Clinical and laboratory characteristics in juvenile-onset systemic lupus erythematosus across age groups.

BACKGROUND: Systemic lupus erythematous (SLE) is a systemic autoimmune/inflammatory condition. Approximately 15-20% of patients develop symptoms before their 18th birthday and are diagnosed with juvenile-onset SLE (JSLE). Gender distribution, clinical presentation, disease courses and outcomes vary significantly between JSLE patients and individuals with adult-onset SLE. This study aimed to identify age-specific clinical and/or serological patterns in JSLE patients enrolled to the UK JSLE Cohort Study. METHODS: Patient records were accessed and grouped based on age at disease-onset: pre-pubertal (≤7 years), peri-pubertal (8-13 years) and adolescent (14-18 years). The presence of American College of Rheumatology (ACR) classification criteria, laboratory results, disease activity [British Isles Lupus Assessment Group (BILAG) and Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2 K) scores] and damage [Systemic Lupus International Collaborating Clinics (SLICC) damage index] were evaluated at diagnosis and last follow up. RESULTS: A total of 418 JSLE patients were included in this study: 43 (10.3%) with pre-pubertal disease onset; 240 (57.4%) with peri-pubertal onset and 135 (32.3%) were diagnosed during adolescence. At diagnosis, adolescent JSLE patients presented with a higher number of ACR criteria when compared with pre-pubertal and peri-pubertal patients [pBILAG2004 scores: 9(4-20] vs. 7(3-13] vs. 7(3-14], respectively, p = 0.015] with increased activity in the following BILAG domains: mucocutaneous (p = 0.025), musculoskeletal (p = 0.029), renal (p = 0.027) and cardiorespiratory (p = 0.001). Furthermore, adolescent JSLE patients were more frequently ANA-positive (p = 0.034) and exhibited higher anti-dsDNA titres (p = 0.001). Pre-pubertal individuals less frequently presented with leukopenia (p = 0.002), thrombocytopenia (p = 0.004) or low complement (p = 0.002) when compared with other age groups. No differences were identified in disease activity (pBILAG2004 score), damage (SLICC damage index) and the number of ACR criteria fulfilled at last follow up. CONCLUSIONS: Disease presentations and laboratory findings vary significantly between age groups within a national cohort of JSLE patients. Patients diagnosed during adolescence exhibit greater disease activity and "classic" autoantibody, immune cell and complement patterns when compared with younger patients. This supports the hypothesis that pathomechanisms may vary between patient age groups.

A new generation IRMA for ACTH with improved specificity: validation in various physiological and pathological conditions.

OBJECTIVE: Measurement of plasma ACTH is a key step for the exploration of hypothalamic-pituitary-adrenal disorders. To further improve ACTH recognition a new generation of ACTH IRMA was developed using antibodies directed towards succinylated ACTH (sACTH IRMA). DESIGN: The usefulness of this assay was compared with that of another commercially available ACTH IRMA assay using intact ACTH (ELSA-ACTH) in various pathophysiological situations: patients with low ACTH plasma levels, high ACTH plasma levels with normal or tumoural pituitaries, or ectopic ACTH syndrome, and pregnant women with high proopiomelanocortin (POMC) plasma levels. METHODS: All plasma samples were assayed simultaneously with the two different IRMAs. Comparisons were assessed by plotting the results along the theoretical line of identical values, and by the graphical method of Bland and Altman. RESULTS: In the ELSA-ACTH IRMA, CLIP (or ACTH18-39) showed true cross-reactivity, and alpha-melanocyte-stimulating hormone and purified POMC both interfered and induced falsely lower ACTH results; in the sACTH IRMA no peptide showed any cross-reactivity, and only extremely high values of CLIP (50 000 pg/ml) interfered and induced falsely lower ACTH results. In ACTH hypersecretory syndromes, of tumoural (Cushing's disease, ectopic ACTH secretion) or non-tumoural (Addison's disease, congenital adrenal hyperplasia) origins a good agreement between the two assays was observed except for very high ACTH plasma values (above 1000 pg/ml) and in some tumours where the sACTH IRMA yielded lower results; in some cases, the presence of circulating CLIP, demonstrated by HPLC studies, may contribute to this discrepancy. It is also likely that the calibration of the ELSA-ACTH kit itself generates higher ACTH values. In normal pregnant women both IRMAs gave highly correlated values, yet lower results were obtained with the sACTH IRMA. CONCLUSION: These data show that the sACTH IRMA has improved qualities of specificity and usefulness for rapid assessment of ACTH plasma levels.

Loss of expression of the ubiquitous transcription factor cAMP response element-binding protein (CREB) and compensatory overexpression of the activator CREMtau in the human adrenocortical cancer cell line H295R.

The pituitary hormone ACTH, acting through the cAMP pathway, plays a key role in proliferation and differentiation of the adrenal cortex. CAMP response element (CRE)-binding protein (CREB) is an ubiquitous transcription factor that binds to the CRE present in the promoter of numerous genes and mediates transcription stimulation by cAMP. Characterization of CRE-binding proteins was performed in the H295R cell line, which is considered a model for human adrenocortical tumor studies. Western blot and RT-PCR studies demonstrated that CREB is not expressed in the human adrenocortical cancer cell line H295R, whereas it is expressed in normal adrenal. During transient transfection experiments, cAMP stimulation of two reporter genes containing canonical CRE was maintained. Cotransfection of the dominant negative inhibitor A-CREB, which prevents transcription factors containing a CREB-like leucine zipper domain to bind DNA, completely inhibited cAMP-induced stimulation of CRE activity. Western blot and RT-PCR studies showed that activating transcription factor-1 (ATF-1), CRE modulator-alpha/gamma (CREMalpha/gamma), and CREMtau2alpha are expressed in H295R cells. High amounts of CREM proteins were present in H295R, demonstrating an overexpression of this transcription factor in the absence of CREB. Furthermore, expression of the activator isoform CREMtau was very high in H295R compared to normal adrenal cortex. Transfection assays demonstrated that CREMtau2alpha is a potent stimulator of CRE activity in H295R. Finally, gel retardation assays showed that CREM and ATF-1 are the nuclear proteins that specifically bind the CRE in H295R cells, whereas CREM binding to CRE is not observed in a CREB-expressing cell line. H295R cells are the first established nontransgenic cell line that does not express the ubiquitous transcription factor CREB. H295R demonstrates that CREMtau up-regulation can compensate for CREB deficiency to maintain CRE regulation by cAMP and is a model of compensation mechanisms between the members of the CREB/ CREM/ATF-1 family of transcription factors. This loss of CREB expression and the overexpression of CREM could be linked to cellular transformation, as the normal adrenal cortex express high levels of CREB and no or low levels of CREMtau.

High precursor level in maternal blood results from the alternate mode of proopiomelanocortin processing in human placenta.

OBJECTIVE: ACTH-producing non-pituitary tumours are often associated with altered precursor processing, particularly in the most aggressive ones. Since placental tissue is characterized by its ability to express the proopiomelanocortin (POMC) gene and rapid cellular proliferation, we examined whether intact POMC could be released physiologically during human gestation. SUBJECTS: One hundred and fifty six normal pregnant women, 12 with multiple pregnancies, and 23 non-pregnant controls. Twenty-eight women were studied in the immediate postpartum period. MEASUREMENTS: We measured plasma POMC levels with a specific immunoradiometric assay (IRMA) using a combination of antibodies directed against ACTH and beta endorphin. Results obtained with this first IRMA were confirmed in 22 subjects with a second assay using the same beta endorphin antibody and a more distal antibody directed against the N-terminal fragment of POMC. Reverse transcription-PCR detected full length, pituitary-like, POMC mRNA in human placenta. RESULTS: Plasma POMC was undetectable (< 60 U/ml) in 23 normal subjects. In normal monofetal pregnancies, POMC became detectable in most women by the third month and then increased steadily until midgestation: 168 +/- 108 (U/ml; mean +/- SD) between 12 and 15 weeks, 190 +/- 103 between 16 and 19 weeks, 324 +/- 180 between 20 and 23 weeks, 276 +/- 171 between 24 and 27 weeks, 292 +/- 177 between 28 and 31 weeks, 290 +/- 235 between 32 and 35 weeks and 308 +/- 210 between 36 weeks and parturition. Plasma POMC was significantly higher in multiple pregnancies with very high levels in three triplet-bearing mothers: 671, 941, and 1731 U/ml at 31, 33 and 32 weeks, respectively. POMC levels felt quickly in post partum, becoming undetectable in five of 13 women on day 1, seven of eight on day 2 and five of six on day 3. Plasma POMC displayed no diurnal variation, was not suppressed by glucocorticoid administration and did not correlate with plasma ACTH or cortisol. In contrast, plasma POMC positively correlated with plasma CRH. CONCLUSIONS: Pregnancy is the only condition in which POMC is produced and released physiologically, similar in some respects to the ectopic ACTH syndrome. POMC is derived solely from the placenta, with no interference from maternal pituitary secretion, and is thus a new and specific placental marker.

Regulated expression of mature human insulin in the liver of transgenic mice.

Transgenic mice expressing either human proinsulin cDNA or mutated proinsulin cDNA in the liver were created. The human proinsulin cDNA was mutated to generate a protein cleavable by the ubiquitous prohormone convertase furin, thus leading to mature insulin peptide. All transgenic lines expressed human C-peptide in the blood, whose level varied according to nutritional conditions. High performance liquid chromatography fractionation of mouse serum revealed that mutant proinsulin was effectively processed into mature insulin in vivo. This transgenic mouse model provides a useful tool for further prospects of gene therapy of insulin-dependent diabetes mellitus.

High plasma proopiomelanocortin in aggressive adrenocorticotropin-secreting tumors.

A specific propiomelanocortin (POMC) immunoradiometric assay was developed using antibodies directed against ACTH and beta-endorphin (beta end). Partially purified standard POMC was prepared from the human small cell lung carcinoma cell line DMS-79 culture medium. Ten units (U) POMC had the same displacement ability as one pg beta end in a C-terminal beta end radioimmunoassay and thus were close if not equal to 10 pg POMC. This POMC assay was used to investigate patients with ACTH-dependent Cushing's syndrome. Plasma POMC was undetectable (< 60 U/mL) in 17 normal controls and in 4 patients with Addison's disease (concomitant ACTH plasma levels between 362 and 1058 pg/mL). Forty-two patients with Cushing's disease were studied, either before (n = 25) or after (n = 17) bilateral adrenalectomy: 7 patients with highly invasive macroadenomas had high POMC plasma levels, between 240 and 4200 U/ml (concomitant ACTH plasma levels between 77 and 5730 pg/mL); 35 patients, including one with an invasive macroadenoma, had undetectable POMC plasma levels (concomitant ACTH plasma levels between 31 and 2820 pg/mL). Among 20 patients with histologically proven ectopic ACTH syndrome, 16 had high POMC plasma levels, between 80 and 8000 U/mL (concomitant ACTH plasma levels between 45 and 9265 pg/mL); all those tumors were malignant, and the highest POMC/ACTH plasma levels ratios (taken as an index of altered POMC processing) were observed in the 3 patients with small cell carcinomas of the lung; in one of these patients, ACTH and POMC plasma levels both decreased during the course of chemotherapy, in parallel with the reduction of the tumoral mass. Four patients with ectopic ACTH syndrome had undetectable POMC plasma levels (concomitant ACTH plasma levels between 78 and 335 pg/mL): they were all typical bronchial carcinoids. These data show that high POMC plasma level is neither specific for nor constant in ectopic ACTH syndrome. Rather it should be considered as a marker of tumor aggressivity, in pituitary- and non-pituitary tumors. Its diagnostic help appears limited for the most frequent cause of occult ectopic ACTH syndrome, the typical bronchial carcinoids.

Phosphorylated forms of adrenocorticotropin and corticotropin-like intermediary lobe peptide in human tumors.

Many peptides contribute to the heterogeneity of immunoreactive adrenocorticotropin (ACTH) in man. The use of a radioimmunoassay (RIA) specifically directed against the C-terminal end of ACTH allowed us to study precisely the following four peptides: ACTH itself, corticotropin-like intermediary lobe peptide (CLIP) or ACTH (18-39) and their phosphorylated forms on Ser31. We have set up a high-performance liquid chromatography system that separates these four molecules in a single run, to establish their relative distributions in tumors responsible for Cushing's disease or for the ectopic ACTH syndrome, and to evaluate the possible interference of phospho-Ser31 on various RIA or immunoradiometric assay (IRMA) recognition systems for ACTH. In this system, alkaline phosphatase treatment shifted the retention time of the phosphorylated peptides to that of their non-phosphorylated counterparts. In three tumors responsible for the ectopic ACTH syndrome, CLIP peptides were predominant in two and phosphorylated molecules represented between 22% and 50% of immunoreactive materials. In five pituitary tumors responsible for Cushing's disease, ACTH peptides were predominant and the phosphorylated molecules varied between 35% and 75% in four of them. In the same tumor the ratios of phosphorylated to non-phosphorylated CLIP or ACTH were identical. The presence of phospho-Ser31 did not affect the recognition ability of two mid-ACTH and two C-terminal ACTH RIAs, nor of the ACTH IRMA (Allegro, Nichols).

Pituitary-like proopiomelanocortin transcripts in human Leydig cell tumors.

Proopiomelanocortin is a polypeptide precursor molecule, the processing of which generates ACTH, beta-endorphin, the beta- and gamma-lipotropins, the joining peptide, and the NH2-terminal fragment. Anterior pituitary corticotrophs are the major site of proopiomelanocortin gene expression in man and the predominant, if not sole source of circulating ACTH. Recent data have established that proopiomelanocortin gene expression also occurs in various normal nonpituitary tissues, one of the best studied being the testis. In this latter organ the dominant gene products are short transcripts of approximately 800 nucleotides, which lack the first two exons of the gene and cannot encode a complete proopiomelanocortin molecule. In this report we show that the mode of proopiomelanocortin gene expression is occasionally modified in human Leydig cell tumors: a 1,200-nucleotide mRNA species identical to that in the pituitary is produced. It results from the usual (pituitary) start site of transcription and thus can encode the complete proopiomelanocortin molecule. In two out of six tumors, large amounts of the 1,200-nucleotide transcript led to a dramatic increase of approximately 1,000-fold in proopiomelanocortin peptide concentrations as compared with the normal and peritumoral testis. Proopiomelanocortin processing in these tumors generates various peptide fragments including ACTH. These results may help to understand the mechanism of proopiomelanocortin expression in nonpituitary tumors and have implications for the more general phenomenon of ectopic hormone secretion.

Corticotrophin-like intermediary lobe peptide as a marker of alternate pro-opiomelanocortin processing in ACTH-producing non-pituitary tumours.

In order to evaluate which of human (h) corticotrophin-like intermediary lobe peptide (CLIP) or h beta-melanocyte stimulating hormone5-22 (h beta MSH5-22) was the better marker of alternate pro-opiomelanocortin (POMC) processing, both peptides were simultaneously sought in the same tissue extracts from a normal human pituitary, six corticotrophic adenomas, and four non-pituitary tumours responsible for an ectopic ACTH syndrome. Human CLIP was detected using a combination of gel exclusion chromatography and two different radioimmunoassays (RIAs): a mid-ACTH RIA which recognized ACTH but not CLIP, and a COOH-ACTH RIA which recognized both molecules. Human beta MSH5-22 had been measured previously. Neither hCLIP nor h beta MSH5-22 were detected in the normal or tumoural pituitaries. The four non-pituitary tumours, in contrast, contained both peptides; the hCLIP and h beta MSH5-22 ratios (CLIP/CLIP + ACTH and h beta MSH5-22/h beta MSH5-22 + h gamma LPH) ranged from 40 to 94% and from 24 to 46%, respectively. In a given tissue the hCLIP ratio was always higher than the h beta MSH5-22 ratio. hCLIP is therefore the better marker of alternate POMC processing.

Microsequencing evidence for the maturation of human proopiomelanocortin into an 18 amino acid beta-melanocyte stimulating hormone [h beta MSH(5-22)] in nonpituitary tissue.

Sixty pmoles of a material with molecular size, immunological, and RP-HPLC characteristics identical to that of h beta MSH(5-22) were purified from a bronchial carcinoid tumor responsible for the ectopic ACTH syndrome. The first 16 cycles of microsequencing revealed the following sequence: Asp-Glu-Gly-Pro-Tyr-Arg-Met-Glu-X-Phe-Arg-Trp-Gly-X-Pro- Pro-, identical to the first 16 amino acids of h beta MSH(5-22). Since this material was recognized by an antibody which requires the free COOH-terminal Asp22 residue, it can be assumed that it is indeed h beta MSH(5-22). We also show that neither the 5 N acetic acid nor the 1 N HCl extraction procedure artefactually generated h beta MSH-like material in normal or tumoral human pituitaries and in nonpituitary tumors. We conclude that h beta MSH(5-22) is a normal maturation product of proopiomelanocortin in the human nonpituitary tissues which express its gene, including the hypothalamus and ACTH-secreting tumors.

Human beta-melanocyte-stimulating hormone revisited.

It is generally accepted that human beta-melanocyte-stimulating hormone (h beta MSH) does not normally exist in humans but was merely an artifactually generated 22-amino acid peptide corresponding to a lipotropin (LPH) fragment (residues 35-56). We examined whether the shorter 18-amino acid peptide h beta MSH-(5-22) could be detected in some human tissues. Normal human pituitaries and hypothalami as well as corticotropin-secreting pituitary and nonpituitary tumors were extracted and chromatographed on Sephadex G-50, and the fractions were measured with two radioimmunoassays using either a COOH-terminal human gamma LPH (h gamma LPH) antiserum that recognized equally h gamma LPH, h beta MSH, and h beta MSH-(5-22) or a mid-portion h gamma LPH antiserum that recognized h gamma LPH and h beta MSH but not h beta MSH-(5-22). Normal pituitaries and pituitary tumors contained a single immunoreactive material coeluting with h gamma LPH. The hypothalami and the nonpituitary tumors all contained h gamma LPH and a smaller molecular weight material that was only detected in the COOH-terminal h gamma LPH radioimmunoassay; its elution volume (Ve/V, 0.75) was identical to that of h beta MSH-(5-22) but different from that of h beta MSH (Ve/V, 0.60); on reversed-phase HPLC, it coeluted with synthetic h beta MSH-(5-22) with a retention time different from that of h beta MSH. It is concluded that h beta MSH-(5-22) that corresponds to the 18-amino acid peptide h beta LPH-(39-56), flanked by two pairs of basic amino acids within the h beta LPH molecule, is a normal maturation product of proopiomelanocortin in human nonpituitary tissues.

Plasma and urinary melatonin in male infants during the first 12 months of life.

Plasma and urinary melatonin, testosterone and luteinizing hormones were radioimmunologically assayed in 26 male babies during the first year of life. The results show that plasma melatonin levels are low during the phase of postnatal elevation of testosterone and luteinizing hormones. They subsequently increase when testicular activity decreases. Urinary elimination of melatonin does not vary during this period, suggesting the existence of variations in the synthesis or utilization of melatonin.

Determination of blood urea by gas-liquid chromatography. A comparison with two other methods.

An original method for the quantitative determination of plasmatic urea by gas-liquid chromatography (GLC) is described. It is based upon the transformation of urea into urethane by alcoholic deamination in a warm and strictly anhydrous medium. Compared with the two other usual methods (enzymatic and colorimetric), the GLC technique is extremely reliable and specific, and can easily be adapted to biological fluids.