Early-Life Microbial Restitution Reduces Colitis Risk Promoted by Antibiotic-Induced Gut Dysbiosis in Interleukin 10-/- Mice.

BACKGROUND & AIMS: Perturbations in the early-life gut microbiome are associated with increased risk for complex immune disorders like inflammatory bowel diseases. We previously showed that maternal antibiotic-induced gut dysbiosis vertically transmitted to offspring increases experimental colitis risk in interleukin (IL) 10 gene deficient (IL10-/-) mice, a finding that may result from the loss/lack of essential microbes needed for appropriate immunologic education early in life. Here, we aimed to identify key microbes required for proper development of the early-life gut microbiome that decrease colitis risk in genetically susceptible animals. METHODS: Metagenomic sequencing followed by reconstruction of metagenome-assembled genomes was performed on fecal samples of IL10-/- mice with and without antibiotic-induced dysbiosis to identify potential missing microbial members needed for immunologic education. One high-value target strain was then engrafted early and/or late into the gut microbiomes of IL10-/- mice with antibiotic-induced dysbiosis. RESULTS: Early-, but not late-, life engraftment of a single dominant Bacteroides strain of non-antibiotic-treated IL10-/- mice was sufficient to restore the development of the gut microbiome, promote immune tolerance, and prevent colitis in IL10-/- mice that had antibiotic-induced dysbiosis. CONCLUSIONS: Restitution of a keystone microbial strain missing in the early-life antibiotic-induced gut dysbiosis results in recovery of the microbiome, proper development of immune tolerance, and reduced risk for colitis in genetically prone hosts.

Impact of BRCA mutation on the survival and risk of contralateral breast cancer in Asian breast cancer patients.

PURPOSE: Breast cancer is increasing around the globe, including Asia. We aimed to examine the survival and risk of contralateral breast cancer (CBC) in Asian breast cancer patients with BRCA mutations. METHODS: A total of 128 breast cancer patients with germline BRCA mutations and 4,754 control breast cancer patients were enrolled. Data on clinical-pathologic characteristics, survival, and CBC were collected from the medical record. The rates of survival and CBC were estimated by Kaplan-Meier method. RESULTS: The mean age of onset in BRCA mutation carriers was significantly younger than control patients (BRCA vs. Non-BRCA: 43.9 vs. 53.2 years old). BRCA mutation carriers had a higher proportion of triple-negative breast cancer (TNBC) (52%) than control patients (12%, p < 0.001). The risk of CBC was significantly higher in BRCA mutation patients than in control cases (hazard ratio (HR) = 3.95, 95% CI 2.71-5.75); when stratified by genotype, the HRs (95%CI) were 4.84 (3.00-7.82) for BRCA1 and 3.13 (1.78-5.49) for BRCA2 carriers, respectively. Moreover, BRCA1 mutation patients with triple-negative breast cancer (TNBC) as their first breast cancer had the highest risk of CBC (HR = 5.55, 95% CI 3.29-9.34). However, we did not observe any differences in relapse-free survival and overall survival between mutation carriers and control patients. CONCLUSION: Our study suggest that BRCA patients had a significantly higher risk of developing CBC, particularly for BRCA1 mutation carriers with TNBC as the first breast cancer.

Aquagenic wrinkling of the palms: review of the literature.

Aquagenic wrinkling of the palms (AWP) is a rare dermatological disease characterized by development of rapid and excessive wrinkling and oedema of the palms and transient whitish or yellowish papules without erythema on the palmar surfaces after immersion in water. This phenomenon can be accompanied by pain and/or pruritus. The most common treatment of AWP involves aluminium-based topicals. This article discusses the associations, pathological mechanisms and treatment options of AWP.

A Case of Lemierre-Like Syndrome in a Pediatric Patient.

Lemierre-like syndrome is a rare, systemic sequelae following a persistent oropharyngeal infection, leading to septic thrombophlebitis of the internal jugular vein (IJV). Lemierre syndrome is caused by the obligate anaerobic organism Fusobacterium necrophorum, innate to the oropharyngeal tract. Lemierre-like syndrome is due to infections caused by other organisms, including methicillin-resistant Staphylococcus aureus (MRSA). We are reporting a case of a five-month-old male who presented with one week of fever that was not alleviated by acetaminophen, bilateral otitis media, and left-sided cervical lymphadenopathy not alleviated with medical therapy. The patient's clinical course continued to deteriorate as he developed respiratory distress that progressed to acute respiratory failure requiring mechanical ventilation support. Extensive laboratory investigation ruled out the causes of primary and secondary immunodeficiencies. Blood cultures were positive for MRSA, and he was treated initially with vancomycin, then switched to linezolid per ENT recommendations, and ultimately needed daptomycin and ceftaroline therapy. A computed tomography (CT) scan of the neck and chest showed deep neck space infection, bilateral loculated pleural empyema, and mediastinitis. The patient required a decortication video-assisted thoracoscopic surgery (VATS), multiple drains, and a mediastinal washout to control the MRSA infection. This report emphasizes that the rapid progression and spread of septic thrombus can become detrimental to a patient's recovery and survival; therefore, it should be recognized early and treated promptly.

Improving Resident Hospital Discharge Communication by Changing Electronic Health Record Templates to Enhance Primary Care Provider Satisfaction.

BACKGROUND AND OBJECTIVES: Despite use of standardized electronic health record templates, the structure of discharge summaries may hinder communication from inpatient settings to primary care providers (PCPs). We developed an enhanced electronic discharge summary template to improve PCP satisfaction with written discharge summaries targeting diagnoses, medication reconciliation, laboratory test results, specialist follow-up, and recommendations. METHODS: Resident template usage was measured using statistical process control charts. PCP reviewers' discharge summary satisfaction was surveyed using 5-point Likert scales analyzed using the Mann-Whitney U test. Residents were surveyed for satisfaction. RESULTS: Resident template usage increased from 61% initially to 72% of discharge summaries at 6 months. The PCP reviewers reported increased satisfaction for summaries using the template compared with those without (4.3 vs 3.9, P = .003). Surveyed residents desired template inclusion in the default electronic discharge summary (93%). CONCLUSIONS: This system-level resident-initiated quality improvement initiative created a novel discharge summary template that achieved widespread usage among residents and significantly increased outpatient PCP satisfaction.

Angiopoietin-like 4 (Angptl4) protein is a physiological mediator of intracellular lipolysis in murine adipocytes.

Intracellular triacylglycerol (TG) hydrolysis and fatty acid release by the white adipose tissue (WAT) during a fast is stimulated by counter-regulatory factors acting in concert, although how adipocytes integrate these lipolytic inputs is unknown. We tested the role of angiopoietin-like 4 (Angptl4), a secreted protein induced by fasting or glucocorticoid treatment, in modulating intracellular adipocyte lipolysis. Glucocorticoid receptor blockade prevented fasting-induced tissue Angptl4 expression and WAT TG hydrolysis in mice, and TG hydrolysis induced by fasts of 6 or 24 h was greatly reduced in mice lacking Angptl4 (Angptl4(-/-)). Glucocorticoid treatment mimicked the lipolytic effects of fasting, although with slower kinetics, and this too required Angptl4. Thus, fasting-induced WAT TG hydrolysis requires glucocorticoid action and Angptl4. Both fasting and glucocorticoid treatment also increased WAT cAMP levels and downstream phosphorylation of lipolytic enzymes. Angptl4 deficiency markedly reduced these effects, suggesting that Angptl4 may stimulate lipolysis by modulating cAMP-dependent signaling. In support of this, cAMP levels and TG hydrolysis were reduced in primary Angptl4(-/-) murine adipocytes treated with catecholamines, which stimulate cAMP-dependent signaling to promote lipolysis, and was restored by treatment with purified human ANGPTL4. Remarkably, human ANGPTL4 treatment alone increased cAMP levels and induced lipolysis in these cells. Pharmacologic agents revealed that Angptl4 modulation of cAMP-dependent signaling occurs upstream of adenylate cyclase and downstream of receptor activation. We show that Angptl4 is a glucocorticoid-responsive mediator of fasting-induced intracellular lipolysis and stimulates cAMP signaling in adipocytes. Such a role is relevant to diseases of aberrant lipolysis, such as insulin resistance.

Real-world outcomes associated with vonoprazan-based versus proton pump inhibitor-based therapy for Helicobacter pylori infection in Japan.

Background: Japanese guidelines recommend triple therapy with vonoprazan or a proton pump inhibitor (PPI) in combination with antibiotics to treat Helicobacter pylori (H. pylori) infection. While studies have shown improved eradication rates and reduced costs with vonoprazan versus PPIs, there is little data describing healthcare resource use (HCRU) and treatment patterns. Objectives: To compare patients treated with a vonoprazan-based or PPI-based regimen for H. pylori infection in Japan in terms of their characteristics, HCRU, healthcare costs, clinical outcomes, and treatment patterns. Design: Retrospective matched cohort. Methods: We used data from the Japan Medical Data Center claims database (July 2014-January 2020) to identify adult patients with H. pylori infection and a first observed use of vonoprazan or a PPI in 2015 or later (index date). Patients prescribed a vonoprazan-based or a PPI-based regimen were matched 1:1 using propensity score matching. HCRU, healthcare costs, diagnostic tests, a proxy for H. pylori eradication (i.e. no triple therapy with amoxicillin in combination with metronidazole or clarithromycin >30 days after the index date), and second-line treatment were described during the 12-month follow-up period. Results: Among 25,389 matched pairs, vonoprazan-treated patients had fewer all-cause and H. pylori-related inpatient stays and outpatient visits than PPI-treated patients, resulting in lower all-cause healthcare costs [185,378 Japanese yen (JPY) versus 230,876 JPY, p < 0.001]. Over 80% of patients received a post-treatment test for H. pylori. Fewer vonoprazan-treated than PPI-treated patients subsequently received an additional triple regimen for H. pylori infection (7.1% versus 20.0%, p < 0.001) or a prescription for vonoprazan or a PPI as monotherapy (12.4% versus 26.4%, p < 0.001) between 31 days and 12 months after the index date. Conclusion: Patients with H. pylori infection who were treated with vonoprazan-based therapy had lower rates of subsequent H. pylori treatment, lower overall and H. pylori-related HCRU, and lower healthcare costs than patients treated with PPI-based therapy.

Diverse genetic spectrum among patients who met the criteria of hereditary breast, ovarian and pancreatic cancer syndrome.

OBJECTIVE: Genetic high-risk assessment combines hereditary breast, ovarian and pancreatic cancer into one syndrome. However, there is a lack of data for comparing the germline mutational spectrum of the cancer predisposing genes between these three cancers. METHODS: Patients who met the criteria of the hereditary breast, ovarian and pancreatic cancer were enrolled and received multi-gene sequencing. RESULTS: We enrolled 730 probands: 418 developed breast cancer, 185 had ovarian cancer, and 145 had pancreatic cancer. Out of the 18 patients who had two types of cancer, 16 had breast and ovarian cancer and 2 had breast and pancreatic cancer. A total of 167 (22.9%) patients had 170 mutations. Mutation frequency in breast, ovarian and pancreatic cancer was 22.3%, 33.5% and 17.2%, respectively. The mutation rate was significantly higher in patients with double cancers than those with a single cancer (p<0.001). BRCA1 and BRCA2 were the most dominant genes associated with hereditary breast and ovarian cancer, whereas ATM was the most prevalent gene related to hereditary pancreatic cancer. Genes of hereditary colon cancer such as lynch syndrome were presented in a part of patients with pancreatic or ovarian cancer but seldom in those with breast cancer. Families with a history of both ovarian and breast cancer were associated with a higher mutation rate than those with other histories. CONCLUSION: The mutation spectrum varies across the three cancer types and family histories. Our analysis provides guidance for physicians, counsellors, and counselees on the offer and uptake of genetic counseling.

Direct health care costs associated with COVID-19 in the United States.

BACKGROUND: Data on the real-world health care burden of COVID-19 in the United States are limited. OBJECTIVE: To compare health care resource use (HRU), direct health care costs, and long-term COVID-19-related complications between patients with vs patients without COVID-19 diagnoses. METHODS: Using IBM MarketScan Commercial Claims and Encounters and Medicare Supplemental and Coordination of Benefits administrative claims databases (January 1, 2018, to March 1, 2021), this retrospective, matched cohort study compared patients with a recorded COVID-19 diagnosis to control subjects with no recorded diagnosis for COVID-19, personal history of COVID-19, or pneumonia due to COVID-19. To capture typical health care utilization, the control group was analyzed in 2019 (prepandemic); their index date was assigned as 1 year before the index date (first observed COVID-19 diagnosis) of their matched COVID-19 patient. All patients had continuous health plan coverage for at least 6 months pre-index (baseline) and at least 6 months post-index (allowing censoring during month 6). Separately for commercial and Medicare cohorts, COVID-19 and control patients were matched 1:1 using propensity scores, number of followup months, and indicator of age 18 years or older. During each month of the 6-month follow-up, all-cause HRU, health care costs, and COVID-19-related complications were compared between patients with COVID-19 and controls. RESULTS: After matching COVID-19 and control patients 1:1, a total of 150,731 commercial matched pairs and 1,862 Medicare matched pairs were retained; baseline characteristics were similar between patients with COVID-19 and controls. Patients with COVID-19 and controls had mean ages of 38.9 and 39.7 years in the commercial cohort and 74.3 and 75.3 years in the Medicare cohort, respectively. In month 1 of follow-up, patients with COVID-19 relative to controls were significantly more likely to have at least 1 inpatient admission (commercial: 6.9% vs 0.5%; Medicare: 29.1% vs 1.3%; both P < 0.001) and at least 1 emergency department visit (commercial: 37.3% vs 3.4%; Medicare: 26.2% vs 4.1%; both P < 0.001). Total health care costs in month 1 were significantly higher among patients with COVID-19 than controls (mean differences: $3,706 for commercial; $10,595 for Medicare; both P < 0.001), driven by inpatient costs. Though the incremental HRU and cost burden of COVID-19 decreased over time, patients with COVID-19 continued to have significantly higher total costs through month 5 (all P < 0.001 for both commercial and Medicare). During follow-up, patients with COVID-19 had significantly higher rates of complications than controls (commercial: 52.8% vs 29.0% with any; Medicare: 74.5% vs 47.9% with any; both P < 0.001), most commonly cough, dyspnea, and fatigue. CONCLUSIONS: COVID-19 was associated with significant economic and clinical burden, both in the short-term and over 6 months following diagnosis. DISCLOSURES: Jessica K DeMartino is an employee of Janssen Scientific Affairs, LLC. Elyse Swallow, Debbie Goldschmidt, Karen Yang, Marta Viola, Tyler Radtke, and Noam Kirson are employees of Analysis Group, Inc., which has received consulting fees from Janssen Scientific Affairs, LLC. This study was funded by Janssen Scientific Affairs, LLC. The sponsor was involved in the study design, interpretation of the results, manuscript review, and the decision to publish the article.

Indirect comparison of apomorphine sublingual film and levodopa inhalation powder for Parkinson's disease 'OFF' episodes.

Aim: To compare efficacy of apomorphine sublingual film (APL) and levodopa inhalation powder (CVT-301) for 'on-demand' treatment of Parkinson's disease 'OFF' episodes. Patients & methods: Patient-level data from an APL pivotal study were re-weighted to match average baseline characteristics from a CVT-301 study (SPAN-PD). Placebo-adjusted treatments were compared at week 12. Results: Improvements in predose Unified Parkinson's Disease Rating Scale Part III scores were significantly larger for APL versus CVT-301 at 60 min postdose (least squares mean difference-in-difference: -8.82; p = 0.002); difference at 30 min favored APL but was not statistically significant (-4.46; p = 0.103). Total daily 'OFF' time reductions were significantly larger for APL versus CVT-301 (-1.31 h; p = 0.013). Conclusion: Results suggest APL treatment may lead to improved efficacy versus CVT-301.

High-fat diet disrupts REG3γ and gut microbial rhythms promoting metabolic dysfunction.

Gut microbial diurnal oscillations are important diet-dependent drivers of host circadian rhythms and metabolism ensuring optimal energy balance. However, the interplay between diet, microbes, and host factors sustaining intestinal oscillations is complex and poorly understood. Here, using a mouse model, we report the host C-type lectin antimicrobial peptide Reg3γ works with key ileal microbes to orchestrate these interactions in a bidirectional manner and does not correlate with the intestinal core circadian clock. High-fat diet is the primary driver of microbial oscillators that impair host metabolic homeostasis, resulting in arrhythmic host Reg3γ expression that secondarily drives abundance and oscillation of key gut microbes. This illustrates transkingdom coordination of biological rhythms primarily influenced by diet and reciprocal sensor-effector signals between host and microbial components, ultimately driving metabolism. Restoring the gut microbiota's capacity to sense dietary signals mediated by specific host factors such as Reg3γ could be harnessed to improve metabolic dysfunction.

Crossroads between membrane trafficking machinery and copper homeostasis in the nerve system.

Imbalanced copper homeostasis and perturbation of membrane trafficking are two common symptoms that have been associated with the pathogenesis of neurodegenerative and neurodevelopmental diseases. Accumulating evidence from biophysical, cellular and in vivo studies suggest that membrane trafficking orchestrates both copper homeostasis and neural functions-however, a systematic review of how copper homeostasis and membrane trafficking interplays in neurons remains lacking. Here, we summarize current knowledge of the general trafficking itineraries for copper transporters and highlight several critical membrane trafficking regulators in maintaining copper homeostasis. We discuss how membrane trafficking regulators may alter copper transporter distribution in different membrane compartments to regulate intracellular copper homeostasis. Using Parkinson's disease and MEDNIK as examples, we further elaborate how misregulated trafficking regulators may interplay parallelly or synergistically with copper dyshomeostasis in devastating pathogenesis in neurodegenerative diseases. Finally, we explore multiple unsolved questions and highlight the existing challenges to understand how copper homeostasis is modulated through membrane trafficking.

Medical costs for managing chronic kidney disease and related complications in patients with chronic kidney disease and type 2 diabetes.

OBJECTIVE: To provide cost estimates for chronic kidney disease (CKD) management and major CKD complications among patients with CKD and type 2 diabetes (T2D). STUDY DESIGN: A retrospective cohort study of 52,599 adults with CKD and T2D using Optum Clinformatics claims data from 2014 to 2019. METHODS: Medical costs associated with CKD management, renal replacement therapies (RRTs), major CKD complications (eg, myocardial infarction, stroke, heart failure, atrial fibrillation, and hyperkalemia), and death were estimated using generalized estimating equations adjusting for baseline demographics, complications, and medical costs. Costs for CKD management, RRT, and major CKD complications were assessed in 4-month cycles. Mortality costs were assessed in the month before death. RESULTS: The estimated 4-month CKD management costs ranged from $7725 for stage I to II disease to $11,879 for stage V (without RRT), with high additional costs for dialysis and kidney transplantation ($87,538 and $124,271, respectively). The acute event costs were $31,063 for heart failure, $21,087 for stroke, and $21,016 for myocardial infarction in the first 4 months after the incident event, which all decreased substantially in subsequent 4-month cycles. The acute event costs of atrial fibrillation and hyperkalemia were $30,500 and $31,212 with hospitalization, and $5162 and $1782 without. The costs associated with cardiovascular-related death, renal-related death, and death from other causes were $17,031, $12,605, and $9900, respectively. CONCLUSIONS: Management of CKD and its complications incurs high medical costs for patients with CKD and T2D. Results from this study can be used to quantify the economic profile of emerging treatments and inform decision-making.

Circulating Tumor DNA as a Predictive Marker of Recurrence for Patients With Stage II-III Breast Cancer Treated With Neoadjuvant Therapy.

BACKGROUND: Patients with stage II to III breast cancer have a high recurrence rate. The early detection of recurrent breast cancer remains a major unmet need. Circulating tumor DNA (ctDNA) has been proven to be a marker of disease progression in metastatic breast cancer. We aimed to evaluate the prognostic value of ctDNA in the setting of neoadjuvant therapy (NAT). METHODS: Plasma was sampled at the initial diagnosis (defined as before NAT) and after breast surgery and neoadjuvant therapy(defined as after NAT). We extracted ctDNA from the plasma and performed deep sequencing of a target gene panel. ctDNA positivity was marked by the detection of alterations, such as mutations and copy number variations. RESULTS: A total of 95 patients were enrolled in this study; 60 patients exhibited ctDNA positivity before NAT, and 31 patients exhibited ctDNA positivity after NAT. A pathologic complete response (pCR) was observed in 13 patients, including one ER(+)Her2(-) patient, six Her2(+) patients and six triple-negative breast cancer (TNBC) patients. Among the entire cohort, multivariate analysis showed that N3 classification and ctDNA positivity after NAT were independent risk factors that predicted recurrence (N3, hazard ratio (HR) 3.34, 95% confidence interval (CI) 1.26 - 8.87, p = 0.016; ctDNA, HR 4.29, 95% CI 2.06 - 8.92, p < 0.0001). The presence of ctDNA before NAT did not affect the rate of recurrence-free survival. For patients with Her2(+) or TNBC, patients who did not achieve pCR were associated with a trend of higher recurrence (p = 0.105). Advanced nodal status and ctDNA positivity after NAT were significant risk factors for recurrence (N2 - 3, HR 3.753, 95% CI 1.146 - 12.297, p = 0.029; ctDNA, HR 3.123, 95% CI 1.139 - 8.564, p = 0.027). Two patients who achieved pCR had ctDNA positivity after NAT; one TNBC patient had hepatic metastases six months after surgery, and one Her2(+) breast cancer patient had brain metastasis 13 months after surgery. CONCLUSIONS: This study suggested that the presence of ctDNA after NAT is a robust marker for predicting relapse in stage II to III breast cancer patients.

The Effect of Numbered Jerseys on Directed Commands, Teamwork, and Clinical Performance During Simulated Emergencies.

ABSTRACT: Communication and teamwork are essential during inpatient emergencies such as cardiac arrest and rapid response (RR) codes. We investigated whether wearing numbered jerseys affect directed commands, teamwork, and performance during simulated codes. Eight teams of 6 residents participated in 64 simulations. Four teams were randomized to the experimental group wearing numbered jerseys, and four to the control group wearing work attire. The experimental group used more directed commands (49% vs. 31%, p < .001) and had higher teamwork score (25 vs. 18, p < .001) compared with control group. There was no difference in time to initiation of chest compression, bag-valve-mask ventilation, and correct medications. Time to defibrillation was longer in the experimental group (190 vs. 140 seconds, p = .035). Using numbered jerseys during simulations was associated with increased use of directed commands and better teamwork. Time to performance of clinical actions was similar except for longer time to defibrillation in the jersey group.

Real-World Treatment Profiles, Clinical Outcomes, and Healthcare Resource Utilization of Patients with Migraine Prescribed Erenumab: A Multicenter Chart-Review Study of US Headache Centers.

INTRODUCTION: Erenumab, a first-in-class monoclonal antibody targeting the calcitonin gene-related peptide pathway, was approved by the US Food and Drug Administration in 2018 for the prevention of migraine in adults. There is limited data available on its impact in real-world settings. The study aim was to characterize the real-world treatment profiles, clinical outcomes, and healthcare resource utilization of patients prescribed erenumab from select major US headache centers. METHODS: A retrospective chart review of patients with migraine treated with erenumab for at least 3 months across five major headache centers was conducted. Data was collected from patient charts between April 2019 and April 2020 and included patient and clinical characteristics, migraine medication use, and outpatient visits. The date of the first prescription fill of erenumab was defined as the index date. The baseline period comprised the 3 months prior to the index date and the study period comprised the at least 3 months on erenumab treatment. RESULTS: Data from a total of 1034 patients with chronic migraine with a mean of 9.3 months of erenumab treatment were analyzed. Patients were on average 48 years old, 86% were female, and 79% were white. Patients had a mean of 5 preventive treatment failures prior to erenumab initiation. Patients used a mean of 2 preventive treatments (excluding erenumab) and 2 acute treatments during baseline and study periods. Among patients with effectiveness data, 45% of patients had improvement in physician-reported migraine severity and 35% experienced at least 50% reduction in mean headache/migraine days per month. The average number of monthly outpatient visits was 0.43 and 0.30 before and after erenumab initiation, respectively. CONCLUSION: In this predominantly refractory chronic migraine population treated in select headache centers, patients had fewer headache/migraine days per month and outpatient visits after initiating erenumab. However, patients largely continued to be managed via a polypharmacy approach after erenumab initiation.

Quantifying the Oligomeric States of Membrane Proteins in Cells through Super-Resolution Localizations.

Transitions between different oligomeric states of membrane proteins are essential for proper cellular functions. However, the quantification of their oligomeric states in cells is technically challenging. Here we developed a new method to quantify oligomeric state(s) of highly expressed membrane proteins using the probability density function of molecule density ( PDFMD) calculated from super-resolution localizations. We provided the theoretical model of PDFMD, discussed the effects of protein concentration, cell geometry, and photophysics of fluorescent proteins on PDFMD, and provided experimental criteria for proper quantification of oligomeric states. This method was further validated using simulated single-molecule fluorescent movies and applied to two membrane proteins, UhpT and SbmA in E. coli. The study shows that PDFMD is useful in quantifying oligomeric states of membrane proteins in cells that can help in understanding cellular tasks. Potential applications to proteins with higher oligomeric states under high concentration and limitations of our methodology were also discussed.

A highly stable polyethylene glycol-conjugated human single-chain antibody neutralizing granulocyte-macrophage colony stimulating factor at low nanomolar concentration.

GM-CSF (granulocyte-macrophage colony stimulating factor) plays a central role in inflammatory processes. Treatment with antibodies neutralizing murine GM-CSF showed significant therapeutic effects in mouse models of inflammatory diseases. We constructed by phage display technology a human scFv, which could potently neutralize human GM-CSF. At first, a human V(L) repertoire was combined with the V(H) domain of a parental GM-CSF-neutralizing rat antibody. One dominant rat/human scFv clone was selected, neutralizing human GM-CSF with an IC50 of 7.3 nM. The human V(L) of this clone was then combined with a human V(H) repertoire. The latter preserved the CDR 3 of the parental rat V(H) domain to retain binding specificity. Several human scFvs were selected, which neutralized human GM-CSF at low nanomolar concentrations (IC50 > or = 2.6 nM). To increase serum half-life, a branched 40 kDa PEG-polymer was coupled to the most potent GM-CSF-neutralizing scFv (3077) via an additional C-terminal cysteine. PEG conjugation had a negligible effect on the in vitro neutralizing potential of the scFv, although it caused a significant drop in binding affinity owing to a reduced on-rate. It also significantly increased the stability of the scFv at elevated temperatures. In mouse experiments, the PEGylated scFv 3077 showed a significantly prolonged elimination half-life of 59 h as compared with 2 h for the unconjugated scFv version. PEGylated scFv 3077 is a potential candidate for development of a novel antibody therapy to treat pro-inflammatory human diseases.

Antitumor activity of poly(ethylene glycol)-camptothecin conjugate: the inhibition of tumor growth in vivo.

Antitumor effect of poly(ethylene glycol)-camptothecin conjugate (PEG-CPT) was studied in the nude mouse model of human colon cancer xenografts. The animals were treated intravenously with 15 mg/kg of camptothecin (CPT) or PEG-CPT conjugate at equivalent CPT dose. Antitumor activity, apoptosis induction and caspase-dependent signaling pathways were studied 12, 24, 48 and 96 h after single injection. In addition, pharmacokinetics, tumor distribution and accumulation of PEG polymer labeled with green fluorescence protein (GFP) were studied. The data obtained showed that the conjugation of low molecular weight anticancer drug CPT with low solubility to high molecular weight water-soluble PEG polymer provides several advantages over the native drug. First, the conjugation improves drug pharmacokinetics in the blood and tumor. Second, such conjugation provides passive tumor targeting by the Enhanced Permeability and Retention (EPR) effect, increasing drug concentration in the tumor. Third, the coupling increases the bioavailability of CPT, induces apoptosis in tumor and, therefore, enhances anticancer activity of PEG-CPT. Thus, the use of macromolecular conjugate provided passive tumor targeting of the drug, improved pharmacokinetics and increased the stability of the drug during circulation. It offered better uptake by the targeted tumor cells and substantially enhanced apoptosis and antitumor activity of the conjugated drug in the tumor and decreased apoptosis in liver and kidney as compared with the native drug. All these characteristics make PEG-CPT conjugate an attractive anticancer drug for the effective chemotherapy of solid tumors.