Harry M. Georgiou

Nuclear Factor Kappa B Regulation of Proinflammatory Cytokines in Human Gestational Tissues In Vitro

Abstract Proinflammatory cytokines are implicated in the initiation and progression of human labor and delivery, particularly in relation to infection-induced preterm labor. In nongestational tissues, the nuclear factor kappa B (NF-κB) transcription pathway is a key regulator of proinflammatory cytokine release. In these tissues, sulfasalazine (SASP), through its ability to inhibit NF-κB activation, inhibits release of interleukin (IL)-2, IL-12, and tumor necrosis factor (TNF)-α. Therefore, the aim of this study was to investigate whether or not NF-κB activation regulates the formation of proinflammatory cytokines in human gestational tissues. Human placenta, amnion, and choriodecidua (n = 9 separate placentas) were incubated with 10 μg/ml of lipopolysaccharide (LPS) in the absence (control) or presence of SASP (0.1, 1, 5, or 10 mM). After 6 h of incubation, the tissues were collected, and NF-κB DNA binding activity in nuclear extracts was assessed by electromobility shift binding assay. The incubation medium was collected and the release of IL-6, IL-8, and TNF-α was quantified by ELISA. Treatment of placenta, amnion, and choriodecidua with SASP at concentrations 5 mM or greater significantly inhibited the release of IL-6, IL-8, and TNF-α, and NF-κB activation (ANOVA, P < 0.05). The data presented in this study demonstrate that the NF-κB transcription pathway is a key regulator of LPS-stimulated IL-6, IL-8, and TNF-α release from human gestational tissues. The control of NF-κB activation may therefore provide an alternative therapeutic strategy for reducing the release of proinflammatory mediators in infection associated preterm labor.

Transgenic Expression of Mouse Proinsulin II Prevents Diabetes in Nonobese Diabetic Mice

IDDM in humans and in nonobese diabetic (NOD) mice is a T-cell–dependent autoimmune disease in which the β-cells of the pancreatic islets are destroyed. Several putative β-cell autoantigens have been identified, but insulin and its precursor, proinsulin, are the only ones that are β-cell specific. (Pro)insulin may be a key autoantigen in IDDM. To address the role of proinsulin in the development of IDDM, we generated NOD mice transgenic for the mouse proinsulin II gene driven off a major histocompatibility complex (MHC) class II promoter to direct expression of the transgene to MHC class II bearing cells, including those in the thymus, with the aim of deleting proinsulin-reactive T-cells. The mononuclear cell infiltration of the islets (insulitis) is almost completely absent, and diabetes is prevented in these transgenic NOD mice. The mononuclear cell infiltration of the salivary glands (sialitis) and immune responses to ovalbumin (OVA) are not altered, indicating that the protective effect of the transgene is specific for islet pathology and not due to general immunosuppression. We conclude that autoimmunity to proinsulin plays a pivotal role in the development of IDDM.

Altered Placental Oxidative Stress Status in Gestational Diabetes Mellitus

Oxidative stress has been clearly linked to type 2 diabetes mellitus, however, limited data are available on the involvement of oxidative stress in gestational diabetes mellitus (GDM), a disease of similar pathophysiology. The aim of this study was to investigate the status of placental oxidative stress in healthy pregnant women and women with GDM. The hypothesis to be tested was that tissue markers of oxidative stress are significantly increased in GDM compared to normal placental tissues. Markers of oxidative stress measured were the release of 8-isoprostane (8-epi-prostaglandin F(2alpha)) from human term placental explants (n=11), the activity of the antioxidant enzymes superoxide dismutase and glutathione peroxidase (n=10), and protein carbonyl content (n=12). Placental release of 8-isoprostane was 2-fold greater from women with GDM (P<0.001) compared to healthy pregnant women. Superoxide dismutase activity and protein carbonyl content were elevated in placentae obtained from women with GDM (P<0.04 and P<0.004 respectively), whilst there was no significant difference in the activity of glutathione peroxidase. These data demonstrate the presence of oxidative stress in the placenta from women with GDM, in addition to the induction of a key antioxidant, collectively indicating a state of existing oxidative stress in this condition.

Glucose-induced release of tumour necrosis factor- alpha from human placental and adipose tissues in gestational diabetes mellitus

Aims  The cytokine tumour necrosis factor‐alpha (TNF‐α) has been implicated in the pathogenesis of insulin resistance in Type 2 diabetes mellitus, but limited data are available in relation to gestational diabetes mellitus (GDM), a disease in which similar biochemical abnormalities exist. We investigated the effect of exogenous glucose on the release of TNF‐α from placental and adipose (omental and subcutaneous) tissue obtained from normal pregnant women, and women with GDM.

Proteomic analysis of human plasma: failure of centrifugal ultrafiltration to remove albumin and other high molecular weight proteins.

Determination of specific low abundance proteins, usually by radiolabelled or enzyme‐linked immunoassays in serum or plasma is widely used in diagnostic medicine. Substitution of these assays by a proteomic approach has been suggested, but this methodology has far from realised its potential as a diagnostic tool. The main protein fractions of plasma represent more than 80% of total protein, making the hundreds or even thousands of other proteins difficult to detect by two‐dimensional electrophoresis (2‐DE). Thus, loading sufficient sample to detect trace proteins invariably means excessive loading of albumin and other high abundance proteins. The aim of this study was to determine whether centrifugal ultrafiltration of whole plasma could be used to eliminate proteins exceeding a desired molecular weight cut‐off. Cellulose filters with a 30 kDa molecular weight cut‐off were used with whole plasma, and total protein was determined before and after ultrafiltration. Samples were processed by routine methods for 2‐DE using 18 cm, pH 3–10 isoelectric focusing strips for the first dimension and 7–15% gradient gels for the second dimension followed by silver staining. Gel analysis of the retentate fraction (30 kDa) revealed a typical 2‐DE plasma profile with most of the major landmark proteins in place and as expected, the gels lacked many of the smaller ( 30 kDa) proteins. Comparison with gels of the filtrate fraction ( 30 kDa) revealed very little difference. Not only were many of the higher molecular weight proteins still present, but some of the smaller 30 kDa landmark proteins were absent. Overall, gels of both the retentate and filtrate fractions were less informative than gels of whole plasma.

Proteomic analysis and characterisation of human cervico-vaginal fluid proteins

Aim:  Cervico‐vaginal fluid (CVF) may provide insight into the biochemical pathways of human reproduction and parturition. The aim of this study was to establish a 2‐D electrophoretic map of human CVF in healthy, pregnant women at term.

Protective effect of CTLA4Ig secreted by transgenic fetal pancreas allografts.

BACKGROUND Pancreas allotransplantation offers a cure for insulin-dependent diabetes mellitus. Systemic immunosuppression used to prevent immune destruction of the graft has side-effects, including increased susceptibility to infection and neoplasia. These unwanted effects may be limited by engineering the graft to secrete immunomodulatory molecules, to achieve local immunosuppression. Several studies have shown that transient local CTLA4Ig results in partial protection of allogeneic grafts. Our intent has been to determine whether sustained secretion of transgenic CTLA4Ig from pancreatic islets is able to protect against allograft rejection. METHODS AND RESULTS Mouse CTLA4 (test=CTLA4Ig) or CD5 leader sequence (control=CD5LIg) was fused to the Fc of mouse IgG2c, and expressed transgenically under the control of the rat insulin promoter in C57BL/6 mice carrying the bml mutation of H-2K(b) (B6.C-H-2(bm1)). This resulted in expression in pancreatic islets. We used ELISA quantification of transgene products secreted into the supernatants of cultured fetal pancreata to select high (CTLA4Ig(hi)) and low (CTLA4Ig(lo)) expresser transgenic mice. Cultured fetal pancreata were transplanted under the kidney capsule of wholly allogeneic CBA recipient mice. CTLA4Ig(hi) but not CTLA4Ig(lo) expresser grafts showed enhanced survival compared with control CD5LIg grafts at 6 weeks posttransplant, provided the recipient mice were transiently depleted of CD4 T cells (by a single low-dose injection of GK1.5) before transplantation. CONCLUSIONS Sustained local secretion of CTLA4Ig from transgenic grafts in combination with transient systemic CD4 T-cell depletion can enhance allograft acceptance.

Omentin-1 Is Decreased in Maternal Plasma, Placenta and Adipose Tissue of Women with Pre-Existing Obesity

Objective The aim of this study was to determine (i) the effect of maternal obesity and gestational diabetes mellitus (GDM) on (i) the circulating levels of omentin-1 in cord and maternal plasma, and (ii) gene expression and release of omentin-1 from human placenta and adipose tissue. The effect of pregnancy on circulating omentin-1 levels was also determined. Design Omentin-1 levels were measured in maternal and cord plasma from obese and non-obese normal glucose tolerant women (NGT; n = 44) and women with GDM (n = 39) at the time of term elective Caesarean section. Placenta and adipose tissue expression and release of omentin-1 was measured from 22 NGT and 22 GDM women collected at the time of term elective Caesarean section. Omentin-1 levels were also measured in maternal plasma from 13 NGT women at 11 and 28 weeks gestation and 7 weeks postpartum. Results Maternal obesity was associated with significantly lower omentin-1 levels in maternal plasma; however, there was no effect of maternal obesity on cord omentin levels. Omentin-1 gene expression was lower in placenta and adipose tissue obtained from women with pre-existing obesity. In addition to this, adipose tissue release of omentin-1 was significantly lower from obese pregnant women. Omentin-1 levels were significantly lower in non-obese GDM compared to non-obese NGT women. However, there was no difference in omentin-1 levels between obese NGT and obese GDM women. There was no effect of GDM on cord omentin levels, and placental and adipose tissue omentin-1 expression. Maternal omentin-1 levels were negatively correlated with fetal birthweight and fetal ponderal index. Conclusions The data presented in this study demonstrate that pre-existing maternal obesity is associated with lower omentin-1 expression in placenta, adipose tissue and maternal plasma. Alteration in omentin-1 in pregnancy may influence the development of metabolic disorders in offspring later in life.

New biomarkers for the prediction of spontaneous preterm labour in symptomatic pregnant women: a comparison with fetal fibronectin.

To identify cervicovaginal fluid (CVF) biomarkers predictive of spontaneous preterm birth in women with symptoms of preterm labour.

Induction of Insulitis in Athymic (Nude) Mice: The Effect of NOD Thymus and Pancreas Transplantation

The NOD mouse is a model for human insulin-dependent diabetes mellitus. The disease is thought to have an autoimmune etiology because it is T-cell dependent and is characterized by mononuclear cell infiltration in and around the pancreatic islets of Langerhans. The mechanism by which autoreactive T-cells are generated is not fully understood, but it has been postulated that there is a breakdown in self-tolerance induction during intrathymic T-cell maturation. The aim of these studies was to determine whether transplantation of NOD thymus into diabetes-resistant mouse strains would generate islet-reactive T-cells. Neonatal thymus was pretreated either by irradiation or culture in 2-deoxyguanosine (dGua) and then transplanted into athymic BALB/c, CBA, and C57BL/6 nude mice. Generally, insulitis was not seen in the CBA or C57BL/6 recipients, but was found in 56% of BALB/c mice transplanted with an irradiated NOD thymus and in 46% BALB/c mice with a dGua-treated thymus. Similar experiments in which a NOD fetal pancreas was transplanted into nude BALB/c mice before NOD thymus transplantation showed a similar frequency and severity of insulitis in both the host pancreas and grafted NOD pancreas. This suggests that NOD islets are no more prone than the host islets to autoimmune attack and do not exacerbate insulitis. Overall, the data suggest that a defect of thymic origin (and correlating with the thymic epithelium) in the NOD mouse can lead to the development of autoreactive T-cells and specific islet cell damage. Autoreactivity appears to be restricted to the H-2Kd allele that is shared by NOD and BALB/c mice.