Inflammatory agents partially explain associations between cortical thickness, surface area, and body mass in adolescents and young adulthood.

BACKGROUND/OBJECTIVES: Excessive body mass index (BMI) has been linked to a low-grade chronic inflammation state. Unhealthy BMI has also been related to neuroanatomical changes in adults. Research in adolescents is relatively limited and has produced conflicting results. This study aims to address the relationship between BMI and adolescents' brain structure as well as to test the role that inflammatory adipose-related agents might have over this putative link. METHODS: We studied structural MRI and serum levels of interleukin-6, tumor necrosis factor alpha (TNF-α), C-reactive protein and fibrinogen in 65 adolescents (aged 12-21 years). Relationships between BMI, cortical thickness and surface area were tested with a vertex-wise analysis. Subsequently, we used backward multiple linear regression models to explore the influence of inflammatory parameters in each brain-altered area. RESULTS: We found a negative association between cortical thickness and BMI in the left lateral occipital cortex (LOC) and the right precentral gyrus as well as a positive relationship between surface area and BMI in the left rostral middle frontal gyrus and the right superior frontal gyrus. In addition, we found that higher fibrinogen serum concentrations were related to thinning within the left LOC (ß = -0.45, p < 0.001), while higher serum levels of TNF-α were associated to a greater surface area in the right superior frontal gyrus (ß = 0.32, p = 0.045). Besides, we have also identified a trend that negatively correlates the cortical thickness of the left fusiform gyrus with the increases in BMI. It was also associated to fibrinogen (ß = -0.33, p = 0.035). CONCLUSIONS: These results suggest that adolescents' body mass increases are related with brain abnormalities in areas that could play a relevant role in some aspects of feeding behavior. Likewise, we have evidenced that these cortical changes were partially explained by inflammatory agents such as fibrinogen and TNF-α.

[Presentation of two cases of Crouzon syndrome: allelic cranio-stenotic conditions of FGFR genes]. / Síndrome de Crouzon: a propósito de 2 casos. Entidades craneoestenóticas alélicas de los genes FGFR.

INTRODUCTION: Craniosynostosis is an abnormal and premature fusion of any cranial suture. Twenty per cent of them involve any specific syndrome with Mendelian transmission; the other 80% are "non syndromic", although but 10-14% of them are genetically transmitted. Using the experience of two patients with Crouzon syndrome, a clinical and genetic review is performed. PATIENTS AND METHODS: Patient 1: girl of 35 days of age with progressive macrocephaly, protrusion of fontanel, ocular proptosis, hypertelorism and divergent strabismus. Cranial RX with sagittal synostosis. Surgical operation was performed with 3 months and 8 months of age due to development of pansynostosis. Patient 2: boy of 3 years 8 months of age with headaches of migrainous type of one year onset. He had acanthosis nigricans. Cranial RX and cerebral CT with evident digital markings and fundus of eye with undefined papillary limits, but 18 month later oedematous papilla were evident and pansynostosis was detected, so surgery was performed. RESULTS: We present a patient with classical Crouzon syndrome (patient 1) and another with acanthosis nigricans (patient 2), both diagnosed by the description of characteristic clinical features. CONCLUSIONS: Ten craniosynostotic clinical forms are currently known as allelic variations of the FGFR genes, and as such have reviewed them. As in our two cases, in syndromic types is very important the accurate study of the phenotype to orientate the diagnosis, although the molecular study will confirm it in many patients and genetic counselling offered.

[Ovarian granulosa cell tumors: an unusual cause of precocious thelarche]. / Tumor ovárico de la granulosa: causa infrecuente de telarquia prematura.

Precocious thelarche usually results from a physiological process but can sometimes be the first sign of precocious pseudopuberty. Ovarian granulosa cell tumors are highly unusual in childhood, appearing as precocious puberty in most prepuberal patients. During adolescence these tumors may cause menstrual irregularities, virilization and abdominal pain. Their malignancy is low and surgical treatment is usually curative if the tumors are limited to the ovaries. More advanced stages require chemotherapy, are difficult to cure and produce high mortality. We present the case of a 16-month-old girl with a granulosa cell tumor who presented with progressive precocious thelarche over 1 month that was satisfactorily resolved after resective surgery. This case demonstrates that other causes of puberal development should be investigated when precocious thelarche with fast progression is observed, with special attention paid to tumoral disease in the differential diagnosis.

Identification of an import signal for, and the nuclear localization of, human lactoferrin.

Many different unique functions have been attributed to lactoferrin (Lf), including DNA and RNA binding, and transport into the nucleus, where Lf binds to specific sequences and activates transcription. A pentapeptide, Gly-Arg-Arg-Arg-Arg, corresponding to a region of the N-terminal portion of human Lf rich in basic amino acids, was synthesized and its intracellular localization was investigated. Peptide internalization was assayed using the rhodaminated form of the same molecule. This N-terminal peptide sequence is able to be internalized within less than 10 min at concentration as low as 1 microM, and its intracellular localization is nuclear, mainly nucleolar. Similar behaviour was observed using peptides composed of either all l or d amino acids, the last one being a retro-inverse peptide. The internalization process does not involve an endocytotic pathway, since no inhibition of the uptake was observed at 4 degrees C. The kinetics of peptide internalization was also evaluated. The internalization properties of such a short Lf pentapeptide have been assayed for its ability to transport peptide nucleic acids (PNAs) inside cells in order to improve their efficacy. The abundant transmembrane transport and nuclear localization of the proposed peptide, deriving from hLf and, for the first time, identified as a nuclear localization signal, could be used as an alternative strategy to tackle the unsolved problem of intracellular accumulation of antisense and antigene drugs and for the development of new pharmacological tools.

Granulocyte-macrophage colony-stimulating factor activity in cerebrospinal fluid.

OBJECTIVES: The purpose of this study was to analyse the presence of the granulocyte-macrophage colony-stimulating factor (GM-CSF) in human cerebrospinal fluid (SF) of patients affected by multiple sclerosis (MS) in comparison with non-inflammatory neurological diseases. MATERIAL AND METHODS: All SFs were collected from 59 patients for diagnostic purpose. The presence of GM-CSF was revealed by measuring its activity and by immunoassay. The data obtained were statistically evaluated. RESULTS: We found that GM-CSF is constitutively present in human SF; this presence was confirmed by its stimulating activity of colony-forming-unit granulocyte-macrophage (CFU-GM) production. No significant changes of the GM-CSF concentration in the SFs were observed among different neurological disorders (degenerative or vascular) and MS. CONCLUSION: Our data suggest that GM-CSF is a constitutive component of human SF, relatively uninfluenced by the different morbid conditions of the nervous system.

Lactoferrin expression in human breast cancer.

We analyzed lactoferrin expression in 78 samples from patients with sporadic breast cancer and found 31/78 negative for mRNA expression. Similar results were obtained by immuno-histochemical localization of the lactoferrin protein. We did not find relationship between lactoferrin expression and clinical parameters. We investigated for the absent lactoferrin expression in some cases of breast cancer. In 68 of the samples analyzed, we found an inverse correlation between estrogen receptor expression and lactoferrin expression (P < 0,0001), thus indicating that regulation by the estrogen receptor is not the main element responsible for the expression of lactoferrin in breast cancer. Analysis of methylation of the lactoferrin genomic DNA extracted from the same patients revealed that the degree of methylation does not explain the observed absence of lactoferrin. The 937 bp lactoferrin promoter was investigated for possible mutations. By single-strand conformation polymorphism analysis one polymorphic site was found and characterized.

An upstream negative regulatory element in human granulocyte-macrophage colony-stimulating factor promoter is recognised by AP1 family members.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine involved in haematopoiesis and host defence. Production of GM-CSF has been detected in tumour cells including the U87MG astrocytoma cell line. Previous studies have been focused on the regulatory role of the proximal region of the GM-CSF promoter. Our studies on the distal region of the promoter in U87MG cells identify a negative cis element (-1377/-1298) which contains a AP1-like site able to bind c-jun and c-fos transcription factors, according to the results of DNA/protein binding assays. Mutagenesis of the AP1-like site eliminates AP1 binding and the negative effect on promoter activity.

Pancreatitis associated with pleural-mediastinal pseudocyst, panniculitis and polyarthritis.

We describe two patients with pancreatitis. One patient had acute pancreatitis of biliary origin and presented with small joint polyarthritis and panniculitis lesions. The other patient was originally hospitalised for dyspnoea with bilateral pleural effusion, and subsequently developed migratory polyarthritis. During his hospital stay he developed panniculitis lesions and a monoclonal IgG disorder of unknown significance. Very few patients with pancreatitis develop polyarthritis and panniculitis. The appearance of pseudocysts in the pleural and mediastinal cavity in the course of pancreatitis is an infrequent complication.

c-Rel and p65 subunits bind to an upstream NF-kappaB site in human granulocyte macrophage-colony stimulating factor promoter involved in phorbol ester response in 5637 cells.

To further clarify the complex transcriptional regulation of the human GM-CSF gene, which was extensively investigated in activated T cells, we have studied the role of an upstream NF-kappaB like site in the 5637 non-lymphoid cell line, which derives from a bladder carcinoma and constitutively produces GM-CSF. This sequence, named the A element, has an active role on GM-CSF transcription and is responsive to the tumor promoter PMA in transient transfection experiments. We describe here a heterodimeric binding complex of NF-kappaB subunits (c-Rel and p65) which is identical to the one obtained using the HIV-LTR-kappaB site as recognition sequence and different from the one (c-Rel and p50) observed with nuclear extracts from Mo T-lymphoid HTLV-II infected cells.

[Burkitt's lymphoma: atypical localization]. / Linfoma de Burkitt: Localización atípica.

We present a case of Burkitt's lymphoma, American type, with massive abdominal involvement, in a 11 years old boy, who presented with mucous-cutaneous paleness, cephalalgia and melaena. The localization of the lesion in the gallbladder and the rectum is stressed as being exceptional. We emphasize the importance of a rapid diagnosis to start early chemotherapy because this lesion grows rapidly. In our case after eight days of chemotherapy treatment there was a 75% reduction of the tumor mass.

An upstream positive regulatory element in human GM-CSF promoter is recognized by NF-kappa B/Rel family members.

To further extend the previous analysis of cis-acting elements and cognate trans-acting factors that contribute to GM-CSF transcriptional regulations, we have examined a promoter region between -1742 and -2010. DNase I footprinting assays showed four protected sequences named A, B, C and D. DNA transfections in the T-lymphoid Mo cell line, which constitutively expresses GM-CSF, indicated that the A element, located between -2002 and -1984, has a positive role on transcription. Further characterization by electrophoretic mobility shift assays in the presence of different competitor oligonucleotides showed that this element binds a factor of the NF-kappa B/Rel family.

Characterization of a distal 5'-flanking region (-2010/-630) of human GM-CSF.

The 5'-flanking region of the human GM-CSF gene was subcloned from the phagic clone lambda J1-16 to detect cis-elements involved in GM-CSF constitutive expression. We determined and sequenced an uncharacterized promoter region of 1381 bp (-2010 to -630), named pPF2000. Putative binding sites of several transcriptional factors were found. Progressive deletion mutants of the PF2000 were analyzed by measuring the linked CAT activities, in constitutive (5637) and inducible (PEU) GM-CSF-producing cells. A positive distal sequence (268 bp), between -2010 and -1742, responsible for the high constitutive expression of GM-CSF in 5637 carcinoma cell line was found.

Lactoferrin down-modulates the activity of the granulocyte macrophage colony-stimulating factor promoter in interleukin-1 beta-stimulated cells.

The human neutrophil lactoferrin (Lf), a cationic iron-binding glycoprotein, has an inhibitor role on granulocyte macrophage colony-stimulating factor (GM-CSF) production via interleukin-1 (IL-1). The nuclear localization of Lf suggests that it may be involved in the transcriptional regulation of GM-CSF gene expression. To explore this possibility, the effect of Lf on GM-CSF gene expression was investigated in various cell lines and in primary cultures of fibroblasts. Down-regulation of GM-CSF mRNA level was observed in Lf-transfected embryonic fibroblasts induced to produce GM-CSF by IL-1 beta. In 5637 cell-line and in embryonic fibroblasts, co-transfection experiments, in which an Lf expression vector was used together with a vector carrying a reporter gene linked to the GM-CSF promoter, revealed that Lf reduces the activity of the GM-CSF promoter. This effect is marked in IL-1 beta-stimulated cells. These findings suggest that Lf plays a negative role in GM-CSF expression at the transcriptional level, perhaps through the mediation of IL-1 beta.

Cytokine expression in human primary and metastatic melanoma cells: analysis in fresh bioptic specimens.

In recent years, several studies have documented that melanoma cell lines produce various cytokine/growth factors and their receptors. Since cell lines can acquire altered properties, such as changes in growth requirements, we studied constitutive cytokine gene expression in melanoma cells from 20 fresh surgical specimens: seven primary melanomas and 13 metastases (12 lymph-node metastases and one subcutaneous metastasis). After tumour cell isolation by discontinuous gradient, we tested for mRNA expression by means of reverse-transcriptase polymerase chain reaction. Most melanoma cells tested expressed growth factors: basic fibroblast growth factor (bFGF), interleukin (IL)1 alpha, IL-1 beta, IL-6 and IL-8 and, in five cases out of 20, expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) (two out of five were also positive for GM-CSF receptor). Our results do not point to a direct correlation between cytokine expression and clinical stage at the time when the bioptic specimen was obtained. However, they allow us to suggest a possible metastatic tumour cell phenotype, in which autogenous GM-CSF expression could modulate immune response against the tumour cell itself or could potentiate metastatic colonization properties.

H and L ferritin gene expression in U937 cells induced to macrophage differentiation.

Ferritin is an ubiquitous protein that has been shown to regulate cell differentiation in several experimental systems. In this study we have investigated the expression of ferritin genes encoding the heavy (H) and light (L) chains in t'B U937 cell line, induced to differentiate to macrophage-like cells by 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA) or 1-beta-D-arabinofuranosylcytosine (Ara-C). An increase in the level of H ferritin mRNA was detected in U937 cells that had been incubated with Ara-C. Treatment of U937 cells with Actinomycin D suggested that the H ferritin mRNA increase was mediated by post-transcriptional mechanisms. The L ferritin mRNA level increased only following stimulation of U937 cells with RA. Immunophenotypic and cytochemical analyses showed that Ara-C was the strongest inducer of the macrophagic differentiation of U937 cells. These results suggest that the increase of H ferritin mRNA expression may represent a sensitive marker of myeloid cells differentiating along the monocyte-macrophage lineage.

Lactoferrin binding sites and nuclear localization in K562(S) cells.

Lactoferrin, a single chain cationic glycoprotein, present in the secondary granules of neutrophils, acts as a negative feedback regulator of myelopoiesis. Specific receptors for lactoferrin were detected on the surface of different hematopoietic cell types. The influence of lactoferrin on cell growth in culture has been reported. Interactions of lactoferrin with DNA were also demonstrated. In the present paper we confirm the presence of lactoferrin specific binding sites on K562 cells and we estimate the number of binding sites and the dissociation constant. By Western blotting analysis performed on K562 lysates we find a band of about 120 kDa responsible for specific binding of lactoferrin. We also show that lactoferrin, after binding at the cell surface, is internalized in a temperature dependent way and is immunologically detectable as a DNA-linked protein in nuclear extracts.

Synthesis of a 60 kD nuclear DNA binding protein induced by cytosine arabinoside in the HL 60 leukemic cell line.

Cytosine arabinoside (ara-C) is being employed at low dosage as differentiative rather than a cytotoxic agent in the therapy of leukemias. We have analyzed nuclear proteins from HL 60 leukemic cells treated with ara-C and have observed increased expression of a 60 kD protein in a dose-dependent fashion. This protein is actively synthesized, as assessed by labeled methionine incorporation. Using DNA cellulose affinity chromatography we could also demonstrate DNA binding properties of the 60 kD protein.

Isolation and characterization of a K562 cell line resistant to 1-beta-D-arabinofuranosylcytosine-mediated erythroid induction.

The isolation of a K562 cell line, K562(S)R, resistant to 1-beta-D-arabinofuranosylcytosine (ara-C)-mediated erythroid induction, is described. Ara-C (10-50 microM) inhibits cell growth of K562(S)R cells but is not able to activate the program of erythroid induction. This failure is associated with the lack in the increase of accumulation of epsilon-globin and gamma-globin mRNA sequences in ara-C-treated K562(S)R cells. This cell line could be of interest for studies focused on molecular mechanisms of activation of globin genes in K562 cells.

Characterization, localization, and biosynthesis of acetylcholinesterase in K 562 cells.

K 562 cell acetylcholinesterase (AChE), identifiable by active site labeling with radioactive diisopropylfluorophosphate (DFP), showed a Mr around 55,000 in both a crude lysate and a purified sample. The K 562 AChE was reactive with one polyclonal and two monoclonal antibodies produced against human erythrocyte AChE. Subcellular localization, investigated by assay on cell fractions, showed that AChE is membrane bound and that it is located on the cell surface as well as on microsomal and Golgi membranes. Biosynthesis of new enzyme molecules, after inactivation of the constitutive AChE with the irreversible inhibitor DFP, allowed us to follow the kinetics of reappearance in the intracellular compartment and at the cell surface (4 and 8 h, respectively).