CHK1 overexpression in T-cell acute lymphoblastic leukemia is essential for proliferation and survival by preventing excessive replication stress.

Checkpoint kinase 1 (CHK1) is a key component of the ATR (ataxia telangiectasia-mutated and Rad3-related)-dependent DNA damage response pathway that protect cells from replication stress, a cell intrinsic phenomenon enhanced by oncogenic transformation. Here, we show that CHK1 is overexpressed and hyperactivated in T-cell acute lymphoblastic leukemia (T-ALL). CHEK1 mRNA is highly abundant in patients of the proliferative T-ALL subgroup and leukemia cells exhibit constitutively elevated levels of the replication stress marker phospho-RPA32 and the DNA damage marker γH2AX. Importantly, pharmacologic inhibition of CHK1 using PF-004777736 or CHK1 short hairpin RNA-mediated silencing impairs T-ALL cell proliferation and viability. CHK1 inactivation results in the accumulation of cells with incompletely replicated DNA, ensuing DNA damage, ATM/CHK2 activation and subsequent ATM- and caspase-3-dependent apoptosis. In contrast to normal thymocytes, primary T-ALL cells are sensitive to therapeutic doses of PF-004777736, even in the presence of stromal or interleukin-7 survival signals. Moreover, CHK1 inhibition significantly delays in vivo growth of xenotransplanted T-ALL tumors. We conclude that CHK1 is critical for T-ALL proliferation and viability by downmodulating replication stress and preventing ATM/caspase-3-dependent cell death. Pharmacologic inhibition of CHK1 may be a promising therapeutic alternative for T-ALL treatment.

Effects of a moderate physical training on the leptin synthesis by adipose tissue of adult rats submitted to a perinatal low-protein diet.

The aim of the study was to verify if moderate physical training affects leptin content in visceral and subcutaneous adipose tissue of adult rats subjected to a low-protein diet during the perinatal period. Male Wistar rats were divided into 2 groups according to their mother's diet during gestation and lactation: control (17% casein, C, n=12) and low-protein (8% casein, LP, n=12). On postnatal day 60, half of each group was submitted to moderate physical training (8 wks, 5 d · wk - 1, 60 min · d - 1, at 70% of VO2max, T) or not. After the physical training period, visceral and subcutaneous adipose tissues were removed. Leptin content was evaluated by western blotting. Starting from the fifth week on, T pups showed a reduction in the body weight. Similarly, LP+T offspring showed a lower body weight starting from the sixth week on. Western blotting analysis showed that leptin content in the visceral tissue was higher in the LP rats (p<0.01) and it was reversed in LP+T. No difference was found in the subcutaneous tissue. Moderate physical training attenuated the effects of a perinatal low-protein diet on the leptin content in visceral adipose tissue in adult offspring.

Effective treatment of rat adjuvant-induced arthritis by celastrol.

We have previously reported an increase in interleukin (IL)-1ß and IL-17 levels, and a continuous activation of caspase-1 in early rheumatoid arthritis (RA) patients. These results suggest that drugs targeting IL-1ß regulatory pathways, in addition to tumor necrosis factor (TNF), may constitute promising therapeutic agents in early RA. We have recently used a THP-1 macrophage-like cell line to screen 2320 compounds for those that down-regulate both IL-1ß and TNF secretion. Celastrol was one of the most promising therapeutic candidates identified in that study. Our main goal in the present work was to investigate whether administration of celastrol is able to attenuate inflammation in a rat model of adjuvant-induced arthritis (AIA). Moreover, since IL-1ß is known to play a role in the polarization of Th17 cells, we also investigate whether administration of digoxin, a specific inhibitor of Th17 cells polarization, is able to attenuate inflammation in the same rat model. We found that celastrol administration significantly suppressed joint inflammation. The histological and immunohistochemical evaluation revealed that celastrol-treated rats had a normal joint structure with complete abrogation of the inflammatory infiltrate and cellular proliferation. In contrast, we observed that digoxin administration significantly ameliorated inflammation but only if administrated in the early phase of disease course (after 4days of disease induction), and it was not efficient at inhibiting the infiltration of immune cells within the joint and in preventing damage. Thus, our results suggest that celastrol has significant anti-inflammatory and anti-proliferative properties and can constitute a potential anti-inflammatory drug with therapeutic efficacy in the treatment of immune-mediated inflammatory diseases such as RA. Furthermore, we find that early inhibition of Th17 cells polarization ameliorates arthritis but it is not as effective as celastrol.

Conserved role of a complement-like protein in phagocytosis revealed by dsRNA knockout in cultured cells of the mosquito, Anopheles gambiae.

We characterize a novel hemocyte-specific acute phase glycoprotein from the malaria vector, Anopheles gambiae. It shows substantial structural and functional similarities, including the highly conserved thioester motif, to both a central component of mammalian complement system, factor C3, and to a pan-protease inhibitor, alpha2-macroglobulin. Most importantly, this protein serves as a complement-like opsonin and promotes phagocytosis of some Gram-negative bacteria in a mosquito hemocyte-like cell line. Chemical inactivation by methylamine and depletion by double-stranded RNA knockout demonstrate that this function is dependent on the internal thioester bond. This evidence of a complement-like function in a protostome animal adds substantially to the accumulating evidence of a common ancestry of immune defenses in insects and vertebrates.

Reinnervation of post-arthritic joints in the rat.

OBJECTIVE: To clarify the role of articular innervation during the acute and post-arthritic phases, we investigated the changes taking place over time in the nerve fibers from joint tissues of adjuvant arthritic rats. METHODS: Nerve densities (i.e., the number of nerve fibers observed per unit area of tissue section) were estimated in periarticular tissues, synovium and epiphysis from ankle joints. Nerves were identified by immunofluorescence microscopy using antibodies against general neuronal markers (Protein Gene Product 9.5, synaptophysin, neurofilaments and Schwann cells), and markers specific for either sensory or sympathetic nerves (Substance P, calcitonin gene related peptide, neuropeptide tyrosine and its C-terminal flanking peptide, and the catecholamine synthesising enzyme tyrosine hydroxilase). RESULTS: In arthritic rats, the density of nerve fibers in the periarticular tissues, epiphysis and synovium was significantly reduced when compared to control animals. This decrease was observed using antibodies for both non releasable neuronal products (Protein Gene Product 9.5, synaptophysin, neurofilaments and Schwann cells) and neuropeptides (Substance P, calcitonin gene related peptide, neuropeptide tyrosine and its C-terminal flanking peptide), thus suggesting the existence of a structural nervous lesion with the parallel depletion of peptides. As the clinical arthritis subsided, there was a progressive reinnervation of all the articular structures analysed, which often exceeded the density of nerves in the control animals. CONCLUSION: This work supports a close relationship between the nervous system and arthritis. We propose that cycles of nerve destruction and regeneration may be related to the characteristic periods of remission and activity of some forms of chronic arthritis.

[Diffuse tracheo-bronchial amyloidosis]. / Amiloidose traqueo-brônquica difusa.

The case of a 52 year old man, whose initial clinical manifestations were dyspnea, bloodstained sputum and malaise is reported. After the initial cancer hypothesis, a diagnosis of diffuse primary tracheo-bronchial amyloidosis was made. The amyloid substance present was not of A A type and the plasma cells next to the deposits were polyclonal. The piece-meal removal of the masses by bronchoscopy led to profuse bleeding. The patient died with sepsis. The clinical, pathological and therapeutical aspects of lower respiratory tract amyloidosis are reviewed.