Clinical utility of comprehensive gene panel testing for common and rare causes of skeletal dysplasia and other skeletal disorders: Results from the largest cohort to date.

Molecular genetics enables more precise diagnoses of skeletal dysplasia and other skeletal disorders (SDs). We investigated the clinical utility of multigene panel testing for 5011 unrelated individuals with SD in the United States (December 2019-April 2022). Median (range) age was 8 (0-90) years, 70.5% had short stature and/or disproportionate growth, 27.4% had a positive molecular diagnosis (MDx), and 30 individuals received two MDx. Genes most commonly contributing to MDx were FGFR3 (16.9%), ALPL (13.0%), and COL1A1 (10.3%). Most of the 112 genes associated with ≥1 MDx were primarily involved in signal transduction (n = 35), metabolism (n = 23), or extracellular matrix organization (n = 17). There were implications associated with specific care/treatment options for 84.4% (1158/1372) of MDx-positive individuals; >50% were linked to conditions with targeted therapy approved or in clinical development, including osteogenesis imperfecta, achondroplasia, hypophosphatasia, and mucopolysaccharidosis. Forty individuals with initially inconclusive results became MDx-positive following family testing. Follow-up mucopolysaccharidosis enzyme activity testing was positive in 14 individuals (10 of these were not MDx-positive). Our findings showed that inclusion of metabolic genes associated with SD increased the clinical utility of a gene panel and confirmed that integrated use of comprehensive gene panel testing with orthogonal testing reduced the burden of inconclusive results.

Palliative dialysis: Addressing the need for alternative dialysis delivery modes.

For some patients with kidney failure, particularly those who have limited life expectancy or severe comorbidities, the "standard" dialysis treatment regimen may be perceived as excessively burdensome and may not align well with the patient's own priorities. For such patients, a palliative approach to the provision of dialysis-whereby treatment is tailored to the needs of the individual so as to optimize quality of life and minimize disease-related symptoms, but limit treatment burden-might offer a way to better align the delivery of care with the life goals of the patient. Here, we discuss the fundamental principles of palliative dialysis: the patients who might most benefit from this approach, treatment strategies and considerations for implementation, as well as potential barriers to its provision.

The clinical and economic burden of pneumonia in patients enrolled in Medicare receiving dialysis: a retrospective, observational cohort study.

BACKGROUND: End-stage renal disease (ESRD) patients receiving dialysis are at particular risk for infection. We assessed the clinical and economic burden of pneumonia in a population of Medicare-enrolled ESRD patients with respect to incidence and case fatality rates, rates of all-cause and cardiovascular hospitalization, and costs. METHODS: Patients received dialysis between 01 January 2009 and 31 December 2011 and were enrolled in Medicare Parts A and B. Pneumonia episodes were identified from institutional and supplier claims. Patients were considered at-risk from first date of Medicare coverage and were censored upon transplant, withdrawal from dialysis, recovery of renal function, loss of Medicare benefits, or death. Linear mixed-effects models were used to assess hospitalization rates and costs over the 3 months prior to and 12 months following pneumonia episodes. RESULTS: The pneumonia incidence rate for the study period was 21.4 events/100 patient-years; the majority of episodes (90.1%) required inpatient treatment. The 30-day case fatality rate was 10.7%. Compared to month -3 prior to event, rates of all-cause and cardiovascular hospitalization were higher in the month of the pneumonia episode (IRR, 4.61 and 4.30). All-cause admission rates remained elevated through month 12; cardiovascular admission rates remained elevated through month 6. Mean per-patient per-month costs were $10,976 higher in the month of index episode compared to month -3, largely driven by increased inpatient costs, and remained elevated through end of 12-month follow-up. CONCLUSION: Pneumonia episodes are frequent among ESRD patients and result in hospitalizations and greater overall costs to Medicare over the following year.

Medicare Advantage associated with lower mortality for incident dialysis patients.

Physicians across the care continuum are increasingly aligned around the belief that coordinated care can improve patient outcomes. As the principal caregivers for one of the most medically fragile patient groups in healthcare, nephrologists are especially attuned to the potential value of integrated care. Medicare Advantage (MA) offers one way to test this hypothesis. By law, end-stage renal disease patients currently cannot enroll into an MA plan, but if they develop ESRD while in such a plan, they may continue to be enrolled. The contrast between these patients and their counterparts who carry Medicare fee for service (MFFS) thereby represents a natural experiment that affords an opportunity to examine whether enrollment in a coordinated care system may improve outcomes. In order to promote (unbiased) comparison of patients in a non-randomized context, we propensity score-matched incident dialysis patients enrolled in MA versus those in MFFS. The data demonstrate that patients who were enrolled in an MA plan upon initiation of dialysis had a 9% lower mortality rate than their MFFS counterparts. This beneficial association of MA enrollment was found to be sustained over the first two years of dialysis treatment.

Improving clinical outcomes among hemodialysis patients: a proposal for a "volume first" approach from the chief medical officers of US dialysis providers.

Addressing fluid intake and volume control requires alignment and coordination of patients, providers, dialysis facilities, and payers, potentially necessitating a "Volume First" approach. This article reports the consensus opinions achieved at the March 2013 symposium of the Chief Medical Officers of 14 of the largest dialysis providers in the United States. These opinions are based on broad experience among participants, but often reinforced by only observational and frequently retrospective studies, highlighting the lack of high-quality clinical trials in nephrology. Given the high morbidity and mortality rates among dialysis patients and the absence of sufficient trial data to guide most aspects of hemodialysis therapy, participants believed that immediate attempts to improve care based on quality improvement initiatives, physiologic principles, and clinical experiences are warranted until such time as rigorous clinical trial data become available. The following overarching consensus opinions emerged. (1) Extracellular fluid status should be a component of sufficient dialysis, such that approaching normalization of extracellular fluid volume should be a primary goal of dialysis care. (2) Fluid removal should be gradual and dialysis treatment duration should not routinely be less than 4 hours without justification based on individual patient factors. (3) Intradialytic sodium loading should be avoided by incorporating dialysate sodium concentrations set routinely in the range of 134-138 mEq/L, avoidance of routine use of sodium modeling, and avoidance of hypertonic saline solution. (4) Dietary counseling should emphasize sodium avoidance.

Bcl-xL gain of function and p19 ARF loss of function cooperate oncogenically with Myc in vivo by distinct mechanisms.

Overexpression of Bcl-xL, loss of p19 ARF, and loss of p53 all accelerate Myc oncogenesis. All three lesions are implicated in suppressing Myc-induced apoptosis, suggesting that this is a common mechanism by which they synergize with Myc. However, using an acutely switchable model of Myc-induced tumorigenesis, we demonstrate that each lesion cooperates with Myc in vivo by a distinct mechanism. While Bcl-xL blocks Myc-induced apoptosis, inactivation of p19 ARF enhances it. However, this increase in apoptosis is matched by increased Myc-induced proliferation. p53 inactivation shares features of both lesions, partially suppressing apoptosis while augmenting proliferation. Bcl-xL and p19 ARF loss together synergize to further accelerate Myc oncogenesis. Thus, differing lesions cooperate oncogenically with Myc by discrete mechanisms that can themselves synergize with each other.

Biosymbiotic haptic feedback - Sustained long term human machine interfaces.

Haptic technology permeates diverse fields and is receiving renewed attention for VR and AR applications. Advances in flexible electronics, facilitate the integration of haptic technologies into soft wearable systems, however, because of small footprint requirements face challenges of operational time requiring either large batteries, wired connections or frequent recharge, restricting the utility of haptic devices to short-duration tasks or low duty cycles, prohibiting continuously assisting applications. Currently many chronic applications are not investigated because of this technological gap. Here, we address wireless power and operation challenges with a biosymbiotic approach enabling continuous operation without user intervention, facilitated by wireless power transfer, eliminating the need for large batteries, and offering long-term haptic feedback without adhesive attachment to the body. These capabilities enable haptic feedback for robotic surgery training and posture correction over weeks of use with neural net computation. The demonstrations showcase that this device class expands use beyond conventional brick and strap or epidermally attached devices enabling new fields of use for imperceptible therapeutic and assistive haptic technologies supporting care and disease management.

Use of incremental peritoneal dialysis: impact on clinical outcomes and quality of life measure.

BACKGROUND: Incremental peritoneal dialysis (PD) can be defined as a PD prescription that is less than the standard, full dose prescription and is typically used for patients initiating PD with residual kidney function. It has been suggested that use of incremental peritoneal dialysis may help preserve residual kidney function and may offer better quality of life due to the lower treatment burden, however published evidence is limited. In this study we assessed the associations between incremental peritoneal dialysis use and both clinical outcomes and quality of life measures in a large cohort of incident peritoneal dialysis patients in the US. METHODS: We considered adult patients initiating peritoneal dialysis between 31 July, 2015 and 31 May, 2019 within a single dialysis organization. Patients with body weight < 40 kg, amputation, or an estimated glomerular filtration rate > 20 mL/min during the first 4 weeks on peritoneal dialysis were excluded. Patients were assigned to exposure groups based on peritoneal dialysis prescription during dialysis weeks 5-8. Incremental peritoneal dialysis was defined by treatment frequency, number of exchanges/day, and exchange volume (for continuous ambulatory peritoneal dialysis patients) or by treatment frequency and presence/absence of last fill (for automated peritoneal dialysis patients). Analyses were performed separately for continuous ambulatory peritoneal dialysis and automated peritoneal dialysis. For each analysis, incremental peritoneal dialysis patients were propensity score matched to eligible full-dose peritoneal dialysis patients. Patients were followed for a maximum of 12 months until censoring for loss to follow-up or study end. Outcomes were compared using Poisson models (mortality, hospitalization, peritoneal dialysis discontinuation), linear mixed models (estimated glomerular filtration rate), and paired t tests (KDQOL domain scores). RESULTS: Among continuous ambulatory peritoneal dialysis patients, compared to full-dose peritoneal dialysis, incremental peritoneal dialysis use was associated with better KDQOL scores on 3 domains: physical composite score (42.5 vs 37.7, p = 0.03), burden of kidney disease (60.2 vs 45.6, p = 0.003), effects of kidney disease (79.4 vs 72.3, p = 0.05). Hospitalization and mortality rates were numerically lower (0.77 vs 1.12 admits/pt-year, p = 0.09 and 5.0 vs 10.2 deaths/100 pt-years, p = 0.22), while no associations were found with estimated glomerular filtration rate or peritoneal dialysis discontinuation rate. Use of incremental peritoneal dialysis was not associated with any discernable effects on outcomes in automated peritoneal dialysis patients. CONCLUSION: These results suggest that there may be benefits of using incremental PD in the context of continuous ambulatory peritoneal dialysis, particularly with respect to quality of life as a prescription strategy when initiating peritoneal dialysis. While no significant benefits of incremental peritoneal dialysis were detected among patients initiating automated peritoneal dialysis, no detrimental effects of using incremental schedules were observed for either peritoneal dialysis type.

Phosphate binder pill burden, adherence, and serum phosphorus control among hemodialysis patients converting to sucroferric oxyhydroxide.

BACKGROUND: Phosphate binders are widely used to achieve serum phosphorus control in patients with end-stage renal disease. However, the large pill burden associated with these medications may decrease adherence to therapy. In clinical trials, sucroferric oxyhydroxide (SO) demonstrated equivalent control of serum phosphorus to sevelamer, with a lower daily pill burden. We examined changes in phosphate binder pill burden, medication possession ratio (MPR), and phosphorus control among in-center hemodialysis (ICHD) patients converting to SO from another phosphate binder as part of routine care. MATERIALS AND METHODS: Patients included in this retrospective analysis (N=490) were ≥18 years old, received ICHD at a large dialysis organization (LDO), and were enrolled in the LDO's pharmacy service. Patients converting to SO were those who had supply of another phosphate binder, received a first prescription fill for SO, and subsequently did not refill the non-SO phosphate binder. Patients were followed over the 6 months before and 6 months following the first SO fill and were censored from the analysis upon modality change, loss to follow-up, discontinuation of SO, or fill of a prescription for another phosphate binder after SO initiation (number censored=361). Outcome measures assessed were total phosphate binder pill burden and MPR, serum phosphorus, and percentage of patients with serum phosphorus ≤5.5 mg/dL. RESULTS: Among patients converting to SO, mean phosphate binder pill burden was 10.8 pills/day during baseline; this decreased to 5.5 pills/day during follow-up (P<0.001). The percentage of patients with serum phosphorus ≤5.5 mg/dL increased from 22.0% to 30.0% (P<0.001). Among patients not using the LDO pharmacy's automated refill management service (N=30), mean phosphate binder MPR increased from 0.68 during baseline to 0.80 during follow-up (P=0.01). CONCLUSION: In a cohort of ICHD patients, conversion to SO was associated with a reduction in pill burden, better adherence, and improvements in phosphorus control.

Malignant mesothelioma cells are rapidly sensitized to TRAIL-induced apoptosis by low-dose anisomycin via Bim.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) holds promise for the treatment of tumors; however, many tumors are resistant to TRAIL alone. We previously showed that resistant malignant mesothelioma cells are sensitized to TRAIL-induced apoptosis by diverse toxic insults including chemotherapy, irradiation, or protein translation inhibitors such as cycloheximide. In seeking nontoxic sensitizers for TRAIL, we tested the protein translation inhibitor anisomycin at subtoxic concentrations 10- to 100-fold below those reported to inhibit protein translation. At these low concentrations (25 ng/mL), anisomycin potently and rapidly sensitized mesothelioma cells to TRAIL-induced apoptosis. Moreover, such sensitization occurred in malignant but not in nonmalignant mesothelial cells. Sensitization by anisomycin was dependent on Bid, indicating a role for mitochondrial amplification in the apoptotic synergy with TRAIL signaling. Consistent with this, we found that anisomycin induces rapid accumulation of the BH3-only protein Bim; moreover, small interfering RNA knockdown of Bim inhibits anisomycin-induced sensitization. Bim accumulation seems not to be transcriptional; instead, it is associated with Bim phosphorylation and increased stability, both consistent with the activation of c-jun NH2-terminal kinase signals by anisomycin. Overall, our data indicate that the rapid and selective sensitization by anisomycin in mesothelioma cells is mediated by posttranslational potentiation of Bim, which primes the cells for apoptosis via the death receptor pathway. Such subtoxic approaches to sensitization may enhance the value of TRAIL in cancer therapy.

c-Myc functionally cooperates with Bax to induce apoptosis.

c-Myc promotes apoptosis by destabilizing mitochondrial integrity, leading to the release of proapoptotic effectors including holocytochrome c. Candidate mediators of c-Myc in this process are the proapoptotic members of the Bcl-2 family. We show here that fibroblasts lacking Bak remain susceptible to c-Myc-induced apoptosis whereas bax-deficient fibroblasts are resistant. However, despite this requirement for Bax, c-Myc activation exerts no detectable effects on Bax expression, localization, or conformation. Moreover, susceptibility to c-Myc-induced apoptosis can be restored in bax-deficient cells by ectopic expression of Bax or by microinjection of a peptide comprising a minimal BH3 domain. Microinjection of BH3 peptide also restores sensitivity to c-Myc-induced apoptosis in p53-deficient primary fibroblasts that are otherwise resistant. By contrast, there is no synergy between BH3 peptide and c-Myc in fibroblasts deficient in both Bax and Bak. We conclude that c-Myc triggers a proapoptotic mitochondrial destabilizing activity that cooperates with proapoptotic members of the Bcl-2 family.

Clinical and economic burden of bloodstream infections in critical care patients with central venous catheters.

PURPOSE: Bloodstream infections (BSIs) complicate the management of intensive care unit (ICU) patients. We assessed the clinical and economic impact of BSI among patients of a managed care provider group who had a central venous catheter (CVC) placed in the ICU. METHODS: We considered hospitalizations occurring between January 1, 2011, and September 30, 2014, that involved an ICU stay during which a CVC was placed. Comparisons were made between episodes where the patient did vs did not develop BSI after CVC insertion. Length of stay, costs of index hospitalization, and total costs over the 180 days after discharge were compared using linear mixed models. Inhospital mortality and 30-day readmission rates were compared using negative binomial regression models. RESULTS: Development of BSI was associated with longer hospital stay (+7 days), more than 3-fold increase in risk of inhospital death, and an additional $129 000 in costs for the index hospitalization. No statistically significant differences in 30-day readmission rates or costs of care over the 180-day period after discharge from the index admission were observed. CONCLUSION: Bloodstream infections after CVC placement in ICU patients are associated with significant increases in costs of care and risk of death during the index hospitalization but no differences in readmissions or costs after discharge.

Comparative Effectiveness of Dialyzers: A Longitudinal, Propensity Score-Matched Study of Incident Hemodialysis Patients.

Differences in dialyzer design may have consequences for patient outcomes. We evaluated the comparative effectiveness of commonly used dialyzers with respect to measures of dialysis treatment, anemia management, inflammation, and dialyzer clotting. Patients receiving hemodialysis between January 1, 2009, and December 31, 2013, and using polyarylethersulfone-polyvinylpyrrolidone (PAS-PVP; Polyflux Revaclear) or polysulfone (PS; Optiflux 160 or Optiflux 180) dialyzers were followed for 1 year or until end of study or censoring for dialyzer switch, modality change, or loss to follow-up. For each comparison, eligible patients were propensity score-matched 1:1 on a range of baseline characteristics. Outcomes were assessed using generalized linear mixed models. Dialysis adequacy was similar in both dialyzer groups. Erythropoiesis-stimulating agent (ESA) doses were lower for patients using PAS-PVP versus patients using PS-160 (difference range: 75-589 units/treatment; statistically significant in months 1-5 and 7) and for patients using PAS-PVP versus patients using PS-180 (difference range: 27-591 unit/treatment; statistically significant in months 1-9). Intravenous iron doses trended lower for patients using PAS-PVP versus patients using PS, but hemoglobin concentrations were equivalent. In conclusion, use of PAS-PVP versus PS dialyzers was associated with equivalent dialysis adequacy, lower ESA doses, modestly lower Intravenous iron doses, and equivalent hemoglobin concentrations.

Use of the Tego needlefree connector is associated with reduced incidence of catheter-related bloodstream infections in hemodialysis patients.

BACKGROUND AND OBJECTIVES: Catheter-related bloodstream infections (CRBSIs) are common in hemodialysis patients using central venous catheters, and catheter occlusion also occurs frequently. The Tego needlefree connector was developed to reduce the incidence of these complications; however, existing studies of its effectiveness and safety are limited. MATERIALS AND METHODS: This retrospective analysis compared outcomes among patients of a large dialysis organization receiving in-center hemodialysis using a central venous catheter with either the Tego connector or standard catheter caps between October 1 and June 30, 2013. Incidence rates for intravenous (IV) antibiotic starts, receipt of an IV antibiotic course, positive blood cultures, mortality, and missed dialysis treatments were calculated, and incidence-rate ratios (IRRs) were estimated using Poisson regression models. Utilization of erythropoiesis-stimulating agents (ESAs) and thrombolytics was described for each patient-month and compared using mixed linear models. Models were run without adjustment, adjusted for covariates that were imbalanced between cohorts, or fully adjusted for all potential confounders. RESULTS: The analysis comprised 10,652 Tego patients and 6,493 controls. Tego use was independently associated with decreased risk of CRBSI, defined by initiation of IV antibiotics (adjusted IRR 0.92, 95% confidence interval [CI] 0.87-0.97) or initiation of IV antibiotic course (adjusted IRR 0.89, 95% CI 0.84-0.95). Tego use was independently associated with decreased rate of missed dialysis treatments (adjusted IRR 0.98, 95% CI 0.97-1.00); no significant difference between Tego and control cohorts was observed with respect to mortality. Tego use was associated with decreased likelihood of thrombolytic use (adjusted per-month probability of 5.6% versus 6.2% for controls) and lower utilization of ESAs in study months 7-9. CONCLUSION: Use of the Tego connector may reduce the risk of CRBSI and result in lower utilization of thrombolytics, antibiotics, and ESAs, as well as fewer missed dialysis treatments.

Down-regulation of the dopamine receptor D2 in mice lacking ataxin 1.

Ataxin 1 (Atxn1) is a protein of unknown function associated with spinocerebellar ataxia type 1 (SCA1), a neurodegenerative disease of late onset with variable degrees of cerebellar ataxia, ophthalmoplegia and neuropathy. SCA1 is caused by the toxic effects triggered by an expanded polyglutamine (polyQ) within Atxn1 resulting in neurodegeneration in the cerebellum, brain stem and spinocerebellar tracts. To gain insights into Atxn1 function, we have analysed the cerebellar gene expression profiles by microarray analysis in Atxn1-null mice, and identified alterations in expression of genes regulated by Sp1-dependent transcription, including the dopamine receptor D2 (Drd2), retinoic acid/thyroid hormone and Wnt-signalling. Interestingly, Drd2 expression levels are reduced in both Atxn1-null and transgenic mice expressing a pathogenic human Atxn1 with an expanded polyglutamine in cerebellar Purkinje cells. Our co-transfection experiments in human neuroblastoma SH-SY5Y cells and luciferase assays provide evidence for transcriptional regulation of Drd2 by Atxn1 and its AXH module. We show that Atxn1 occupies at the Drd2 promoter in vivo, and interacts and functions synergistically with the zinc-finger transcription factor Sp1 to co-regulate Drd2 expression. The interaction and transcriptional effects are mediated by the AXH domain within Atxn1 and are abrogated by the expanded polyQ within Atxn1. Therefore, this study identifies novel molecular targets that are regulated by Atxn1 which might contribute to the motor deficits in SCA1, and provides new insights into the mechanisms by which Atxn1 co-regulates transcription.

The rat ARF protein is translated from two closely spaced ATG start codons and can transcriptionally activate p53 in the absence of p53 protein stabilization.

We have analyzed the transcriptional start sites of the rat ARF gene and the amino acid sequence of the rat ARF tumor suppressor protein. The 5' end of the ratARF gene is similar to that of a number of cellular housekeeping genes in that it is CG-rich and does not contain an upstream TATA box motif to define a precise transcriptional start site. The transcription of the rat ARF gene is initiated at multiple start sites with one major start site accounting for 41% of transcription. The rat ARF protein contains two methionine ATG codons at its amino terminus separated by 10 amino acids. The translation of the major endogenous ARF protein species is initiated from the upstream methionine ATG codon. The upstream methionine ATG codon is predominantly used, despite the fact that it is both very close to the major transcriptional start site (6 bases downstream) and is in a less favorable nucleic acid sequence context than the downstream ATG, relative to the ideal sequence postulated for efficient initiation of translation. The downstream, inefficient rat ARF ATG is equivalent to the major mouse ARF ATG start codon. Both of these closely spaced ATGs can be utilized as a translational start codon to produce a nucleolar-localized ARF protein which can induce a p53-dependent inhibition of cell division and transcriptional activation of p53 in the absence of p53 stabilization.

Bid mediates apoptotic synergy between tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and DNA damage.

The death ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), has shown great promise for inducing apoptosis selectively in tumors. Although many tumor cells are resistant to TRAIL-induced apoptosis alone, they can often be sensitized by co-treatment with DNA-damaging agents such as etoposide. However, the molecular mechanism underlying this therapeutically important synergy is unknown. We explored the mechanism mediating TRAIL-DNA damage apoptotic synergy in human mesothelioma cells, a tumor type particularly refractory to existing therapies. We show that Bid, a cytoplasmic Bcl-2 homology domain 3-containing protein activated by caspase 8 in response to TRAIL ligation, is essential for TRAIL-etoposide apo-ptotic synergy and, furthermore, that exposure to DNA damage primes cells to induction of apoptosis by otherwise sublethal levels of activated Bid. Finally, we show that the extensive caspase 8 cleavage seen during TRAIL-etoposide synergy is a consequence and not a cause of the apoptotic cascade activated downstream of Bid. These data indicate that TRAIL-etoposide apoptotic synergy arises because DNA damage increases the inherent sensitivity of cells to levels of TRAIL-activated Bid that would otherwise be insufficient for apoptosis. Such studies indicate how the adroit combination of differing proapoptotic and sublethal signals can provide an effective strategy for treating refractory tumors.

IL-4 regulation of p38 MAPK signalling is dependent on cell type.

p38 MAPK was originally characterized as a stress-induced kinase, along with JNK. Subsequently, p38 MAPK was found to be activated by stimuli other than cellular stress, such as growth factors and mitogens, like interleukin (IL)-2, IL-7 and IL-3. A notable exception was IL-4, as studies in mast cells showed no activation of p38 MAPK by this cytokine. In this study we show that the regulation of p38 MAPK is cell type dependent. Like other cytokines that signal through the gamma (gamma)(c), IL-4 can activate p38 MAPK in the CT6 T-cell line and BA/F3 pro-B-cells. However, IL-4 was unable to activate p38 MAPK in the murine macrophage cell line, RAW 264.7 and, indeed, prolonged exposure of cells to IL-4 results in suppression of LPS-induced MAPK activation. This result correlates with the well defined inhibitory effect of IL-4 on tumour necrosis factor alpha (TNFalpha) production. In contrast, studies in primary human monocytes showed that prolonged exposure to IL-4 resulted in enhanced activation of LPS-stimulated p38 MAPK; this correlated with an enhanced TNFalpha production. These data highlight the complexity of IL-4 signalling mechanisms, the diversity that can exist in the regulation of a given signalling pathway by a given cytokine and, furthermore, indicate the problems that can arise from extrapolation between different cell systems.

Minimal BH3 peptides promote cell death by antagonizing anti-apoptotic proteins.

The pro-apoptotic "BH3 domain-only" proteins of the Bcl-2 family (e.g. Bid and Bad) transduce multiple death signals to the mitochondrion. They interact with the anti-apoptotic Bcl-2 family members and induce apoptosis by a mechanism that requires the presence of at least one of the multidomain pro-apoptotic proteins Bax or Bak. Although the BH3 domain of Bid can promote the pro-apoptotic assembly and function of Bax/Bak by itself, other BH3 domains do not function as such. The latter point raises the question of whether, and how, these BH3 domains induce apoptosis. We show here that a peptide comprising the minimal BH3 domain from Bax induces apoptosis but is unable to stimulate the apoptotic activity of microinjected recombinant Bax. This relies on the inability of the peptide to directly induce Bax translocation to mitochondria or a change in its conformation. This peptide nevertheless interferes with Bax/Bcl-xL interactions in vitro and stimulates the apoptotic activity of Bax when combined with Bcl-xL. Similarly, a peptide derived from the BH3 domain of Bad stimulates Bax activity only in the presence of Bcl-xL. Thus, BH3 domains do not necessarily activate multidomain pro-apoptotic proteins directly but promote apoptosis by releasing active multidomain pro-apoptotic proteins from their anti-apoptotic counterparts.

A novel mechanism for TNF-alpha regulation by p38 MAPK: involvement of NF-kappa B with implications for therapy in rheumatoid arthritis.

TNF-alpha is a key factor in a variety of inflammatory diseases. This study examines the role of p38 MAPK in the regulation of TNF-alpha in primary human cells relevant to inflammation, e.g., macrophages and rheumatoid synovial cells. Using a dominant negative variant (D168A) of p38 MAPK and a kinase inhibitor, SB203580, we confirm in primary human macrophages that p38 MAPK regulates TNF-alpha production using a posttranscriptional mechanism requiring the 3' untranslated region of the gene. However, in LPS-activated primary human macrophages we also detect a second previously unidentified mechanism, the p38 MAPK modulation of TNF-alpha transcription. This is mediated through p38 MAPK regulation of NF-kappaB. Interestingly this mechanism was not observed in rheumatoid synovial cells. Importantly however, the dominant negative mutant of p38 MAPK, but not SB203580 was effective at inhibiting spontaneous TNF-alpha production in these ex vivo rheumatoid synovial cell cultures. These data indicate there are potential major differences in the role of p38 MAPK in inflammatory signaling that have a bearing on the use of this kinase as a target for therapy. These results indicate despite disappointing results with p38 MAPK inhibitors in the clinic, this kinase is a valid target in rheumatoid disease.