Generation and characterization of a laforin nanobody inhibitor

ObjectivesMutations in the gene encoding the glycogen phosphatase laforin result in the fatal childhood dementia Lafora disease (LD). A cellular hallmark of LD is cytoplasmic, hyper-phosphorylated, glycogen-like aggregates called Lafora bodies (LBs) that form in nearly all tissues and drive disease progression. Additional tools are needed to define the cellular function of laforin, understand the pathological role of laforin in LD, and determine the role of glycogen phosphate in glycogen metabolism. In this work, we present the generation and characterization of laforin nanobodies, with one being a laforin inhibitor.Design and methodsWe identify multiple classes of specific laforin-binding nanobodies and determine their binding epitopes using hydrogen deuterium exchange (HDX) mass spectrometry. Using para-nitrophenyl phosphate (pNPP) and a malachite gold-based assay specific for glucan phosphatase activity, we assess the inhibitory effect of one nanobody on laforin’s catalytic activity.ResultsSix families of laforin nanobodies are characterized and their epitopes mapped. One nanobody is identified and characterized that serves as an inhibitor of laforin’s phosphatase activity.ConclusionsThe six generated and characterized laforin nanobodies, with one being a laforin inhibitor, are an important set of tools that open new avenues to define unresolved glycogen metabolism questions.     

Lung-protective mechanical ventilation does not protect against acute kidney injury in patients without lung injury at onset of mechanical ventilation

Introduction

Preclinical and clinical studies suggest that mechanical ventilation contributes to the development of acute kidney injury (AKI), particularly in the setting of lung-injurious ventilator strategies.

Objective

To determine whether ventilator settings in critically ill patients without acute lung injury (ALI) at onset of mechanical ventilation affect the development of AKI.

Design, Setting, and Patients

Secondary analysis of a randomized controlled trial (N = 150), comparing conventional tidal volume (V_T, 10 mL/kg) with low tidal volume (V_T, 6 mL/kg) mechanical ventilation in critically ill patients without ALI at randomization. During the first 5 days of mechanical ventilation, the RIFLE class was determined daily, whereas neutrophil gelatinase–associated lipocalin and cystatin C levels were measured in plasma collected on days 0, 2, and 4.

Results

Eighty-six patients had no AKI at inclusion, and 18 patients (21%) subsequently developed AKI, but without significant difference between ventilation strategies. (Cumulative hazard, 0.26 vs 0.23; P = .88.) The courses of neutrophil gelatinase–associated lipocalin and cystatin C plasma levels did not differ significantly between randomization groups.

Conclusion

In the present study in critically patients without ALI at onset of mechanical ventilation, lower tidal volume ventilation did not reduce the development or worsening of AKI compared with conventional tidal volume ventilation.     

Multiplex detection of KRAS mutations by a matrix-assisted laser desorption/ionization-time of flight mass spectrometry assay

Objectives

Mutations of the Kirsten rat sarcoma viral oncogene (KRAS) gene are known to be important in the pathogenesis of a variety of cancers. Patients with mutant KRAS tumors do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Testing for KRAS mutations is now being recommended as a tailored therapeutic strategy prior to anti-EGFR treatment; however, the low sensitivity of direct sequencing frequently leads to failure of detection of KRAS mutations in clinical samples.

Design and methods

We developed restriction fragment mass polymorphism (RFMP) assays, to detect KRAS mutations in codons 12, 13, and 61. We performed RFMP analysis for KRAS on DNA isolated from eight different KRAS mutant cell lines and 34 formalin-fixed paraffin-embedded (FFPE) lung cancer tissues.

Results

Overall, the RFMP assay was in good concordance with direct sequencing analysis in the detection of KRAS mutations. By using dilutions of KRAS mutant DNA in wild type DNA from mutation cell lines with a known KRAS status, we confirmed that the RFMP assay has a higher analytical sensitivity, requiring only 3% of cells in a sample to have mutant alleles, compared to direct sequencing, which had a detection threshold of ~ 25%. In the FFPE samples, RFMP successfully detected KRAS genotypes in codons 12, 13, and 61.

Conclusion

The RFMP might be efficiently applicable for the detection of KRAS mutations in a clinical setting, particularly for tumor samples containing abundant non-neoplastic cells.     

Measurement of lower limb volume: Agreement between the vertically oriented perometer and a tape measure method

Objective

To investigate the agreement between lower limb volume measurements for the vertically oriented perometer and a tape measure method.

Design

Between-methods agreement and test–retest reliability study.

Setting

University setting.

Participants

Thirty university staff and students were recruited using convenience sampling with no participant drop-outs.

Interventions

All participants’ dominant lower limb volumes were measured using the vertically oriented perometer and a tape measure method.

Main outcome measures

The Bland–Altman difference plot was reported for determining the bias between the two methods and its 95% confidence interval. Test–retest reliability and the measurement error for the perometer method were determined using the ICC(2,1) model and within-subject standard deviation (s_w) respectively.

Results

There was a lack of agreement between the tape measure and perometer method. The Bland–Altman difference plot showed that the tape measure method overestimated limb volume by 157 ml compared to the perometer method. A 95% confidence interval of −834 ml to 519 ml was observed. The test–retest reliability of the perometer method was ICC(2,1) = 0.99. The measurement error of the perometer method was clinically acceptable (s_w = 121 ml).

Conclusions

There was a lack of agreement between the two methods investigated. Therefore the perometer and tape measure methods are not interchangeable. It is recommended that future validity and reliability studies for the vertically oriented perometer are conducted on clinical populations.     

Rapid detection of K-, N-, H-RAS, and BRAF hotspot mutations in thyroid cancer using the multiplex primer extension

Objectives

The objective of this study is to develop a multiplex PCR and primer extension to detect K-, N-, H-RAS, and BRAF mutations.

Design and methods

DNA samples were isolated from 76 thyroid cancer patients. Multiplex amplification of exons 2 and 3 of three RAS genes and exon 15 of the BRAF gene using three pairs of primers was performed in a single tube. The products were split into three tubes. First, we used nine different-sized N-RAS and BRAF primers to detect base changes in N-RAS and BRAF. The other two tubes used seven separate different-sized K-RAS and H-RAS primers to detect base changes.

Results

We compared these results with direct sequencing. The two methods generated identical results, but our method was superior to direct sequencing in terms of the amount of work and time involved.

Conclusions

We present a rapid method to detect mutations of K-, N-, H-RAS, and BRAF in human cancers.     

Food Poisoning Associated with Kudoa Septempunctata

Background

Kudoa septempunctata is a recently identified cause of food poisoning. We report three cases of food poisoning due to ingestion of this parasite.

Case Reports

Among the 358 people exposed during the same catered meal, 94 (including our 3 patients) developed vomiting and diarrhea within 1–9 h after ingestion of raw muscle from contaminated aquacultured olive flounders (Paralichthys olivaceus). These symptoms occurred frequently but were temporary; only 1 patient was hospitalized for dehydration and was discharged 2 days later.

Conclusion

In Japan, cases of food poisoning due to eating olive flounder have increased during recent years. This increase should prompt heightened awareness among clinicians diagnosing food poisoning.     

An assessment of resuscitation quality in the television drama Emergency Room: Guideline non-compliance and low-quality cardiopulmonary resuscitation lead to a favorable outcome?

Objective

Two earlier studies found that outcome after cardiopulmonary resuscitation (CPR) in the television medical drama Emergency Room (ER) is not realistic. No study has yet evaluated CPR quality in ER.

Design

Retrospective analysis of CPR quality in episodes of ER.

Setting

Three independent board-certified emergency physicians trained in CPR and the American Heart Association (AHA) guidelines reviewed ER episodes in two 5-year time-frames (2001–2005 and 2005–2009). Congruency with the corresponding 2000 and 2005 AHA guidelines was determined for each CPR scene.

Patients

None.

Interventions

None.

Main outcome measures

To evaluate whether CPR is in agreement with the specific algorithms of the AHA guidelines. Fisher's exact test and Mann–Whitney-U-test were used to evaluate statistical significance (P < 0.05).

Results

A total of 136 on-screen cardiac arrests occurred in 174 episodes. Trauma was the leading cause of cardiac arrest (56.6%), which was witnessed in 80.1%. Return of spontaneous circulation occurred in 38.2%. Altogether, 19.1% of patients survived until ICU admission, and 5.1% were discharged alive.

Conclusions

Only one CPR scene was in agreement with the published AHA guidelines. However, low-quality CPR and non-compliance with the guidelines resulted in favorable outcomes.     

Patient-specific decision modeling to guide the use of drotrecogin α (activated) in patients with severe sepsis

Purpose

The expected benefit of treating severe sepsis with drotrecogin α (activated) for an individual patient may depend upon several clinical factors including disease severity. Our objective was to create a decision support tool incorporating patient-specific inputs to estimate the balance between treatment risks and benefits for individual patients with severe sepsis.

Materials and Methods

Logistic regression models were developed to calculate patient-specific mortality risk with and without treatment, which were then used as inputs into a 75-state Markov model. Patient-specific inputs included patient age, sex, and 12 readily available clinical characteristics.

Results

The expected benefit from drotrecogin α (activated) treatment was most dependent upon the underlying disease severity. For example, for a 56-year-old white man with severe sepsis and a 28-day mortality risk of 29%, the model predicted a treatment-related gain of 1.2 quality-adjusted life years (17.3 vs 16.1). Probabilistic sensitivity analyses demonstrated that this patient would benefit from therapy 85% of the time.

Conclusions

A customizable decision model using patient-specific inputs can be used to inform the treatment decision when considering the use of drotrecogin α (activated) therapy by weighing the risks vs the benefits of therapy in the treatment of severe sepsis.     

Investigation of critical care unit utilization and mortality in patients infected with Clostridium difficile

Background

A nationwide increase in the rate and severity of Clostridium difficile–associated disease may reflect infection with a virulent strain characterized by polymerase chain reaction as ribotype 027 (NAP1/B1).

Hypothesis

The high prevalence of ribotype 027 at our institution would allow investigation of the risk of mortality and admission to the intensive care unit (ICU) associated with C difficile infection.

Methods

In a retrospective cohort study, we identified 108 patients with positive enzyme-linked immunosorbant assay tests for C difficile toxins over a 6-month period and compared them to 108 patients who were suspected to have C difficile but with negative toxin assays. Proportions of all-cause mortality and ICU admission were compared using χ², and odds ratios (ORs) were estimated using logistic regression to adjust for potential confounders. Mean log lengths of stay were compared using t test.

Results

Comparing patients with C difficile to those without, mortality (20% vs 8%) and ICU admission (32% vs 17%) were significantly higher (P = .02 for both), whereas log length of stay was not (P = .29). Adjusting for potential confounders, the OR for mortality was 6.8 (95% confidence interval, 1.8-25.4; P = .01), whereas for ICU admission, the association was no longer observed (OR, 1.0; 95% confidence interval, 0.4-2.5; P = .97).

Conclusion

C difficile infection was associated with increased all-cause mortality. An observed association with ICU admission and C difficile infection was identified through univariate analysis but was not significant in multivariate analysis. Although we did not strain-type isolates for patients infected with C difficile, the institutional prevalence of ribotype 027 C difficile infection was known to be high. These results document a strong association between ribotype 027 C difficile infection and mortality and underscore the need to identify effective C difficile preventive strategies.     

Meningococcal Epiglottitis in a Diabetic Adult Patient: A Case Report

Background

After deployment of the Haemophilus influenzae vaccination, the range of pathogens causing acute epiglottitis has changed, as has the epidemiology from a primarily pediatric syndrome towards more frequent adult onset.

Objectives

We present a case of acute-onset meningococcal epiglottitis in an adult patient, to our knowledge one of a few reported cases in the medical literature. We review the historic changes and outcomes of similar episodes.

Case Report

A 37-year-old diabetic man presented to our Emergency Department in acute respiratory distress. Examination revealed epiglottitis; his airway subsequently closed rapidly and was secured by surgical cricothyroidotomy; blood cultures showed the primary pathogen to be Neisseria meningitidis type C.

Conclusion

Neisseria meningitidis has been found to be an emerging cause of acute epiglottitis in adult patients over the last decade, possibly having worsened outcomes compared to other etiologies.     

Chest Pain and Diarrhea: A Case of Campylobacter Jejuni-Associated Myocarditis

Background

Diarrhea and chest pain are common symptoms in patients presenting to the emergency department (ED). However, rarely is a relationship between these two symptoms established in a single patient.

Objective

Describe a case of Campylobacter-associated myocarditis.

Case Report

A 43-year-old man with a history of hypertension presented to the ED with angina-like chest pain and a 3-day history of diarrhea. Electrocardiogram revealed ST-segment elevation in the lateral leads. Coronary angiogram revealed no obstructive coronary artery disease. Troponin T rose to 1.75 ng/mL. Cardiac magnetic resonance imaging showed subepicardial and mid-myocardial enhancement, particularly in the anterolateral wall and interventricular septum, consistent with a diagnosis of myocarditis. Stool studies were positive for Campylobacter jejuni.

Conclusions

Campylobacter-associated myocarditis is rare, but performing the appropriate initial diagnostic testing, including stool cultures, is critical to making the diagnosis. Identifying the etiology of myocarditis as bacterial will ensure that appropriate treatment with antibiotics occurs in addition to any cardiology medications needed for supportive care.     

FoxP3 demethylation is increased in human colorectal cancer and rat cholangiocarcinoma tissue

Objectives

FoxP3 expression is a marker for Tregs which are known to be involved in tumor immunity. We aimed to evaluate FoxP3 promoter demethylation in human colorectal cancer (CRC) and rat intrahepatic cholangiocarcinoma (ICC).

Design and methods

Bisulfite-treated genomic DNA templates of shock frozen paired samples were studied from 13 anonymous CRC patients and from 10 male rats (n = 6 ICC induced by thioacetamide and n = 4 age-matched controls). Real-time PCR was carried out using a LightCycler 480 system. Human FoxP3 and CD3 promoter demethylations were estimated using previously described assays; and rat FoxP3 promoter demethylation using a newly developed assay.

Results

A significant 3.5-fold increase of the demethylation in FoxP3 promoter region was found in human CRC and rat ICC (P < 0.05). The average frequency of cells with FoxP3 demethylation in patients suffering from CRC was 0.26% in normal tissue and 0.92% in tumor tissue (n = 11 paired samples). Although, no significant difference was found between the mean frequency of CD3 demethylation in normal tissue (4.80%, n = 6) and in tumor tissue (4.14%, n = 6) from CRC patients, the ratio of demethylated CD3/FoxP3 promoter areas was significantly lower in tumor specimens (P < 0.05). Using our novel assay, we found a significant increase in mean frequencies of cells with FoxP3 demethylation in rats with ICC (7.42%, n = 6) in comparison to controls (2.14%, n = 4).

Conclusion

FoxP3 seems to be an interesting biomarker for immune response to epithelial tumors. Functional consequences from the increase of Tregs remain to be demonstrated. Further studies with outcome data are necessary.     

Contamination of 0.2-micrometer infusion filters by N,N-dimethylacrylamide

Purpose

Infusion filters, 0.2 μm, are commonly used in intensive care units as in-line filters to minimize particle and microbiological burden on patients. These filters usually contain either a positively charged or an uncharged membrane. The aim of the present study was to identify and to quantify an additive causing an unexpected maximum at 234.5 nm in the ultraviolet spectrum of a filtered drug solution using a filter.

Materials and Methods

For identification and quantification of the substance, water for injection was used as eluent. Measurements were done by mass spectrometry and ultraviolet spectroscopy.

Results

The unexpected additive in the filter was found to be N,N-dimethylacrylamide. The eluate of 2 filter batches had a mean N,N-dimethylacrylamide concentration of 3.9 and 2.5 μg/mL, respectively. Further investigations showed that after infusion breaks of different duration, the N,N-dimethylacrylamide concentration at the beginning of the next infusion cycle reaches a higher level than that at the end of the preceding infusion cycle.

Conclusions

The type of filter examined in this study is primarily used in premature infants, newborns, and infants. These patients are vulnerable to neurotoxic substances having a long-term effect. Therefore, N,N-dimethylacrylamide should be completely removed from the final filter product.     

Effectiveness of the antibiotic lock therapy for the treatment of port-related enterococci,Gram-negative,or Gram-positive bacilli bloodstream infections

Objective

The purpose of this study is to evaluate the efficacy of antibiotic lock therapy to treat port-related enterococci, Gram-negative, or Gram-positive bacilli bloodstream infections.

Patients and Methods

We conducted a prospective observational study including all patients with port-related bacteremia diagnosed at the Clinica Universitaria de Navarra, Pamplona, Spain. During a 36-month period, 110 patients were diagnosed with port-related bacteremia. Of these patients, 18 met criteria to be enrolled in the study. They were treated with a combination of systemic and antibiotic lock therapy (12–24 h/day during 7–14 days). Treatment effectiveness was assessed by clinical and microbiologic criteria.

Results

Treatment was associated with clinical and microbiologic success in 88.8% of our patients (2/2 of the Propionibacterium acnes, 5/5 of the Corynebacterium spp., 6/7 of the Gram-negative bacillus, and 3/4 of the Enterococcus faecium port-related bloodstream infections). Mean increase of port life span for all patients after bacteremia was 288 days (range, 0–1403 days).

Conclusion

Antibiotic lock therapy combined with systemic antibiotics appears to be a safe and effective treatment of port-related bacteremia caused by enterococci, Gram-negative, or Gram-positive bacilli if the patient is stable and no septic syndrome is associated.     

Association study of APC polymorphisms with colorectal cancer in Han Chinese

Objectives

Colorectal cancer (CRC) is a common malignancy with worldwide prevalence. Familial adenomatous polyposis (FAP), a predisposition syndrome of CRC, is caused by germ line mutations in the APC gene. Mutations in APC are thought to be an early event in colorectal tumorigenesis. We hypothesized that common variants in APC might be associated with CRC.

Design and methods

A case-control study genotyping ten SNPs was conducted in 312 CRC patients and 270 normal controls in the Chinese Han population.

Results

The genotype frequency of rs2019720 showed a significant difference between cases and controls (p = 0.046, after Bonferroni correction). For the three pairs of SNPs in strong LD, we carried out haplotype analyses but no significant association was detected.

Conclusion

Our results suggest that APC polymorphisms might be associated with CRC in the Chinese Han population.     

Primary sternal osteomyelitis with bacteremia and distal seeding

Background

Primary sternal osteomyelitis is a rare disease, whereas secondary sternal osteomyelitis is relatively well known as a complication in post-sternotomy patients.

Objective

Describe a case of primary Staphylococcus aureus sternal osteomyelitis in an immunocompetent man and its diagnostic challenges, treatment, and complications.

Case Report

A 46-year-old man presented with prolonged sternal pain and swelling, fatigue, and fever. He is diagnosed with primary sternal osteomyelitis with concurrent bacteremia and complicated by local and distant abscess formation. The diagnosis was aided by computed tomography, and treatment consisted of incision and drainage and long-term antibiotics.

Conclusion

Primary sternal osteomyelitis is uncommon and often difficult to diagnose. Complications include abscess formation and distal seeding. Diagnosis can be aided by a detailed history and radiography, and treatment may consist of a prolonged course of antibiotics and incision and drainage.     

Correlation of MIC value and disk inhibition zone diameters in clinical Legionella pneumophila serogroup 1 isolates

Objectives

Routine use of disk diffusion tests for detecting antibiotic resistance in Legionella pneumophila has not been described. The goal of this study was to determine the correlation of MIC values and inhibition zone diameter (MDcorr) in clinical L. pneumophila isolates.

Methods

Inhibition zone diameter of 183 L. pneumophila clinical isolates were determined for ten antimicrobials. Disk diffusion results were correlated with MICs as determined earlier with E-tests.

Results

Overall the correlation of MIC values and inhibition zone diameters (MDcorr) of the tested antimicrobials is good, and all antimicrobials showed a WT distribution. Of the tested fluoroquinolones levofloxacin showed the best MDcorr. All macrolides showed a wide MIC distribution and good MDcorr. The MDcorr for cefotaxim, doxycycline and tigecycline was good, while for rifampicin and moxifloxacin, they were not.

Conclusion

Overall good correlation between MIC value and disk inhibition zone were found for the fluoroquinolones, macrolides and cefotaxim.     

The Utility of Wet Prep in Predicting Neisseria gonorrhoeae and Chlamydia trachomatis

Background

Diagnosing Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) cervical infections can be difficult in the Emergency Department without real-time testing, as historical and physical elements are known to be unreliable.

Objective

To evaluate the utility of the vaginal wet mount preparation (wet prep) in predicting an infection with NG or CT.

Methods

A retrospective chart review was performed on 12 months of data from September 2007 to August 2008 on patients aged 18 years and above who had a chief complaint requiring a pelvic examination and had concurrent testing for NG/CT and a wet prep. Wet preps were analyzed and reported as quantity of white cells and clue cells present (none, few, moderate, or many) as well as the presence of Trichomonas vaginalis (TV). Wet prep results were evaluated to see if there was a correlation with NG/CT.

Results

There were 2439 patient encounters reviewed. A total of 373/2439 (15.3%) patient encounters were positive for NG or CT; 272/2439 (11.2%) were positive for TV, whereas 966/2439 (39.6%) had white cells and 995/2439 (40.8%) had clue cells on wet prep. Clue cells and TV did not correlate with the presence of NG or CT. Only the presence of “moderate” and “many” white cells correlated with NG or CT (odds ratio [OR] 1.58, 95% confidence interval [CI] 1.12–2.22 and OR 2.47, 95% CI 1.86–3.27, respectively).

Conclusion

In patients who are diagnosed with NG or CT, the presence of TV or clue cells on wet prep is an unreliable marker for diagnosis. However, having moderate or many white cells present on wet prep does increase the probability of concurrent NG or CT infection and may be used in cases where the clinical suspicion is equivocal.