Determination of age-dependent bone marrow normocellularity.

OBJECTIVES: Determination of bone marrow cellularity is a key part of bone marrow examination because it provides a small window into a patient's current state of hematopoietic well-being. Traditionally, bone marrow cellularity is estimated semiquantitatively through microscopic examination of core biopsy specimens harvested from the iliac crest of the pelvic bone. Bone marrow cellularity is then designated as hypercellular, normocellular, or hypocellular based on the patient's age. This assessment can have significant clinical impact, but the variation in the age-adjusted normocellularity range is not sufficiently characterized because of a lack of study data, especially in older patients (those older than 70 years of age). This study further established the normal range of bone marrow cellularity, particularly in older adults. METHODS: In this study, 570 benign staging and healthy donor bone marrows from patients 1 year to 93 years of age were analyzed for cellularity. RESULTS: Linear regression modeling demonstrates that cellularity in adults declines approximately 3% per decade, including after the seventh decade of life. The 90% reference interval for normocellularity in United States is 30% to 75% for those aged 18 to 90 years. CONCLUSIONS: The findings revealed a more stable and slower rate of decline in cellularity with age in adults than the widely used linear model of "100% minus the patient age in decades." Normocellularity is better modeled based on age group. In those younger than 20 years of age, normocellularity ranges from 45% to 85% (mean [SD], 65% [20%]), as defined by Friebert et al in 1998. Based on our study finding of a little less than 3% decline per decade of age, the following is our recommendation for normocellularity range: For individuals 20 to 40 years of age, it ranges from 40% to 70% (mean [SD], 55% [15%]); for individuals 40 to 60 years of age, it ranges from 35% to 65% (mean [SD], 50% [15%]); and for individuals older than 60 years of age, it ranges from 30% to 60% (mean [SD], 45% [15%]). Interestingly, those older than 70 years of age do not show a significant decrease from those aged 60 to 69 years.

Carfilzomib combined with rituximab, ifosfamide, carboplatin, and etoposide for relapsed or refractory DLBCL.

The CORAL study highlighted the need to develop novel salvage regimens in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) previously treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. Carfilzomib (CFZ) can overcome rituximab chemotherapy resistance in lymphoma preclinical models by targeting the ubiquitin-proteasome system. We conducted an investigator initiated, single-center, open-label, prospective phase 1 study evaluating the safety and efficacy of CFZ in combination with rituximab, ifosfamide, carboplatin, and etoposide (C-R-ICE) in high-dose chemotherapy with autologous stem cell transplant (HDC-ASCT) eligible patients with R/R DLBCL (NCT01959698). In the dose-escalation phase, 18 patients were enrolled at 6 dose levels with no dose-limiting toxicities noted. CFZ 45 mg/m2 was selected as the recommended dose for expansion. Eleven additional patients were enrolled in the dose-expansion phase. Overall response rate (ORR) was 66% (48% CR; 17% PR); 52% patients underwent HDC-ASCT. An ORR of 85% was observed in patients with nongerminal center B-cell-like (non-GCB) DLBCL compared with only 13% in those with GCB DLBCL. Median progression-free survival (PFS) was 15.2 months (5.1 months, not reached [NR]), and median overall survival (OS) was 22.6 months (6.8 months, NR). Patients with non-GCB subtype had a significantly longer PFS (NR vs 6.6 months; P = .0001) and OS (NR vs 6.6 months; P = .001) than those with GCB subtype. C-R-ICE is well tolerated in patients with R/R DLBCL with toxicities comparable to rituximab, ifosfamide, carboplatin, and etoposide therapy. Our data show that patients with non-GCB DLBCL benefit significantly from incorporating CFZ into second-line therapy and HDC-ASCT.

Relative frequency and clinicopathologic characteristics of MYC-rearranged follicular lymphoma.

MYC rearrangement is a relatively rare genetic abnormality in follicular lymphoma (FL). In this study, we evaluated the relative frequency of MYC rearrangement in 522 cases of FL and studied their clinicopathologic, cytogenetic, and molecular characteristics. Fluorescence in situ hybridization studies for MYC (break-apart probe), MYC/IGH, IGH/BCL2, and BCL6 rearrangements were performed on tissue microarrays. Immunohistochemical stains for CD10, BCL2, BCL6, and MYC were performed and scored on MYC-rearranged cases. On 4 FL cases, a custom targeted panel of 356 genes was used for mutation analysis. Ten cases (1.9%) were positive for MYC rearrangement. Histologically, 6 of 10 cases were grade 1-2, and 4 cases were grade 3A. By immunohistochemistry, 9 of 9 tested cases were CD10+, all cases were BCL6+, and 9/10 cases were BCL2+. MYC protein staining was low in all cases tested. IGH/BCL2 rearrangement was detected in 5 of 9 cases, whereas BCL6 rearrangement was detected in 3 of 7 tested cases and 4 of 10 cases showed MYC/IGH rearrangement. The most commonly detected mutations in the MYC-positive cases included HLA-B, TNFRSF14, and KMT2D. MYC and/or B2M abnormalities were detected in 2 cases. In conclusion, MYC rearrangement is uncommon in FL and these cases do not appear to have specific histologic characteristics. Molecular analysis showed abnormalities in genes associated with transformation, namely MYC and B2M. Larger studies are needed to evaluate if MYC-rearrangement in FL has prognostic significance.

Cost effectiveness analysis of a polygenic risk tailored breast cancer screening programme in Singapore.

BACKGROUND: This study aimed to evaluate the cost-effectiveness of a breast cancer screening programme that incorporates genetic testing using breast cancer associated single nucleotide polymorphisms (SNPs), against the current biennial mammogram-only screening programme to aid in its implementation into the current programme in Singapore. METHODS: A Markov model was used to compare the costs and health outcomes of the current screening programme, against a polygenic risk-tailored screening programme, which can advise a long-term screening strategy depending on the individual's polygenic risk. The model took the perspective of the healthcare system, with a time horizon of 40 years, following women from the age of 35 to 74. Epidemiological and cost data were taken from Asian studies, and an annual discount rate of 3% was used. The model outcome was the incremental cost-effectiveness ratio (ICER), calculated from the difference in costs per quality-adjusted life year (QALY). Scenarios with varying risk thresholds for each polygenic risk group were examined. One-way and probabilistic sensitivity analyses were performed to assess parameter uncertainty. RESULTS: The ICER for a polygenic risk-tailored breast cancer screening programme, compared with the current biennial mammogram-only screening programme, was - 3713.80 SGD/QALY, with incremental costs < 0 and incremental effects > 0. The scenario analysis of different polygenic risk cutoffs showed that the ICERs remain negative, with all ICERs falling within the south-east quadrant of the cost-effectiveness plane, indicating that tailored screening is more cost effective than mammogram-only screening, with lower costs and higher QALYs to be gained. This suggests that a polygenic risk-tailored breast cancer screening programme is cost effective, entailing lower cost than the current mammogram-only programme, while causing no additional harm to women. CONCLUSION: Results from this cost-effectiveness analysis show that polygenic risk-tailored screening is cost effective with an ICER of - 3713.80 SGD/QALY. Tailored screening remains cost effective even across varying percentile cutoffs for each risk group. While the results look promising for incorporating polygenic risk into the current breast cancer screening programme, further studies should be conducted to address various limitations.

Clinical outcomes of different therapeutic options for COVID-19 in two Chinese case cohorts: A propensity-score analysis.

BACKGROUND: The timing of administration of agents and use of combination treatments in COVID-19 remain unclear. We assessed the effectiveness of therapeutics in cohorts in Hong Kong SAR and Anhui, China. METHODS: We conducted propensity-score analysis of 4771 symptomatic patients from Hong Kong between 21st January and 6th December 2020, and 648 symptomatic patients from Anhui between 1st January and 27th February 2020. We censored all observations as at 13st December 2020. Time from hospital admission to discharge, and composite outcome of death, invasive mechanical ventilation or intensive care unit admission across 1) all therapeutic options including lopinavir-ritonavir, ribavirin, umifenovir, interferon-alpha-2b, interferon-beta-1b, corticosteroids, antibiotics, and Chinese medicines, and 2) four interferon-beta-1b combination treatment groups were investigated. FINDINGS: Interferon-beta-1b was associated with an improved composite outcome (OR=0.55, 95%CI 0.38, 0.80) and earlier discharge (-8.8 days, 95%CI -9.7, -7.9) compared to those not administered interferon-beta-1b. Oral ribavirin initiated within 7 days from onset was associated with lower risk of the composite outcome in Hong Kong (OR=0.51, 95%CI 0.29, 0.90). Lopinavir-ritonavir, intravenous ribavirin, umifenovir, corticosteroids, interferon-alpha-2b, antibiotics or Chinese medicines failed to show consistent clinical benefit. Interferon-beta-1b co-administered with ribavirin was associated with improved composite outcome (OR=0.50, 95%CI 0.32, 0.78) and earlier discharge (-2.35 days, 95%CI -3.65, -1.06) compared to interferon-beta-1b monotherapy. INTERPRETATION: Our findings support the early administration of interferon-beta-1b alone or in combination with oral ribavirin for COVID-19 patients. FUNDING: Hong Kong Health and Medical Research Fund; Hong Kong Innovation and Technology Commission; Chinese Fundamental Research Funds for the Central Universities.

Quantification of Early-Stage Myeloid-Derived Suppressor Cells in Cancer Requires Excluding Basophils.

Myeloid derived suppressor cells (MDSC) are a heterogeneous group of immature cells that accumulate in the peripheral blood and tumor microenvironment and are barriers to cancer therapy. MDSCs serve as prognostic biomarkers and are targets for therapy. On the basis of surface markers, three subsets of MDSCs have been defined in humans: granulocytic, monocytic, and early stage (e-MDSC). The markers attributed to e-MDSCs overlap with those of basophils, which are rare circulating myeloid cells with unrecognized roles in cancer. Thus, we asked whether e-MDSCs in circulation and the tumor microenvironment include basophils. On average, 58% of cells with e-MDSC surface markers in blood and 36% in ascites from patients with ovarian cancer were basophils based on CD123high expression and cytology, whereas cells with immature features were rare. Circulating and ascites basophils did not suppress proliferation of stimulated T cells, a key feature of MDSCs. Increased accumulation of basophils and basogranulin, a marker of basophil degranulation, were observed in ascites compared to serum in patients with newly diagnosed ovarian cancer. Basophils recruited to the tumor microenvironment may exacerbate fluid accumulation by their release of proinflammatory granular constituents that promote vascular leakage. No significant correlation was observed between peripheral basophil counts and survival in patients with ovarian cancer. Our results suggest that studies in which e-MDSCs were defined solely by surface markers should be reevaluated to exclude basophils. Both immaturity and suppression are criteria to define e-MDSCs in future studies.

RIPK1 Mediates TNF-Induced Intestinal Crypt Apoptosis During Chronic NF-κB Activation.

BACKGROUND AND AIMS: Tumor necrosis factor (TNF) is a major pathogenic effector and a therapeutic target in inflammatory bowel disease (IBD), yet the basis for TNF-induced intestinal epithelial cell (IEC) death is unknown, because TNF does not kill normal IECs. Here, we investigated how chronic nuclear factor (NF)- κB activation, which occurs in human IBD, promotes TNF-dependent IEC death in mice. METHODS: Human IBD specimens were stained for p65 and cleaved caspase-3. C57BL/6 mice with constitutively active IKKß in IEC (Ikkß(EE)IEC), Ripk1D138N/D138N knockin mice, and Ripk3-/- mice were injected with TNF or lipopolysaccharide. Enteroids were also isolated from these mice and challenged with TNF with or without RIPK1 and RIPK3 inhibitors or butylated hydroxyanisole. Ripoptosome-mediated caspase-8 activation was assessed by immunoprecipitation. RESULTS: NF-κB activation in human IBD correlated with appearance of cleaved caspase-3. Congruently, unlike normal mouse IECs that are TNF-resistant, IECs in Ikkß(EE)IEC mice and enteroids were susceptible to TNF-dependent apoptosis, which depended on the protein kinase function of RIPK1. Constitutively active IKKß facilitated ripoptosome formation, a RIPK1 signaling complex that mediates caspase-8 activation by TNF. Butylated hydroxyanisole treatment and RIPK1 inhibitors attenuated TNF-induced and ripoptosome-mediated caspase-8 activation and IEC death in vitro and in vivo. CONCLUSIONS: Contrary to common expectations, chronic NF-κB activation induced intestinal crypt apoptosis after TNF stimulation, resulting in severe mucosal erosion. RIPK1 kinase inhibitors selectively inhibited TNF destructive properties while preserving its survival and proliferative properties, which do not require RIPK1 kinase activity. RIPK1 kinase inhibition could be a potential treatment for IBD.

Mature neutrophils suppress T cell immunity in ovarian cancer microenvironment.

Epithelial ovarian cancer (EOC) often presents with metastases and ascites. Granulocytic myeloid-derived suppressor cells are an immature population that impairs antitumor immunity. Since suppressive granulocytes in the ascites of patients with newly diagnosed EOC were morphologically mature, we hypothesized that PMN were rendered suppressive in the tumor microenvironment (TME). Circulating PMN from patients were not suppressive but acquired a suppressor phenotype (defined as ≥1 log10 reduction of anti-CD3/CD28-stimulated T cell proliferation) after ascites supernatant exposure. Ascites supernatants (20 of 31 supernatants) recapitulated the suppressor phenotype in PMN from healthy donors. T cell proliferation was restored with ascites removal and restimulation. PMN suppressors also inhibited T cell activation and cytokine production. PMN suppressors completely suppressed proliferation in naive, central memory, and effector memory T cells and in engineered tumor antigen-specific cytotoxic T lymphocytes, while antigen-specific cell lysis was unaffected. Inhibition of complement C3 activation and PMN effector functions, including CR3 signaling, protein synthesis, and vesicular trafficking, abrogated the PMN suppressor phenotype. Moreover, malignant effusions from patients with various metastatic cancers also induced the C3-dependent PMN suppressor phenotype. These results point to PMN impairing T cell expansion and activation in the TME and the potential for complement inhibition to abrogate this barrier to antitumor immunity.

Epidemiology and Pathology of T- and NK-Cell Lymphomas.

PURPOSE: This review will describe and update readers on the recent changes in the 2017 WHO classification regarding peripheral T-cell lymphomas. RECENT FINDINGS: Signficant advances in molecular studies have resulted in revisions to the classification as well as introduction to provisional entities such as breast implant-associated ALCL and nodal PTCL with T-follicular helper phenotype. SUMMARY: Major advances in molecular and gene expression profiling has expanded our knowledge of these rare and aggressive diseases.

Elevated A20 promotes TNF-induced and RIPK1-dependent intestinal epithelial cell death.

Intestinal epithelial cell (IEC) death is a common feature of inflammatory bowel disease (IBD) that triggers inflammation by compromising barrier integrity. In many patients with IBD, epithelial damage and inflammation are TNF-dependent. Elevated TNF production in IBD is accompanied by increased expression of the TNFAIP3 gene, which encodes A20, a negative feedback regulator of NF-κB. A20 in intestinal epithelium from patients with IBD coincided with the presence of cleaved caspase-3, and A20 transgenic (Tg) mice, in which A20 is expressed from an IEC-specific promoter, were highly susceptible to TNF-induced IEC death, intestinal damage, and shock. A20-expressing intestinal organoids were also susceptible to TNF-induced death, demonstrating that enhanced TNF-induced apoptosis was a cell-autonomous property of A20. This effect was dependent on Receptor Interacting Protein Kinase 1 (RIPK1) activity, and A20 was found to associate with the Ripoptosome complex, potentiating its ability to activate caspase-8. A20-potentiated RIPK1-dependent apoptosis did not require the A20 deubiquitinase (DUB) domain and zinc finger 4 (ZnF4), which mediate NF-κB inhibition in fibroblasts, but was strictly dependent on ZnF7 and A20 dimerization. We suggest that A20 dimers bind linear ubiquitin to stabilize the Ripoptosome and potentiate its apoptosis-inducing activity.

New mitochondrial DNA synthesis enables NLRP3 inflammasome activation.

Dysregulated NLRP3 inflammasome activity results in uncontrolled inflammation, which underlies many chronic diseases. Although mitochondrial damage is needed for the assembly and activation of the NLRP3 inflammasome, it is unclear how macrophages are able to respond to structurally diverse inflammasome-activating stimuli. Here we show that the synthesis of mitochondrial DNA (mtDNA), induced after the engagement of Toll-like receptors, is crucial for NLRP3 signalling. Toll-like receptors signal via the MyD88 and TRIF adaptors to trigger IRF1-dependent transcription of CMPK2, a rate-limiting enzyme that supplies deoxyribonucleotides for mtDNA synthesis. CMPK2-dependent mtDNA synthesis is necessary for the production of oxidized mtDNA fragments after exposure to NLRP3 activators. Cytosolic oxidized mtDNA associates with the NLRP3 inflammasome complex and is required for its activation. The dependence on CMPK2 catalytic activity provides opportunities for more effective control of NLRP3 inflammasome-associated diseases.

Selenzyme: enzyme selection tool for pathway design.

Summary: Synthetic biology applies the principles of engineering to biology in order to create biological functionalities not seen before in nature. One of the most exciting applications of synthetic biology is the design of new organisms with the ability to produce valuable chemicals including pharmaceuticals and biomaterials in a greener; sustainable fashion. Selecting the right enzymes to catalyze each reaction step in order to produce a desired target compound is, however, not trivial. Here, we present Selenzyme, a free online enzyme selection tool for metabolic pathway design. The user is guided through several decision steps in order to shortlist the best candidates for a given pathway step. The tool graphically presents key information about enzymes based on existing databases and tools such as: similarity of sequences and of catalyzed reactions; phylogenetic distance between source organism and intended host species; multiple alignment highlighting conserved regions, predicted catalytic site, and active regions and relevant properties such as predicted solubility and transmembrane regions. Selenzyme provides bespoke sequence selection for automated workflows in biofoundries. Availability and implementation: The tool is integrated as part of the pathway design stage into the design-build-test-learn SYNBIOCHEM pipeline. The Selenzyme web server is available at http://selenzyme.synbiochem.co.uk. Supplementary information: Supplementary data are available at Bioinformatics online.

Phosphorylation and degradation of αB-crystallin during enterovirus infection facilitates viral replication and induces viral pathogenesis.

Protein quality control (PQC) plays a key role in maintaining cardiomyocyte function and homeostasis, and malfunction in PQC is implicated in various forms of heart diseases. Molecular chaperones serve as the primary checkpoint for PQC; however, their roles in the pathogenesis of viral myocarditis, an inflammation of the myocardium caused by viral infection, are largely unknown. AlphaB-crystallin (CryAB) is the most abundant chaperone protein in the heart. It interacts with desmin and cytoplasmic actin to prevent protein misfolding and aggregation and to help maintain cytoskeletal integrity and cardiac function. Here we showed that coxsackievirus infection induced desminopathy-like phenotype of the myocardium, as characterized by the accumulation of protein aggregates and the disruption of desmin organization. We further demonstrated that CryAB was phosphorylated during early and downregulated at later stages of infection. Moreover, we showed that phosphorylated CryAB had a shorter half-life and was targeted to the ubiquitin-proteasome system for degradation. Lastly, we found that overexpression of CryAB significantly attenuated viral protein production and progeny release, indicating an anti-viral function for CryAB. Together, our results suggest a mechanism by which coxsackieviral infection induces CryAB degradation and loss-of-function, resulting in desmin aggregation, ultimately contributing to compromised cytoskeletal integrity and viral cardiomyopathy.

GFI1 downregulation promotes inflammation-linked metastasis of colorectal cancer.

Inflammation is frequently associated with initiation, progression, and metastasis of colorectal cancer (CRC). Here, we unveil a CRC-specific metastatic programme that is triggered via the transcriptional repressor, GFI1. Using data from a large cohort of clinical samples including inflammatory bowel disease and CRC, and a cellular model of CRC progression mediated by cross-talk between the cancer cell and the inflammatory microenvironment, we identified GFI1 as a gating regulator responsible for a constitutively activated signalling circuit that renders CRC cells competent for metastatic spread. Further analysis of mouse models with metastatic CRC and human clinical specimens reinforced the influence of GFI1 downregulation in promoting CRC metastatic spread. The novel role of GFI1 is uncovered for the first time in a human solid tumour such as CRC. Our results imply that GFI1 is a potential therapeutic target for interfering with inflammation-induced CRC progression and spread.

NF-κB Restricts Inflammasome Activation via Elimination of Damaged Mitochondria.

Nuclear factor κB (NF-κB), a key activator of inflammation, primes the NLRP3-inflammasome for activation by inducing pro-IL-1ß and NLRP3 expression. NF-κB, however, also prevents excessive inflammation and restrains NLRP3-inflammasome activation through a poorly defined mechanism. We now show that NF-κB exerts its anti-inflammatory activity by inducing delayed accumulation of the autophagy receptor p62/SQSTM1. External NLRP3-activating stimuli trigger a form of mitochondrial (mt) damage that is caspase-1- and NLRP3-independent and causes release of direct NLRP3-inflammasome activators, including mtDNA and mtROS. Damaged mitochondria undergo Parkin-dependent ubiquitin conjugation and are specifically recognized by p62, which induces their mitophagic clearance. Macrophage-specific p62 ablation causes pronounced accumulation of damaged mitochondria and excessive IL-1ß-dependent inflammation, enhancing macrophage death. Therefore, the "NF-κB-p62-mitophagy" pathway is a macrophage-intrinsic regulatory loop through which NF-κB restrains its own inflammation-promoting activity and orchestrates a self-limiting host response that maintains homeostasis and favors tissue repair.

Dysferlin deficiency confers increased susceptibility to coxsackievirus-induced cardiomyopathy.

Coxsackievirus infection can lead to viral myocarditis and its sequela, dilated cardiomyopathy, which represent major causes of cardiovascular mortality worldwide in children. Yet, the host genetic susceptible factors and the underlying mechanisms by which viral infection damages cardiac function remain to be fully resolved. Dysferlin is a transmembrane protein highly expressed in skeletal and cardiac muscles. In humans, mutations in the dysferlin gene can cause limb-girdle muscular dystrophy type 2B and Miyoshi myopathy. Dysferlin deficiency has also been linked to cardiomyopathy. Defective muscle membrane repair has been suggested to be an important mechanism responsible for muscle degeneration in dysferlin-deficient patients and animals. Using both naturally occurring and genetically engineered dysferlin-deficient mice, we demonstrated that loss of dysferlin confers increased susceptibility to coxsackievirus infection and myocardial damage. More interestingly, we found that dysferlin is cleaved following coxsackieviral infection through the proteolytic activity of virally encoded proteinases, suggesting an important mechanism underlying virus-induced cardiac dysfunction. Our results in this study not only identify dysferlin deficiency as a novel host risk factor for viral myocarditis but also reveal a key mechanism by which coxsackievirus infection impairs cardiac function, leading to the development of dilated cardiomyopathy.

Interleukin-17 receptor a signaling in transformed enterocytes promotes early colorectal tumorigenesis.

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine linked to rapid malignant progression of colorectal cancer (CRC) and therapy resistance. IL-17A exerts its pro-tumorigenic activity through its type A receptor (IL-17RA). However, IL-17RA is expressed in many cell types, including hematopoietic, fibroblastoid, and epithelial cells, in the tumor microenvironment, and how IL-17RA engagement promotes colonic tumorigenesis is unknown. Here we show that IL-17RA signals directly within transformed colonic epithelial cells (enterocytes) to promote early tumor development. IL-17RA engagement activates ERK, p38 MAPK, and NF-κB signaling and promotes the proliferation of tumorigenic enterocytes that just lost expression of the APC tumor suppressor. Although IL-17RA signaling also controls the production of IL-6, this mechanism makes only a partial contribution to colonic tumorigenesis. Combined treatment with chemotherapy, which induces IL-17A expression, and an IL-17A neutralizing antibody enhanced the therapeutic responsiveness of established colon tumors. These findings establish IL-17A and IL-17RA as therapeutic targets in colorectal cancer.

Genes related to emphysema are enriched for ubiquitination pathways.

BACKGROUND: Increased small airway resistance and decreased lung elasticity contribute to the airflow limitation in chronic obstructive pulmonary disease (COPD). The lesion that corresponds to loss of lung elasticity is emphysema; the small airway obstruction is due to inflammatory narrowing and obliteration. Despite their convergence in altered physiology, different mechanisms contribute to these processes. The relationships between gene expression and these specific phenotypes may be more revealing than comparison with lung function. METHODS: We measured the ratio of alveolar surface area to lung volume (SA/V) in lung tissue from 43 smokers. Two samples from 21 subjects, in which SA/V differed by >49 cm2/mL were profiled to select genes whose expression correlated with SA/V. Significant genes were tested for replication in the 22 remaining subjects. RESULTS: The level of expression of 181 transcripts was related to SA/V ( p < 0.05). When these genes were tested in the 22 remaining subjects as a replication, thirty of the 181 genes remained significantly associated with SA/V (P < 0.05) and the direction of association was the same in 164/181. Pathway and network analysis revealed enrichment of genes involved in protein ubiquitination, and western blotting showed altered expression of genes involved in protein ubiquitination in obstructed individuals. CONCLUSION: This study implicates modified protein ubiquitination and degradation as a potentially important pathway in the pathogenesis of emphysema.