Differentiation of human dendritic cell subsets for immune tolerance induction.

OBJECTIVES: Since no further progress was achieved, in order to improve the long-term organ transplantation outcome, the immune tolerance appears as an interesting therapeutic goal. Dendritic cells (DCs) are specialized cells participating in the homeostasis of the immune response. Moreover, subsets of DCs, identified in humans, appear to have their respective competences in immune response modulation. Our objective is to purify from PBMC or to differentiate DC subsets from monocytes using several strategies and evaluate their IL10 secretion. METHODS: CD14+ cells were purified from peripheral blood mononuclear cell (PBMC) by affinity beads and cultured with cytokines up to 7 days. The pDCs were purified with anti-BDCA-2 beads from PBMC fraction enriched by Percoll® gradient. The moDCs, pDCs and moLCs subsets were analyzed by phenotype labelling and FACS analyses and IL10 secretion measured by ELISA. RESULTS: The moDCs were characterized by the CD209 expression and a lower expression of CD1a markers. Expression of CD207 and CD1a markers characterized moLCs and CD123+/BDCA-2+ pDCs. Variable IL-10 secretions were shown between the three DC subsets, both at basal and activated levels. CONCLUSIONS: As the several DC populations studied have different capacities of IL-10 synthesis, they might play, among others, distinct roles in the induction of immune tolerance.

Effect of oestrous cycle and early pregnancy on uterine production and expression of immune regulatory factors in gilts.

This study was undertaken to characterize uterine immune factors involved in the establishment of pregnancy in gilts. Thirty crossbred Yorkshire-Landrace gilts of similar age and weight were observed twice a day for oestrous behaviour with intact boars. On the day of first standing oestrus (Day 0) and 12h later, 15 gilts were inseminated with pooled semen from Duroc boars of proven fertility. Pregnant gilts were slaughtered either on Days 10, 15 or 25 of gestation (n=5 per day). The other 15 gilts were not inseminated and were slaughtered on either Days 0, 10 or 15 of the oestrous cycle (n=5 per day). Immediately after slaughter, endometrial tissue samples from the mesometrial side were removed for gene expression using RNase protection assay and in situ hybridization methodologies. The other uterine horn was flushed with 20 ml of PBS to collect the uterine fluid. In pregnant gilts, endometrial interleukin (IL)-6 mRNA expression was higher on Day 15 than on Days 10 and 25 (P<0.01 and P<0.1, respectively). On Day 15, IL-6 expression was also significantly higher (P<0.01) in pregnant gilts than in cyclic gilts. In both pregnant and cyclic gilts, transforming growth factor (TGF)-beta2 in uterine fluid was significantly higher (P<0.0001) on Day 15 than on Day 10. At the gene expression level, TGF-beta2 also increased between Days 10 and 15 in both cyclic and pregnant gilts but differences were not significant. On Day 15, concentrations of interferon-gamma and prostaglandin E(2) (PGE(2)) in uterine fluid were markedly higher (P<0.001) in pregnant gilts than in cyclic gilts, whereas the total amount of TGF-beta2 in uterine fluid and its endometrial expression were approximately 70% higher although this increase was not significant. Finally, tumour-necrosis factor-alpha and granulocyte-macrophage/colony-stimulating factor mRNA expressions were undetectable in all endometrial samples. In conclusion, production and/or expression of uterine TGF-beta2, IL-6 and PGE(2) increased during the embryonic attachment period and are coincidental with embryonic interferon-gamma production.

Stimulating effect of glycoprotein hormone free alpha-subunit and daily gonadotropin releasing hormone treatment on prolactin release from 50-day ovine foetal pituitary explants.

The aim of our study was to determine whether free alpha of glycoprotein hormones (free alpha) plays a role in lactotroph function during early pituitary development in the sheep foetus. Detection and quantification of free alpha, luteinzing hormone beta-subunit (LHbeta) and prolactin immunolabelling were determined by immunocytochemistry at days 32, 37, 42, 50 and 63 of gestation. Free alpha- and LHbeta-containing cells were first detected in the ovine foetal pituitary gland on day 37 of gestation, while prolactin-containing cells were first identified on day 42. Analysis of serial sections suggested that free alpha immunoreactive cells were also LHbeta-positive, indicating that free alpha was mainly synthesized by gonadotrophs. In early foetal stages, free alpha occurred in the antero-medio ventral region of the pituitary gland, whereas prolactin-containing cells were more dorsally and more caudally localized. The free alpha-, LHbeta- and prolactin-immunostained area increased markedly between days 50 and 63 of gestation. To evaluate a possible functional relationship between gonadotrophs and lactotrophs, the effects of free alpha or gonadotropin releasing hormone (GnRH) on prolactin release were assayed. Chronic treatment of pituitary explants from male and female 42-day-old ovine foetuses for 8 days with 10-9 or 10-7 M ovine free alpha did not affect prolactin release. By contrast, free alpha administration on pituitary explants from male and female 50-day-old foetuses resulted in enhanced prolactin release. At this age, a daily (2 h per day) treatment with 10-8 M GnRH had similar stimulatory effect to free alpha whereas a 'first day' treatment (24 h on the first day) reduced prolactin release throughout the culture in males and had no effect in females. These results indicate that, despite early detection of free alpha at day 37 in the ovine foetal pituitary, its stimulatory effect on prolactin release occurs from day 50 of gestation, corresponding to the first period of lactotroph development in vivo. A daily treatment with GnRH mimics the effect of free alpha on prolactin release.

Two free isoforms of ovine glycoprotein hormone alpha-subunit strongly differ in their ability to stimulate prolactin release from foetal pituitaries.

alpha-Subunit dissociated from glycoprotein hormones has been previously shown to stimulate rat pituitary lactotroph differentiation and proliferation. However, whether the free form of the alpha-subunit (free alpha) can also play such a role is not known. To test whether free alpha may act on prolactin (PRL) release from ovine foetal pituitaries, this molecule was purified and two major isoforms, alphaA and alphaB were isolated. Free alphaA was found to be more acidic and more hydrophobic than both free alphaB and ovine LH alpha-subunit (oLHalpha). Free alphaA and oLHalpha exhibited a molecular mass of 14 kDa as determined by mass spectrometry, whereas free alphaB displayed a molecular mass of only 13.5 kDa because of its truncated N-terminus. All three alpha molecules bear mature-type N-linked saccharide chains including Nacetyl galactosamine residues but none of them contains O-linked oligosaccharide. The free alphaA isoform, more than the oLHalpha, was able to stimulate PRL release from ovine foetal pituitary explants in culture, whereas the free alphaB isoform displayed no activity. Moreover, the free alphaA and alphaB isoforms were able to recombine with the ovine LH beta-subunit (oLHbeta). The free alphaB/oLHbeta, and the oLHalpha/oLHbeta dimer were 4-fold more active than the free alphaA/oLHbeta dimer in a specific LH radioreceptor assay and in the stimulation of testosterone release from rat Leydig cells. The present study demonstrates that the two free alpha isoforms of ovine glycoprotein hormones exhibit distinct efficiencies in stimulating PRL release from ovine foetal pituitaries. Moreover, despite their identical ability to recombine with the oLHbeta, the free alpha isoform, which is the most efficient on PRL release, is the least efficient in conferring LH activity on the alpha/beta dimer.

Ectopic ACTH Cushing's syndrome: V3 vasopressin receptor but not CRH receptor gene expression in a pulmonary carcinoid tumor.

In the etiological diagnosis of ACTH-dependent Cushing's syndrome, it may be difficult to distinguish pituitary disease from ectopic ACTH production, specially when this is due to a benign neuroendocrine tumor. We describe a patient with partial dexamethasone suppression consistent with Cushing's disease, an absent response to CRH suggesting ectopic ACTH production and an atypical, apparent circadian rhythm. Bilateral cavernous sinus catheterization suggested a nonpituitary source of ACTH and, in the search of an ectopic tumor, somatostatin receptor scintigraphy, abdominal CT scan, and duodenopancreatic endoscopic echography were performed and failed to reveal any abnormality. Thoracic CT scan disclosed a tiny right lung nodule that showed a definite tracer uptake on MIBG scintigraphy. After resection, the nodule proved to be an 8-mm typical pulmonary carcinoid, with positive immunostaining for the classical neuroendocrine markers and for ACTH, and showing tissue expression of the POMC gene. However, the CRH receptor gene was not expressed, explaining the absent CRH response in vivo, whereas the V3 vasopressin receptor gene was expressed in the tumor tissue. The latter feature appears to be characteristic of benign carcinoids and may contribute to explaining the CRH-independent circadian rhythm observed in this case.

Recurrent pneumatosis intestinalis in young infants.

In 67 infants with necrotizing enterocolitis (NEC) at Children's Hospital of Michigan from 1987-1990, three had recurrent pneumatosis intestinalis (PI) after the neonatal period and after hospital discharge. All three infants were premature (26-34 weeks), and one had an additional risk factor of gastroschisis. All three had their initial episode of NEC within the first month of life. Two had bowel resections, and one was treated medically. 15 h to seven months after discharge from the hospital, all three infants developed recurrent PI at ages ranging from 2.5 to 9 months. Two of the infants had free intraperitoneal air. Though all three infants had PI, only one had true recurrent NEC with ischemic bowel and died. This baby was the only one with rotazyme positive stools. The second infant, who had surgery for gastroschisis, had incomplete obstruction secondary to adhesions resulting in PI. Since surgery this infant has thrived. The third infant had extensive PI of the colon with free air. At surgery there was no evidence of bowel perforation. The free air was attributed to rupture of one of the many colonic cysts of PI. Subsequently, the child has done well. Recurrent PI in infants who have had NEC is unusual and the causes are varied. Because more neonates are surviving NEC, the pediatric radiologist needs to be aware of this delayed complication.

[Control of diabetes and late complications]. / Equilibre du diabète et complications tardives.

When insulin was introduced in medical therapy in 1922, it permitted to save diabetics from premature death; however, it has allowed, after a certain period of time, for the appearance of a cohort of chronic complications connected more or less specifically to the degree of hyperglycemia. After a short review of the pathophysiology of the microangiopathy, the authors have tried to demonstrate, on the basis of numerous prospective and retrospective studies in the human as well as in the animal, that an important relationship exists between the degree of glycemic control and the severity of the classical complications, retinopathy, neuropathy and nephropathy. However, the most recent studies have stressed the role of some other factors, not well established in the past, as for example the potential negative impact on retinopathy of rapid normalization of glycemia, following a long period of poor metabolic control. Likewise, high blood pressure, smoking, genetic background, as well as probably also excess of protein intake, do play an important etiopathogenic role. Thus, the simplistic equation hyperglycemia = complications is not completely valid. Microangiopathic risk in insulin-dependent diabetics seems to be low as long as their HbAlc is below 7.5%, and they do not have hypertension and do not abuse tobacco. Finally, the general approach to therapy is redefined: Try to get as close as possible to near-normoglycemia by multiple insulin injections, without causing, however, major hypoglycemia; this should be done very early after the onset of the disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Oral hypoglycemic agent update.

The treatment of diabetes is still a problem more than a half-century after the discovery of insulin. Patients are now living significantly longer but until the development of oral hypoglycemic agents, the only direct treatment modalities were exercise, diet, and insulin. Before evaluating the effectiveness of treatment, a therapeutic goal must be determined. While there are no absolutely "hard" facts proving that "good control" is beneficial in preventing chronic complications of diabetes, increasing accumulation of "soft" data strongly suggests that normal blood glucose levels are most desirable, when possible, but not at the cost of severe or disabling hypoglycemic reactions. The development of the oral agents was a great public health advance in that many persons with early diabetes, but fearful of insulin injections, had less dread of "the pills" and sought treatment. The oral agents simplified care but this very simplification process often undermined the need for proper diet and good fundamental care. This often led to mediocre diabetes care. While useful, the oral agents have marked limitations and in some are effective only temporarily. The presently available oral agents are sulfonylureas and require a viable beta-cell system for success. This limits the number of diabetics responsive to such treatment. The general indications for tolbutamide, chlorpropamide, acetohexamide and tolazamide are in maturity-onset diabetics, generally beyond the age of 40 with diabetes of less than 10 years. They are contraindicated in juvenile-onset diabetics, in pregnant women, and usually in patients undergoing major surgery, and can become ineffective during periods of extreme stress or during severe infection. They can lower blood glucose levels if used in proper doses in properly selected patients. Contrary to several decades of documentation, it has become popular to suggest that the oral agents are not effective. They can be effective but for many reasons apparently were not in their use by the U.G.D.P. researchers. This might not be the fault of the oral agent used. If ineffective, they should be discontinued. Many, but not all, patients may respond to diet therapy, which is then the treatment of choice. Obviously insulin, though difficult to use for many persons and in itself able to induce several severe reactions if not used properly, is the only treatment (with diet) for the severe diabetic. There is a large spectrum of patients inbetween in whom the oral agents may be useful. The use of phenformin (phenethyl-biguanide) has been effectively curtailed because of many reported cases of lactic acidosis, and while it is doubtful that phenformin alone, in the absence of complicating factors, is the causative factor, it is capable of being an augmenting influence when other conditions, such as decreased kidney function, prevail...