Site-dependent differences in both prelamin A and adipogenic genes in subcutaneous adipose tissue of patients with type 2 familial partial lipodystrophy.

BACKGROUND: Type 2 familial partial lipodystrophy (FPLD2) is characterised by loss of fat in the limbs and buttocks and results from mutations in the LMNA gene. AIM: To evaluate the role of several genes involved in adipogenesis in order to better understand the underlying mechanisms of regional loss of subcutaneous adipose tissue (scAT) in patients with FPLD2. METHODS: In total, 7 patients with FPLD2 and 10 healthy control participants were studied. A minimal model was used to calculate the insulin sensitivity (IS). scAT was obtained from abdomen and thigh by biopsy. Relative gene expression was quantified by real-time reverse transcription PCR in a thermal cycler. Prelamin A western blot analysis was carried out on scAT and prelamin A nuclear localisation was determined using immunofluorescence. Adipocyte nuclei were examined by electron microscopy. RESULTS: Patients with FPLD2 were found to have significantly lower IS. The expression of LMNA was similar in both groups. The expression of PPARG2, RB1, CCND3 and LPL in thigh but not in abdomen scAT was significantly reduced (67%, 25%, 38% and 66% respectively) in patients with FPLD2. Significantly higher levels of prelamin A were found in peripheral scAT of patients with FPLD2. Defects in the peripheral heterochromatin and a nuclear fibrous dense lamina were present in the adipocytes of patients with FPLD2. CONCLUSIONS: In FPLD2 participants, prelamin A accumulation in peripheral scAT is associated with a reduced expression of several genes involved in adipogenesis, which could perturb the balance between proliferation and differentiation in adipocytes, leading to less efficient tissue regeneration.

Prevalence of mutations in TSHR, GNAS, PRKAR1A and RAS genes in a large series of toxic thyroid adenomas from Galicia, an iodine-deficient area in NW Spain.

OBJECTIVE: Toxic thyroid adenoma (TA) is a common cause of hyperthyroidism. Mutations in the TSH receptor (TSHR) gene, and less frequently in the adenylate cyclase-stimulating G alpha protein (GNAS) gene, are well established causes of TA in Europe. However, genetic causes of TA remain unknown in a small percentage of cases. We report the first study to investigate mutations in TSHR, GNAS, protein kinase, cAMP-dependent, regulatory, type I alpha (PRKAR1A) and RAS genes, in a large series of TA from Galicia, an iodine-deficient region in NW Spain. DESIGN AND METHODS: Eighty-five TA samples were obtained surgically from 77 hyperthyroid patients, operated on for treatment of non-autoimmune toxic nodular goitre. After DNA extraction, all coding exons of TSHR, GNAS and PRKAR1A genes, and exons 2 and 3 of HRAS, KRAS and NRAS were amplified by PCR and sequenced. Previously unreported mutants were cloned in expression vectors and their basal constitutive activities were determined by quantification of cAMP response element (CRE)-luciferase activity in CO7 cells transfected with wild-type and mutant plasmids. RESULTS: TSHR gene mutations were found in 52 (61.2%) samples, GNAS gene mutations in 4 (4.71%) samples and no PRKAR1A or RAS mutations were found. Only three previously unreported mutations were found, two affecting the TSHR, A623F and I635V, and one affecting the G-protein alpha-subunit (Gsalpha), L203P. All mutant proteins showed higher CRE-luciferase activity than their wild-type counterparts. CONCLUSIONS: TA in a hyperthyroid population living in Galicia, a Spanish iodine-deficient region, harbours elevated frequencies of TSHR and GNAS mutations activating the cAMP pathway. However, the genetic cause of TA was undetermined in 34% of the TA samples.

Identification of molecular mechanisms related to nonthyroidal illness syndrome in skeletal muscle and adipose tissue from patients with septic shock.

OBJECTIVE: Septic shock is one of various causes of nonthyroidal illness syndrome (NTIS). In humans, the molecular mechanisms involved in NTIS are mostly unknown. The aim of this study was to investigate, in patients with NTIS secondary to septic shock, changes in the expression of genes involved in the actions of thyroid hormones and in the activity of deiodinase enzymes, in two tissues important for protein and energy metabolism, skeletal muscle (SM) and subcutaneous adipose tissue (SAT). DESIGN: Hospitalized patients were divided into a control and a septic shock NTIS group. MEASUREMENT: Serum collection for biochemical measurements, and SM and SAT biopsies for mRNA expression analysis of thyroid hormone receptors (THRB1, THRA1), retinoid X receptors (RXRA, RXRB, RXRG), nuclear receptor corepressor (NCOR1), silencing mediator of retinoid and thyroid hormone receptor (SMRT), steroid receptor coactivator (SRC1), type 1 and 2 deiodinases (D1, D2), monocarboxylate transporter 8 (MCT8), SECIS binding protein 2 (SBP2) and uncoupling protein 3 (UCP3) as well as D1, D2 and D3 enzyme activity measurements. RESULTS: The NTIS group had lower serum TSH, and free T3 and higher rT3 than controls. D1 and D3 were detected in SAT, with no differences found between the two groups; SM had very low D2 activity and again no differences were found between groups; D3 activity in SM was higher in NTIS than controls. SM expression of THRB1, RXRG and D2 was lower and RXRA higher in NTIS than controls. SAT from NTIS patients had lower MCT8, THRB1, THRA1, RXRG and SMRT, and higher UCP3 expression than controls. CONCLUSIONS: In patients with septic shock NTIS tissue responses are orientated to decrease production and increase degradation (muscle) or decrease uptake (adipose tissue) of T3, as well as to decrease thyroid hormone actions.

Alterations induced by chronic stress in lymphocyte subsets of blood and primary and secondary immune organs of mice.

BACKGROUND: The immune system is particularly sensitive to stress. Although acute stress generally has positive effects, chronic stress typically provokes immunosuppression. The elucidation of the mechanisms involved in immunosuppression are of interest for the design of therapeutic approaches to avoid the appearance of stress disorders. This study aimed to investigate chronic stress-induced alterations on lymphocyte subset distribution and percentages. The experiments were performed with C57BL/6 mice subjected to chronic immobilization stress. RESULTS: Stress caused a marked increase in apoptosis inside the thymus, and a reduction in the total number of thymocytes. Furthermore, the proportion of immature thymocytes declined significantly suggesting that the increased apoptosis mainly affected cells of immature phenotype. In blood, the total number of lymphocytes diminished but not all lymphocyte populations showed the same tendency: while the relative proportion of B cells declined slightly, the relative proportion of circulating CD3+ cells, and particularly some T cell subsets showing an immature phenotype (CD3+PNA+), increased under stress. The spleen and lymph nodes show a marked reduction in cellularity, but the relative proportion of T cells increased, while no change or only a slight reduction was observed in the relative proportion of B cells. Similarly, the relative proportion of T cells increased in bone marrow. CONCLUSIONS: Detailed data on the alterations of lymphoid cell subsets occurring under immobilization stress, both in the bloodstream and in different lymphoid tissues, are obtained. In general, T cells are more affected than B cells and, in particular, a marked increase in the percentage of a subset of circulating PNA+CD3+ T cells is observed.

Role of pre-T cells and chemoattractants on stress-associated thymus involution.

Male C57BL/6 mice were stressed by immobilization for 1, 2, 3, or 5 h per day for 14 days, with subsequent assessment of (a) thymic involution, (b) in vitro migration of stressed mice bone marrow cells toward thymocyte culture supernatants from neonates and from control or stressed mice, (c) composition of the bone marrow cell population, and (d) in vitro migration of normal bone marrow cells toward stressed mice thymocyte culture supernatants. The results obtained support the view that the reduced repopulation of thymus by precursor T cells contributes to thymus involution associated with stress. It is further shown that this effect could be owing to a reduction in the number of precursor T cells in the bone marrow, and/or to a diminished production of precursor T-cell chemoattractants.

Modulation of stress-induced murine lymphoid tissue involution by age, sex and strain: role of bone marrow.

C57BL/6 and Balb C male and female mice of various ages were stressed by immobilization for 1 h/day (1, 2, 3, 5, 8, 11 or 14 consecutive days). The animals were then killed for determination of total body weight and the weights of the thymus, spleen and axillary lymph nodes. In addition, the total number of cells in the thymus and the proportion of lymphoid cells in the bone marrow cell population was defined. The effects of stress were modulated by age, sex and strain. Stress-induced involution of the thymus was generally more pronounced in older animals, while for the spleen was the opposite. Involution of the thymus was higher in males than in females, but there were no marked differences between the sexes in the response of the spleen. In general C57BL/6 mice were more sensitive to stress than Balb C mice. However, for the involution induced by stress on lymph nodes there were not a clear trend with age, sex or strain. In male and female mice of all ages and both strains, stress led to statistically significant reductions in the absolute number of cells inside the thymus and spleen and in the proportion of lymphoid cells in the bone marrow.

Age-related changes in primary and secondary immune organs of the mouse.

The present work describes the murine immune tissue evolution with age with special emphasis on the bone marrow. To that effect we monitored the weights of the thymus, spleen and axillary lymph nodes over the first year of life in C57BL/6 male and female mice. In addition, we monitored the relative proportions of erythroid, lymphoid and myeloid cells in the bone marrow, and performed in vitro migration assays of bone marrow cells to thymic supernatants, with the aim of determining whether the migration of such cells or the thymic attractive capacity are affected by age. Before puberty, a remarkable decline in the relative weight of the thymus, spleen and lymph nodes was observed; after that stage, however, only the thymus showed an involution. The proportion of myeloid cells in the bone marrow showed an increase with age. Furthermore, the migration of myeloid cells to thymic supernatants increased with age and paralleled the time-course of the myeloid cell increase found in the bone marrow. More interestingly, the proportion of lymphoid cells to total bone marrow cells showed a clear decline with age. The time-course of this decline closely paralleled that of thymus weight, suggesting that the involution of the thymus may be related to changes in the cell composition of the bone marrow.

Time-course of the murine lymphoid tissue involution during and following stressor exposure.

Groups of 35-day-old male C57BL/6 mice were stressed 1 hour per day by immobilization for 1, 2, 3, 5, 8, 11 or 14 consecutive days. Control groups were left undisturbed. The animals were then killed and body weight and the weights of the thymus, spleen and axillary lymph nodes determined. Chronic immobilization stress caused involution of the thymus, spleen and lymph nodes to an extent depending on the number of days of stress. The thymus showed the fastest response: thymus weight was significantly lower in stressed animals than in controls by the third day of stress while significant effects on spleen and lymph node weight were not observed until day 5. Fast recovery of lymphoid organ weight was observed after the stress period. The thymus recovered most quickly: control values were re-attained approximately 8 days after cessation of stress, and indeed by day 20 thymus weight was about 12% higher than in normal animals. The spleen and lymph nodes recuperated weight more slowly, re-attaining control values after about 20 days.

Lymphocyte protein synthesis: evidence that murine T cells are more affected by stress than B cells.

B and T cells present in the spleen and other sites of the immune system play a crucial role in the protection of individuals by mounting a specific primary and secondary immune response. Disturbances in their signaling and functioning could lead to a deterioration of the defense mechanisms and thereby lead to infections, pathologies or diseases. In this work, we studied the effects of stress on the protein synthesis and metabolism of mouse splenocytes. The study was done by radiolabeling the entire mouse with 35S-methionine (in vivo procedure) or by culturing spleen cells under various conditions of stimulation in the presence of 35S-methionine (in vitro labeling). The stimulus was lipopolysaccharide (LPS), LPS + interleukin-4 (IL-4) and concanavalin A (on A). Samples from immobilization stressed and control animals were studied in parallel. The results showed minimum alterations due to stress on B cells, though a small decrease in proliferative capacity was observed. In contrast, T cells are profoundly affected by stress. More than 100 new proteins are expressed on 2D-gel patterns of T cells from stressed animals as compared with controls, some of which could be implicated in different signaling, or could have appeared because of an alteration in a pathway of synthesis, or even as a consequence of a change in the composition of T cell populations induced by stress.

Arteether, a new antimalarial drug: synthesis and antimalarial properties.

Arteether (6) has been prepared from dihydroquinghaosu (3) by etherification with ethanol in the presence of Lewis acid and separated from its chromatographically slower moving alpha-dihydroqinghaosu ethyl ether (7). The absolute stereochemistry at C-12 has been determined by 1H NMR data (J11,12, NOESY). Ethyl ethers 6 and 7 showed potent in vitro inhibition of Plasmodium falciparum, and both compounds were highly potent antimalarials in mice infected with a drug-sensitive strain of Plasmodium berghei. Crystalline arteether (6) and its oily epimer 7 were 2-3 times more potent schizontocides than quinghaosu (1), but deoxy compounds 8, 9, and 11 were 100-300 times less potent in vitro than their corresponding peroxy precursors. Pharmacological studies have shown arteether(6) to have antimalarial activity in animals comparable to artesunate (2) and artemether (4), both of which are fast-acting blood schizontocides in humans. Arteether (6) has now been chosen for a clinical evaluation in high-risk malaria patients.