pattern of IGF-I induction in astrocytes during demyelination and the expression of the IGF-I receptor in regenerating oligodendrocytes during recovery suggest that IGF-I functions in the regulation of oligodendrocyte and myelin metabolism in vivo.     

Insulin-like growth factor I receptors on erythrocytes in NIDDM

We examined 125I-insulin-like growth factor I (125I-IGF-I) binding to erythrocytes from 24 normal and 21 non-insulin-dependent diabetic (NIDDM) subjects. 125I-IGF-I binding to human erythrocytes was specifically inhibited by unlabeled IGF-I, and Scatchard analysis indicated a curvilinear plot. There was a significant difference in IGF-I binding between normal and diabetic subjects (7.78 +/- 0.42 vs. 5.80 +/- 0.33%/2.4 x 10(9) cells/ml, P less than 0.001). Among diabetic patients, IGF-I binding to erythrocytes from those with retinopathy and those without retinopathy was comparable. These results suggested that decreased IGF-I binding might be a factor responsible for some pathological features such as delayed wound healing in diabetic patients.     

Insulin-like growth factor I in suckling rat gastric contents

The small intestinal mucosa of the neonatal rat expresses primarily lactase activity until just prior to weaning when lactase falls to low levels and a full complement of adult digestive enzymes appears. Insulin-like growth factor 1 (IGF-I) is a normal component of maternal milk of humans and experimental animals. Experiments were performed to examine the concentrations of IGF-I in dam milk and the gastric content of suckling pups. Lactase activity in 1-day-old neonates was 0.66 µmol glucose formed/mg protein/hr (unit) and fell progressively until day 25, whereas sucrase activity at day 1 postpartum was 0.07 units and rose progressively to 0.21 units at day 25. The IGF-I content of dam milk was measured at 1, 5, 10, 15, 18, and 20 days postpartum by radioreceptor assay (RRA). Milk contained 1.02 pmol IGF-I/ml milk at one day postpartum, peaked at day 18 with 5.08 pmol IGF-I/ml, and fell to 2.31 pmol/ml at day 20. By day 25, dams were dry. The IGF-I content of the neonate gastric lumen was also measured by RRA. At day 1 the gastric lumen contained 2.63 pmol IGF-I/ml of luminal contents, fell to 1.06 pmol IGF-I/ml at day 5, and then rose again to peak at 3.37 pmol/ml at day 15 just prior to weaning. Two days after weaning, the level of luminal IGF-I had fallen to 1.15 pmol/ml. These data demonstrate the concentration of IGF-I in maternal milk is reflected in the concentration of the peptide in gastric contents of suckling pups and that the concentration in the gastric lumen may be high enough to affect epithelial cell proliferation and differentiation.This work was supported by grants to ER Seidel from the NIH (DK 34110) and the North Carolina Institute of Nutrition.     

Insulin-like growth factor I (IGF-I)-binding protein complex is a better mitogen than free IGF-I

The insulin-like growth factors (IGF)-I and -II are bound to specific carrier proteins in the circulation. For investigation of their physiological role, the acid-stable subunit of the major binding protein (SmBP) was isolated from human plasma Cohn fraction IV. Its effect on the mitogenic activity of IGF-I was studied with baby hamster kidney fibroblasts (BHK-21) and human skin fibroblasts. While free IGF-I had no effect on thymidine incorporation into DNA with BHK-21 cells and only a moderate effect with human fibroblasts under standard conditions, DNA synthesis was significantly enhanced with both cell lines if IGF-I was complexed with SmBP before the experiment. The enhancement was optimal at an approximately equimolar ratio of both peptides. In contrast to experiments in which large concentrations of IGF-I were added at the beginning, repeated addition of small quantities of free IGF-I at hourly intervals clearly stimulated DNA synthesis in BHK-21 cells. Binding studies with radiolabeled SmBP revealed no evidence for direct interaction with either cell line. It is concluded that SmBP acts as a reservoir, releasing continuously low amounts of IGF-I and thereby creating a steady state situation of receptor occupancy, which appears to be a better mitogenic stimulus than temporary large concentrations of IGF-I.     

Insulin-like growth factor 2 expressed in a novel fetal liver cell population is a growth factor for hematopoietic stem cells

Hematopoietic stem cells (HSCs) undergo dramatic expansion during fetal liver development, but attempts to expand their numbers ex vivo have failed. We hypothesized that unidentified fetal liver cells produce growth factors that support HSC proliferation. Here we describe a novel population of CD3+ and Ter119- day-15 fetal liver cells that support HSC expansion in culture, as determined by limiting dilution mouse reconstitution analyses. DNA array experiments showed that, among other proteins, insulin-like growth factor 2 (IGF-2) is specifically expressed in fetal liver CD3+ cells but not in several cells that do not support HSCs. Treatment of fetal liver CD3+Ter119- cells with anti-IGF-2 abrogated their HSC supportive activity, suggesting that IGF-2 is the key molecule produced by these cells that stimulates HSC expansion. All mouse fetal liver and adult bone marrow HSCs express receptors for IGF-2. Indeed, when combined with other growth factors, IGF-2 supports a 2-fold expansion of day-15 fetal liver Lin-Sca-1+c-Kit+ long-term (LT)-HSC numbers. Thus, fetal liver CD3+Ter119- cells are a novel stromal population that is capable of supporting HSC expansion, and IGF-2, produced by these cells, is an important growth factor for fetal liver and, as we show, adult bone marrow HSCs.     

Insulin-like growth factor I is essential for terminal end bud formation and ductal morphogenesis during mammary development.

Previous studies from this laboratory have emphasized the essential role of GH in pubertal mammary development and shown that insulin-like growth factor I (IGF-I) was capable of substituting for GH in this process in rats and mice. The present study shows that, even when GH is present, no mammary development is possible unless IGF-I is present. IGF-I(-/-) null female animals were found to have significantly less mammary development than age matched wild-type controls (P <0.006) using several endpoints including the number of terminal end buds or TEBs (1.3 vs. 7.3), percent of the fat pad occupied by glandular elements (6.5 vs. 100), and number of ducts (15 vs. too numerous to count). That the deficiency in mammary gland development was related to the absence of IGF-I was underscored by the observation that des (1-3) IGF-I administration to IGF-I(-/-) null animals for 5 days caused significant mammary gland development as measured by TEB formation and branching of ducts. The number of TEBs rose from a mean of 1.3 in controls to 20.5 without added E2 (P < 0.009), and from 1.7 to 21 when des (1-3) IGF-I was given together with E2 (P < 0.006). The number of ducts increased significantly from a mean of 12 to 27 in response to IGF-I and E2, and from 15 to 24.5 with IGF-I alone. In contrast, administration of human GH with E2 had no stimulatory effect on mammary development in these animals, indicating that the full effect of GH in this process is mediated by IGF-I. To determine whether IGF-I was also responsible for further ductal morphogenesis, we administered des (1-3) IGF-I + E2 to the knockout animals for 14 days and compared the effects of this combination of hormones on mammary development with those observed after 5 days. We found that there was a significant increase from 5 to 14 days in the number of TEBs (mean: 21 vs. 41) and the area of the mammary fat pad occupied by glands (mean: 10 vs. 20%). There was elongation and thickening of the ducts which accounted for the increased area that was occupied by ductal structures. There was no significant increase in the number of ducts. However, there was the appearance of a large number of buds along the length of the ductal structures, suggesting the beginning of side branching. These results suggest that IGF-I, when given along with E2, is responsible for ductal morphogenesis.     

Antileishmanial activities of caffeic acid phenethyl ester loaded PLGA nanoparticles against Leishmania infantum promastigotes and amastigotes in vitro

Objective: To investigate and compare the antileishmanial effects of CAPE and(CAPE)PLGA NPs on Leishmania infantum(L.infantum) promastigotes and amastigotes in vitro,Methods: Efficacies of CAPE,(CAPE)PLGA NPs and free PLGA nanoparticles(NPs) on promastigotes were evaluated using MTT and promastigote count assays,and their anti-amastigote effects were determined via infection index analysis,Griess reaction was also performed to calculate nitric oxide production of macrophages exposed to investigated molecules,Results: It was determined that CAPE and(CAPE)PLGA NPs demonstrated significant inhibitory effects on L.infantum promastigotes and amastigotes,while free NPs did not exhibit any meaningful antileishmanial effectiveness,The IC50 values of CAPE for L.infantum promastigotes and amastigotes were assessed as(51.0±0.8) and(19.0±1.4) μg/m L,respectively(P0.05),On the other side,it was revealed that(CAPE)PLGA NPs had superior antileishmanial activity on both forms of parasites since its IC50 values for L.infantum promastigotes and amastigotes were(32.0±1.3) and(8.0±0.9) μg/m L,respectively(P0.05),It was also determined that both agents strongly stimulated nitric oxide production of macrophages,Conclusions: The obtained results show that(CAPE)PLGA NPs have a great potential to be especially used in treatment of visceral leishmaniasis; however,in vivo antileishmanial screening of these molecules should be performed in the near future.