Shiva Reddy

Insulin-Like Growth Factor-I and Binding Proteins 1, 2, and 3 in Bovine Nuclear Transfer Pregnancies

Abstract In cloned pregnancies, placental deficiencies, including increased placentome size, reduced placentome number, and increased accumulation of allantoic fluid, have been associated with low cloning efficiency. To assess differences in paracrine and endocrine growth regulation in cloned versus normal bovine placentomes and pregnancies, we have examined the expression of insulin-like growth factor (IGF)-I and -II and their binding proteins (IGFBP)-1 through -3 in placentomes of artificially inseminated (AI), in vitro-produced (IVP), and nuclear transfer (NT) pregnancies at Days 50, 100, and 150 of gestation. Fetal, maternal, and binucleate cell counts in representative placentomes were performed on Days 50–150 of gestation in all three groups. Increased numbers of fetal, maternal, and binucleate cells were present in NT placentomes at all stages of gestation examined. Immunolocalization studies showed that spatial and temporal patterns of expression of IGFBP-2 and -3 were markedly altered in the placentomes of NT pregnancies compared to AI/IVP controls. Concentrations of IGF-I in fetal plasma, as determined by RIA, were significantly higher (P = 0.001) in NT pregnancies (mean ± SEM, 30.3 ± 2.3 ng/ml) compared with AI (19.1 ± 5.5 ng/ml) or IVP (24.2 ± 2.5 ng/ml) pregnancies on Day 150 of gestation. Allantoic fluid levels of IGFBP-1 were also increased in NT pregnancies. These findings suggest that endocrine and paracrine perturbations of the IGF axis may modulate placental dysfunction in NT pregnancies. Furthermore, increased cell numbers in NT placentomes likely have significant implications for fetomaternal communication and may contribute to the placental overgrowth observed in the NT placentomes.

Altered Placental Lactogen and Leptin Expression in Placentomes from Bovine Nuclear Transfer Pregnancies

Abstract Appropriate growth, development, and function of the placenta is central to the success of nutrient partitioning between the mother, placenta, and fetus. Hormones such as placental lactogen (PL) and leptin are produced in the bovine placenta and play an important role in nutrient partitioning and regulation of placental and fetal growth. Nuclear transfer pregnancies are associated with a number of fetal and placental abnormalities, including increased placental growth and macrosomia, and hence represent a unique situation to gain insight into fetoplacental growth regulation. We have examined the expression of bovine PL (bPL) and leptin in placentomes of artificially inseminated (AI), in vitro produced (IVP), and nuclear transfer (NT) pregnancies at Days 50, 100, and 150 of gestation in the cow. Immunolocalization studies showed that spatial and temporal patterns of expression of bPL and leptin were markedly altered in the placentomes of NT pregnancies compared with AI or IVP controls. Concentrations of bPL in allantoic fluid, as determined by radioimmunoassay (RIA), were significantly higher (P ≤ 0.001) in NT pregnancies (17.9 ± 3.2 ng/ml; mean ± SD) compared with AI (2.03 ± 1.5 ng/ml), but not IVP (23.4 ± 12.8 ng/ ml) pregnancies on Day 150 of gestation. In contrast, amniotic fluid levels of bPL were significantly decreased in NT pregnancies at Day 150 gestation. Leptin mRNA expression, as determined by real-time reverse transcription-PCR, was increased 2.4- to 3.0-fold in NT placentomes compared with AI controls at all gestational ages examined. We speculate that the observed dysregulation of expression of bPL and leptin in NT placentomes could contribute to aberrations in cell migration and invasion and subsequently to alterations in placental metabolism and transfer of nutrients to the fetus, thus leading to increased placental and fetal macrosomia in NT pregnancies.

Quantitative proteomic profiling identifies new renal targets of copper(II)-selective chelation in the reversal of diabetic nephropathy in rats.

This study aimed to identify new diabetic nephropathy (DN)‐related proteins and renal targets of the copper(II)‐selective chelator, triethylenetetramine (TETA) in streptozotocin‐diabetic rats. We used the recently developed iTRAQ™ technology to compare renal protein profiles among non‐diabetic, diabetic, and TETA‐treated diabetic rats. In diabetic kidneys, tubulointerstitial nephritis antigen (TINag), voltage‐dependent anion‐selective channel (VDAC) 1, and VDAC2 were up‐regulated in parallel with alterations in expression of proteins with functions in oxidative stress and oxidative phosphorylation (OxPhos) pathways. By contrast, mitochondrial HSP 60, Cu/Zn‐superoxide dismutase, glutathione S‐transferase α3 and aquaporin‐1 were down‐regulated in diabetic kidneys. Following TETA treatment, levels of D‐amino acid oxidase‐1, epoxide hydrolase‐1, aquaporin‐1, and a number of mitochondrial proteins were normalized, with concomitant amelioration of albuminuria. Changes in levels of TINag, collagen VIα1, actinin 4α, apoptosis‐inducing factor 1, cytochrome C, histone H3, VDAC1, and aquaporin‐1 were confirmed by Western blotting or immunohistochemistry. Changes in expression of proteins related to tubulointerstitial function, podocyte structure, and mitochondrial apoptosis are implicated in the mechanism of DN and their reversal by TETA. These findings are consistent with the hypothesis that this new experimental therapy may be useful for treatment of DN.

“Iron-saturated” bovine lactoferrin improves the chemotherapeutic effects of tamoxifen in the treatment of basal-like breast cancer in mice

BackgroundTamoxifen is used in hormone therapy for estrogen-receptor (ER)-positive breast cancer, but also has chemopreventative effects against ER-negative breast cancers. This study sought to investigate whether oral iron-saturated bovine lactoferrin (Fe-Lf), a natural product which enhances chemotherapy, could improve the chemotherapeutic effects of tamoxifen in the treatment of ER-negative breast cancers.MethodsIn a model of breast cancer prevention, female Balb/c mice treated with tamoxifen (5 mg/Kg) were fed an Fe-Lf supplemented diet (5 g/Kg diet) or the base diet. At week 2, 4T1 mammary carcinoma cells were injected into an inguinal mammary fat pad. In a model of breast cancer treatment, tamoxifen treatment was not started until two weeks following tumor cell injection. Tumor growth, metastasis, body weight, and levels of interleukin 18 (IL-18) and interferon γ (IFN-γ) were analyzed.ResultsTamoxifen weakly (IC50 ~ 8 μM) inhibited the proliferation of 4T1 cells at pharmacological concentrations in vitro. In the tumor prevention study, a Fe-Lf diet in combination with tamoxifen caused a 4 day delay in tumor formation, and significantly inhibited tumor growth and metastasis to the liver and lung by 48, 58, and 66% (all P < 0.001), respectively, compared to untreated controls. The combination therapy was significantly (all P < 0.05) more effective than the respective monotherapies. Oral Fe-Lf attenuated the loss of body weight caused by tamoxifen and cancer cachexia. It prevented tamoxifen-induced reductions in serum levels of IL-18 and IFN-γ, and intestinal cells expressing IL-18 and IFN-γ. It increased the levels of Lf in leukocytes residing in gut-associated lymphoid tissues. B, T and Natural killer (NK) cells containing high levels of Lf were identified in 4T1 tumors, suggesting they had migrated from the intestine. Similar effects of Fe-Lf and tamoxifen on tumor cell viability were seen in the treatment of established tumors.ConclusionsThe results indicate that Fe-Lf is a potent natural adjuvant capable of augmenting the chemotherapeutic activity of tamoxifen. It could have application in delaying relapse in tamoxifen-treated breast cancer patients who are at risk of developing ER-negative tumors.

Erratum to: Analysis of peri-islet CD45-positive leucocytic infiltrates in long-standing type 1 diabetic patients

Aims/hypothesis The role of peri-islet CD45-positive leucocytes, as one component of insulitis, in beta cell death during human type 1 diabetes remains unclear. We undertook a case study, comparing and quantifying leucocytes in the peri- and intra-islet areas in insulin-positive and -negative islets, to assess whether peri-islet leucocytes are pathogenic to beta cells during type 1 diabetes.

Prevention of cyclophosphamide-induced accelerated diabetes in the NOD mouse by nicotinamide or a soy protein-based infant formula.

Spontaneous diabetes in the NOD mouse can be prevented by nicotinamide or by an infant formula diet in which the protein source is replaced with casein hydrolysate (Pregestimil) or soy protein (Prosobee). NOD mice maintained on the standard diet (chow and water) and given cyclophosphamide (Cy) at day 95 develop accelerated and synchronised diabetes within 14 days. Here, we compared the ability of oral nicotinamide or Prosobee, either given alone or concurrently, from weaning, in preventing diabetes in the Cy model. The resulting insulitis and the expression of intra-islet inducible nitric oxide synthase (iNOS) were examined at days 0, 4, 7, 11 and 14 following Cy administration. Intra-islet CD4 and CD8 cells and macrophages were also enumerated at day 11. In mice given the standard diet and injected with Cy at day 95 (group 5), diabetes developed in 7/11 mice, 14 days later. Mice exposed to oral nicotinamide (group 2), Prosobee (group 3) or both (group 4), did not develop the disease during this period and until a further 30 days (p = 0.03). In mice exposed to the standard diet and without Cy treatment (group 1) the insulitis scores increased slowly until day 11 and then declined slightly at day 14 whereas mice exposed to the same diet but given Cy at day 95, showed a sharp decline at day 4 followed by a rapid increase between day 7–14. However, in mice given either nicotinamide, Prosobee or both, the insulitis scores at most time-points were generally lower than in Cy-teated animals on the standard diet. In the latter group, CD4 and CD8 cells and macrophages were also higher at day 11 than all other 4 groups (CD4: p < 0.05; CD8: p < 0.05; macrophages: p < 0.0001). The number of iNOS labelled cells increased progressively in mice on the standard diet and given Cy and were significantly higher at days 4, 7 and 11 than in the 3 dietary groups. Thus, oral nicotinamide or Prosobee, either alone or together, prevents Cy induced diabetes in the NOD mouse. The protective diets suppress Cyinduced intra-islet immune cell influx and iNOS expression.

Immunohistochemical Demonstration of Nitrotyrosine, a Biomarker of Oxidative Stress, in Islet Cells of the NOD Mouse

Abstract: Reactive oxygen species and nitric oxide generated within the beta cell or by intraislet immune cells may be major mediators of beta cell death during insulin‐dependent diabetes mellitus. Here, the intraislet expression of nitrotyrosine, a biomarker of oxidative damage, and its cellular sources were examined in the islets of NOD mice at various stages of spontaneous and cyclophosphamide‐accelerated diabetes. At day 30, nitrotyrosine‐positive cells were undetectable; they were rare at day 40, being expressed in only a few beta cells and in macrophages located in the periphery of some islets. At day 90 and at onset of diabetes, an increasing number of macrophages and beta cells were nitrotyrosine positive. In the cyclophosphamide group at day 4, the number of beta cells and macrophages with positive immunolabeling declined slightly from day 0 (day of cyclophosphamide injection = day 90). This pattern of immunolabeling increased gradually by day 7 but increased markedly by days 11 and 14 after cyclophosphamide administration. In the spontaneous and cyclophosphamide groups, macrophages were the predominant source of nitrotyrosine and were present within the intraislet and periislet regions. Nitrotyrosine labeling was observed in a significant number of beta cells but less than in immunolabeled macrophages. We conclude that protein nitration in beta cells and macrophages is an important feature of the diabetogenic process in the NOD mouse. Whether this deleterious process also occurs during the very early stages of the disease and preceding insulitis is unclear.

Histopathological Changes in Insulin, Glucagon and Somatostatin Cells in the Islets of NOD Mice During Cyclophosphamide-accelerated Diabetes: A Combined Immunohistochemical and Histochemical Study

SummaryThe cyclophosphamide model of accelerated diabetes in the NOD mouse is a useful model of insulin-dependent diabetes mellitus (IDDM). Knowledge on the progressive destruction of beta cells and the fate of other islet endocrine cell-types in this model is sparse. We employed immunohistochemistry and histochemistry, to study temporal changes in islet cell populations, insulitis and glucose transporter-2 expression during cyclophosphamide administration. Cyclophosphamide was administered to day 95 female NOD mice and the pancreas studied at days 0 ( = day 95), 4, 7, 11 and 14 after treatment and in age-matched control mice. At day 0, a majority of the endocrine cells were insulin-positive. Glucagon and somatostatin cells were mostly in the islet periphery and also internally. In the cyclophosphamide group, insulitis was moderate at day 0, declined at day 4 but increased progressively from day 7. The extent of insulitis in treated mice which were diabetes-free at day 14 was comparable to age-matched control mice. From day 11, the marked increase in insulitis correlated with a reciprocal decline in the extent of insulin immunostained islet area. At day 14, the mean insulin area per islet was markedly less in diabetic mice than in age-matched non-diabetic treated and controls. At diabetes, some islets showed co-expression of glucagon and insulin. Our studies suggest that the mean number of glucagon or somatostatin cells per islet does not vary during the study. Glucose transporter-2 immunolabelling was restricted to beta cells but declined in those adjacent to immune cells. We conclude that in the cyclophosphamide model, there is specific and augmented destruction of beta cells immediately prior to diabetes onset. We speculate that the selective loss of glucose transporter-2 shown in this study suggests the existence of a deleterious gradient close to the immune cell and beta cell surface boundary.

Fas and Fas Ligand Immunoexpression in Pancreatic Islets of NOD Mice during Spontaneous and Cyclophosphamide-Accelerated Diabetes

Abstract: During insulin‐dependent diabetes mellitus, beta cell destruction may involve activation of the Fas‐Fas ligand (Fas‐FasL) system. Here, we employed dual‐label immunohistochemistry to examine the intra‐islet expression, distribution, and cellular sources of Fas and FasL in the NOD mouse. Pancreatic tissues were studied during spontaneous diabetes (days 21, 40, and 90) and following acceleration of diabetes with cyclophosphamide (days 0, 4, 7, 11, and 14 after cyclophosphamide administration). Our results show that FasL was expressed constitutively in most beta cells of NOD mice and in nondiabetes‐prone mice, but not in glucagon or somatostatin cells or in islet inflammatory cells. It paralleled the loss of insulin immunolabeling with advancing disease. Immunolabeling for Fas was first observed in extra‐islet macrophages and those close to the islet in NOD and nondiabetes‐prone mice. During spontaneous and cyclophosphamide diabetes, it was observed in a higher proportion of islet infiltrating macrophages than in CD4 and CD8 cells. In the cyclophosphamide group, Fas expression in intra‐islet CD4 and CD8 cells showed an increase close to the onset of diabetes. At days 11 and 14, several intra‐islet macrophages with immunolabeling for Fas also coexpressed interleukin‐1β and inducible nitric oxide synthase. Fas was not detected in beta cells and other endocrine cells during spontaneous and cyclophosphamide diabetes. We show constitutive expression of FasL in beta cells in the NOD mouse and predominant expression of Fas in intra‐islet macrophages and to a lesser extent in T cells prior to diabetes onset. The role of Fas‐FasL in beta cell destruction in the NOD mouse requires further clarification.

Immunolocalization of Caspase-3 in Pancreatic Islets of NOD Mice during Cyclophosphamide-Accelerated Diabetes

Abstract: Apoptosis may be a major mechanism of beta cell loss during insulin‐dependent diabetes mellitus. Caspase‐3 is a key enzyme involved in the terminal steps of this death process. Here, the intra‐islet expression of caspase‐3 in the NOD mouse was examined immunohistochemically following acceleration of the disease with cyclophosphamide. Female NOD mice were treated at day 95 with cyclophosphamide, and caspase‐3 expression in pancreatic sections was studied at days 0, 4, 7, 11, and 14 and compared with age‐matched control tissue. In the treated group at day 0, caspase‐3 labeling was seen in several peri‐islet macrophages and only extremely rarely in beta cells. At day 4, only a few beta cells weakly expressed the enzyme. From day 7, caspase‐3 expression began to increase in intra‐islet macrophages and reached a peak at days 11 and 14, when a small number of CD4 and CD8 T cells also showed positive labeling. Beta cell expression of caspase‐3 at days 11 and 14 was rare. At this stage, several intra‐islet immune cells with positive labeling for the enzyme coexpressed either Fas or interleukin‐1β. Only a small proportion of intra‐islet caspase‐3 cells showed apoptotic nuclei judged by terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL). We conclude that, during cyclophosphamide‐accelerated diabetes, the predominant caspase‐3 immunolabeling in intra‐ and extra‐islet macrophages suggests that apoptosis of macrophages may be an important mechanism for their elimination. The virtual absence of caspase‐3 immunolabeling in most beta cells even during the height of beta cell loss supports the need for developing other markers of early beta cell apoptosis in the NOD mouse.