Cancer-associated mesothelial cells are regulated by the anti-Müllerian hormone axis.

Cancer-associated mesothelial cells (CAMCs) in the tumor microenvironment are thought to promote growth and immune evasion. We find that, in mouse and human ovarian tumors, cancer cells express anti-Müllerian hormone (AMH) while CAMCs express its receptor AMHR2, suggesting a paracrine axis. Factors secreted by cancer cells induce AMHR2 expression during their reprogramming into CAMCs in mouse and human in vitro models. Overexpression of AMHR2 in the Met5a mesothelial cell line is sufficient to induce expression of immunosuppressive cytokines and growth factors that stimulate ovarian cancer cell growth in an AMH-dependent way. Finally, syngeneic cancer cells implanted in transgenic mice with Amhr2-/- CAMCs grow significantly slower than in wild-type hosts. The cytokine profile of Amhr2-/- tumor-bearing mice is altered and their tumors express less immune checkpoint markers programmed-cell-death 1 (PD1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4). Taken together, these data suggest that the AMH/AMHR2 axis plays a critical role in regulating the pro-tumoral function of CAMCs in ovarian cancer.

Direct renin inhibition improves parasympathetic function in diabetes.

AIM: The renin-angiotensin-aldosterone system (RAAS) and autonomic nervous system regulate the cardiovascular system. Blockade of the RAAS may slow the progression of end-organ damage. Direct renin inhibition offers a means for blocking the RAAS. The objective of this study was to examine the effect of direct renin inhibition on cardiovascular autonomic function. METHODS: In this double-blind, placebo-controlled trial, 60 individuals with diabetes were randomly assigned to 300 mg of aliskiren or placebo once daily for 6 weeks. The primary end point was a change in tests of cardiovascular autonomic function. Autonomic function was assessed by power spectral analysis and RR-variation during deep breathing [i.e. mean circular resultant (MCR), expiration/inspiration (E/I) ratio]. The MCR and E/I ratio assess parasympathetic function. Secondary measures included change in biochemical parameters [e.g. plasma renin activity, leptin and interleukin-6]. Change in cardiovascular autonomic function and blood analytes were analysed by a mixed effects model for repeated measures. RESULTS: Baseline characteristics were similar between treatment groups. In response to aliskiren compared with placebo, blood pressure was reduced as well as plasma renin activity [from 2.4 ± 3.8 (mean ± standard deviation) to 0.5 ± 0.4 µg/l/h, p < 0.001]. There was a significant interaction (aliskiren × visit) for MCR (p = 0.003) and E/I ratio (p = 0.003) indicating improvement in MCR and E/I ratio for those on aliskiren. MCR means, baseline vs. follow-up, were 41.8 ± 19.7 vs. 50.8 ± 26.1 (aliskiren) and 38.2 ± 23.6 vs. 37.5 ± 24.1 (placebo). CONCLUSIONS: Parasympathetic function (i.e. MCR and E/I ratio) was enhanced by downregulation of the RAAS.

Autonomic imbalance: prophet of doom or scope for hope?

It has long been recognized that cardiac autonomic neuropathy increases morbidity and mortality in diabetes and may have greater predictive power than traditional risk factors for cardiovascular events. Significant morbidity and mortality can now be attributable to autonomic imbalance between the sympathetic and parasympathetic nervous system regulation of cardiovascular function. New and emerging syndromes include orthostatic tachycardia, orthostatic bradycardia and an inability to use heart rate as a guide to exercise intensity because of the resting tachycardia. Recent studies have shown that autonomic imbalance may be a predictor of risk of sudden death with intensification of glycaemic control. This review examines an association of autonomic dysregulation and the role of inflammatory cytokines and adipocytokines that promote cardiovascular risk. In addition, conditions of autonomic imbalance associated with cardiovascular risk are discussed. Potential treatment for restoration of autonomic balance is outlined.

A detailed examination of cytokine abnormalities in Major Depressive Disorder.

Recent technological advances offer an opportunity to further elucidate the complex cytokine network in Major Depressive Disorder (MDD). Twenty cytokines were simultaneously assessed in 49 individuals with MDD and 49 age and gender matched controls. Multiple pro-inflammatory and two anti-inflammatory cytokines were significantly elevated in the MDD sample, including an antidepressant naïve subset. These data support a generalized chronic inflammatory state in MDD, and implicate additional cytokines and chemokines previously linked to cardiovascular disease.

Diminished lifespan and acute stress-induced death in DNA-PKcs-deficient mice with limiting telomeres.

An adequate and appropriate response to physiological and pathophysiological stresses is critical for long-term homeostasis and viability of the aging organism. Previous work has pointed to the immune system, telomeres and DNA repair pathways as important and distinct determinants of a normal healthy lifespan. In this study, we explored the genetic interactions of telomeres and DNA-PKcs, a protein involved in non-homologous end-joining (NHEJ) and immune responses, in the context of a key aspect of aging and lifespan--the capacity to mount an acute and appropriate immune-mediated stress response. We observed that the combination of DNA-PKcs deficiency and telomere dysfunction resulted in a shortened lifespan that was reduced further following viral infection or experimental activation of the innate immune response. Analysis of the innate immune response in the DNA-PKcs-deficient mice with short dysfunctional telomeres revealed high basal serum levels of tumor necrosis factor alpha (TNFalpha) and hyper-active cytokine responses upon challenge with polyinosinic-polycytidylic acid (poly-IC). We further show that serum cytokine levels become elevated in telomere dysfunctional mice as a function of age. These results raise speculation that these genetic factors may contribute to misdirected immune responses of the aged under conditions of acute and chronic stress.

K-ras activation generates an inflammatory response in lung tumors.

Activating mutations in K-ras are one of the most common genetic alterations in human lung cancer. To dissect the role of K-ras activation in bronchial epithelial cells during lung tumorigenesis, we created a model of lung adenocarcinoma by generating a conditional mutant mouse with both Clara cell secretory protein (CC10)-Cre recombinase and the Lox-Stop-Lox K-ras(G12D) alleles. The activation of K-ras mutant allele in CC10 positive cells resulted in a progressive phenotype characterized by cellular atypia, adenoma and ultimately adenocarcinoma. Surprisingly, K-ras activation in the bronchiolar epithelium is associated with a robust inflammatory response characterized by an abundant infiltration of alveolar macrophages and neutrophils. These mice displayed early mortality in the setting of this pulmonary inflammatory response with a median survival of 8 weeks. Bronchoalveolar lavage fluid from these mutant mice contained the MIP-2, KC, MCP-1 and LIX chemokines that increased significantly with age. Cell lines derived from these tumors directly produced MIP-2, LIX and KC. This model demonstrates that K-ras activation in the lung induces the elaboration of inflammatory chemokines and provides an excellent means to further study the complex interactions between inflammatory cells, chemokines and tumor progression.

Strength of medial structures of the knee joint are decreased by isolated injury to the medial collateral ligament and subsequent joint immobilization.

Past studies of the healing of the medial collateral ligament (MCL) in animal models have been conducted over a variety of healing intervals, some as early as 1 week. One concern with testing at early healing intervals is the difficulty in identifying and isolating the tissues that carry load. The purpose of this study was to determine if isolation of the MCL and healing time are critical factors in the assessment of structural strength in this model. Furthermore, the effect of immobilization on these critical factors was investigated. Our approach was to calculate the load-sharing ratio between the MCL and the MCL plus capsule. A 4 mm gap was created in the midsubstance of both hindlimb MCLs of 52 female New Zealand White rabbits (n=104). Of these, 29 rabbits had their right hindlimb pin immobilized (immobilized group), leaving the left hindlimb non-immobilized. Testing was performed at 3 (n=12), 6 (n=22), and 14 (n=24) weeks. The remaining 23 rabbits, which had both limbs non-immobilized (non-immobilized group), were tested at 3 (n=10), 6 (n=12), 14 (n=12), and 40 (n=12) weeks. For both groups, half of the specimens at each healing interval were used to test the MCL alone and half to test the MCL plus capsule, except for 3 week immobilized joints where only the MCL plus capsule was tested. Additionally, MCL (n=12), MCL plus capsule (n=6), and capsule alone (n=5) were tested from normal animals. The load-sharing ratio at MCL failure for the normal joint was 89%, suggesting an MCL-dominated response. For the non-immobilized group, the load-sharing ratio was 24% at 3 weeks of healing, suggesting a capsule-dominated response. At and after 6 weeks of healing, an MCL-dominated response was observed, with the ratio being 68% or greater. Thus, at less than 6 weeks of healing, the structural strength capabilities of the joint may be better represented by the medial structures rather than the isolated MCL. Immobilization delayed the transition from a capsule-dominated response to an MCL-dominated response in this model.

Do plasminogen activator inhibitor (PAI-1) or tissue plasminogen activator PAI-1 complexes predict complications in Type 1 diabetes: the Pittsburgh Epidemiology of Diabetes Complications Study.

AIMS: To examine the predictive power of plasminogen activator inhibitor-1 (PAI-1) and the complexes it forms with tissue plasminogen activator (tPA-PAI-1) for the two major Type 1 diabetes (T1D) complications (coronary artery disease (CAD) and overt nephropathy) in the context of standard risk factors. METHODS: Observational prospective study of 454 participants with childhood onset (< 17 years) T1D, aged 18+ years at baseline. PAI-1 and tPA-PAI-1 were determined using ELISA methodology. Follow-up (6 years) was limited to 382 individuals for CAD and 294 individuals for overt nephropathy, after excluding baseline cases. Total, HDL and LDL-cholesterol, triglycerides, HbA1, blood pressure, body mass index (BMI), waist-hip ratio (WHR), leucocyte count, Beck depression score and fibrinogen were also examined. RESULTS: The 56 incident cases of CAD had marginally lower PAI-1 and higher tPA-PAI-1 levels compared with those free of CAD. However, marginally higher PAI-1 and significantly higher tPA-PAI-1 (P = 0.04) levels were seen in those who developed nephropathy. After controlling for age, both PAI-1 and tPA-PAI-1 showed significant negative correlations with HDL-cholesterol, and positive correlations with triglycerides, WHR, HbA1 and fibrinogen. tPA-PAI-1 was also positively correlated with total and LDL-cholesterol. In multivariate analyses, neither PAI-1 nor tPA-PAI-1 was an independent predictor of CAD or overt nephropathy. CONCLUSIONS: These results suggest little association between PAI-1 and later CAD in patients with T1D. However, tPA-PAI-1 complexes may be involved in the pathogenesis of overt nephropathy.

Obesity is not a confounding factor for performing autonomic function tests in individuals with diabetes mellitus.

AIM: Cardiovascular autonomic neuropathy is a serious complication of diabetes mellitus. Previous studies have revealed conflicting results with regard to the role of obesity and its effect on the performance of tests (e.g. RR-variation during deep breathing) for the determination of the presence of cardiovascular autonomic dysfunction. The objective of this study was to determine if obesity affects the performance and the reproducibility of autonomic function tests. METHODS: This cross-sectional study included 159 diabetic individuals. Autonomic function tests included: RR-variation during deep breathing and the Valsalva ratio. These tests were assessed using the ANS2000 ECG Monitor and Respiration Pacer. RR-variation was measured by vector analysis (i.e. mean circular resultant, MCR). Reproducibility of the autonomic function tests was assessed by determining the coefficient of variation (CV) on repeat testing. RESULTS: Using cut-off points to describe normal weight (body mass index (b.m.i.) < or = 25 kg/m(2)), overweight (b.m.i. 25.01-30 kg/m(2)), obese (b.m.i. 30.01-40 kg/m(2)), and morbidly obese (b.m.i. > or = 40.1 kg/m(2)), no difference was found for the MCR, Valsalva ratio, CV of the MCR, or CV of the Valsalva ratio among the various weight levels for individuals with type 1 or type 2 diabetes. CONCLUSIONS: The results of this study indicate that obesity is not a confounding factor in the performance of autonomic function tests. Likewise, the reproducibility of autonomic function testing is not affected by obesity. Assessment of autonomic function is important for obese and non-obese individuals given that reduced RR-variation is associated with exercise intolerance, intraoperative cardiovascular lability and increased risk of mortality.

Isolation of Bcl-2 binding proteins that exhibit homology with BAG-1 and suppressor of death domains protein.

The Bcl-2 oncoprotein is a potent inhibitor of apoptosis and is overexpressed in a variety of different malignancies. Bcl-2 function is regulated through heterodimerization with other members of the Bcl-2 protein family. In addition, several proteins that are not members of the Bcl-2 family can bind to Bcl-2, including BAG-1 protein. In this study, we screened for proteins that bind to Bcl-2, and isolated two additional members of the BAG-1 protein family, BAG-3 and BAG-4. The BAG-4 protein that we cloned also corresponds to the recently isolated suppressor of death domains (SODD) protein, a molecule that binds and inhibits signaling by tumor necrosis factor receptor 1 (TNFR1). Both BAG-3 and BAG-4/SODD were found to physically associate with Bcl-2, and both proteins are well conserved from human to mouse. A region of homology, comprising 68 amino acids, is present in the carboxyl termini of BAG-3 and BAG-4/SODD, and this region corresponds with sequences termed BAG domains that are found in other members of the BAG-1 protein family. In BAG-3 and BAG-4/SODD, the BAG domains appear to constitute the Bcl-2 binding regions of these molecules. BAG-3 and BAG-4/SODD, like BAG-1, were also shown to bind to Hsp70 inside the cell. Moreover, BAG-3 overexpression modestly inhibited apoptosis resulting from cytokine deprivation of IL-3-dependent 32D cells. Together, our findings demonstrate that other members of the BAG-1 protein family, namely BAG-3 and BAG-4/SODD, bind to Bcl-2 and provide a potential link between pathways regulated by Bcl-2 and pathways regulated by Hsp70, as well as TNFR1.

Mre11 complex and DNA replication: linkage to E2F and sites of DNA synthesis.

We show that the Mre11 complex associates with E2F family members via the Nbs1 N terminus. This association and Nbs1 phosphorylation are correlated with S-phase checkpoint proficiency, whereas neither is sufficient individually for checkpoint activation. The Nbs1 E2F interaction occurred near the Epstein-Barr virus origin of replication as well as near a chromosomal replication origin in the c-myc promoter region and was restricted to S-phase cells. The Mre11 complex colocalized with PCNA at replication forks throughout S phase, both prior to and coincident with the appearance of nascent DNA. These data suggest that the Mre11 complex suppresses genomic instability through its influence on both the regulation and progression of DNA replication.

Polycystin-1 transforms the cAMP growth-responsive phenotype of M-1 cells.

BACKGROUND: Polycystic kidney disease (PKD) is characterized by the abnormal proliferation of tubular epithelial cells. It was recently shown that the growth of PKD cyst-lining cells is stimulated by cyclic adenosine monophosphate (cAMP), whereas the growth of normal human kidney cortex cells is inhibited. METHODS: We have examined the effects of overexpressing the C-terminal cytosolic tail of mouse polycystin-1, as a membrane-targeted fusion protein, on cAMP-responsive cell proliferation in stably transfected M-1 cortical collecting duct cells. Two cell lines that express high levels of the polycystin-1 fusion protein and two control cell lines that do not express the fusion protein were tested. RESULTS: Growth of parental M-1 cells and the control cell lines was inhibited by 8-Br-cAMP and by a variety of cAMP agonists. In contrast, growth of the polycystin-1-expressing clones was stimulated by cAMP. Consistent with this, the protein kinase A (PKA) inhibitor H-89 caused either a positive or a negative growth effect depending on the primary response to cAMP. PD98059 blocked the cAMP stimulation of cell proliferation, indicating that the pathway is MEK1 dependent. CONCLUSIONS: Expression of the polycystin-1 C-terminal tail disrupts normal cellular signaling and transforms the stably transfected M-1 cells to an abnormal PKD cell proliferation phenotype.

An alternative mode of translation permits production of a variant NBS1 protein from the common Nijmegen breakage syndrome allele.

Nijmegen breakage syndrome (NBS) is a rare chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity and radioresistant DNA synthesis-S phase checkpoint deficiency, which results in the failure to suppress DNA replication origins following DNA damage. Approximately 90% of NBS patients are homozygous for the 657del5 allele, a truncating mutation of NBS1 that causes premature termination at codon 219. Because null mutations in MRE11 and RAD50, which encode binding partners of NBS1, are lethal in vertebrates, and mouse Nbs1-null mutants are inviable, we tested the hypothesis that the NBS1 657del5 mutation was a hypomorphic defect. We showed that NBS cells contain the predicted 26-kD amino-terminal protein fragment, NBS1p26, and a 70-kD NBS1 protein (NBS1p70) lacking the native N terminus. The NBSp26 protein is not physically associated with the MRE11 complex, whereas the p70 species is physically associated with it. NBS1p70 is produced by internal translation initiation within the NBS1 mRNA using an open reading frame generated by the 657del5 frameshift. We propose that the common NBS1 allele encodes a partially functional protein that diminishes the severity of the NBS phenotype.

Analysis of the role of conserved cysteine residues in the bcl-2 oncoprotein.

The Bcl-2 oncoprotein is an integral membrane protein localized primarily to the outer membrane of the mitochondria. The precise molecular mechanism responsible for the antiapoptotic action of Bcl-2 remains unknown. Two cysteine residues are found in Bcl-2 and these residues are well-conserved across species. The first cysteine (cys(155)) is located in the alpha5 domain, a region important for the ion channel properties of Bcl-2, while the second cysteine (cys(226)) is located in the carboxyl-terminal membrane anchor domain. In this study, we found that replacement of both cysteines with serine residues generated a mutant protein that retained the ability to homodimerize and heterodimerize with proapoptotic Bax protein in vitro. In whole cells, the mutant protein efficiently heterodimerized with Bax, but exhibited impaired homodimerizationrelative to wild-type Bcl-2. The mutant protein was also less efficient than wild-type Bcl-2 at suppressing caspase activation, DNA fragmentation, and loss of viability during IL-3 withdrawal-induced apoptosis. Together, the data indicate that the cysteine residues in Bcl-2 contribute, but are not absolutely essential, to the ability of Bcl-2 to homodimerize, heterodimerize with Bax, and suppress apoptosis.

Radiosensitivity in Nijmegen Breakage Syndrome cells is attributable to a repair defect and not cell cycle checkpoint defects.

Cells derived from Nijmegen Breakage Syndrome (NBS) patients display radiosensitivity and cell cycle checkpoint defects. Here, we examine whether the radiosensitivity of NBS cells is the result of a repair defect or whether it can be attributed to impaired checkpoint arrest. We report a small increased fraction of unrejoined double strand breaks and, more significantly, increased chromosome breaks in noncycling NBS cells at 24 h after irradiation. One of the NBS lines examined (347BR) was atypical in showing a nearly normal checkpoint response. In contrast to the mild checkpoint defect, 347BR displays marked y-ray sensitivity similar to that shown by other NBS lines. Thus, the gamma-ray sensitivity correlates with the repair defect rather than impaired checkpoint control. Taken together, the results provide direct evidence for a repair defect in NBS cells and are inconsistent with the suggestion that the radiosensitivity is attributable only to impaired checkpoint arrest. 347BR also displays elevated spontaneous damage that cannot be attributed to impaired G2-M arrest, suggesting a function of Nbsl in decreasing or limiting the impact of spontaneously arising double strand breaks.

Cardiovascular autonomic neuropathy (expiration and inspiration ratio) in type 1 diabetes. Incidence and predictors.

The pathogenesis of diabetic neuropathy is poorly understood. In this prospective study, we investigated the incidence rate and potential predictors for cardiovascular autonomic neuropathy (CAN) in a cohort of childhood-onset type 1 diabetic patients. Subjects from the Epidemiology of Diabetes Complications Study were examined at baseline and then biennially. CAN was diagnosed by abnormal (200 ug/min) (RR=2.46, p=0.0001) to be significant independent predictors. Hypertension was, however, predictive if nephropathy was not included in the model. We conclude that beyond age and poor glycemic control, nephropathy is a significant risk factor for CAN and this association may explain some of the increased mortality seen in CAN.

ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway.

The rare diseases ataxia-telangiectasia (AT), caused by mutations in the ATM gene, and Nijmegen breakage syndrome (NBS), with mutations in the p95/nbs1 gene, share a variety of phenotypic abnormalities such as chromosomal instability, radiation sensitivity and defects in cell-cycle checkpoints in response to ionizing radiation. The ATM gene encodes a protein kinase that is activated by ionizing radiation or radiomimetic drugs, whereas p95/nbs1 is part of a protein complex that is involved in responses to DNA double-strand breaks. Here, because of the similarities between AT and NBS, we evaluated the functional interactions between ATM and p95/nbs1. Activation of the ATM kinase by ionizing radiation and induction of ATM-dependent responses in NBS cells indicated that p95/nbs1 may not be required for signalling to ATM after ionizing radiation. However, p95/nbs1 was phosphorylated on serine 343 in an ATM-dependent manner in vitro and in vivo after ionizing radiation. A p95/nbs1 construct mutated at the ATM phosphorylation site abrogated an S-phase checkpoint induced by ionizing radiation in normal cells and failed to compensate for this functional deficiency in NBS cells. These observations link ATM and p95/nbs1 in a common signalling pathway and provide an explanation for phenotypic similarities in these two diseases.

The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder.

We show that hypomorphic mutations in hMRE11, but not in ATM, are present in certain individuals with an ataxia-telangiectasia-like disorder (ATLD). The cellular features resulting from these hMRE11 mutations are similar to those seen in A-T as well as NBS and include hypersensitivity to ionizing radiation, radioresistant DNA synthesis, and abrogation of ATM-dependent events, such as the activation of Jun kinase following exposure to gamma irradiation. Although the mutant hMre11 proteins retain some ability to interact with hRad50 and Nbs1, formation of ionizing radiation-induced hMre11 and Nbs1 foci was absent in hMRE11 mutant cells. These data demonstrate that ATM and the hMre11/hRad50/Nbs1 protein complex act in the same DNA damage response pathway and link hMre11 to the complex pathology of A-T.

Over-the-counter yellow ultraviolet light protective lenses: any benefit for individuals with diabetes mellitus?

The use of yellow UV protective lenses did improve some individual's ability to see even under artificial and controlled circumstances. Each test, although given in random order, cannot be interpreted to demonstrate the full range of benefit. The improvement for most was modest. Any improvement was most likely due to increased contrast. Light of a short wavelength is scattered more than long wavelength light. The yellow UV protective lenses block light of a short wavelength, thus reducing scatter and increasing contrast. Therefore, patients with visual problems of increased scatter would be expected to demonstrate the greatest improvement. We did not test for the duration of benefit. Subjective reports, from other patients, who have routinely used these lenses (only yellow lens-naive patients were included in the trial) suggest that the benefit increased with duration of use. Patients who routinely use the yellow UV protective lenses state that due to the increased contrast, they squint less. This seems to be most true at dusk. These regular users note that both their eyes are less tired and driving, in particular, is less stressful with the use of yellow lenses. The small benefit might conceivably be magnified in a real world setting. Given that the lenses cost only $10 to $15 and can be purchased in any sporting goods store, even our small measured improvement is likely to be worthwhile. Finally, patients were tested with "off-the-shelf" yellow lenses. To benefit patients the study was designed for their convenience and the low purchase price. The color of the lenses can be chosen to maximize the desired effect. We did not test various wavelength yellow lenses. Consequently, there may be better, albeit more expensive, yellow/orange lenses which might be designed explicitly for this purpose. In summary, the use of these yellow "sunglasses" might provide some improvement in sight for diabetic patients while keeping expense to a minimum.