Coagulation Activation in Children with Sickle Cell Disease Is Associated with Cerebral Small Vessel Vasculopathy

Background

Thrombotic complications in Sickle Cell Disease (SCD) arise since infancy, but the role of the coagulation system in children has been poorly explored. To determine its role in the development of clinical complications in childhood we measured coagulation and endothelial parameters in children with SCD at steady state.

Methods

Markers of thrombin generation, fibrin dissolution and endothelial activation were evaluated in 38 children with SS-Sβ°, 6 with SC disease and 50 age and blood group matched controls. Coagulation variables were correlated with markers of hemolysis and inflammation, with the presence of cerebral and lung vasculopathy and with the frequency of clinical complications.

Results

SS-Sβ° patients presented higher levels of factor VIII, von Willebrand factor antigen (VWF:Ag) and collagen binding activity, tissue plasminogen activator antigen (t-PA:Ag), D-dimer, p-selectin, prothrombin fragment1+2 (F1+2) and lower ADAMTS-13:activity/VWF:Ag (p<0.05) compared to controls and SC patients. In SS-Sβ° patients coagulation variables correlated positively with markers of inflammation, hemolysis, and negatively with HbF (p<0.05). Patients with cerebral silent infarcts showed significant decrease in t-PA:Ag and ADAMTS-13 Antigen and a tendency toward higher D-dimer, F1+2, TAT compared to patients without them. D-dimer was associated with a six fold increased risk of cerebral silent infarcts. No correlation was found between coagulation activation and large vessel vasculopathy or other clinical events except for decreased t-PA:Ag in patients with tricuspid Rigurgitant Velocity >2.5m/sec.

Conclusions

SS-Sβ° disease is associated with extensive activation of the coagulation system at steady state since young age. ADAMTS-13 and t-PA:Ag are involved in the development of cerebral silent infarcts.     

Elevated Pulse Pressure is Associated with Hemolysis,Proteinuria and Chronic Kidney Disease in Sickle Cell Disease

A seeming paradox of sickle cell disease is that patients do not suffer from a high prevalence of systemic hypertension in spite of endothelial dysfunction, chronic inflammation and vasculopathy. However, some patients do develop systolic hypertension and increased pulse pressure, an increasingly recognized major cardiovascular risk factor in other populations. Hence, we hypothesized that pulse pressure, unlike other blood pressure parameters, is independently associated with markers of hemolytic anemia and cardiovascular risk in sickle cell disease. We analyzed the correlates of pulse pressure in patients (n  =  661) enrolled in a multicenter international sickle cell trial. Markers of hemolysis were analyzed as independent variables and as a previously validated hemolytic index that includes multiple variables. We found that pulse pressure, not systolic, diastolic or mean arterial pressure, independently correlated with high reticulocyte count (beta  =  2.37, p  =  0.02) and high hemolytic index (beta  =  1.53, p = 0.002) in patients with homozygous sickle cell disease in two multiple linear regression models which include the markers of hemolysis as independent variables or the hemolytic index, respectively. Pulse pressure was also independently associated with elevated serum creatinine (beta  =  3.21, p  =  0.02), and with proteinuria (beta  =  2.52, p  =  0.04). These results from the largest sickle cell disease cohort to date since the Cooperative Study of Sickle Cell Disease show that pulse pressure is independently associated with hemolysis, proteinuria and chronic kidney disease. We propose that high pulse pressure may be a risk factor for clinical complications of vascular dysfunction in sickle cell disease. Longitudinal and mechanistic studies should be conducted to confirm these hypotheses.     

Anti-Cyclic Citrullinated Peptide Antibody Is Associated with Interstitial Lung Disease in Patients with Rheumatoid Arthritis

Objective

Patients with rheumatoid arthritis (RA) are at risk to develop RA-associated interstitial lung disease (RA-ILD). This retrospective study aimed to investigate the potential association of the positivity of serum anti-cyclic citrullinated peptide antibody (anti-CCP2) and rheumatoid factor (RF) with RA-ILD in RA patients.

Methods

A total of 285 RA patients were recruited at the inpatient service of Peking Union Medical College Hospital in China between 2004 and 2013. Individual patients were evaluated for the evidence of ILD. The concentrations of serum anti-CCP2 and RF in individual patients were measured. The potential risk factors for ILD in RA patients were assessed by univariate and multivariate models.

Results

There were 71 RA patients with RA-ILD, accounting for 24.9% in this population. The positive rates of anti-CCP2 and RF in the patients with RA-ILD were significantly higher than that in the patients with RA-only (88.7% vs. 67.3%, p<0.001; 84.5% vs. 70.6%, p = 0.02, respectively). Univariate and multivariate logistic regression analysis revealed that RA patients with positive serum anti-CCP2, but not RF, were associated with an increased risk of ILD (crude odds ratio [cOR] 3.83, 95% confidence interval [CI] 1.74–8.43, p<0.001; adjusted odds ratio [aOR] 3.50, 95% CI 1.52–8.04, p<0.001).

Conclusion

Our findings suggest that positive serum anti-CCP2, but not RF, may be associated with RA-ILD in RA patients.     

Hydroxyurea-Increased Fetal Hemoglobin Is Associated with Less Organ Damage and Longer Survival in Adults with Sickle Cell Anemia

Background

Adults with sickle cell anemia (HbSS) are inconsistently treated with hydroxyurea.

Objectives

We retrospectively evaluated the effects of elevating fetal hemoglobin with hydroxyurea on organ damage and survival in patients enrolled in our screening study between 2001 and 2010.

Methods

An electronic medical record facilitated development of a database for comparison of study parameters based on hydroxyurea exposure and dose. This study is registered with ClinicalTrials.gov, number NCT00011648.

Results

Three hundred eighty-three adults with homozygous sickle cell disease were analyzed with 59 deaths during study follow-up. Cox regression analysis revealed deceased subjects had more hepatic dysfunction (elevated alkaline phosphatase, Hazard Ratio = 1.005, 95% CI 1.003–1.006, p<0.0.0001), kidney dysfunction (elevated creatinine, Hazard Ratio = 1.13, 95% CI 1.00–1.27, p = 0.043), and cardiopulmonary dysfunction (elevated tricuspid jet velocity on echocardiogram, Hazard Ratio = 2.22, 1.23–4.02, p = 0.0082). Sixty-six percent of subjects were treated with hydroxyurea, although only 66% of those received a dose within the recommended therapeutic range. Hydroxyurea use was associated with improved survival (Hazard Ratio = 0.58, 95% CI 0.34–0.97, p = 0.040). This effect was most pronounced in those taking the recommended dose of 15–35 mg/kg/day (Hazard Ratio 0.36, 95% CI 0.17–0.73, p = 0.0050). Hydroxyurea use was not associated with changes in organ function over time. Further, subjects with higher fetal hemoglobin responses to hydroxyurea were more likely to survive (p = 0.0004). While alkaline phosphatase was lowest in patients with the best fetal hemoglobin response (95.4 versus 123.6, p = 0.0065 and 96.1 versus 113.6U/L, p = 0.041 at first and last visits, respectively), other markers of organ damage were not consistently improved over time in patients with the highest fetal hemoglobin levels.

Conclusions

Our data suggest that adults should be treated with the maximum tolerated hydroxyurea dose, ideally before organ damage occurs. Prospective studies are indicated to validate these findings.     

Addition of Valproic Acid to CHO Cell Fed-Batch Cultures Improves Monoclonal Antibody Titers

Improving the productivity of a biopharmaceutical Chinese hamster ovary (CHO) fed-batch cell culture can enable cost savings and more efficient manufacturing capacity utilization. One method for increasing CHO cell productivity is the addition of histone deacetylase (HDAC) inhibitors to the cell culture process. In this study, we examined the effect of valproic acid (VPA, 2-propylpentanoic acid), a branched-chain carboxylic acid HDAC inhibitor, on the productivity of three of our CHO cell lines that stably express monoclonal antibodies. Fed-batch shake flask VPA titrations on the three different CHO cell lines yielded cell line-specific results. Cell line A responded highly positively, cell line B responded mildly positively, and cell line C did not respond. We then performed factorial experiments to identify the optimal VPA concentration and day of addition for cell line A. After identifying the optimal conditions for cell line A, we performed verification experiments in fed-batch bioreactors for cell lines A and B. These experiments confirmed that a high dose of VPA late in the culture can increase harvest titer >20 % without greatly changing antibody aggregation, charge heterogeneity, and N-linked glycosylation profiles. Our results suggest that VPA is an attractive and viable small molecule enhancer of protein production for biopharmaceutical CHO cell culture processes.     

Expression of the Stem Cell Factor Nestin in Malignant Pleural Mesothelioma Is Associated with Poor Prognosis

Background

The epithelioid and sarcomatoid histologic variants of malignant pleural mesothelioma (MPM) can be considered as E- and M-parts of the epithelial-mesenchymal transition (EMT) axis; the biphasic being an intermediate. EMT is associated with an increase of stem cell (SC) traits. We correlated the neural crest SC marker nestin and the EMT marker periostin with histology, type of neo-adjuvant chemotherapy (CT) and overall survival (OS) of MPM patients.

Patients and Methods

Tumor tissues of a historic cohort 1 (320 patients) and an intended induction chemotherapy followed by extrapleural pneumonectomy (EPP) cohort 2 (145 patients) were immunohistochemically H-scored (intensity of immunoreactivity multiplied by frequency of stained cells). Paired chemo-naïve biopsies and -treated surgical specimens were available for 105/145 patients. CT included platinum/gemcitabine (Pla/Gem) or platinum/pemetrexed (Pla/Pem).

Results

Expression of any cytosolic nestin progressively increased from epithelioid to biphasic to sarcomatoid MPM in cohort 1, whereas the diagnostic markers calretinin and podoplanin decreased. In cohort 2, Pla/Pem CT increased the expression level of nestin in comparison to Pla/Gem, whereas the opposite was found for periostin. In Pla/Pem treated patients, nestin was higher in biphasic MPM compared to epithelioid. In addition to non-epithelioid histology, any expression of nestin in chemo-naïve biopsies (median overall survival: 22 vs. 17 months) and chemo-treated surgical specimens (18 vs. 12 months) as well as high periostin in biopsies (23 vs. 15 months) were associated with poor prognosis. In the multivariate survival analysis, any nestin expression in chemo-naïve biopsies proved to be an independent prognosticator against histology. In both pre- and post-CT situations, the combination of nestin or periostin expression with non-epithelioid histology was particularly/ dismal (all p-values <0.05).

Conclusions

The SC marker nestin and the EMT marker periostin allow for further prognostic stratification among histologic variants of MPM. Their expression level is influenced by neo-adjuvant chemotherapy.     

Increased Expression of Pregnancy Up-Regulated Non-Ubiquitous Calmodulin Kinase Is Associated with Poor Prognosis in Clear Cell Renal Cell Carcinoma

Purpose

The aims of this study were to evaluate the clinical significance and potential prognostic value of pregnancy up-regulated non-ubiquitous calmodulin kinase (PNCK) in clear cell renal cell carcinoma (ccRCC) patients.

Materials and Methods

The expression of PNCK mRNA was determined in 24 paired samples of ccRCCs and adjacent normal tissues using real-time RT-PCR. The expression of PNCK was determined in 248 samples of ccRCCs and 92 paired samples of adjacent normal tissues by immunohistochemical analysis. Statistical analysis was performed to define the relationship between PNCK expression and the clinical features of ccRCC.

Results

The mRNA level of PNCK was significantly higher in tumorous tissues than in the adjacent non-tumorous tissues (p<0.001). An immunohistochemical analysis of 92 paired tissue specimens showed that PNCK expression was higher in tumorous tissues than in the adjacent non-tumorous tissues (p<0.001). Moreover, there was a significant correlation between the PNCK expression and various clinicopathological parameters such as Fuhrman grade (p = 0.011), tumor size (p<0.001), T stage (p<0.001) and N stage (p = 0.015). Patients with higher PNCK expression had shorter overall survival time than those with lower PNCK expression (p<0.001). Multivariate analysis indicated that PNCK expression was an independent predictor for poor survival of ccRCC patients.

Conclusions

To our knowledge, this is the first study that determines the relationship between PNCK and prognosis in ccRCC. We found that increased PNCK expression is associated with poor prognosis in ccRCC. PNCK may represent a novel prognostic marker for ccRCC.     

Lethal Antibody Enhancement of Dengue Disease in Mice Is Prevented by Fc Modification

Immunity to one of the four dengue virus (DV) serotypes can increase disease severity in humans upon subsequent infection with another DV serotype. Serotype cross-reactive antibodies facilitate DV infection of myeloid cells in vitro by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody-dependent enhancement (ADE). However, despite decades of investigation, no in vivo model for antibody enhancement of dengue disease severity has been described. Analogous to human infants who receive anti-DV antibodies by transplacental transfer and develop severe dengue disease during primary infection, we show here that passive administration of anti-DV antibodies is sufficient to enhance DV infection and disease in mice using both mouse-adapted and clinical DV isolates. Antibody-enhanced lethal disease featured many of the hallmarks of severe dengue disease in humans, including thrombocytopenia, vascular leakage, elevated serum cytokine levels, and increased systemic viral burden in serum and tissue phagocytes. Passive transfer of a high dose of serotype-specific antibodies eliminated viremia, but lower doses of these antibodies or cross-reactive polyclonal or monoclonal antibodies all enhanced disease in vivo even when antibody levels were neutralizing in vitro. In contrast, a genetically engineered antibody variant (E60-N297Q) that cannot bind FcγR exhibited prophylactic and therapeutic efficacy against ADE-induced lethal challenge. These observations provide insight into the pathogenesis of antibody-enhanced dengue disease and identify a novel strategy for the design of therapeutic antibodies against dengue.     

Expression of Sirtuin 1 and 2 Is Associated with Poor Prognosis in Non-Small Cell Lung Cancer Patients

Background

Sirtuin 1 (SIRT1) and sirtuin 2 (SIRT2) are NAD+-dependent protein deacetylases involved in the regulation of key cancer-associated genes. In this study we evaluated the relevance of these deacetylases in lung cancer biology.

Material and Methods

Protein levels of SIRT1 and SIRT2 were determined in non-small cell lung cancer (NSCLC) cell lines and primary tumors from 105 patients. Changes in proliferation were assessed after SIRT1 and SIRT2 downregulation in lung cancer cell lines using siRNA-mediated technology or tenovin-1, a SIRT1 and SIRT2 inhibitor.

Results

High SIRT1 and SIRT2 protein levels were found in NSCLC cell lines compared with non-tumor lung epithelial cells. The expression of SIRT1 and SIRT2 proteins was also significantly higher in lung primary tumors than in normal tissue (P<0.001 for both sirtuins). Stronger nuclear SIRT1 staining was observed in adenocarcinomas than in squamous cell carcinomas (P=0.033). Interestingly, in NSCLC patients, high SIRT1 and SIRT2 expression levels were associated with shorter recurrence-free survival (P=0.04 and P=0.007, respectively). Moreover, the combination of high SIRT1 and SIRT2 expression was an independent prognostic factor for shorter recurrence-free survival (P=0.002) and overall survival (P=0.022). In vitro studies showed that SIRT1 and/or SIRT2 downregulation significantly decreased proliferation of NSCLC.

Conclusions

Our results support the hypothesis that SIRT1 and SIRT2 have a protumorigenic role in lung cancer, promoting cell proliferation. Moreover, the expression of these proteins is associated with poor prognosis in NSCLC patients and may help to identify those NSCLC patients with high risk of recurrence that could benefit from adjuvant therapy after resection.     

Monocarboxylate Transporter 4 Facilitates Cell Proliferation and Migration and Is Associated with Poor Prognosis in Oral Squamous Cell Carcinoma Patients

Monocarboxylate transporter 4 (MCT4) is a cell membrane transporter of lactate. Recent studies have shown that MCT4 is over-expressed in various cancers; however, its role in cancer maintenance and aggressiveness has not been fully demonstrated. This study investigated the role of MCT4 in oral squamous cell carcinoma (OSCC), and found that it is highly expressed in OSCC patients by using immunohistochemistry. Moreover, this over-expression of MCT4 was closely associated with tumor size, TNM classification, lymphatic metastasis, distant metastasis and tumor recurrence, and also poor prognosis. To further study mechanisms of MCT4 in vitro, we used small-interfering RNA to silence its expression in OSCC cell lines. The results showed that knock-down of MCT4 decreased cell proliferation, migration, and invasion. The inhibition of proliferation was associated with down-regulation of p-AKT and p-ERK1/2, while decreased cell migration and invasion may be caused by down-regulation of integrin β4-SRC-FAK and MEK-ERK signaling. Together, these findings provide new insight into the critical role of MCT4 in cell proliferation and metastasis in OSCC.     

EIF4EBP1 Overexpression Is Associated with Poor Survival and Disease Progression in Patients with Hepatocellular Carcinoma

ObjectiveEIF4EBP1 acts as a crucial effector in mTOR signaling pathway. Studies have suggested that EIF4EBP1 plays a critical role in carcinogenesis. However, the clinical significance and biological role of EIF4EBP1 in hepatocellular carcinoma (HCC) have not been elucidated. Therefore, we aimed to investigate the clinical significance of EIF4EBP1 in HCC.MethodsTotal 128 cases of HCCs were included in this study. EIF4EBP1 expression in HCC tissues was detected by qRT-PCR, Western blot and immunohistochemistry, respectively. Then the relationships between EIF4EBP1 expression and clinical features as well as survival were analyzed.ResultsThe expression level of EIF4EBP1 mRNA is significantly higher in 60% (24/40) of fresh HCC tissues than that in the matched adjacent nontumor liver (NCL) tissues (P = 0.044). Similarly, EIF4EBP1 protein is notably upregulated in 8 HCC tissues (randomly selected from the 40 HCCs) measured by Western blot and is significantly increased in another 88 paraffin-embedded HCCs (53%, 47/88) by immunohistochemistry compared with the matched NCLs (P < 0.001). EIF4EBP1 protein expression in HCC tissues is significantly correlated with serum AFP (P = 0.003) and marginally significantly associated with pathological grade (P = 0.085), tumor number (P = 0.084), tumor embolus (P = 0.084) and capsulation (P = 0.073). Patients with higher EIF4EBP1 protein expression have a much worse 5-year overall survival (40.3% vs 73.6%) and 5-year disease-free survival (33.0% vs 49.0%) than those with low expression. Furthermore, Cox regression analysis shows that EIF4EBP1 protein is an independent prognostic factor for overall survival (HR, 2.285; 95% CI, 1.154–4.527; P = 0.018) and disease-free survival (HR, 1.901; 95% CI, 1.067–3.386; P = 0.029) in HCC patients.ConclusionsOur results demonstrate for the first time that EIF4EBP1 mRNA and protein are markedly up-regulated in HCC tissues, and the protein overexpression is significantly associated with poor survival and progression, which provide a potential new prognostic marker and therapeutic target for HCC patients.     

Mild Concussion,but Not Moderate Traumatic Brain Injury,Is Associated with Long-Term Depression-Like Phenotype in Mice

Mild traumatic brain injuries can lead to long-lasting cognitive and motor deficits, increasing the risk of future behavioral, neurological, and affective disorders. Our study focused on long-term behavioral deficits after repeated injury in which mice received either a single mild CHI (mCHI), a repeated mild CHI (rmCHI) consisting of one impact to each hemisphere separated by 3 days, or a moderate controlled cortical impact injury (CCI). Shams received only anesthesia. Behavioral tests were administered at 1, 3, 5, 7, and 90 days post-injury (dpi). CCI animals showed significant motor and sensory deficits in the early (1–7 dpi) and long-term (90 dpi) stages of testing. Interestingly, sensory and subtle motor deficits in rmCHI animals were found at 90 dpi. Most importantly, depression-like behaviors and social passiveness were observed in rmCHI animals at 90 dpi. These data suggest that mild concussive injuries lead to motor and sensory deficits and affective disorders that are not observed after moderate TBI.     

An Experimental-Computational Approach to Quantify Blood Rheology in Sickle Cell Disease

In sickle cell disease, aberrant blood flow due to oxygen-dependent changes in red cell biomechanics is a key driver of pathology. Most studies to date have focused on the potential role of altered red cell deformability and blood rheology in precipitating vaso-occlusive crises. Numerous studies, however, have shown that sickle blood flow is affected even at high oxygen tensions, suggesting a potentially systemic role for altered blood flow in driving pathologies, including endothelial dysfunction, ischemia, and stroke. In this study, we applied a combined experimental-computation approach that leveraged an experimental platform that quantifies sickle blood velocity fields under a range of oxygen tensions and shear rates. We computationally fitted a continuum model to our experimental data to generate physics-based parameters that capture patient-specific rheological alterations. Our results suggest that sickle blood flow is altered systemically, from the arterial to the venous circulation. We also demonstrated the application of this approach as a tool to design patient-specific transfusion regimens. Finally, we demonstrated that patient-specific rheological parameters can be combined with patient-derived vascular models to identify patients who are at higher risk for cerebrovascular complications such as aneurysm and stroke. Overall, this study highlights that sickle blood flow is altered systemically, which can drive numerous pathologies, and this study demonstrates the potential utility of an experimentally parameterized continuum model as a predictive tool for patient-specific care.     

TLR9 Promoter Polymorphism Is Associated with Both an Increased Susceptibility to Gastric Carcinoma and Poor Prognosis

Objective

Genetic polymorphisms of Toll-like receptors (TLRs) may influence the effects of H. pylori infection and play important roles in gastric carcinogenesis. The aim of this study was to determine whether the polymorphisms of TLR4 and TLR9 are associated with susceptibility to gastric carcinoma and its prognosis.

Methods

This study consisted of 314 patients with gastric cancer and 314 healthy controls. The polymorphisms were assessed using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis. Survival was analyzed by Kaplan–Meier survival curves.

Results

No variant genotypes of TLR4+896A/G, TLR4+1196C/T, or TLR9 -1237T/C were detected. For TLR9 -1486 T/C, multiple logistic regression analyses revealed that compared with the TT homozygote, patients with both the TC variant (adjusted odds ratio (OR) = 1.47, 95% confidence interval (CI) = 1.04–2.10) and the CC variant (adjusted OR = 1.63, 95% CI = 1.01–2.64) had higher risks of gastric cancer. Further stratification analyses revealed that an increased risk of gastric cancer associated with C carriers was evident among females (adjusted OR = 1.84, 95%CI = 1.02–3.33), in younger subjects aged less than 60 years old (adjusted OR = 1.86, 95%CI = 1.15–3.00), and subjects with H. pylori infection (adjusted OR = 1.53, 95% CI = 1.03–2.27). We also observed a significant association between C carriers and noncardia gastric cancer (adjusted OR = 1.51, 95% CI = 1.03–2.20). In addition, we demonstrated that the C carrier genotype and H. pylori infection may have a synergistic effect and conferred an OR of 2.44 for developing gastric cancer. TLR9 -1486C was also identified as an independent marker of poor survival of carcinoma.

Conclusions

Our results suggest that TLR9 -1486C carriers are associated with an increased risk and poor prognosis of gastric carcinoma in the Chinese population.     

FoxM1 Is Associated with Poor Prognosis of Non-Small Cell Lung Cancer Patients through Promoting Tumor Metastasis

Background

FoxM1 has been reported to be important in initiation and progression of various tumors. However, whether FoxM1 has any indication for prognosis in non-small cell lung cancer patients remains unclear.

Methodology/Principal Findings

In this study, FoxM1 expression in tumor cells was examined first by immunohistochemistry in 175 NSCLC specimens, the result of which showed that FoxM1 overexpression was significantly associated with positive smoking status (P = 0.001), poorer tissue differentiation (P = 0.0052), higher TNM stage (P<0.0001), lymph node metastasis (P<0.0001), advanced tumor stage (P<0.0001), and poorer prognosis (P<0.0001). Multivariable analysis showed that FoxM1 expression increased the hazard of death (hazard ratio, 1.899; 95% CI, 1.016–3.551). Furthermore, by various in vitro and in vivo experiments, we showed that targeted knockdown of FoxM1 expression could inhibit the migratory and invasive abilities of NSCLC cells, whereas enforced expression of FoxM1 could increased the invasion and migration of NSCLC cells. Finally, we found that one of the cellular mechanisms by which FoxM1 promotes tumor metastasis is through inducing epithelial-mesenchymal transition (EMT) program.

Conclusions

These results suggested that FoxM1 overexpression in tumor tissues is significantly associated with the poor prognosis of NSCLC patients through promoting tumor metastasis.     

Chronic Obstructive Pulmonary Disease Is Not Associated with KRAS Mutations in Non-Small Cell Lung Cancer

Mutations in epithelial growth factor receptor (EGFR), as well as in the EGFR downstream target KRAS are frequently observed in non-small cell lung cancer (NSCLC). Chronic obstructive pulmonary disease (COPD), an independent risk factor for developing NSCLC, is associated with an increased activation of EGFR. In this study we determined presence of EGFR and KRAS hotspot mutations in 325 consecutive NSCLC patients subjected to EGFR and KRAS mutation analysis in the diagnostic setting and for whom the pulmonary function has been determined at time of NSCLC diagnosis. Information about age at diagnosis, sex, smoking status, forced vital capacity (FVC) and forced expiratory volume in 1 sec (FEV₁) was collected. Chronic obstructive pulmonary disease(COPD) was defined according to 2013 GOLD criteria. Chi-Square, student t-test and multivariate logistic regression were used to analyze the data. A total of 325 NSCLC patients were included, 193 with COPD and 132 without COPD. COPD was not associated with presence of KRAS hotspot mutations, while EGFR mutations were significantly higher in non-COPD NSCLC patients. Both female gender (HR 2.61; 95% CI: 1.56–4.39; p<0.001) and smoking (HR 4.10; 95% CI: 1.14–14.79; p = 0.03) were associated with KRAS mutational status. In contrast, only smoking (HR 0.11; 95% CI: 0.04–0.32; p<0.001) was inversely associated with EGFR mutational status. Smoking related G>T and G>C transversions were significantly more frequent in females (86.2%) than in males (61.5%) (p = 0.008). The exon 19del mutation was more frequent in non-smokers (90%) compared to current or past smokers (36.8%). In conclusion, KRAS mutations are more common in females and smokers, but are not associated with COPD-status in NSCLC patients. EGFR mutations are more common in non-smoking NSCLC patients.     

The Physical Foundation of Vasoocclusion in Sickle Cell Disease

The pathology of sickle cell disease arises from the occlusion of small blood vessels because of polymerization of the sickle hemoglobin within the red cells. We present measurements using a microfluidic method we have developed to determine the pressure required to eject individual red cells from a capillary-sized channel after the cell has sickled. We find that the maximum pressure is only ∼100 Pa, much smaller than typically found in the microcirculation. This explains why experiments using animal models have not observed occlusion beginning in capillaries. The magnitude of the pressure and its dependence on intracellular concentration are both well described as consequences of sickle hemoglobin polymerization acting as a Brownian ratchet. Given the recently determined stiffness of sickle hemoglobin gels, the observed obstruction seen in sickle cell disease as mediated by adherent cells can now be rationalized, and surprisingly suggests a window of maximum vulnerability during circulation of sickle cells.Human capillaries are narrower than the erythrocytes they convey. In sickle cell disease, red cells can become rigid in those capillaries, because the hemoglobin inside the red cell will aggregate into stiff polymers. This happens once the molecules deliver their oxygen, and led to the long-held view that capillary occlusion was central to the pathophysiology of the disease (1,2). This was challenged when microscopic study of animal model tissues perfused with sickle blood revealed blockages that began further downstream, in the somewhat larger venules (3–5), at the site of adherent red or white cells which diminished the vessel lumen without fully obstructing the flow. Yet no rationale has been presented for the failure of the prior assumption of capillary blockage. Microfluidic methods (6) are ideally suited to discover why cells don’t get stuck in the capillaries, yet occlude subsequent vessels, and we have constructed a system to address this question. Our measurements show that the pressure differences across capillaries in vivo can easily dislodge a cell sickled within a capillary, giving an experimental answer to the question of why sickled cells don’t stick in capillaries. It turns out that the pressure a cell can withstand is quantitatively explained by the Brownian ratchet behavior of sickle hemoglobin polymerization.We constructed single-cell channels in transparent polydimethylsiloxane, with a cross section (1.5 μm × 4 μm) that is smaller than the resting diameter of red cells (Fig. 1). These channels are much narrower than those that have been employed in other recent studies of the sickling process (7,8), and they resemble human capillaries in permitting only one cell at a time to pass through them. We used a laser photolysis method to create ligand free (deoxygenated) cells, and this requires that the hemoglobin bind CO, which can then be readily removed by strong illumination, in contrast to bound O₂ which is released with far lower efficiency than CO. The microfluidic chips were enclosed in a gas-tight chamber flushed with CO to avoid introduction of oxygen and keep the cells fully ligated before photolysis. The profiles of the channels were confirmed by microscopic observation. To confirm that liquid did not pass around the cells when they were trapped in the channels, fluorescent beads were introduced into some cell solutions. The beads did not pass the cells, nor did they approach the cell when it was occluded, verifying that no significant flow occurred around the cell when it was stuck.Open in a separate windowFigure 1An erythrocyte enters a channel (moving left to right) and is positioned in the center, where it will be photolyzed. The channel cross section is 1.5 μm × 4 μm, smaller than a resting red cell diameter.Optical measurements were carried out on a microspectrophotometer constructed on an optical table. The system employed ×32 LWD objectives (Leitz, Wetzlar, Germany), which were autofocused during collection of absorption spectra to minimize aberrations. Spectra were obtained using a series 300 camera (Photometrics, Tucson, AZ); video imaging was done with a high-speed camera (Photron, San Diego, CA). Photolysis was provided by a 2020 Argon Ion laser (Spectra Physics, Houston, TX). Sickle cells were obtained from patients at the Marian Anderson Sickle Cell Center at St. Christopher''s Hospital for Children, Philadelphia, PA by phlebotomy into EDTA-containing tubes. The blood was centrifuged at 5°C at 1200g for 4 min, and then the pellet was washed 4× with 15 volumes of buffer (120 mM NaCl, 2 mM KCl, 10 mM dibasic Na Phosphate, 7 mM monobasic Na Phosphate, 3.4 mM Na Bicarbonate, and 6 mM Dextrose) by repeated suspension and centrifugation at 30g for 4 min. This minimizes fibrinogen and platelets in the final suspension, to insure that these studies are controlled by the mechanical properties of the cells themselves.Our experiment began by parking a cell in the center of a channel (Fig. 1). The cell, its hemoglobin, and the microchannel environment all were saturated with CO. Because the thickness of the channel is known, we were able to determine the hemoglobin concentration inside the cell from its absorption spectrum (Fig. 2 A). Steady-state laser illumination then removed the CO, allowing the hemoglobin to polymerize, in which condition it remained while the laser was kept on. Removal of CO was confirmed by observing the spectral difference between COHb and deoxyHb. Photolysis of COHb generates negligible heating (9–11). During illumination, hydrostatic pressure was applied until the cell broke free.Open in a separate windowFigure 2(A) Absorption of the cell (points), fit to a standard spectrum (9). (B) Pressure to dislodge a cell sickled in the microchannel, as a function of intracellular concentration. Note that typical intracellular concentrations are ∼32 g/dL. (Line) Brownian-ratchet theory described in the text. The coefficient of friction (0.036) is within the observed range, and is the only parameter varied.The magnitude of the dislodging pressure, measured by simple height difference between input and output cell reservoirs, is shown in Fig. 2 B. The pressure needed to dislodge the cell increased with increasing intracellular Hb concentration, implying that an increased mass of polymerized hemoglobin is more difficult to dislodge. A clear concentration threshold for capture is apparent. While there is a well-known solubility below which polymers cannot form (18.5 g/dL for the 22°C of this experiment (12)), the threshold here is significantly higher.Central to explaining these observations is a Brownian ratchet mechanism (13) which derives from the metastable nature of this polymerization process. Unless disrupted, as by centrifugation, polymerization in sickle hemoglobin terminates before the thermodynamic limit of monomer solubility is reached (14,15). This arises from the fact that polymers only grow at their ends, which are easily occluded in the dense mass of polymers that form. This end obstruction leaves the system in a metastable state and fluctuations accordingly provide polymers with space into which they can incrementally grow. This Brownian ratchet has been shown to lead to dramatic fiber buckling when individual fibers are isolated in sickle cells (16). The force can be simply expressed as f = (kT/δ) ln S(c), where k is Boltzmann’s constant, T the absolute temperature, δ the net spatial elongation from addition of a single monomer, and S is the supersaturation of the solution when the metastable limit is reached, at monomer concentration c. In this calculation, c is taken as the terminal concentration, computed from our empirical finding (15) that in this metastable system the amount of polymerized hemoglobin Δ is Δ(∞) = 2/3 (c_o-c_s), rather than the expected thermodynamic limit c_o-c_s, where c_o is the initial concentration and c_s is the solubility.For determining the net force, the total number of fibers must be known, and can be calculated based on the double nucleation mechanism (17) which has been quantitatively successful in describing polymerization. The concentration of polymers [p(t)] initially grows exponentially, described by[p(t)]=(AB2J)exp(Bt),where A and B are parameters related to nucleation, and J is the polymer elongation rate, as described in Ferrone et al. (17). Because A and B are both extremely concentration-dependent (9), they will drop dramatically once monomers begin to add to polymers in any significant numbers, and thereby diminish the remaining monomer pool. Thanks to the extreme concentration dependence of the reaction, this rapidly shuts off further polymerization. This happens at approximately the 10th time (the time when the reaction has reached 1/10 of its maximum). Thus, the [p(t_1/10)] ≈ [p(∞)]. Moreover, at one-tenth of the reaction,Δ(t1/10)=12Aexp(Bt1/10)=Δ(∞)10,and thus[p(∞)]=(BJ)(Δ(∞)10)=(BJ)((co−cs)15).For computing the number of fibers, the volume of the cell was taken as 90 μm³. This calculation shows, as expected, that the number of polymers in the cell is highly concentration-dependent, and very few fibers are produced at concentrations just above solubility, but the number grows sharply as concentration rises. This is the main contribution to the threshold in holding force shown by the data.With the force per fiber, and the total number of fibers, the net force against the wall is known. With a coefficient of friction, this reveals the force that a trapped cell can withstand. If the force is divided by the cross-sectional area across which the force is applied, we get a prediction of the dislodging pressure, which can be compared to the data. For a quantitative comparison with the results, two further corrections, of order unity, were applied. Because only normal force will contribute to friction, the calculated force was determined by integrating cos θ. This integration is not over all angles (π) because of the possibility that large incidence angles of the fibers against the wall will lead to fiber runaway (18). Therefore, the integration described is taken to the runaway threshold, here ∼1 rad. Finally, it is necessary to assign a coefficient of friction. Known values span the range of 0.03–0.06 (19). We therefore selected a value within the range, 0.036, as the best match for the data. The predicted pressures match the measurements well, as the line in Fig. 2 B shows.Because the flow resistance is comparable for red cells traversing glass channels and endothelial-lined capillaries (20), we conclude that in vivo the pressures a sickled cell inside a capillary can withstand are no more than hundreds of Pa. This is significantly smaller than typical arteriovenous pressure differentials that have been measured, which range from 0.7 kPa (in hamster skin (21)) to 7.9 kPa (in rat mesentery (22)).Our measurements coupled with recent determination of the stiffness of sickle hemoglobin gels (23) provide the missing physical basis for the processes of vasoocclusion seen in ex vivo tissue and animal models of sickle cell disease, arguing that these observations indeed represent fundamental behavior of sickle cell disease. We now understand this behavior in terms of three possible outcomes, all intimately connected with kinetics:

• 1.Certain escape: A cell that does not polymerize until after passing the obstruction can reach the lungs where it reoxygenates and resets its polymerization clock.
• 2.Possible escape: A cell that polymerizes within the capillary will assume an elongated sausage shape. The forces that it can exert against the wall cannot hold it there, and it will emerge into the postcapillary venule. There it has some chance of passing a subsequent obstruction, though it might also obstruct flow were it to rotate before reaching the adherent cell, so as to present its long dimension to the reduced space it must traverse.
• 3.Certain occlusion: A cell that does not polymerize in the capillary reassumes a larger diameter as soon as it escapes. If the cell then polymerizes before it encounters a cell attached to the venule wall, this rigidified cell will not be able to squeeze past the adherent cell, because that kind of deformation takes MPa (23). This would precipitate the type of blockage that is observed. This suggests that there is a window of greatest vulnerability, toward which therapies might be addressed.