Vascular calcification and mineral bone disorder in chronic kidney disease

Chronic Kidney Disease (CKD), osteoporosis and mild hyponatremia are all prevalent chronic conditions that may coexist and are often under-recognized. Mineral-Bone Disorder begins early in the natural history of CKD and results in complex abnormalities of bone which ultimately confers a well-established increased risk of fragility fractures in End Stage Kidney Disease. Hyponatremia is a novel, usually renal mediated metabolic perturbation, that most commonly occurs independently of the stage of renal dysfunction but which may also predispose to increased fracture risk. The extent -if any- to which either early stages of renal dysfunction or the presence of hyponatremia contribute to fracture occurrence in the general population, independently of osteoporosis, is unclear. Renal transplantation is the treatment of choice for ESKD and although it restores endogenous renal function it typically fails to normalize either the long term cardiovascular or fracture risk. One potential mechanism contributing to these elevated long-term risks and to diminished Health Related Quality of Life is persistent, post-transplant hyperparathyroidism. In this study we retrospectively examine the association of renal function and serum sodium with Bone Mineral Density and fracture occurrence in a retrospective cohort of 1930 female members of the general population who underwent routine DXA scan. We then prospectively recruited a cohort of 90 renal transplant recipients in order to examine the association of post transplant parathyroid hormone (PTH) level with measures of CKD Mineral Bone Disorder, including, DXA Bone Mineral Density, Vascular Calcification (assessed using both abdominal radiography and CT techniques, as well as indirectly by carotid-femoral Pulse Wave Velocity) and Quality of Life (using the Short Form-12 and a PTH specific symptom score). In the retrospective DXA cohort, moderate CKD (eGFR 30-59ml/min/1.73m2) and hyponatremia (<135mmol/L) were associated with fracture occurrence, independently of BMD, with an adjusted Odds Ratio (95% Confidence Interval), of 1.37 (1.0, 1.89) and 2.25 (1.24, 4.09) respectively. In the renal transplant study, PTH was independently associated with the presence of osteoporosis, adjusted Odds Ratio (95% Confidence Interval), 1.15 (per 10ng/ml increment), (1.04, 1.26). The presence of osteoporosis but not PTH was independently associated with measures of vascular calcification, adjusted ß (95% Confidence Interval), 12.45, (1.16, 23.75). Of the eight quality-of-life domains examined, post-transplant PTH (per 10ng/ml increment), was only significantly and independently associated with reduced Physical Functioning, (95% Confidence Interval), 1.12 (1.01, 1.23). CKD and hyponatremia are both common health problems that may contribute to fracture occurrence in the general population, a major on-going public health concern. PTH and decreased Bone Mineral Density may signal sub-optimal long-term outcomes post renal transplantation, influencing bone and vascular health and to a limited extent long term Health Related Quality of Life

Role of KLF4 in regulation of myocardin induced SMC differentiation in human smooth muscle stem progenitor cells (hSMSPC)

The differentiation of stem cells into multiple lineages has been explored in vascular regenerative medicine. However, in the case of smooth muscle cells (SMC), issues exist concerning inefficient rates of differentiation. In stem cells, multiple repressors potentially downregulate myocardin, the potent SRF coactivator induced SMC transcription including Krüppel like zinc finger transcription factor-4 (KLF4). This thesis aimed to explore the role of KLF4 in the regulation of myocardin gene expression in human smooth muscle stem/progenitor cells (hSMSPC), a novel circulating stem cell identified in our laboratory which expresses low levels of myocardin and higher levels of KLF4. hSMSPC cells cultured in SmGM2 1% FBS with TGF-β1 (5 ng/ml “differentiation media”) show limited SMC cell differentiation potential. Furthermore, myocardin transduced hSMSPC cells cultured in differentiation media induced myofilamentous SMC like cells with expression of SM markers. Five potential KLF4 binding sites were identified in silico within 3.9Kb upstream of the translational start site of the human myocardin promoter. Chromatin immunoprecipitation assays verified that endogenous KLF4 binds the human myocardin promoter at -3702bp with Respect to the translation start site (-1). Transduction of lentiviral vectors encoding either myocardin cDNA (LV_myocardin) or KLF4 targeting shRNA (LV_shKLF4 B) induced human myocardin promoter activity in hSMSPCs. Silencing of KLF4 expression in differentiation media induced smooth muscle like morphology by day 5 in culture and increased overtime with expression of SMC markers in hSMSPCs. Implantation of silastic tubes into the rat peritoneal cavity induces formation of a tissue capsule structure which may be used as vascular grafts. Rat SMSPCs integrate into, strengthen and enhance the SMC component of such tubular capsules. These data demonstrate that KLF4 directly represses myocardin gene expression in hSMSPCs, which when differentiated, provide a potential source of SMCs in the development of autologous vascular grafts in regenerative medicine.

The role of mitogen activated protein kinase phosphatase-1 in neurotoxic and inflammatory-induced changes in the development of midbrain dopaminergic neurons: relevance to Parkinson’s disease

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterised by the loss of midbrain dopaminergic neurons from the substantia nigra pars compacta(SNpc), which results in motor, cognitive and psychiatric symptoms. Evidence supports a role for the mitogen-activated protein kinase p38 in the demise of dopaminergic neurons, while mitogen-activated protein kinase phosphatase-1 (MKP-1), which negatively regulates p38 activity, has not yet been investigated in this context. Inflammation may also be associated with the neuropathology of PD due to evidence of increased levels of proinflammatory cytokines such as interleukin-1β (IL-1β) within the SNpc. Because of the specific loss of dopaminergic neurons in a discreet region of the brain, PD is considered a suitable candidate for cell replacement therapy but challenges remain to optimise dopaminergic cell survival and morphological development. The present thesis examined the role of MKP-1 in neurotoxic and inflammatory-induced changes in the development of midbrain dopaminergic neurons. We show that MKP-1 is expressed in dopaminergic neurons cultured from embryonic day (E) 14 rat ventral mesencephalon (VM). Inhibition of dopaminergic neurite growth induced by treatment of rat VM neurons with the dopaminergic neurotoxin 6- hydroxydopamine (6-OHDA) is mediated by p38, and is concomitant with a significant and selective decrease in MKP-1 expression in these neurons. Dopaminergic neurons transfected to overexpress MKP-1 displayed a more complex morphology and contributed to neuroprotection against the effects of 6-OHDA. Therefore, MKP-1 expression can promote the growth and elaboration of dopaminergic neuronal processes and can help protect them from the neurotoxic effects of 6-OHDA. Neural precursor cells (NPCs) have emerged as promising alternative candidates to fetal VM for cell replacement strategies in PD. Here we show that phosphorylated (and thus activated) p38 and MKP-1 are expressed at basal levels in untreated E14 rat VM NPCs (nestin, DCX, GFAP and DAT-positive cells) following proliferation as well as in their differentiated progeny (DCX, DAT, GFAP and βIII-tubulin) in vitro. Challenge with 6-OHDA or IL-1β changed the expression of endogenous phospho-p38 and MKP-1 in these cells in a time-dependent manner, and so the dynamic balance in expression may mediate the detrimental effects of neurotoxicity and inflammation in proliferating and differentiating NPCs. We demonstrate that there was an up-regulation in MKP-1 mRNA expression in adult rat midbrain tissue 4 days post lesion in two rat models of PD; the 6-OHDA medial forebrain bundle (MFB) model and the four-site 6-OHDA striatal lesion model. This was concomitant with a decrease in tyrosine hydroxylase (TH) mRNA expression at 4 and 10 days post-lesion in the MFB model and 10 and 28 days post-lesion in the striatal lesion model. There was no change in mRNA expression of the pro-apoptotic gene, bax and the anti-apoptotic gene, bcl-2 in the midbrain and striatum. These data suggest that the early and transient upregulation of MKP-1 mRNA in the midbrain at 4 days post-6-OHDA administration may be indicative of an attempt by dopaminergic neurons in the midbrain to protect against the neurotoxic effects of 6-OHDA at later time points. Collectively, these findings show that MKP-1 is expressed by developing and adult dopaminergic neurons in the midbrain, and can promote their morphological development. MKP-1 also exerts neuroprotective effects against dopaminergic neurotoxins in vitro, and its expression in dopaminergic neurons can be modulated by inflammatory and neurotoxic insults both in vitro and in vivo. Thus, these data contribute to the information needed to develop therapeutic strategies for protecting midbrain dopaminergic neurons in the context of PD.

Alcohol income and health: a complicated but desirable mix

The aim of this thesis is to examine if a difference exists in income for different categories of drinkers in Ireland using the 2007 Slán data set. The possible impact of alcohol consumption on health status and health care utilisation is also examined. Potential endogeneity and selection bias is accounted for throughout. Endogeneity is where an independent variable included in the model is determined within the context of the model (Chenhall and Moers, 2007). An endogenous relationship between income and alcohol and between health and alcohol is accounted for by the use of separate income equations and separate health status equations for each category of drinker similar to what was done in previous studies into the effects of alcohol on earnings (Hamilton and Hamilton, 1997; Barrett, 2002). Sample selection bias arises when a sector selection is non-random due to individuals choosing a particular sector because of their personal characteristics (Heckman, 1979; Zhang, 2004). In relation to alcohol consumption, selection bias may arise as people may select into a particular drinker group due to the fact that they know that by doing so it will not have a negative effect on their income or health (Hamilton and Hamilton, 1997; Di Pietro and Pedace, 2008; Barrett, 2002). Selection bias of alcohol consumption is accounted for by using the Multinomial Logit OLS Two Step Estimate as proposed by Lee (1982), which is an extension of the Heckman Probit OLS Two Step Estimate. Alcohol status as an ordered variable is examined and possible methods of estimation accounting for this ordinality while also accounting for selection bias are looked at. Limited Information Methods and Full Information Methods of estimation of simultaneous equations are assessed and compared. Findings show that in Ireland moderate drinkers have a higher income compared with abstainers or heavy drinkers. Some studies such as Barrett (2002) argue that this is as a consequence of alcohol improving ones health, which in turn can influence ones productivity which may ultimately be reflected in earnings, due to the fact that previous studies have found that moderate levels of alcohol consumption are beneficial towards ones health status. This study goes on to examine the relationship between health status and alcohol consumption and whether the correlation between income and the consumption of alcohol is similar in terms of sign and magnitude to the correlation between health status and the consumption of alcohol. Results indicate that moderate drinkers have a higher income than non or heavy drinkers, with the weekly household income of moderate drinkers being €660.10, non drinkers being €546.75 and heavy drinkers being €449.99. Moderate Drinkers also report having a better health status than non drinkers and a slightly better health status than heavy drinkers. More non-drinkers report poor health than either moderate or heavy drinkers. As part of the analysis into the effect of alcohol consumption on income and on health status, the relationship between other socio economic variables such as gender, age, education among others, with income, health and alcohol status is examined.

Ireland's knowledge economy policy: beliefs, drivers and prospects

This dissertation critically examines Ireland’s knowledge economy policy, the country’s basis for economic recovery and growth, to enhance future policy decisions and debate. Much has been written internationally on the ‘knowledge economy’ with its emergence closely related to globalisation and technological progression in the 1990s. Since the late 1990s, Irish policy-makers have been firmly committed to positioning Ireland as a leading knowledge economy. Transforming the country’s competitive base to a knowledge economy is pivotal, directly shaping the course of Ireland’s economy and society. Given Ireland’s current economic crisis, limited resources, global competition from leaders in science and technology and growing challenges from emerging economies, a systematic study of Ireland’s major competitive policy is imperative. Above all, this study explores the processes behind the policy and the multiple actors from different institutions who follow and seek to influence decisions. The advocacy coalition framework is used to identify the advocacy coalition operating in the knowledge economy policy subsystem. The theoretical insights of this framework are also combined with other public policy approaches, providing complementary insights into the policy process. The research is framed around three elements - the beliefs underpinning the policy; who is driving the policy; and the prospects of the policy. Primary information is collected by way of semi-structured in-depth interviews with 49 Irish elites (politicians, senior bureaucrats, academics and business leaders) involved in the formation and implementation of the policy. This study finds that a strong advocacy coalition has formed in this policy subsystem whose members are collectively driving the policy. Both exogenous and endogenous forces help frame a common perception of the problems the policy addresses and the solutions it offers. Evidence suggests that this policy is a sustainable option for Ireland’s economic future and the study concludes with policy recommendations for advancing Ireland’s knowledge economy.

Akt signal transduction dysfunction in the 3xTg-AD mouse model of Alzheimer's disease

Alzheimer’s disease (AD) is an incurable neurodegenerative disorder, accounting for over 60% of all cases of dementia. The primary risk factor for AD is age, however several genetic and environmental factors are also involved. The pathological characteristics of AD include extracellular deposition of the beta-amyloid peptide (Aβ) and intraneuronal accumulation of neurofibrillary tangles (NFTs) made of aggregated paired helical filaments (PHFs) of the hyperphosphorylated tau protein, along with synaptic loss and neuronal death. There are numerous biochemical mechanisms involved in AD pathogenesis, however the reigning hypothesis points to toxic oligomeric Aβ species as the primary causative factor in a cascade of events leading to neuronal stress and dyshomeostasis that initiate abnormal regulation of tau. The insulin and IGF-1 receptors (IR, IGF-1R) are the primary activators of PI3- K/Akt through which they regulate cell growth, development, glucose metabolism, and learning and memory. Work in our lab and others shows increased Akt activity and phosphorylation of its downstream targets in AD brain, along with insulin and insulin-like growth factor-1 signalling (IIS) dysfunction. This is supported by studies of AD models in vivo and in vitro. Our group and others hypothesise that Aβ activates Akt through IIS to initiate a negative feedback mechanism that desensitises neurons to insulin/IGF-1, and sustains activation of Akt. In this study the functions of endogenous Akt, IR, and the insulin receptor substrate (IRS-1) were examined in relationship to Aβ and tau pathology in the 3xTg-AD mouse model, which contains three mutant human transgenes associated with familial AD or dementia. The 3xTg-AD mouse develops Aβ and tau pathology in a spatiotemporal manner that best recapitulates the progression of AD in human brain. Western blotting and immunofluorescent microscopy techniques were utilised in vivo and in vitro, to examine the relationship between IIS, Akt, and AD pathology. I first characterised in detail AD pathology in 3xTg-AD mice, where an age-related accumulation of intraneuronal Aβ and tau was observed in the hippocampal formation, amygdala, and entorhinal cortex, and at late stages (18 months), extracellular amyloid plaques and NFTs, primarily in the subiculum and the CA1 layer of the hippocampal formation. Increased activity of Akt, detected with antibody to phosphoSer473-Akt, was increased in 3xTg-AD mice compared to age-matched non-transgenic mice (non-Tg), and in direct correlation to the accumulation of Aβ and tau in neuronal somatodendritic compartments. Akt phosphorylates tau at residue Ser214 within a highly specific consensus sequence for Akt phosphorylation, and phosphoSer214-tau strongly decreases microtubule (MT) stabilisation by preventing tau-MT binding. PhosphoSer214-tau increased concomitantly with this in the same age-related and region-specific fashion. Polarisation of tau phosphorylation was observed, where PHF-1 (tauSer396/404) and phosphoSer214-tau both appeared early in 3xTg-AD mice in distinct neuronal compartments: PHF-1 in axons, and phosphoSer214-tau in neuronal soma and dendrites. At 18 months, phosphoSer214-tau strongly colocalised with NFTs positive for the PHF- 1 and AT8 (tauSer202/Thr205) phosphoepitopes. IR was decreased with age in 3xTg-AD brain and in comparison to age-matched non-Tg, and this was specific for brain regions containing Aβ, tau, and hyperactive Akt. IRS-1 was similarly decreased, and both proteins showed altered subcellular distribution. Phosphorylation of IRS-1Ser312 is a strong indicator of IIS dysfunction and insulin resistance, and was increased in 3xTg-AD mice with age and in relation to pathology. Of particular note was our observation that abberant IIS and Akt signalling in 3xTg-AD brain related to Aβ and tau pathology on a gross anatomical level, and specifically localised to the brain regions and circuitry of the perforant path. Finally, I conducted a preliminary study of the effects of synthetic Aβ oligomers on embryonic rat hippocampus neuronal cultures to support these results and those in the literature. Taken together, these novel findings provide evidence for IIS and Akt signal transduction dysfunction as the missing link between Aβ and tau pathogenesis, and contribute to the overall understanding of the biochemical mechanisms of AD.

Redox biology of myeloid leukaemias

Internal tandem duplication of FMS-like receptor tyrosine kinase (FLT3-ITD) has been associated with an aggressive AML phenotype. FLT3-ITD expressing cell lines have been shown to generate increased levels of reactive oxygen species (ROS) and DNA double strand breaks (dsbs). However, the molecular basis of how FLT3-ITD-driven ROS leads to the aggressive form of AML is not clearly understood. Herein, we observe that the majority of H2O2 in FLT3-ITD-expressing MV4-11 cells colocalises to the endoplasmic reticulum (ER). Furthermore, ER localisation of ROS in MV4-11 cells corresponds to the localisation of p22phox, a small membrane-bound subunit of NOX complex. Furthermore, we show that 32D cells, a myeloblast-like cell line transfected with FLT3-ITD, possess higher steady protein levels of p22phox than their wild type FLT3 (FLT3-WT)-expressing counterparts. Moreover, the inhibition of FLT3-ITD, using various FLT3 tyrosine kinase inhibitors, uniformly results in a posttranslational downregulation of p22phox. We also show that depletion of NOX2 and NOX4 and p22phox, but not NOX1 proteins causes a reduction in endogenous H2O2 levels. We show that genomic instability induced by FLT3-ITD leads to an increase in nuclear levels of H2O2. The presence of H2O2 in the nucleus is largely reduced by inhibition of FLT3-ITD or NOX. Furthermore, similar results are also observed following siRNA knockdowns of p22phox or NOX4. We demonstrate that 32D cells transfected with FLT3-ITD have a higher level of DNA damage than 32D cells transfected with FLT3-WT. Additionally, inhibition of FLT3-ITD, p22phox and NOX knockdowns decrease the number of DNA dsbs. In summary, this study presents a novel mechanism of genomic instability generation in FLT3-ITD-expressing AML cells, whereby FLT3-ITD activates NOX complexes by stabilising p22phox. This in turn leads to elevated generation of ROS and DNA damage in these cells.

Self-organized vegetation patterning as a fingerprint of climate and human impact on semi-arid ecosystems

Spatially periodic vegetation patterns are well known in arid and semi-arid regions around the world. Mathematical models have been developed that attribute this phenomenon to a symmetry-breaking instability. Such models are based on the interplay between competitive and facilitative influences that the vegetation exerts on its own dynamics when it is constrained by arid conditions, but evidence for these predictions is still lacking. Moreover, not all models can account for the development of regularly spaced spots of bare ground in the absence of a soil prepattern. We applied Fourier analysis to high-resolution, remotely sensed data taken at either end of a 40-year interval in southern Niger. Statistical comparisons based on this textural characterization gave us broad-scale evidence that the decrease in rainfall over recent decades in the sub-Saharan Sahel has been accompanied by a detectable shift from homogeneous vegetation cover to spotted patterns marked by a spatial frequency of about 20 cycles km-1. Wood cutting and grazing by domestic animals have led to a much more marked transition in unprotected areas than in a protected reserve. Field measurements demonstrated that the dominant spatial frequency was endogenous rather than reflecting the spatial variation of any pre-existing heterogeneity in soil properties. All these results support the use of models that can account for periodic vegetation patterns without invoking substrate heterogeneity or anisotropy, and provide new elements for further developments, refinements and tests. This study underlines the potential of studying vegetation pattern properties for monitoring climatic and human impacts on the extensive fragile areas bordering hot deserts. Explicit consideration of vegetation self-patterning may also improve our understanding of vegetation and climate interactions in arid areas. © 2006 The Authors.

Essays on Vietnam’s Financial Reforms: Foreign Exchange Statistics and Evidence of Long-Run Equilibrium

In this paper, we examine exchange rates in Vietnam’s transitional economy. Evidence of long-run equilibrium are established in most cases through a single co-integrating vector among endogenous variables that determine the real exchange rates. This supports relative PPP in which ECT of the system can be combined linearly into a stationary process, reducing deviation from PPP in the long run. Restricted coefficient vectors ß’ = (1, 1, -1) for real exchange rates of currencies in question are not rejected. This empirics of relative PPP adds to found evidences by many researchers, including Flre et al. (1999), Lee (1999), Johnson (1990), Culver and Papell (1999), Cuddington and Liang (2001). Instead of testing for different time series on a common base currency, we use different base currencies (USD, GBP, JPY and EUR). By doing so we want to know the whether theory may posit significant differences against one currency? We have found consensus, given inevitable technical differences, even with smallerdata sample for EUR. Speeds of convergence to PPP and adjustment are faster compared to results from other researches for developed economies, using both observed and bootstrapped HL measures. Perhaps, a better explanation is the adjustment from hyperinflation period, after which the theory indicates that adjusting process actually accelerates. We observe that deviation appears to have been large in early stages of the reform, mostly overvaluation. Over time, its correction took place leading significant deviations to gradually disappear.

IL-12 inhibits in vitro immunoglobulin production by human lupus peripheral blood mononuclear cells (PBMC).

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by polyclonal B cell activation and by the production of anti-double-stranded (ds) DNA antibodies. Given the inhibitory effects of IL-12 on humoral immune responses, we investigated whether IL-12 displayed such an activity on in vitro immunoglobulin production by SLE PBMC. Spontaneous IgG, IgG1, IgG2, IgG3 and IgM antibody production was dramatically reduced by addition of IL-12. These results were confirmed by Elispot assays detecting IgG- and anti-dsDNA-secreting cells. While IL-6 and TNF titres measured in PBMC supernatants were not modified by addition of IL-12, interferon-gamma (IFN-gamma) titres were up-regulated and IL-10 production down-regulated. Since addition of IFN-gamma did not down-regulate immunoglobulin production and since the inhibitory activity of IL-12 on immunoglobulin synthesis was not suppressed by anti-IFN-gamma antibody, we concluded that the effect of IL-12 on immunoglobulin production was not mediated through IFN-gamma. Our data also argue against the possibility that down-regulation of endogenous IL-10 production was responsible for the effect of IL-12. Thus, inhibition of IL-10 production by IFN-gamma was not accompanied by inhibition of immunoglobulin production, and conversely, restoration of IL-10 production by anti-IFN-gamma antibody did not suppress the inhibitory activity exerted by IL-12 on immunoglobulin production. Taken together, our data indicate that reduction of excessive immunoglobulin and anti-dsDNA antibody production by lupus PBMC can be achieved in vitro by IL-12, independently of IFN-gamma and IL-10 modulation.

Unpredictable Cases of Complicated Ovarian Hyperstimulation in IVF

Objective-To characterize a subpopulation of complicated cases of ovarian hyperstimulation syndrome (OHSS). Method-Descriptive retrospective study. Results-0.75% of our IVF-ET population suffered from OHSS. Among this group, 33% did not exhibit any recognized risk criteria of OHSS in terms of infertility characteristics and ovarian response to exogenous gonadotrophins. Only severe (ascites) OHSS cases were considered (n = 5) in this study. Previous IVF-ET attempts had been uneventful and during the complicated trial, estradiol peak levels and numbers of oocytes retrieved remained below 2,500 pg/mL (conversion factor to SI unit, 3.671) and 10, respectively. In all cases, the luteal phase was supplemented by hCG and all patients became pregnant. A threshold level of exogenous and/or endogenous hCG seems to be responsible for the occurrence of OHSS. Conclusion-One-third of the patients developing an ovarian hyperstimulation syndrome after IVF-ET had not previously shown risk criteria. A causal role of exogenous and/or endogenous hCG is suggested.

Agglomeration effects in foreign direct investment and the pollution haven hypothesis

Does environmental regulation impair international competitiveness of pollution-intensive industries to the extent that they relocate to countries with less stringent regulation, turning those countries into "pollution havens"? We test this hypothesis using panel data on outward foreign direct investment (FDI) flows of various industries in the German manufacturing sector and account for several econometric issues that have been ignored in previous studies. Most importantly, we demonstrate that externalities associated with FDI agglomeration can bias estimates away from finding a pollution haven effect if omitted from the analysis. We include the stock of inward FDI as a proxy for agglomeration and employ a GMM estimator to control for endogenous time-varying determinants of FDI flows. Furthermore, we propose a difference estimator based on the least polluting industry to break the possible correlation between environmental regulatory stringency and unobservable attributes of FDI recipients in the cross-section. When accounting for these issues we find robust evidence of a pollution haven effect for the chemical industry. © 2008 Springer Science+Business Media B.V.

Weighted and two-stage least squares estimation of semiparametric truncated regression models

This paper provides a root-n consistent, asymptotically normal weighted least squares estimator of the coefficients in a truncated regression model. The distribution of the errors is unknown and permits general forms of unknown heteroskedasticity. Also provided is an instrumental variables based two-stage least squares estimator for this model, which can be used when some regressors are endogenous, mismeasured, or otherwise correlated with the errors. A simulation study indicates that the new estimators perform well in finite samples. Our limiting distribution theory includes a new asymptotic trimming result addressing the boundary bias in first-stage density estimation without knowledge of the support boundary. © 2007 Cambridge University Press.

Gait and behavior in an IL1β-mediated model of rat knee arthritis and effects of an IL1 antagonist.

Interleukin-1 beta (IL1β) is a proinflammatory cytokine that mediates arthritic pathologies. Our objectives were to evaluate pain and limb dysfunction resulting from IL1β over-expression in the rat knee and to investigate the ability of local IL1 receptor antagonist (IL1Ra) delivery to reverse-associated pathology. IL1β over-expression was induced in the right knees of 30 Wistar rats via intra-articular injection of rat fibroblasts retrovirally infected with human IL1β cDNA. A subset of animals received a 30 µl intra-articular injection of saline or human IL1Ra on day 1 after cell delivery (0.65 µg/µl hIL1Ra, n = 7 per group). Joint swelling, gait, and sensitivity were investigated over 1 week. On day 8, animals were sacrificed and joints were collected for histological evaluation. Joint inflammation and elevated levels of endogenous IL1β were observed in knees receiving IL1β-infected fibroblasts. Asymmetric gaits favoring the affected limb and heightened mechanical sensitivity (allodynia) reflected a unilateral pathology. Histopathology revealed cartilage loss on the femoral groove and condyle of affected joints. Intra-articular IL1Ra injection failed to restore gait and sensitivity to preoperative levels and did not reduce cartilage degeneration observed in histopathology. Joint swelling and degeneration subsequent to IL1β over-expression is associated limb hypersensitivity and gait compensation. Intra-articular IL1Ra delivery did not result in marked improvement for this model; this may be driven by rapid clearance of administered IL1Ra from the joint space. These results motivate work to further investigate the behavioral consequences of monoarticular arthritis and sustained release drug delivery strategies for the joint space.

Differential inhibition of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and TZM-bl cells by endotoxin-mediated chemokine and gamma interferon production.

Bacterial lipopolysaccharide (endotoxin) is a frequent contaminant of biological specimens and is also known to be a potent inducer of beta-chemokines and other soluble factors that inhibit HIV-1 infection in vitro. Though lipopolysaccharide (LPS) has been shown to stimulate the production of soluble HIV-1 inhibitors in cultures of monocyte-derived macrophages, the ability of LPS to induce similar inhibitors in other cell types is poorly characterized. Here we show that LPS exhibits potent anti-HIV activity in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) but has no detectable anti-HIV-1 activity in TZM-bl cells. The anti-HIV-1 activity of LPS in PBMCs was strongly associated with the production of beta-chemokines from CD14-positive monocytes. Culture supernatants from LPS-stimulated PBMCs exhibited potent anti-HIV-1 activity when added to TZM-bl cells but, in this case, the antiviral activity appeared to be related to IFN-gamma rather than to beta-chemokines. These observations indicate that LPS stimulates PBMCs to produce a complex array of soluble HIV-1 inhibitors, including beta-chemokines and IFN-gamma, that differentially inhibit HIV-1 depending on the target cell type. The results also highlight the need to use endotoxin-free specimens to avoid artifacts when assessing HIV-1-specific neutralizing antibodies in PBMC-based assays.