Endometrial secretions: creating a stimulatory microenvironment within the human early placenta and implications for the aetiopathogenesis of preeclampsia.

Endometrial glands represent an important source of nutrients for the conceptus during the first trimester. Their secretions are enriched with carbohydrates, and glycogen accumulates within the syncytiotrophoblast of the placenta. It has been assumed that fetal and placental metabolism follow adult pathways, although it is now appreciated that early development occurs in a low-oxygen environment. In past decades, a novel family of putative insulin mediators, inositol phosphoglycans (IPGs), was discovered. These molecules act as allosteric activators and/or inhibitors of enzymes and transduction proteins involved in the control of cell signalling and metabolic pathways, and determine the specificity of responses after activation of the insulin receptor. One member, IPG P-type, activates pyruvate dehydrogenase phosphatase (PDH-Pase), glycogen synthase phosphatase, and glycerol-3-phosphate acyltransferase. Activation of key phosphatases play a major role in the regulation of glucose disposal by oxidative metabolism via PDH, and the non-oxidative storage by glycogen synthesis, both pathways classically known to be regulated by insulin. High concentrations of IPG P-type in amniotic fluid suggest a role in the regulation of carbohydrate metabolism in the fetal-placental unit. Glycogen accumulation in the syncytiotrophoblast also occurs in preeclamptic pregnancies, and is consistently associated with higher placental levels of IPG P-type. Here, we explore the relationship between nutrients provided by the endometrial glands during early pregnancy, IPG P-type and fetal metabolic requirements. We also discuss whether a disconnect between the placental/fetal metabolic state and oxygen tension could lead to a preeclamptic-type syndrome via leakage of Warburg/IPG mediators into the maternal circulation.

Insight into the role of inositol phosphoglycans in insulin response and the regulation of glucose and lipid metabolism illustrated by the response of adipocytes from two strains of rats.

Differences in biochemical and hormone profiles between two strains of rats provide insights into the relationships between insulin response, inositol phosphoglycans and lipid metabolism in adipose tissue. The results suggest the apparent anomaly of a higher rate of lipogenesis and response to insulin with a lower fat pad weight in the Charles River vs. Harlan Olac group relates to: (i) enzyme pre-programming with IPG-A, (ii) faster turnover of lipid, (iii) effects of leptin and cAMP.

Urinary excretion of inositol phosphoglycan P-type in gestational diabetes mellitus.

OBJECTIVE: The mechanisms underlying insulin resistance during normal pregnancy, and its further exacerbation in pregnancies complicated by gestational diabetes mellitus (GDM), are generally unknown. Inositolphosphoglycan P-type (P-IPG), a putative second messenger of insulin, correlates with the degree of insulin resistance in diabetic subjects. An increase during normal pregnancy, in maternal and fetal compartments, has recently been reported. METHODS: A cross-sectional study was carried out in 48 women with GDM and 23 healthy pregnant women. Urinary levels of P-IPG were assessed spectrophotometrically by the activation of pyruvate dehydrogenase phosphatase in urinary specimens and correlated with clinical parameters. RESULTS: Urinary excretion of P-IPG was higher in GDM than in control women (312.1 +/- 151.0 vs. 210.6 +/- 82.7 nmol NADH/min/mg creatinine, P < 0.01) with values increasing throughout pregnancy in control subjects (r2 = 0.34, P < 0.01). P-IPG correlated with blood glucose levels (r(2) = 0.39, P < 0.01 for postprandial glycaemia and r2 = 0.18 P < 0.01 for mean glycaemia) and birthweight in the diabetic group (r2 = 0.14, P < 0.01). CONCLUSIONS: Increased P-IPG urinary excretion occurs in GDM and positively correlates with blood glucose levels. P-IPG may play a role in maternal glycaemic control and, possibly, fetal growth in GDM.

P-type inositol phosphoglycans in serum and amniotic fluid in active pre-eclampsia.

OBJECTIVES: Abnormal secretion of P-type inositol phosphoglycans (IPG-P) has been described in maternal urine of pre-eclamptic women. The aim of this study was to determine the origin of production of IPG-P. We examined the IPG-P content of maternal and fetal serum, maternal urine and amniotic fluid in both normal pregnancy and pre-eclampsia. DESIGN: Established extraction and bioactivity assay techniques were used to compare total IPG-P levels in serum samples, and a polyclonal-antibody-based ELISA to assay the amniotic fluid and urine samples in matched pairs of women. SUBJECTS: Eleven women with pre-eclampsia requiring caesarean section (subjects), 11 pregnant women requiring elective caesarean section for reasons other than pre-eclampsia (controls). RESULTS: Our data confirm the abnormal level of IPG-P in maternal urine during pre-eclampsia. Moreover, IPG-P levels were higher in umbilical sera than in maternal sera samples. Amniotic fluid as well as urine ELISA results were significantly higher in the pre-eclamptic group compared with normal controls. Total IPG-P bioactivity in serum did not vary between serum compartments in normal pregnancy. Uterine vein IPG-P levels were lower in pre-eclampsia when compared with normal pregnancy. A possible correlation was observed between urine and amniotic fluid levels in normal women. No correlation was observed between measured blood levels and those in urine and amniotic fluid. CONCLUSIONS: It is hypothesized that steady state equilibrium of IPG-P in serum in normal pregnancy is disrupted in pre-eclampsia. Additionally, an abnormal IPG-P sub-fraction, detectable in urine and amniotic fluid, may be present and involved in the pathophysiology of the syndrome, although sites of production of this abnormal form remain unclear.

Improvement of glucose homeostasis in obese diabetic db/db mice given Plasmodium yoelii glycosylphosphatidylinositols.

We have previously reported that infection with Plasmodium yoelii, Plasmodium chabaudi, or injection of extracts from malaria-parasitized red blood cells induces hypoglycemia in normal mice and normalizes the hyperglycemia in streptozotocin (STZ)-diabetic mice. P yoelii glycosylphosphatidylinositols (GPIs) were extracted in chloroform:methanol:water (CMW) (10:10:3), purified by high-performance thin layer chromatography (HPTLC) and tested for their insulin-mimetic activities. The effects of P yoelii GPIs on blood glucose were investigated in insulin-resistant C57BL/ks-db/db diabetic mice. A single intravenous injection of GPIs (9 and 30 nmol/mouse) induced a significant dose-related decrease in blood glucose (P < .001), but insignificantly increased plasma insulin concentrations. A single oral dose of 2.7 micromol GPIs per db/db mouse significantly lowered blood glucose (P < .01). P yoelii GPIs in vitro (0.062 to 1 micromol/L) significantly stimulated lipogenesis in rat adipocytes in a dose-dependent manner both in the presence and absence of 10(-8) mol/L insulin (P < .01). P yoelii GPIs stimulated pyruvate dehydrogenase phosphatase (PDH-Pase) and inhibited both cyclic adenosine monophosphate (cAMP)-dependent protein kinase A and glucose-6-phosphatase (G6Pase). P yoelii GPIs had no effect on the activity of the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK). This is the first report of the hypoglycemic effect of P yoelii GPIs in murine models of type 2 diabetes. In conclusion, P yoelii GPIs demonstrated acute antidiabetic effects in db/db mice and in vitro. We suggest that P yoelii GPIs, when fully characterized, may provide structural information for the synthesis of new drugs for the management of diabetes.

Possible involvement of inositol phosphoglycan-P in human parturition.

Preterm labour is a major cause of neonatal morbidity and mortality but the pathophysiology that underlies preterm labour is unknown. Inositolphosphoglycans (IPGs) comprise a ubiquitous family of putative carbohydrate second messengers and they have been linked to the pathogenesis of various conditions, including diabetes and pre-eclampsia. Studying IPG-P levels in normal and pre-eclamptic pregnancies, we noticed a constant rise of urinary IPG-P levels in all women at the time of delivery. A prospective pilot study of urinary IPG-P levels in 23 non-labouring and labouring women with uncomplicated pregnancies has, therefore, been performed. Levels of urinary IPG-P were significantly higher in labour than in the non-labouring group (P<0.0001). These higher levels have been found in both spontaneous and induced labour. The clinical significance of this observation with particular reference to the onset of labour itself is discussed.

Placental GPI-PLD is of maternal origin and its GPI substrate is absent from placentae of pregnancies associated with pre-eclampsia.

Pre-eclampsia (PE) is a disorder affecting 5-10% of all pregnancies and is characterised by abnormal trophoblast invasion, maternal endothelial cell dysfunction and a systemic maternal response. A unifying factor responsible for eliciting these effects remains unknown. However, levels of the autocrine insulin mediators, inositolphosphoglycans (IPG), are elevated 3-fold in pre-eclamptic placentae compared with controls and are also elevated 3-fold in maternal urine of pre-eclamptic women, suggesting an abnormal paracrine role of the mediator in the systemic maternal response. At the placental level, IPGs are metabolic second messengers capable of eliciting some of the characteristic features of PE, such as the 10-fold increase in glycogen synthesis and 16-fold increase in the activity of the IPG-dependent enzyme glycogen synthase. IPGs are derived from their lipidic precursors, the glycosylphosphatidylinositols (GPI), in membrane associated caveolae by the action of a GPI-specific phospholipase D whose activity is regulated by its membrane microenvironment. We show that the lipidic GPI precursor was detected in total placental membrane and microvillous membrane from normal placentae. The presence of GPI could not be detected in PE placentae, suggesting that the GPI/IPG signalling system is dysregulated in this disorder. Equivalent amounts of a proteolytically-cleaved 50 kDa GPI-PLD protein is detected in both normal and PE placentae. However, GPI-PLD mRNA is absent, suggesting a mechanism of uptake from maternal serum. Since GPI-PLD, whose presence is required for hydrolysis of GPI and release of free IPG, is detectable with equal activity in both normal and PE placentae, we postulate that dysregulation of the tubular caveolar structure of the microvilli in pre-eclamptic placentae provides an environment which promotes the unregulated hydrolysis of GPI in this disorder.

Placentally derived prostaglandin E2 acts via the EP4 receptor to inhibit IL-2-dependent proliferation of CTLL-2 T cells.

A number of immunomodulatory molecules are present in the placenta, including cytokines, prostaglandins, progesterone and indoleamine 2,3-dioxygenase. An undefined factor capable of down-regulating T-cell activity has recently been reported [1] as being produced by short-term cultures of placental fragments. By careful repetition of these studies we have confirmed that chorionic villi isolated from term placenta produce a low molecular weight, heat stable factor capable of inhibiting the IL-2-dependent proliferation of mouse CTLL-2 cells. This activity was not due, however, to a previously unknown immunosuppressive molecule, but rather to prostaglandin E2 (PGE2). Expression of cyclooxygenase (COX)-2 was detected in the syncytiotrophoblast of chorionic villi explants using immunohistochemistry. Culture of the explants in the presence of the COX-1/COX--2 inhibitors indomethacin and diclofenac, or with the COX-2-selective inhibitor DFP, blocked the production of the immunosuppressive factor. The immunosuppressive activity was restored by adding PGE2 to the supernatants obtained from diclofenac-inhibited explants. A number of different receptors are involved in mediating the biological effects of prostaglandins. By utilizing selective antagonists of individual receptors, we have established that the immunosuppressive effect of PGE2 on CTLL-2 cells is exerted via the EP4 receptor. Thus, addition of an EP4-selective antagonist, but not of EP1 or EP3 antagonists, abolished the immunosuppressive effect of PGE2 on CTLL-2 cells. This may have implications for attempts to selectively manipulate T-cell responses.

Insulin reduces serum glycosylphosphatidylinositol phospholipase D levels in human type I diabetic patients and streptozotocin diabetic rats.

The enzyme glycosylphosphatidylinositol phospholipase D has a postulated role in the insulin-mimetic signaling pathway of glycosylphosphatidylinositol compounds. We have investigated enzyme activity in the serum of human type I diabetic patients and plasma and tissues of streptozotocin-induced diabetic rats following insulin administration. In the human diabetic patients serum enzyme activity fell by an average of 10.6% (SEM = 2.7; P = 0.008; n = 20) following administration of insulin. In addition serum enzyme activity appeared to be depleted by 27% (SEM = 8.8; P = 0.011; n = 10) compared to nondiabetic controls. In untreated diabetic rats plasma enzyme activity gradually increased 0.3-fold over a 6-week period (P < 0.001; n = 8), this increase was reversed and activity normalized when these animals were treated with insulin. Cloning of the rat glycosylphosphatidylinositol phospholipase D cDNA enabled confirmation of the liver as the principal organ of synthesis. Analysis of mRNA levels in the livers of the diabetic rats showed that gene expression was reduced in the insulin-treated animals compared to the noninsulin-treated controls by 0.7-fold (P = 0.004; n = 4). Tissue enzyme activity was also reduced in the insulin-treated rats; in skeletal muscle enzyme activity was 0.3-fold lower (P = 0.001; n = 4). Insulin therefore decreases glycosylphosphatidylinositol phospholipase D synthesis in diabetic animals resulting in decreased serum enzyme levels, suggesting a relationship between this enzyme and the function of insulin.

Reversal of type 2 diabetes in mice by products of malaria parasites. II. Role of inositol phosphoglycans (IPGs).

We have previously shown that infection with Plasmodium yoelii malaria or injection of extracts from malaria-parasitized red cells induces hypoglycemia in normal mice and normalizes the hyperglycemia in mice made moderately diabetic with streptozotocin. Inositol phosphoglycans (IPGs) are released outside cells by hydrolysis of membrane-bound glycosylphosphatidylinositols (GPIs), and act as second messengers mediating insulin action. The C57BL/Ks-db/db and C57BL/6J-ob/ob mice offer good models for studies on human obesity and Type 2 diabetes. In the present study, we show that a single iv injection of IPG-A or IPG-P extracted from P. yoelii significantly (P < 0.02) lowers the blood glucose in STZ-diabetic, db/db, and in ob/ob mice for at least 4--6 h. Using rat white adipocytes, IPG-P increased lipogenesis by 20--30% in the presence and absence of maximal concentrations of insulin (10(-8) M) (P < 0.01) and stimulated pyruvate dehydrogenase (PDH) phosphatase in a dose-related manner. Both IPG-A and IPG-P inhibited c-AMP-dependent protein kinase (PKA) in a dose-related manner. Compositional analysis of IPGs after 24 h hydrolysis revealed the presence of myo-inositol, phosphorus, galactosamine, glucosamine, and glucose in both IPG-A and IPG-P. However, hydrolysis of IPGs for 4 h highlighted differences between IPG-A and IPG-P. There are some functional similarities between P. yoelii IPGs and those previously described for mammalian liver. However, this is the first report of the hypoglycemic effect of IPGs in murine models of Type 2 diabetes. We suggest that IPGs isolated from P. yoelii, when fully characterized, may provide structural information for the synthesis of new drugs for the management of diabetes mellitus.

Inositolphosphoglycan mediators structurally related to glycosyl phosphatidylinositol anchors: synthesis, structure and biological activity.

The preparation of the pseudopentasaccharide 1a, an inositol-phosphoglycan (IPG) that contains the conserved linear structure of glycosyl phosphatidylinositol anchors (GPI anchors), was carried out by using a highly convergent 2+3-block synthesis approach which involves imidate and sulfoxide glycosylation reactions. The preferred solution conformation of this structure was determined by using NMR spectroscopy and molecular dynamics simulations prior to carrying out quantitative structure--activity relationship studies in connection with the insulin signalling process. The ability of 1a to stimulate lipogenesis in rat adipocytes as well as to inhibit cAMP dependent protein kinase and to activate pyruvate dehydrogenase phosphatase was investigated. Compound 1a did not show any significant activity, which may be taken as a strong indication that the GPI anchors are not the precursors of the IPG mediators.

Structure and expression of the human glycosylphosphatidylinositol phospholipase D1 (GPLD1) gene.

Here we report the structure of the human glycosylphosphatidylinositol phospholipase D1 gene, which covers at least 80 kb on chromosome 6p22. The gene comprises 25 exons and encodes a 5.8 kb mRNA, which was detected only in the liver. Southern blot analysis shows that the human genome contains only one GPLD gene and we could only detect one of the two previously reported cDNAs.

Inositol phosphoglycans and the regulation of the secretion of leptin: in vitro effects on leptin release from adipocytes and the relationship to obesity.

The ratio of two families of inositol phosphoglycans (IPG-A:IPG-P), insulin second messengers, is raised in non-insulin-dependent diabetes mellitus (NIDDM) and obesity. It is shown here that IPG A type inhibits leptin release from adipocytes, contrasting with the action of insulin (stimulation) and IPG P type (no effect). The significance of inhibitory effects of IPG A type on leptin release is important in relation to obesity and NIDDM in view of the action of leptin in promoting Lep expression and fat oxidation in muscle, in addition to its effects on satiety. Energy conservation and oxidation via interorgan regulation by leptin could be compromised by a rise in the IPG-A:IPG-P ratio.

Inositol phosphoglycans and signal transduction systems in pregnancy in preeclampsia and diabetes: evidence for a significant regulatory role in preeclampsia at placental and systemic levels.

Measurements have been made of the urinary content of inositol phosphoglycans IPG P-type and IPG A-type, putative insulin second messengers, in preeclampsia, in type I insulin-treated diabetic pregnant women and their matched control subjects, and nonpregnant women of child-bearing age. The content of IPG P-type and IPG A-type was also measured in the placenta from preeclamptic patients and from normal pregnancies. Pregnancy was associated with an increase, approximately twofold, in urinary output of IPG-P-type relative to nonpregnant controls (P<0.01). The 24-h output of IPG P-type in urine in preeclamptic women was significantly higher (2- to 3-fold) than in pregnant control subjects matched for age, parity, and stage of gestation (P<0.02). In contrast, insulin-dependent diabetic pregnant women did not show any significant change in urinary output of IPG P-type or IPG A-type relative to pregnant control subjects. Evidence for a possible relationship and correlation between the urinary excretion of IPG P-type and markers of preeclampsia, including proteinuria (r = 0.720, P<0.01), plasma aspartate transaminase (r = 0.658, P<0.05), and platelet counts (r = 0.613, P<0.05) is presented. A high yield of IPG P-type was extracted from human placenta, in preeclampsia some 3-fold higher (P = 0.03) than the normal value, whereas no IPG A-type (with lipogenic-stimulating activity) was found. Low concentrations of placental IPG A-type were detected relative to IPG P-type using assay systems dependent upon the effect of this mediator on cAMP-dependent protein kinase or on a proliferation assay using thymidine incorporation into DNA of EGFR T17 fibroblasts. It is postulated that the high urinary excretion IPG P-type in preeclampsia reflects high placental levels and relates to the accumulation of glycogen in the placenta. The paracrine effects of placental IPG P-type (stimulation off other endocrine glands and/or endothelial cells) could contribute to the pathogenesis of the maternal syndrome. A possible theoretical link between elevated placental IPG P-type and apoptosis is proposed.

Inositol phosphoglycans in diabetes and obesity: urinary levels of IPG A-type and IPG P-type, and relationship to pathophysiological changes.

Measurements have been made, in adult male diabetic patients and control subjects, of the urinary content of inositol phosphoglycans (IPGs), the IPG A-type and IPG P-type forms, which, among other actions, regulate pathways of glucose utilization, lipogenesis, triglyceride formation, and pyruvate dehydrogenase (PDH) activity. Urine samples from the entire diabetic group showed a 2- to 3-fold increase in IPG A-type, and a fall in the IPG P-type:IPG A-type ratio relative to the control group. Subdivision of the diabetic patients into lean IDDM and obese NIDDM groups revealed significant differences in the IPG P-type:IPG A-type ratio between these groups, this ratio decreasing with increases in the body mass index (BMI). Analysis of the relationships among IPGs and HbA1, blood pressure, and BMI indicated that a fall in the IPG P-type:IPG A-type ratio correlated with a rise in the HbA1 (indicative of impaired glycemic control), with increased systolic blood pressure and increased obesity, all factors linked to Syndrome X. There was a parallism between the profile of the IPG P-type:IPG A-type ratio and the well-established pattern of insulin resistance and BMI. In vitro studies of the effects of alterations in the IPG P-type:IPG A-type ratio on the activation of the pyruvate dehydrogenase complex (PDH complex) at the PDH phosphatase reaction demonstrated that IPG A-type forms antagonized the stimulation of the PDH phosphatase by IPG P-type forms, thus having a negative effect on the conversion of PDH to the active, dephosphorylated, form. This observation could provide a mechanism whereby the shifts in the IPG P-type:IPG A-type ratio reported above could change the metabolic pattern from one directed to glucose oxidation to one more directed toward energy conservation and lipid storage.

Correlation between IgG anti-type II collagen levels and arthritic severity in murine arthritis.

This study examines the relationship between the serum levels of IgG antibodies to heterologous and homologous type II collagen and the subsequent arthritic severity in a collagen induced arthritis model. Arthritis was induced in DBA-1 mice using intradermal injections of heterologous type II collagen in Freunds complete adjuvant, the time of arthritis onset was noted and the severity was monitored regularly. Serum samples were taken and IgG levels of anti-heterologous and homologous type II collagen were analyzed both pre and post arthritis onset. We observed that post induction/pre arthritic serum IgG anti-heterologous and homologous type II collagen levels showed a significant correlation (both p < 0.01) with the severity of the arthritis that subsequently developed. Mice with early arthritis showed a highly significant correlation (p < 0.002) between sera IgG anti-homologous type II collagen levels and arthritic severity, a lesser correlation was also apparent between anti-heterologous type II collagen titres and arthritic severity (p < 0.05). The high levels of correlation observed in this study between anti-type II collagen titres and arthritic severity before actual onset of arthritis, clearly suggest that the magnitude of the initial humoral response to type II collagen plays a crucial role in determining the resultant arthritic severity. This observation is only apparent due to the use of an arthritis susceptible inbred mouse strain, which removes variables such as H-2 restriction, antigen processing/presentation and possible complement deficiencies, and the early time scale of the analysed sera samples.

Recent advances in glycoconjugate analysis and glycobiology.

Glycoconjugate chemistry and glycobiology were rapidly evolving scientific disciplines in the 1980s. Their impact, however, on understanding fundamental biological processes was not immediately forthcoming. Within the past 12-18 months, there has been a resurgence in the field with major discoveries leading to powerful new insights into the complex role of glycoconjugates in biological processes.

Isolation and partial characterisation of insulin-mimetic inositol phosphoglycans from human liver.

Extracts of human liver were found to contain activities which copurified and coeluted with the two major subtypes of mediators (type A and type P) isolated from insulin-stimulated rat liver. The putative type A mediator from human liver inhibited cAMP-dependent protein kinase from bovine heart, decreased phosphoenolypyruvate carboxykinase mRNA levels in rat hepatoma cells, and stimulated lipogenesis in rat adipocytes. The putative type P mediator stimulated bovine heart pyruvate dehydrogenase phosphatase. Both fractions were able to stimulate proliferation of EGFR T17 fibroblasts and the type A was able to support growth in organotypic cultures of chicken embryo cochleovestibular ganglia. Both activities were resistant to Pronase treatment and the presence of carbohydrates, phosphate, and free-amino groups were confirmed in the two fractions. These properties are consistent with the structure/ function characteristics of the type A and P inositolphosphoglycans (IPG) previously characterized from rat liver. Further, the ability of the human-derived mediators to interact with rat adipocytes and bovine-derived metabolic enzymes suggests similarity in structure between the mediators purified from different species. Galactose oxidase-susceptible membrane-associated glycosylphosphatidylinositols (GPI) have been proposed to be the precursors of IPG. GPI was purified from human liver membranes followed by treatment with galactose oxidase and reduction with NaB3H4. Serial t.l.c. revealed three radiolabeled bands which comigrated with the putative GPI precursors found in rat liver. These galactose-oxidase-reactive lipidic compounds, however, were only partially susceptible to hydrolysis with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis and were resistant to glycosylphosphatidylinositol-specific phospholipase C from Trypanosoma brucei. These data indicate that IPG molecules with insulin-like biological activities are present in human liver.

Reduction in the incidence and severity of collagen-induced arthritis in DBA/1 mice, using exogenous dehydroepiandrosterone.

OBJECTIVE: This study examined the effect of exogenous dehydroepiandrosterone (DHEA) on the onset, incidence, and severity of collagen-induced arthritis (CIA). METHODS: DHEA was administered subcutaneously prior to arthritis induction in DBA/1 mice, and the severity of the subsequent arthritis was monitored. Serum levels of total IgG and IgG isotype-specific anti-murine type II collagen were measured. RESULTS: Repeated administration of DHEA during arthritis induction delayed the onset and decreased the severity of arthritis in male and female DBA/1 mice. DHEA failed to have an observable effect on established arthritis. IgG isotype autoantibody levels were found to be decreased in the sera of DHEA-treated mice. CONCLUSION: Administration of exogenous DHEA offered protection against the development of CIA. These data support the results of human studies in which low DHEA levels have been identified as a potential risk factor for the development of rheumatoid arthritis. These findings also highlight DHEA as a potential therapy worthy of further investigation.

Analysis of murine IgG isotype galactosylation in collagen-induced arthritis.

The galactosylation status of IgG from both control and arthritic DBA-1 mice was determined by exoglycosidase sequencing and an anti-GlcNAc monoclonal based ELISA. Two to three weeks after arthritis onset mice with collagen-induced arthritis (CIA) showed a modest increase in IgG anti-GlcNAc reactivity against controls (0.644 +/- 0.080 and 0.530 +/- 0.087, respectively, mean absorbance +/- SEM n = 4) consistent with previous literature reports. However, the authors were unable to detect any significant changes in the galactosylation of purified IgG from arthritic and control mice using direct sequencing techniques (percentage G0 of 34.5% +/- 1.9 and 37.7% +/- 2.4, respectively, mean +/- SEM n = 4). It has been demonstrated that a correlation exists between percentage G0 and anti-GlcNAc reactivity for purified human IgG and sera samples, respectively; no such correlation was found for murine IgG and sera. The galactosylation of purified polyclonal murine IgG isotypes was analysed on a separate group of arthritic mice selected for their high anti-GlcNAc reactivities. No significant differences were observed between control and arthritic mice. Immunoglobulin G isotype specific differences were found, with IgG1 exhibiting the highest percentage G0 (45-48%) followed by IgG2a (27-37%), IgG3 (20-32%) and IgG2b the lowest (13-17%). The percentage G0 and anti-GlcNAc reactivity of purified IgG1 and IgG2b showed a narrow range of values when compared to those of IgG2a and IgG3 samples. Pooled sera from both arthritic and control mice was used to purify large quantities of IgG3. Which on analysis revealed a fourfold increase in anti-GlcNAc reactivity in the arthritic sample compared to control. Paradoxically these same IgG3 samples contained similar percentage G0 levels as determined by direct sequencing. The results suggest that IgG1 and IgG2b exhibit isotype selective oligosaccharide processing as little sample heterogeneity could be observed. These two purified IgG isotypes displayed a good correlation between percentage G0 and anti-GlcNAc reactivity; this was in contrast to IgG2a and IgG3. Immunoglobulin G3 from arthritic mice may have G0 oligosaccharides selectively paired together with no net increase in percentage G0. This observation is discussed in detail as is the role of agalactosyl IgG in murine type II collagen-induced arthritis.