Microfluidics-based capture of human neutrophils for expression analysis in blood and bronchoalveolar lavage.

Gene expression analysis can be a powerful tool in predicting patient outcomes and identifying patients who may benefit from targeted therapies. However, isolating human blood polymorphonuclear cells (PMNs) for genomic analysis has been challenging. We used a novel microfluidic technique that isolates PMNs by capturing CD66b(+) cells and compared it with dextran-Ficoll gradient isolation. We also used microfluidic isolation techniques for blood and bronchoalveolar lavage (BAL) samples of patients with acute respiratory distress syndrome (ARDS) to evaluate PMN genomic alterations secondary to pulmonary sequestration. PMNs obtained from ex vivo lipopolysaccharide (LPS)-stimulated or -unstimulated whole blood from five healthy volunteers were isolated by either dextran-Ficoll gradient, microfluidics capture, or a combination of the two techniques. Blood and BAL fluid PMNs were also isolated using microfluidics from seven hospitalized patients with ARDS. Gene expression was inferred from extracted RNA using Affymetrix U133 Plus 2.0 GeneChips. All methods of PMN isolation produced similar quantities of high-quality RNA, when adjusted for recovered cell number. Unsupervised analysis and hierarchical clustering indicated that LPS stimulation was the primary factor affecting gene expression patterns among all ex vivo samples. Patterns of gene expression from blood and BAL PMNs differed significantly from each other in the patients with ARDS. Isolation of PMNs by microfluidics can be applied to both blood and BAL specimens from critically ill, hospitalized patients. Unique genomic expression patterns are obtained from the blood and BAL fluid of critically ill patients with ARDS, and these differ significantly from genomic patterns seen after ex vivo LPS stimulation.

Laparoscopic pancreatic surgery: what now and what next?

In recent years, improved laparoscopic skill sets have expanded surgical management of pancreatic disease to encompass pancreatic resection, tumor enucleation, debridement, and drainage. With the aid of radiologically guided drainage catheters, necrosectomy for acute pancreatitis can be delayed and accomplished laparoscopically in a select patient population. Pancreatic pseudocysts from chronic pancreatitis can now be approached via minimally invasive strategies, including emerging combined laparoscopic procedures and natural orifice transluminal endoscopic surgery. It is clear that laparoscopic pancreaticoduodenectomy is possible in experienced hands; pancreatic neoplasms in the body and tail are more suitable for laparoscopic procedures because distal pancreatic resection requires no reconstruction of the biliary or enteric tract. Laparoscopic staging of pancreatic tumors has decreased as preoperative radiographic imaging becomes more sensitive. Similarly, laparoscopic palliative procedures have decreased because of the emergence of other minimally invasive options for relieving gastric outlet obstruction and biliary obstruction. Nonetheless, major advances in minimally invasive pancreatic surgery will continue as technology and skill sets advance.

The effects of outpatient management on hospitalization for ambulatory care sensitive conditions associated with diabetes mellitus.

BACKGROUND: Ambulatory care sensitive conditions (ACSC) related to diabetes mellitus can potentially be prevented by good quality outpatient care. The purpose of this study was to evaluate which characteristics of ambulatory patients with diabetes were associated with ACSC hospitalizations. METHODS: Retrospective chart review of 65 adults with an ACSC diabetes-related hospitalization, and 130 controls with diabetes without ACSC hospitalizations. The primary outcome measure was the difference in hemoglobin A1c between groups. RESULTS: Patients with an ACSC hospitalization had poorer glucose control (mean A1c 9.24 versus 7.68, P < 0.001), but there was no difference in blood pressure or lipid control. CONCLUSIONS: Prevention of diabetes-related hospitalizations related more closely to glycemic control, rather than other important aspects of comprehensive care.

Titanium mesh implantation--a method to stabilize the spine and protect the spinal cord following a multilevel laminectomy in the adult rat.

The development of clinically relevant larger spinal cord injury models is in part limited by the possibility of a widened or multilevel laminectomy causing a spinal cord injury from an unstable spine or from compression of the spinal cord by adjacent soft tissues. In the adult rat, we have developed a method to protect the spinal cord and stabilize the spinal column using a titanium mesh implant following a bilateral, multilevel lumbar laminectomy. For this purpose, bilateral and expanded L1-4 laminectomies were performed with or without the use of a titanium mesh to protect the spinal cord and stabilize the spine. Without titanium mesh protection, the rats developed a severe paraparesis or paraplegia, urinary retention, gross anatomical signs of cord compression, and motoneuron loss. In the titanium mesh treatment group, the rats typically maintained a normal gait and lower urinary tract function, normal gross anatomical features of the spinal cord, and normal motoneuron counts. We propose that the use of a titanium mesh implant may assist in the development of clinically relevant larger spinal cord injury and repair models.

Local pancreatic head resection: the search for optimal indications through quality of life assessments.

BACKGROUND: Local pancreatic head resection (LPHR) for chronic pancreatitis has had limited adoption in the United States perhaps because of sparse outcomes and quality of life data. METHODS: Forty-four patients underwent LPHR and retrospective evaluation of patient outcomes and quality of life assessment was performed. RESULTS: The mean age was 49 ± 11 years (50% men) with chronic alcohol use as the etiology in 79% of patients. One patient (2%) died within 90 days. The intensive care unit stay was 1.8 ± 3.1 days and postoperative length of stay was 12.6 ± 9.4 days with 96% of patients discharged home. Ten (22%) patients had major perioperative complications. Biliary stricture was the most common late complication (14%). Quality of life assessment results showed that global status (47/100) and physical (66/100), cognitive (68/100), and social (52/100) functions were acceptable. Prevalent postoperative symptoms were pain (52/100), insomnia (56/100), and digestive disturbance (60/100). CONCLUSIONS: LPHR is safe and effective for a substantial proportion of patients with chronic pancreatitis. Further refinement in the selection of patients most likely to benefit from this operation is warranted.

Should informed consent be required for laboratory testing for drugs of abuse in medical settings?

Laboratory testing for drugs of abuse is often conducted in medical settings, with little consideration of the technical limitations and the potential for legal and social harm to the patient. We consider several technical problems associated with such testing, including the lack of chain-of-custody procedures, the possibility of false-positive results with screening immunoassays, and the infrequency of confirmatory testing. Important ethical issues arise because of the sensitive nature of drug test results, the ramifications of false-positive results, the limitations of confidentiality protection, and the practice of testing without the patient's knowledge. Taken together, these technical and ethical concerns suggest that drug testing policies in medical settings should specify which conditions require explicit informed consent, as well as create procedures for protecting this sensitive information.

Respiratory manifestations of gastroesophageal reflux disease.

Gastroesophageal reflux disease (GERD) is highly associated with a range of respiratory symptoms, arising from a variety of etiologies. The following commentaries on respiratory manifestations of GERD address evidence for a role of a vagally mediated bronchoconstriction reflex in the development of asthma; the direct effects of airway obstruction on lower esophageal sphincter (LES) pressure and reflux episodes; the mechanisms by which reflux may play roles in chronic cough and airway stenosis; the limited efficacy of laparoscopic antireflux surgery (LARS) in improving GERD-related respiratory symptoms; the search for a marker for microaspiration and reflux-induced airway disease; and the potential of proton pump inhibitor (PPI) treatment for patients presenting with asthma and GERD.

Opioids for the treatment of chronic noncancer pain.

Increasingly, opioids are used to treat chronic noncancer pain. While opioids are well recognized for their effectiveness in treating acute pain, the evidence supporting the benefits for the treatment of chronic pain is less well established. Improvement of both pain and function should be considered goals of therapy. Patients with chronic pain have a higher incidence of preexisting psychological disorders. Adverse effects of opioid therapy include dependence, overdose, and withdrawal. Risk factors for poor outcomes with opioid therapy are identified, and include preexisting mental illness and dose prescribed. Recommended strategies to more safely use opioids are discussed, including tools for identifying high-risk patients. The evidence supporting the use of treatment agreements and urine drug testing to reduce the effects of adverse outcomes is limited.

Construction and screening of attenuated ΔphoP/Q Salmonella typhimurium vectored plague vaccine candidates.

Preclinical studies evaluating plague vaccine candidates have demonstrated that the F1 and V protein antigens of Yersinia pestis confer protection against challenge from virulent strains. Live-attenuated ΔphoP/Q Salmonella typhimurium recombinants were constructed expressing either F1, V antigens, F1 and V antigens, or a F1-V fusion from Asd (+) balanced-lethal plasmids. To improve antigen delivery, genes encoding plague antigens were modified in order to localize antigens to specific bacterial cellular compartments which include cytoplasmic, outer membrane, or secreted. Candidate vaccine strains were evaluated for growth characteristics, full-length lipopolysaccharide (LPS), plasmid stability, and antigen expression in vitro. Plague vaccine candidate strains with favorable in vitro profiles were evaluated in murine or rabbit preclinical oral immunogenicity studies. Attenuated S. typhimurium strains expressing cytoplasmically localized F1-V and V antigen antigens were more immunogenic than strains that secreted or localized plague antigens to the outer membrane. In particular, S. typhimurium M020 and M023, which express Asd(+)-plasmid derived soluble F1-V and soluble V antigen, respectively, at high levels in the bacterial cell cytoplasm were found to induce the highest levels of plague-specific serum antibodies. To further evaluate balanced-lethal plasmid retention capacity, ΔphoP/Q S. typhimurium PurB(+) and GlnA(+) balanced-lethal plasmid systems harboring F1-V were compared with M020 in vitro and in BALB/c mice in a immunogenicity study. Although there was no detectable difference in plague antigen expression in vitro, S. typhimurium M020 was the most immunogenic plague antigen vector strain evaluated, inducing high-titer serum IgG antibodies specific against F1, V and F1-V.

Clinical microfluidics for neutrophil genomics and proteomics.

Neutrophils have key roles in modulating the immune response. We present a robust methodology for rapidly isolating neutrophils directly from whole blood with 'on-chip' processing for mRNA and protein isolation for genomics and proteomics. We validate this device with an ex vivo stimulation experiment and by comparison with standard bulk isolation methodologies. Last, we implement this tool as part of a near-patient blood processing system within a multi-center clinical study of the immune response to severe trauma and burn injury. The preliminary results from a small cohort of subjects in our study and healthy controls show a unique time-dependent gene expression pattern clearly demonstrating the ability of this tool to discriminate temporal transcriptional events of neutrophils within a clinical setting.

Using innate immunity to characterize the host response to microbial invasion in severe sepsis.

Severe sepsis is a life-threatening systemic inflammatory response to microbial infection. Although mortality from severe sepsis has decreased modestly over the past three decades, its incidence is increasing dramatically. Current treatment of severe sepsis relies primarily on identification and appropriate antibiotic coverage of the invading pathogen, with supportive therapies aimed primarily at reducing the risk of associated organ injury. Current efforts to identify the invading pathogens are successful in approximately only 50% of patients with severe sepsis. In this review, we examine host-pathogen interactions through the Toll-like receptors and Nod signaling systems as principal mechanisms of the innate immune response, and explore the application of high-throughput genomic technologies that have permitted a more complete dissection of the innate immune response during severe sepsis. Early controlled studies suggest that these genome-wide tools can readily identify pathogen-specific host responses in macrophages and dendritic cells. However, despite considerable progress, clinical application of these approaches to pathogen recognition in hospitalized patients with severe sepsis is still years away.

Differential distribution of growth associated protein (GAP-43) in the motor nuclei of the adult rat conus medullaris.

GAP-43 is normally produced by neurons during developmental growth and axonal regeneration, but it is also expressed in specific regions of the normal adult nervous system. We studied the protein expression of GAP-43 within the conus medullaris portion of the spinal cord in adult male rats. Immunohistochemistry for choline acetyltransferase (ChAT) was first performed to identify specific efferent autonomic and motor nuclei in lumbosacral segments of the spinal cord. Adjacent sections were then processed for GAP-43 immunoreactivity (IR). We show GAP-43 IR in the superficial portion of the dorsal horn, the intermediolateral nucleus, and the dorsal commissural tract. We also demonstrate a differential distribution of GAP-43 IR between different motor nuclei of the conus medullaris. Using densitometry, the most prominent GAP-43 IR was detected in the dorsolateral and dorsomedial motor nuclei, which represent the human Onuf's nucleus homologue. Confocal microscopy of double immunofluorescent labeling for ChAT and GAP-43 demonstrate GAP-43 IR in the neuropil of the autonomic and motor nuclei, and many of the GAP-43 IR arbors are in close apposition with the efferent cholinergic neurons. We note that the efferent neurons of both the autonomic and somatic nuclei, which are ultimately responsible for the integrated normal control of the lower urinary tract, bowel and sexual functions, are heavily innervated by GAP-43 enriched projections. We speculate that these functionally related neurons retain a physiological GAP-43-associated synaptic plasticity throughout adult life.

Is your patient using cocaine?

Preview Cocaine may bring a feeling of euphoria, but the short-lived high often comes with lasting adverse consequences. Because use of the drug is on the rise, primary care physicians should be familiar with its attendant medical complications. How does cocaine affect the heart? The lungs? The nervous system? Which bodily functions does it inhibit? Or enhance? Dr Warner answers these and other questions in this overview of the medical consequences of cocaine use.

Using medical record reminders to improve pneumococcal vaccination rates.

BACKGROUND: Despite widespread recommendations, the pneumococcal vaccination rate of patients 65 years of age or older is below target goals. A reminder system was instituted at the time of the scheduled primary care visit as a pilot quality improvement project. METHODS: Medical records staff used clinic schedules to identify patients 65 years of age or older, stamping that day's progress note with a reminder about pneumococcal vaccination. The physician could note previous vaccination or could order vaccination at the visit. If the patient was previously vaccinated or received a vaccine during that visit, an indicator was placed on the front of the chart. Staff at both clinics received an educational session on the indications and benefits of pneumococcal vaccination. RESULTS: Before implementing the reminder intervention, the pneumococcal immunization recording was comparable at the reminder clinic and at a second clinic that did not use reminders (x2 = 0.324, NS). At 6 months, 95/112 (85%) and 75/115 (65%) patients at the reminder and the second clinic had documentation of pneumococcal vaccination (x2 = 12.66, p < .005); at 12 months, 76% and 58% had such documentation. At the reminder clinic, the reminder stamp was found in 158/205 (77%) of the charts reviewed, with documentation of pneumococcal vaccination in 140/158 (89%) of these charts. DISCUSSION: The simple and inexpensive method of placing selective reminders in charts for elderly patients eligible for pneumococcal vaccination was highly effective in increasing the pneumococcal vaccination rate.