TCF7L2 lncRNA: a link between bipolar disorder and body mass index through glucocorticoid signaling.

Bipolar disorder (BD) and obesity are highly comorbid. We previously performed a genome-wide association study (GWAS) for BD risk accounting for the effect of body mass index (BMI), which identified a genome-wide significant single-nucleotide polymorphism (SNP) in the gene encoding the transcription factor 7 like 2 (TCF7L2). However, the molecular function of TCF7L2 in the central nervous system (CNS) and its possible role in the BD and BMI interaction remained unclear. In the present study, we demonstrated by studying human induced pluripotent stem cell (hiPSC)-derived astrocytes, cells that highly express TCF7L2 in the CNS, that the BD-BMI GWAS risk SNP is associated with glucocorticoid-dependent repression of the expression of a previously uncharacterized TCF7L2 transcript variant. That transcript is a long non-coding RNA (lncRNA-TCF7L2) that is highly expressed in the CNS but not in peripheral tissues such as the liver and pancreas that are involved in metabolism. In astrocytes, knockdown of the lncRNA-TCF7L2 resulted in decreased expression of the parent gene, TCF7L2, as well as alterations in the expression of a series of genes involved in insulin signaling and diabetes. We also studied the function of TCF7L2 in hiPSC-derived astrocytes by integrating RNA sequencing data after TCF7L2 knockdown with TCF7L2 chromatin-immunoprecipitation sequencing (ChIP-seq) data. Those studies showed that TCF7L2 directly regulated a series of BD risk genes. In summary, these results support the existence of a CNS-based mechanism underlying BD-BMI genetic risk, a mechanism based on a glucocorticoid-dependent expression quantitative trait locus that regulates the expression of a novel TCF7L2 non-coding transcript.

ERICH3: vesicular association and antidepressant treatment response.

Selective serotonin reuptake inhibitors (SSRIs) are standard of care for major depressive disorder (MDD) pharmacotherapy, but only approximately half of these patients remit on SSRI therapy. Our previous genome-wide association study identified a single-nucleotide polymorphism (SNP) signal across the glutamate-rich 3 (ERICH3) gene that was nearly genome-wide significantly associated with plasma serotonin (5-HT) concentrations, which were themselves associated with SSRI response for MDD patients enrolled in the Mayo Clinic PGRN-AMPS SSRI trial. In this study, we performed a meta-analysis which demonstrated that those SNPs were significantly associated with SSRI treatment outcomes in four independent MDD trials. However, the function of ERICH3 and molecular mechanism(s) by which it might be associated with plasma 5-HT concentrations and SSRI clinical response remained unclear. Therefore, we characterized the human ERICH3 gene functionally and identified ERICH3 mRNA transcripts and protein isoforms that are highly expressed in central nervous system cells. Coimmunoprecipitation identified a series of ERICH3 interacting proteins including clathrin heavy chain which are known to play a role in vesicular function. Immunofluorescence showed ERICH3 colocalization with 5-HT in vesicle-like structures, and ERICH3 knock-out dramatically decreased 5-HT staining in SK-N-SH cells as well as 5-HT concentrations in the culture media and cell lysates without changing the expression of 5-HT synthesizing or metabolizing enzymes. Finally, immunofluorescence also showed ERICH3 colocalization with dopamine in human iPSC-derived neurons. These results suggest that ERICH3 may play a significant role in vesicular function in serotonergic and other neuronal cell types, which might help explain its association with antidepressant treatment response.

Pediatric Olecranon Osteochondral Flap Fractures With Subtle Radiographic Abnormality.

Pediatric olecranon osteochondral flap fractures are shear injuries of the humeroulnar joint with elevation of an articular cartilaginous flap from the subchondral bone of the olecranon articular surface. All previously reported cases included an osteochondral flap containing the coronoid, with varying imaging and fixation methods used. We treated 2 pediatric patients with this injury. One of our patients had a large, displaced osteochondral fracture of the medial semilunar notch including the coronoid, in addition to avulsions of the medial flexor mass and distal ulnar collateral ligament. The other patient sustained a displaced, rotated osteochondral fracture including the coronoid with subsequent humeroulnar subluxation. Both fractures were anatomically reduced and fixed with absorbable suture, leading to excellent results at 1 year. A high degree of suspicion, thorough work-up, and anatomical reduction of all injured structures are paramount in treatment of this rare, difficult-to-diagnose injury.

Long Non-Coding RNA ANRIL as a Potential Biomarker of Chemosensitivity and Clinical Outcomes in Osteosarcoma.

Osteosarcoma has a poor prognosis due to chemo-resistance and/or metastases. Increasing evidence shows that long non-coding RNAs (lncRNAs) can play an important role in drug sensitivity and cancer metastasis. Using osteosarcoma cell lines, we identified a positive correlation between the expression of a lncRNA and ANRIL, and resistance to two of the three standard-of-care agents for treating osteosarcoma-cisplatin and doxorubicin. To confirm the potential role of ANRIL in chemosensitivity, we independently inhibited and over-expressed ANRIL in osteosarcoma cell lines followed by treatment with either cisplatin or doxorubicin. Knocking-down ANRIL in SAOS2 resulted in a significant increase in cellular sensitivity to both cisplatin and doxorubicin, while the over-expression of ANRIL in both HOS and U2OS cells led to an increased resistance to both agents. To investigate the clinical significance of ANRIL in osteosarcoma, we assessed ANRIL expression in relation to clinical phenotypes using the osteosarcoma data from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) dataset. Higher ANRIL expression was significantly associated with increased rates of metastases at diagnosis and death and was a significant predictor of reduced overall survival rate. Collectively, our results suggest that the lncRNA ANRIL can be a chemosensitivity and prognosis biomarker in osteosarcoma. Furthermore, reducing ANRIL expression may be a therapeutic strategy to overcome current standard-of-care treatment resistance.

Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli.

Robust gene circuit construction requires use of promoters exhibiting low crosstalk. Orthogonal promoters have been engineered utilizing an assortment of natural and synthetic transcription factors, but design of large orthogonal promoter-repressor sets is complicated, labor-intensive, and often results in unanticipated crosstalk. The specificity and ease of targeting the RNA-guided DNA-binding protein dCas9 to any 20 bp user-defined DNA sequence makes it a promising candidate for orthogonal promoter regulation. Here, we rapidly construct orthogonal variants of the classic T7-lac promoter using site-directed mutagenesis, generating a panel of inducible hybrid promoters regulated by both LacI and dCas9. Remarkably, orthogonality is mediated by only two to three nucleotide mismatches in a narrow window of the RNA:DNA hybrid, neighboring the protospacer adjacent motif. We demonstrate that, contrary to many reports, one PAM-proximal mismatch is insufficient to abolish dCas9-mediated repression, and we show for the first time that mismatch tolerance is a function of target copy number. Finally, these promoters were incorporated into the branched violacein biosynthetic pathway as dCas9-dependent switches capable of throttling and selectively redirecting carbon flux in Escherichia coli We anticipate this strategy is relevant for any promoter and will be adopted for many applications at the interface of synthetic biology and metabolic engineering.

CRISPRi-mediated metabolic engineering of E. coli for O-methylated anthocyanin production.

BACKGROUND: Anthocyanins are a class of brightly colored, glycosylated flavonoid pigments that imbue their flower and fruit host tissues with hues of predominantly red, orange, purple, and blue. Although all anthocyanins exhibit pH-responsive photochemical changes, distinct structural decorations on the core anthocyanin skeleton also cause dramatic color shifts, in addition to improved stabilities and unique pharmacological properties. In this work, we report for the first time the extension of the reconstituted plant anthocyanin pathway from (+)-catechin to O-methylated anthocyanins in a microbial production system, an effort which requires simultaneous co-option of the endogenous metabolites UDP-glucose and S-adenosyl-L-methionine (SAM or AdoMet). RESULTS: Anthocyanin O-methyltransferase (AOMT) orthologs from various plant sources were co-expressed in Escherichia coli with Petunia hybrida anthocyanidin synthase (PhANS) and Arabidopsis thaliana anthocyanidin 3-O-glucosyltransferase (At3GT). Vitis vinifera AOMT (VvAOMT1) and fragrant cyclamen 'Kaori-no-mai' AOMT (CkmOMT2) were found to be the most effective AOMTs for production of the 3'-O-methylated product peonidin 3-O-glucoside (P3G), attaining the highest titers at 2.4 and 2.7 mg/L, respectively. Following modulation of plasmid copy number and optimization of VvAOMT1 and CkmOMT2 expression conditions, production was further improved to 23 mg/L using VvAOMT1. Finally, CRISPRi was utilized to silence the transcriptional repressor MetJ in order to deregulate the methionine biosynthetic pathway and improve SAM availability for O-methylation of cyanidin 3-O-glucoside (C3G), the biosynthetic precursor to P3G. MetJ repression led to a final titer of 51 mg/L (56 mg/L upon scale-up to shake flask), representing a twofold improvement over the non-targeting CRISPRi control strain and 21-fold improvement overall. CONCLUSIONS: An E. coli strain was engineered for production of the specialty anthocyanin P3G using the abundant and comparatively inexpensive flavonol precursor, (+)-catechin. Furthermore, dCas9-mediated transcriptional repression of metJ alleviated a limiting SAM pool size, enhancing titers of the methylated anthocyanin product. While microbial production of P3G and other O-methylated anthocyanin pigments will likely be valuable to the food industry as natural food and beverage colorants, we expect that the strain constructed here will also prove useful to the ornamental plant industry as a platform for evaluating putative anthocyanin O-methyltransferases in pursuit of bespoke flower pigment compositions.

Integrating Whispering Gallery Mode Refractive Index Sensing with Capillary Electrophoresis Separations Using Phase Sensitive Detection.

Whispering gallery mode (WGM) resonators are small, radially symmetric dielectrics that recirculate light through continuous total internal reflection. High-Q resonances are observed that shift in response to changes in surrounding refractive index, leading to many applications in label-free sensing. Surface binding measurements with WGM resonators have demonstrated competitive analytical detection metrics compared to other sensing schemes. Similar figures of merit for detecting bulk refractive index changes, however, have proven more challenging. This has limited their use in applications such as capillary electrophoresis (CE), where their compact footprint and refractive index sensitivity offers advantages in nondestructive, universal detection. Here we couple WGM detection with CE by introducing a modulation scheme to improve detection limits. Phase sensitive WGM (PS-WGM) detection is developed to monitor real-time shifts in the WGM spectrum due to changes in surrounding refractive index. We directly compare phase sensitive detection with spectral measurements normally used to track WGM shifts. We report an improvement in detection limits by almost 300-fold using the PS-WGM method. The integrated CE with PS-WGM approach is demonstrated by detecting the separation of a three-component mixture of cations (Na(+), Li(+), and K(+)).

Integration of microsphere resonators with bioassay fluidics for whispering gallery mode imaging.

Whispering gallery mode resonators are small, radially symmetric dielectrics that trap light through continuous total internal reflection. The resonant condition at which light is efficiently confined within the structure is linked with refractive index, which has led to the development of sensitive label-free sensing schemes based on whispering gallery mode resonators. One resonator design uses inexpensive high index glass microspheres that offer intrinsically superior optical characteristics, but have proven difficult to multiplex and integrate with the fluidics for sample delivery and fluid exchange necessary for assay development. Recently, we introduced a fluorescence imaging approach that enables large scale multiplexing with microsphere resonators, thus removing one obstacle for assay development. Here we report an approach for microsphere immobilization that overcomes limitations arising from their integration with fluidic delivery. The approach is an adaptation of a calcium-assisted glass bonding method originally developed for microfluidic glass chip fabrication. Microspheres bonded to glass using this technique are shown to be stable with respect to fluid flow and show no detectable loss in optical performance. Measured Q-factors, for example, remain unchanged following sphere bonding to the substrate. The stability of the immobilized resonators is further demonstrated by transferring lipid films onto the immobilized spheres using the Langmuir-Blodgett technique. Bilayers of DOPC doped with GM1 were transferred onto immobilized resonators to detect the binding of cholera toxin to GM1. Binding curves generated from shifts in the whispering gallery mode resonance result in a measured Kd of 1.5 × 10(-11) with a limit of detection of 3.3 pM. These results are discussed in terms of future assay development using microsphere resonators.

Minimally Invasive Decompression With Noninstrumented Facet Fusion Versus Minimally Invasive Transforaminal Lumbar Interbody Fusion for Stenosis Associated With Grade 1 Lumbar Degenerative Spondylolisthesis.

STUDY DESIGN: Retrospective matched cohort study. SUMMARY OF BACKGROUND DATA: With a growing interest in minimally invasive spine surgery (MIS), the question of which technique is the most advantageous for patients with low-grade degenerative lumbar spondylolisthesis (DLS) still remains unclear. OBJECTIVE: To compare patient-reported outcomes, perioperative morbidity, and rates of reoperation between MIS decompression with either unilateral noninstrumented facet fusion (MIS-F) or with transforaminal interbody fusion (MIS-T) for grade 1 DLS. METHODS: Twenty patients who underwent MIS-T and 20 patients with MIS-F were matched based on age, sex, and preoperative ODI, VAS back, and VAS leg. All patients had DLS with at least 4 millimeters of translation on standing radiographs. Exclusion criteria included prior level surgery, multilevel instability, disk impinging on the exiting nerve root, spondylolisthesis from significant facet arthropathy, or foraminal compromise from disk collapse. ODI, VAS back, VAS leg, and patient satisfaction measured by the North American Spine Society questionnaire were tracked at 3, 6, 12, and 24 months postoperatively. Minimum clinically important differences and substantial clinical benefits were calculated. RESULTS: MIS-F and MIS-T resulted in decreased ODI at 3, 6, and 12 months following the index procedure. Sixty percent of MIS-F and 83% of MIS-T patients reached minimum clinically important difference at 1 year postoperatively; however, using the threshold of 30% ODI reduction from baseline, 67% of MIS-F and 83% MIS-T ( P = 0.25) achieved this goal. Forty-three percent of MIS-F and 59% of MIS-T patients met substantial clinical benefits. Satisfaction at 1 year, measured by a score of 1 or 2 on the North American Spine Society questionnaire, was 64% for MIS-F and 83% for MIS-T. CONCLUSIONS: MIS-F and MIS-T are effective treatment options for spinal stenosis associated with low-grade DLS. Both techniques result in comparable patient-reported outcomes and satisfaction up to 2 years and have similar long-term reoperation rates. More evidence is required to delineate optimal selection characteristics for MIS-F versus MIS-T.

Vacuum-Assisted Wound Closure Management for Posterior Lumbar Spine Infections.

A postoperative infection involving the tissues deep to the fascia is defined as a deep wound infection (DWI). Management of such infections after lumbar spinal surgery remains a challenge. One strategy is the use of vacuum-assisted wound closure (VAC) which applies negative pressure to the wound to improve microcirculation and to promote the formation of granulation tissue. When combined with antibiotics, VAC has been shown to successfully treat DWIs and is now a common strategy for treating deep postoperative wound infections after spinal surgery. In this article, we review the technique of VAC, its mechanism of action, indications and contraindications, and clinical outcomes in the treatment of DWIs after lumbar spinal surgery.

Minimally Invasive Percutaneous Fasciotomy in Lumbar Paraspinal Compartment Syndrome: A Case Report and Review of Literature.

CASE: Lumbar paraspinal compartment syndrome (LPCS) is a rare but potentially catastrophic pathology caused by increased intracompartmental pressures leading to potential ischemic necrosis of muscle, local denervation, and complications of rhabdomyolysis. A combination of unique clinical history and examination findings, laboratory findings, and radiologic imaging help in diagnosis. CONCLUSION: Early detection and intervention with minimally invasive percutaneous fasciotomy can minimize local soft tissue trauma while providing extensive fascial release with good clinical outcomes. Radiographic follow-up in this case suggests that the muscle at risk in LPCS can be spared with early surgical intervention.

Severe hypotension with loss of motor evoked potentials during cervical surgery prompting immediate cardiovascular resuscitation.

BACKGROUND: Intraoperative neuromonitoring (IONM) is a well-established adjunct to spinal surgery to ensure safety of the neural elements.IONM has extremely high sensitivity and specificity for impending neurologic damage. In very rare instances, hypoperfusion of the cord may lead to a loss of IONM modalities that may be reversed if blood pressure issues responsible for the drop out of potentials are immediately addressed. CASE DESCRIPTION: The authors describe a case in which IONM documented hypoperfusion of the cord intraoperatively due to hypotension. Recognition of this problem and reversal of the hypotension resulted in normalization of postoperative function. CONCLUSION: The use of IONM allowed for quick recognition of an impending neurological insult during spinal deformity surgery. Prompt response to signaling changes allowed for the correction of hypotension and favorable neurologic outcome.

Deep Infection Following Dry Needling in a Young Athlete: An Underreported Complication of an Increasingly Prevalent Modality: A Case Report.

CASE: Dry needling frequently is performed by a variety of practitioners for pain treatment. A 16-year-old boy had dry needling in the posterolateral aspect of the right thigh for treatment of pain after a knee injury. He developed an abscess on the posterolateral distal aspect of the right thigh deep to the site of the dry needling. Treatment included surgical drainage and intravenous antibiotics. CONCLUSION: Deep infection is a rare but serious complication of dry needling. Standardized guidelines for safety and sterile technique with dry needling are needed to minimize the risk of infection.

Anatomy of the heart at multidetector CT: what the radiologist needs to know.

Continued improvements in multidetector computed tomographic (CT) scanners have made cardiac CT an important clinical tool that is revolutionizing cardiac imaging. Multidetector CT with submillimeter collimation and gantry rotation times under 0.5 seconds allows the acquisition of studies with high temporal resolution and isotropic voxels. The volumetric data set that is generated can be analyzed with a depth previously not possible, requiring a solid understanding of the cardiac anatomy and its appearance on CT scans and postprocessed images.

Midshaft Clavicle Fractures: A Critical Review.

The clavicle is the most commonly broken bone in the human body, accounting for up to 5% to 10% of all fractures seen in hospital emergency admissions. Fractures of the middle third, or midshaft, are the most common, accounting for up to 80% of all clavicle fractures. Traditional treatment of midshaft clavicle fractures is usually nonoperative management, using a sling or figure-of-eight bandage. The majority of adults treated nonoperatively for midshaft clavicle fractures will heal completely. However, newer studies have shown that malunion, pain, and deformity rates may be higher than previously reported with traditional management. Recent evidence demonstrates that operative treatment of midshaft clavicle fractures can result in better functional results and patient satisfaction than nonoperative treatment in patients meeting certain criteria. This article provides a review of relevant anatomy, classification systems, and injury mechanisms for midshaft clavicle fractures, as well as a comparison of various treatment options. [Orthopedics.2016; 39(5):e814-e821.].

Recent Advances in Microscale Western Blotting.

Western blotting is a ubiquitous tool used extensively in the clinical and research settings to identify proteins and characterize their levels. It has rapidly become a mainstay in research laboratories due to its specificity, low cost, and ease of use. The specificity arises from the orthogonal processes used to identify proteins. Samples are first separated based on size and then probed with antibodies specific for the protein of interest. This confirmatory approach helps avoid pitfalls associated with antibody cross-reactivity and specificity issues. While the technique has evolved since its inception, the last decade has witnessed a paradigm shift in Western blotting technology. The introduction of capillary and microfluidic platforms has significantly decreased time and sample requirements while enabling high-throughput capabilities. These advances have enabled Western analysis down to the single cell level in highly parallel formats, opening vast new opportunities for studying cellular heterogeneity. Recent innovations in microscale Western blotting are surveyed, and the potential for enhancing detection using advances in label-free biosensing is briefly discussed.

Hydrolysis of Polysorbate 20 and 80 by a Range of Carboxylester Hydrolases.

Degradation of the surfactant polysorbate (PS) by enzyme impurities has been previously suggested as a mechanism for the formation of visible and subvisible particles that affect product quality. Although chemical degradation pathways of PS, such as oxidation and acid/base hydrolysis, have been previously characterized, enzymatic degradation of PS remains poorly understood. In this report, enzyme-mediated hydrolysis of the major components of PS was monitored using an evaporative light scattering detection-high-performance liquid chromatography method. PS20 and PS80 tested contained 99% of laurate and 98% oleate esters, respectively, were heterogeneous with respect to head group, and contained a distribution of ester types. Carboxylester hydrolases tested included those from Pseudomonas cepacia, Thermomyces lanuginosus, Candida antarctica, rabbit liver, and pig pancreas. PS hydrolysis was monitored by observing the change in the peak area of major PS components over time and quantified using a parameter called t50, which was defined as the time required for each peak to reach 50% of its initial value. Time course experiments suggested that PS hydrolysis was dependent on the order of esters (mono-, di-, or triester), the identity of the hydrophilic head group (sorbitan or isosorbide), and the identity of the fatty acid ester tail (C12 vs C18:1). In addition, the pattern of PS hydrolysis was unique to the type of enzyme used. Importantly, we observed that no PS component was completely resistant to the carboxylester hydrolases tested here. Our results illustrate a potential fingerprint approach that could be useful in verifying enzyme-mediated PS degradation in drug substance and provide an improved understanding of the complexity of PS degradation in the presence of enzymes. LAY ABSTRACT: Degradation of the non-ionic surfactant polysorbate (PS) has been reported to lead to the formation of visible and subvisible particles that affect product quality. Chemical degradation pathways of PS, such as oxidation and acid/base hydrolysis, have been previously studied, but enzymatic degradation of PS remains poorly understood. In this study, enzyme-mediated hydrolysis of the major components in a heterogeneous mixture of PS20 or PS80 was monitored using an evaporative light scattering detection-high-performance liquid chromatography method. Carboxylester hydrolases from a broad range of organisms were tested, including enzymes from Pseudomonas cepacia, Thermomyces lanuginosus, Candida antarctica, rabbit liver, and pig pancreas. Time course experiments suggested that PS hydrolysis was dependent on the order of esters (mono-, di-, or triester), the identity of the hydrophilic head group (sorbitan or isosorbide), the identity of the fatty acid ester tail (C12 vs C18:1), and the identity of the enzyme. Importantly, no PS component was completely resistant to all the carboxylester hydrolases tested here. Our results illustrate a potential fingerprint approach that could be useful in verifying or identifying enzyme-mediated PS degradation in drug substance and provide an improved understanding of the complexity of PS degradation in the presence of enzymes.

Contrasting pediatric and adult methicillin-resistant Staphylococcus aureus isolates.

We performed a prospective study of all inpatient and outpatient methicillin-resistant Staphylococcus aureus (MRSA) isolates identified at the University of Chicago Hospitals from November 2003 through November 2004. Differences in resistance to non-beta-lactam antimicrobial drugs were determined after stratification of the 578 MRSA isolates into 4 groups by patient age (pediatric vs. adult) and onset location (community vs. hospital). Non-beta-lactam resistance was significantly greater among the 288 adult than the 177 pediatric community-associated isolates for erythromycin (93.2 vs. 87.0%, p = 0.03), clindamycin (51.8 vs. 7.3%, p<0.001), ciprofloxacin (62.1 vs. 10.7%, p<0.001), gentamicin (11.1 vs. 1.1%, p<0.001), and tetracycline (19.9 vs. 6.4%, p<0.001). In contrast, hospital-associated MRSA isolates from children and adults had similar rates of non-beta-lactam antimicrobial drug resistance. In our region, clindamycin is an appropriate empiric therapy of community-associated MRSA infection in children but should be used with caution in adults.

Cysteinyl leukotrienes regulate Th2 cell-dependent pulmonary inflammation.

The Th2 cell-dependent inflammatory response is a central component of asthma, and the ways in which it is regulated is a critical question. The cysteinyl leukotrienes (cys-LTs) are 5-lipoxygenase pathway products implicated in asthma, in particular, by their function as smooth muscle constrictors of airways and microvasculature. To elucidate additional roles for cys-LTs in the pathobiology of pulmonary inflammation, we used an OVA sensitization and challenge protocol with mice lacking leukotriene C(4) synthase (LTC(4)S), the terminal enzyme for cys-LT generation. Ag-induced pulmonary inflammation, characterized by eosinophil infiltration, goblet cell hyperplasia with mucus hypersecretion, and accumulation and activation of intraepithelial mast cells was markedly reduced in LTC(4)S(null) mice. Furthermore, Ag-specific IgE and IgG1 in serum, Th2 cell cytokine mRNA expression in the lung, and airway hyperresponsiveness to methacholine were significantly reduced in LTC(4)S(null) mice compared with wild-type controls. Finally, the number of parabronchial lymph node cells from sensitized LTC(4)S(null) mice and their capacity to generate Th2 cell cytokines ex vivo after restimulation with Ag were also significantly reduced. In contrast, delayed-type cutaneous hypersensitivity, a prototypic Th1 cell-dependent response, was intact in LTC(4)S(null) mice. These findings provide direct evidence of a role for cys-LTs in regulating the initiation and/or amplification of Th2 cell-dependent pulmonary inflammation.