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author:("Kaur, harmed")
1.  ACR Appropriateness Criteria Right Upper Quadrant Pain 
Acute right upper quadrant pain is a common presenting symptom in patients with acute cholecystitis. When acute cholecystitis is suspected in patients with right upper quadrant pain, in most clinical scenarios, the initial imaging modality of choice is ultrasound. Although cholescintigraphy has been shown to have slightly higher sensitivity and specificity for diagnosis, ultrasound is preferred as the initial study for a variety of reasons, including greater availability, shorter examination time, lack of ionizing radiation, morphologic evaluation, confirmation of the presence or absence of gallstones, evaluation of bile ducts, and identification or exclusion of alternative diagnoses. CT or MRI may be helpful in equivocal cases and may identify complications of acute cholecystitis. When ultrasound findings are inconclusive, MRI is the preferred imaging test in pregnant patients who present with right upper quadrant pain.
The ACR Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed every 2 years by a multidisciplinary expert panel. The guideline development and review include an extensive analysis of current medical literature from peer-reviewed journals and the application of a well-established consensus methodology (modified Delphi) to rate the appropriateness of imaging and treatment procedures by the panel. In those instances in which evidence is lacking or not definitive, expert opinion may be used to recommend imaging or treatment.
doi:10.1016/j.jacr.2013.11.017
PMCID: PMC4332791  PMID: 24485592
Appropriateness Criteria; cholescintigraphy; ultrasound; cholecystitis; abdominal pain; comparative studies
2.  Optimization of MR Imaging for Pretreatment Evaluation of Endometrial and Cervical Cancer 
Endometrial and cervical cancer are the most common gynecologic malignancies in the world. Accurate staging of cervical and endometrial cancer is essential for determining the correct treatment approach. The current FIGO (International Federation of Gynecology and Obstetrics) staging system does not include modern imaging modalities. However, magnetic resonance imaging (MRI) has proven to be the most accurate noninvasive imaging modality for staging of endometrial and cervical carcinomas and often assists in patients risk stratification and treatment decisions. Multiparametric MR imaging is increasingly being used in the evaluation of the female pelvis. This approach combines anatomic T2-weighted imaging with functional imaging, i.e. dynamic contrast enhanced MRI (DCE-MRI) and diffusion weighted imaging (DWI). In endometrial and cervical cancer MR imaging is used to guide treatment decisions through an assessment of the depth of myometrial invasion and cervical stromal involvement in endometrial cancer, and of tumor size and parametrial invasion in cervical cancer. However, the efficacy of MRI in achieving accurate local staging is dependent on technique and image quality. In this article we discuss optimization of the MR imaging protocol for endometrial and cervical cancer. The use of thin section high resolution (HR) multi-planar T2 weighted images with simple modifications such as double oblique T2 weighted images supplemented by diffusion weighted imaging and contrast enhanced MRI are reviewed.
doi:10.1148/rg.344140001
PMCID: PMC4307629  PMID: 25019443
3.  Hepatectomy for Recurrent Colorectal Liver Metastases after Radiofrequency Ablation 
The British journal of surgery  2011;98(7):1003-1009.
STRUCTURED ABSTRACT
Background
The results of surgery for recurrent colorectal liver metastases (CLM) after radiofrequency ablation (RFA) have not been evaluated.
Methods
From 1993 to 2009, data on patients who underwent resection or RFA for recurrent CLM were collected prospectively. Inclusion criteria were: RFA as initial treatment of CLM, resection of recurrent CLM after RFA. Postoperative results and oncologic outcome were analyzed.
Results
Twenty-eight patients (median number of tumours 1 [range = 1 – 3], median size 2.75 [range = 2 – 4] cm) met the inclusion criteria. Of these, 22 had recurrence at the site of RFA only, 2 developed new lesions, while 4 had both recurrent and de novo metastases. At the time of resection, patients had a median number of 1 (range = 1 – 13) CLM and median maximum tumour diameter was 5 (range = 1.8 – 11) cm, significantly larger than at the time of RFA (p = 0.02). Ninety-day postoperative morbidity and mortality rates were 46% and 7% respectively. After median follow-up of 35 (range = 0 – 70) months, Kaplan-Meier analyzed 3-year and 5-year overall survival rates were 60% and 33% respectively. Carcinoembryonic antigen plasma level > 5 ng/mL at the time of resection and a rectal primary tumour were associated with worse survival (p = 0.041 and p = 0.02 respectively).
Conclusions
Resection for recurrence after RFA is associated with significant morbidity and modest long-term benefit.
doi:10.1002/bjs.7506
PMCID: PMC4203671  PMID: 21541936
4.  Association of computed tomography morphological criteria with pathologic response and survival in patients treated with bevacizumab for colorectal liver metastases 
Context
The standard criteria used to evaluate tumor response, the Response Evaluation Criteria in Solid Tumors (RECIST), were developed to assess tumor shrinkage after cytotoxic chemotherapy and may be limited in assessing response to biologic agents, which have a cytostatic mechanism of action..
Objective
To validate novel tumor response criteria based on morphologic changes observed on computed tomography (CT) in patients with colorectal liver metastases (CLM) treated with bevacizumab-containing chemotherapy regimens.
Design, Setting, and Patients
234 CLM were analyzed in 50 patients who underwent hepatic resection after preoperative chemotherapy that included bevacizumab at a comprehensive US cancer center from 2004 to 2007; date of last follow-up was March 2008. All patients underwent routine contrast-enhanced CT at the start and end of preoperative therapy. Three blinded, independent radiologists evaluated images for morphologic response, based upon metastases changing from heterogeneous masses with ill-defined margins into homogeneous lesions with sharp borders. These criteria were validated with a separate cohort of 82 patients with unresectable CLM treated with bevacizumab-containing chemotherapy.
Main Outcome Measures
Response determined using morphologic criteria and RECIST was correlated with pathologic response in resected liver specimens and with patient survival.
Results
Interobserver agreement for scoring morphologic changes was good among three radiologists (κ=0.68–0.78; 95% confidence interval, 0.51–0.93). In resected tumor specimens with morphologic optimal, incomplete, and no response, the median percentages of residual tumor cells were 20% (interquartile range [IQR], 10%–30%), 50% (IQR, 30%–60%), and 70% (IQR, 60%–70%), respectively (P<.001). With RECIST partial response, stable disease, and progressive disease, the median percentages of residual tumor cells were 30% (IQR, 10%–60%), 50% (IQR, 20%–70%), and 70% (IQR, 65%–70%), respectively (P=.04). Among patients who underwent hepatic resection, median overall survival was not yet reached with optimal morphologic response and 35 months (95% CI, 20.2 to 29.8 months) with incomplete or no morphologic response (P=.03). In the validation cohort, patients with optimal morphologic response had median overall survival of 31 months (95% CI, 26.8 to 35.2 months) compared to 19 months (95% CI, 14.6 to 23.4 months) with incomplete or no morphologic response (P=.009). RECIST did not correlate with survival in neither the surgical nor validation cohort.
Conclusions
Among patients with CLM treated with bevacizumab-containing chemotherapy, CT-based morphologic criteria had a statistically significant association with pathologic response and overall survival.
doi:10.1001/jama.2009.1755
PMCID: PMC4139149  PMID: 19952320
5.  SSAT/AHPBA Joint Symposium on Evaluation and Treatment of Benign Liver Lesions 
Background
Benign liver lesions are common incidental radiologic findings.
Methods
Experts convened in 2011 at a Society for Surgery of the Alimentary Tract/ Americas Hepato-Pancreato-Biliary Association joint symposium to discuss the evaluation and treatment of benign liver lesions.
Results
Most benign liver lesions can be accurately diagnosed with high-quality imaging, including ultrasonography, multiphase computed tomography, and magnetic resonance imaging, particularly with hepatocyte-specific contrast agents. Percutaneous biopsy is reserved for lesions that cannot be characterized radiographically, and its accuracy is improved with immunophenotypic markers. Hepatic cysts are the most commonly diagnosed benign liver lesions; these must be distinguished from malignant cystic lesions, which are rare. Among the solid benign liver lesions, hemangiomas and focal nodular hyperplasia seldom require treatment. In contrast, hepatocellular adenomas are associated with a risk for complications. A new classification system for hepatocellular adenomas based on genetic and phenotypic features can help guide patient care. In patients who are symptomatic or at risk for complications, multidisciplinary evaluation and treatment based on clinicopathologic, radiographic, and molecular analysis is needed.
Conclusions
Most benign liver lesions can be accurately diagnosed radiographically and do not require treatment. Treatment is necessary for patients with symptoms or at risk for complications.
doi:10.1007/s11605-013-2153-1
PMCID: PMC3628697  PMID: 23377783
Liver; cyst; hemangioma; adenoma; focal nodular hyperplasia
6.  Human C-reactive protein Accentuates Macrophage Activity in Biobreeding Diabetic rats 
Objective
Type 1 diabetes (T1DM) is a pro-inflammatory state characterized by high C-reactive protein (CRP) levels. However, there is a paucity of data examining the role of CRP in promoting the pro-inflammatory state of diabetes. Thus, we examined the pro-inflammatory effects of human CRP using spontaneously diabetic bio-breeding (BB) rats.
Methods
Diabetic rats (n=9/group) were injected with Human serum albumin (huSA) or Human CRP (hCRP, 20 mg/kg body weight; i.p) for 3 consecutive days. Blood and peritoneal macrophages (MØ) were obtained following euthanasia. Peritoneal macrophages were used for measuring superoxide anion release, NF-κB DNA binding activity, proinflammatory mediator secretion.
Results
hCRP administration resulted in significantly increased superoxide anion production, along with increased release of cytokines/chemokines, and plasminogen activator inhibitor (PAI-1) and Tissue Factor (TF) activity in diabetic rats compared to huSA. hCRP-treated BB rat MØ showed significant induction of protein kinase C (PKC)-alpha, PKC-delta and p47 phox expression and NF-κB compared to huSA.
Conclusions
Thus, our data suggests that human CRP exacerbates in-vivo the pro-inflammatory, pro-oxidant and procoagulant states of diabetes predominantly via increased macrophage activity and this could have implications with respect to vascular complications and anti-inflammatory therapies.
doi:10.1016/j.jdiacomp.2012.03.020
PMCID: PMC3404262  PMID: 22520400
C-reactive protein; NF-κB; Macrophages; Tissue factor; PAI-1; PKC; p47Phox
7.  Genetic Variation in the PNPLA3 Gene and Hepatocellular Carcinoma in USA: Risk and Prognosis Prediction 
Molecular carcinogenesis  2013;52(0):10.1002/mc.22057.
Nonalcoholic fatty liver disease (NAFLD) is an emerging epidemic with high prevalence in Western countries. Genome-wide association studies had reported that a variation in the patatin-like phospholipase domain containing 3 (PNPLA3) gene is associated with high susceptibility to NAFLD. However, the relationship between this variation and hepatocellular carcinoma (HCC) has not been well established. We investigated the impact of PNPLA3 genetic variation (rs738409: C>G) on HCC risk and prognosis in the United States by conducting a case–control study that included 257 newly diagnosed and pathologically confirmed Caucasian patients with HCC (cases) and 494 healthy controls. Multivariate logistics and Cox regression models were used to control for the confounding effects of HCC risk and prognostic factors. We observed higher risk of HCC for subjects with a homozygous GG genotype than for those with CC or CG genotypes, the adjusted odds ratio (OR) was 3.21 (95% confidence interval [CI], 1.68–6.41). We observed risk modification among individuals with diabetes mellitus (OR = 19.11; 95% CI, 5.13–71.20). The PNPLA3 GG genotype was significantly associated with underlying cirrhosis in HCC patients (OR = 2.48; 95% CI, 1.05–5.87). Moreover, GG allele represents an independent risk factor for death. The adjusted hazard ratio of the GG genotype was 2.11 (95% CI, 1.26–3.52) compared with CC and CG genotypes. PNPLA3 genetic variation (rs738409: C>G) may determine individual susceptibility to HCC development and poor prognosis. Further experimental investigations are necessary for thorough assessment of the hepatocarcinogenic role of PNPLA3.
doi:10.1002/mc.22057
PMCID: PMC3808509  PMID: 23776098
molecular epidemiology; genetic susceptibility; case–control; single nucleotide polymorphism
8.  Differentially expressed myo-inositol monophosphatase gene (CaIMP) in chickpea (Cicer arietinum L.) encodes a lithium-sensitive phosphatase enzyme with broad substrate specificity and improves seed germination and seedling growth under abiotic stresses 
Journal of Experimental Botany  2013;64(18):5623-5639.
myo-Inositol monophosphatase (IMP) is an essential enzyme in the myo-inositol metabolic pathway where it primarily dephosphorylates myo-inositol 1-phosphate to maintain the cellular inositol pool which is important for many metabolic and signalling pathways in plants. The stress-induced increased accumulation of inositol has been reported in a few plants including chickpea; however, the role and regulation of IMP is not well defined in response to stress. In this work, it has been shown that IMP activity is distributed in all organs in chickpea and was noticeably enhanced during environmental stresses. Subsequently, using degenerate oligonucleotides and RACE strategy, a full-length IMP cDNA (CaIMP) was cloned and sequenced. Biochemical study revealed that CaIMP encodes a lithium-sensitive phosphatase enzyme with broad substrate specificity, although maximum activity was observed with the myo-inositol 1-phosphate and l-galactose 1-phosphate substrates. Transcript analysis revealed that CaIMP is differentially expressed and regulated in different organs, stresses and phytohormones. Complementation analysis in Arabidopsis further confirmed the role of CaIMP in l-galactose 1-phosphate and myo-inositol 1-phosphate hydrolysis and its participation in myo-inositol and ascorbate biosynthesis. Moreover, Arabidopsis transgenic plants over-expressing CaIMP exhibited improved tolerance to stress during seed germination and seedling growth, while the VTC4/IMP loss-of-function mutants exhibited sensitivity to stress. Collectively, CaIMP links various metabolic pathways and plays an important role in improving seed germination and seedling growth, particularly under stressful environments.
doi:10.1093/jxb/ert336
PMCID: PMC3871819  PMID: 24123252
Ascorbate; gluconeogenesis; inositol; multifunctional; phosphatase; seed germination; stress tolerance.
9.  Footprint-based identification of viral entry inhibitors targeting HIVgp41 
A targeted virtual screen to the N-helix hydrophobic pocket on HIVgp41 was performed using DOCK followed by re-ranking with a new footprint-based scoring function which employed native gp41 C-helix residues as a reference. Of ca. 500,000 small molecules screened, 115 were purchased, and 7 hits were identified with favorable binding (Ki), cell-cell fusion (IC50), and cytotoxicity (CC50) profiles. Three of the seven active compounds would not have been discovered without the use of the footprints, demonstrating the utility of the method for structure-based design when a known reference compound or substrate is available.
doi:10.1016/j.bmcl.2012.02.017
PMCID: PMC3321075  PMID: 22425565
10.  Increased Toll-Like Receptor Activity in Patients With Metabolic Syndrome 
Diabetes Care  2012;35(4):900-904.
OBJECTIVE
The metabolic syndrome (MetS) is highly prevalent and confers an increased risk for diabetes and cardiovascular disease (CVD). While MetS is a proinflammatory state, there is a paucity of data on cellular inflammation in MetS. Toll-like receptors (TLRs) are classical pattern recognition receptors of the innate immune response.
RESEARCH DESIGN AND METHODS
The aim of this study was to examine monocyte TLR2 and TLR4 in MetS patients without diabetes or CVD and control subjects since both of the receptors have been implicated in atherosclerosis and insulin resistance. Fasting blood was obtained for TLR expression and activity.
RESULTS
Circulating levels of high-sensitivity C-reactive protein, interleukin (IL)-1β, IL-6, IL-8, and soluble tumor necrosis factor receptor 1 (sTNFR1) were significantly increased in MetS versus control subjects following adjustment for waist circumference. There was a significant increase in both TLR2 and TLR4 surface expression and mRNA on monocytes after adjustment for waist circumference. In addition to increased nuclear factor-κB nuclear binding, there was significantly increased release of IL-1β, IL-6, and IL-8 in MetS versus control subjects following priming of the monocytes with lipopolysaccharides. While both plasma free fatty acids and endotoxin were increased in MetS, they correlated significantly with TLR4 only.
CONCLUSIONS
In conclusion, we make the novel observation that both TLR2 and TLR4 expression and activity are increased in the monocytes of patients with MetS and could contribute to increased risk for diabetes and CVD.
doi:10.2337/dc11-2375
PMCID: PMC3308307  PMID: 22357188
11.  Pre-treatment high-resolution rectal MRI and treatment response to neoadjuvant chemoradiation 
Diseases of the colon and rectum  2012;55(4):371-377.
Background
Use of rectal MRI evaluation of patients with rectal cancer for primary tumor staging and for identification for poor prognostic features is increasing. MR imaging permits precise delineation of tumor anatomy and assessment of mesorectal tumor penetration and radial margin risk.
Objective
To evaluate the ability of pre-treatment rectal MRI to classify tumor response to neoadjuvant chemoradiation.
Design
Retrospective, consecutive cohort study, central review.
Setting
Tertiary academic hospital.
Patients
62 consecutive patients with locally advanced (stage cII-cIII)rectal cancer who underwent rectal cancer protocol high resolution MRI prior to surgery(12/09-3/11).
Main Outcome Measures
Probability of good (ypT0-2N0) vs. poor (≥ypT3N0) response as a function of mesorectal tumor depth, lymph node status, extramural vascular invasion, and grade assessed by uni- and multi-variate logistic regression.
Results
Tumor response was good in 25, 40.3% and poor in 37, 59.7%.Median interval from MRI to OP was 7.9weeks (IQR: 7.0–9.0). MRI tumor depth was <1 mm in 10 (16.9%), 1–5 mm in 30 (50.8%), and >5 mm in 21(33.9%). LN status was positive in 40 (61.5%) and vascular invasion was present in 16 (25.8%). Tumor response was associated with MRI tumor depth (P=0.001), MRI lymph nodes status (P=<0.001)and vascular invasion (P=0.009). Multivariate regression indicated >5mm MRI tumor depth (OR=0.08, 95% CI=0.01–0.93, p=0.04) and MRI LN positivity (OR=0.12, 95% CI=0.03–0.53, p=0.005) were less likely to achieve a good response to neoadjuvant chemoradiotherapy.
Limitations
Uncertain generalizability in centers with limited experience with MRI staging for rectal cancer.
Conclusion
MRI assessment of tumor depth and lymph node status in rectal cancer is associated to tumor response to neoadjuvant chemoradiotherapy. These factors should therefore be considered for stratification of patients for novel treatment strategies reliant on pathologic response to treatment or for the selection of poor-risk patients for intensified treatment regimens.
doi:10.1097/DCR.0b013e31824678e3
PMCID: PMC3546551  PMID: 22426259
Rectal Cancer; Staging; Magnetic Resonance Imaging; Neoadjuvant Therapy
12.  Development of indole compounds as small molecule fusion inhibitors targeting HIV-1 glycoprotein-41 
Journal of medicinal chemistry  2011;54(20):7220-7231.
Non-peptide inhibition of fusion remains an important goal in anti-HIV research, due to its potential for low cost prophylaxis or prevention of cell–cell transmission of the virus. We report here on a series of indole compounds that have been identified as fusion inhibitors of gp41 through a structure-based drug design approach. Experimental binding affinities of the compounds for the hydrophobic pocket were strongly correlated to fusion inhibitory data (R2 = 0.91), and corresponding inhibition of viral replication confirmed the hydrophobic pocket as a valid target for low molecular weight fusion inhibitors. The most active compound bound to the hydrophobic pocket and inhibited cell-cell fusion and viral replication at sub-µM levels. A common binding mode for the inhibitors in this series was established by carrying out docking studies using structures of gp41 in the Protein Data Bank. The molecules were flexible enough to conform to the contours of the pocket, and the most active compound was able to adopt a structure mimicking the hydrophobic contacts of the D-peptide PIE7. The results enhance our understanding of indole compounds as inhibitors of gp41.
doi:10.1021/jm200791z
PMCID: PMC3234170  PMID: 21928824
gp41; small molecule; fusion inhibitor; docking; indole
13.  Subscapular tumoral calcinosis in a toddler: case report 
Tumoral calcinosis is uncommon in toddlers, and rare within the subscapular area. Although typically benign, tumoral calcinosis is often incorrectly diagnosed prior to biopsy. We present a case of subscapular tumoral calcinosis in a 16-month old girl and discuss the radiological findings on X-ray, ultrasound, computed tomography and magnetic resonance imaging, including the first description of T1-weighted post contrast imaging, which demonstrate the fibrotic components of tumoral calcinosis.
doi:10.3941/jrcr.v6i6.967
PMCID: PMC3558015  PMID: 23378877
Tumoral Calcinosis; Magnetic Resonance Imaging; Computed Tomography; Case Report
14.  Chemotherapy Outcomes for the Treatment of Unresectable Intrahepatic and Hilar Cholangiocarcinoma: A Retrospective Analysis 
ABSTRACT
Background:
Recent clinical trials for “biliary cancers” include a heterogenous group of patients with cholangiocarcinoma, gallbladder, and ampullary cancers. Limited data exist regarding the relative effectiveness of known chemotherapeutic regimens specifically in intrahepatic or hilar cholangiocarcinoma.
Methods:
Records of M D Anderson Cancer Center patients with unresectable intrahepatic and hilar cholangiocarcinoma who received first-line chemotherapy from January 1, 2005, to October 31, 2009, were retrospectively reviewed. The primary objective of this research was to determine overall tumor control rates with chemotherapeutic regimens used for first-line treatment of unresectable intrahepatic and hilar cholangiocarcinoma. Secondary objectives included duration of response, overall survival, and prognostic factors.
Results:
Eighty-five patients met inclusion criteria and were eligible for analysis. The most commonly used regimen was gemcitabine/cisplatin (62%), followed by oxaliplatin and capecitabine (16%). There was no significant difference between tumor control rates with gemcitabine/cisplatin (72% PR + SD) and other regimens (69% PR + SD). There was no significant difference between overall survival with the use of gemcitabine/cisplatin (15.2 months) or alternative regimens (13.9 months). A decrease in overall survival was seen with elevated baseline CA 19–9 (p < .0001), an initial diagnosis of unknown primary tumor (p = .0001), and prior treatment with chemoradiation (p = .0018).
Conclusion:
In this retrospective review, both gemcitabine/cisplatin and alternative doublets (including capecitabine/oxaliplatin, gemcitabine/capecitabine, and gemcitabine/oxaliplatin) were effective regimens in maintaining disease control in intrahepatic and hilar cholangiocarcinoma.
PMCID: PMC3269144  PMID: 22295126
15.  Expression of PDGF Receptor-α in Corneal Myofibroblasts In Situ 
Experimental eye research  2009;89(3):432-434.
The purpose of this study was to investigate the expression of platelet-derived growth factor receptor-alpha (PDGFR–α) in the myofibroblasts of corneas with stromal haze. Central corneal sections from rabbit eyes that had -9 diopter PRK were analyzed by immunocytochemistry (IHC) for the expression of PDGFR-α at 4 week after surgery. PDGFR-α was expressed immediately beneath the epithelial basement membrane in the anterior stroma. Double IHC studies revealed the expression of PDGFR-α in the anterior stroma co-localized with alpha-smooth muscle actin (SMA) marker for myofibroblasts. In vitro studies have suggested that PDGF is important in the development and viability of myofibroblasts after corneal injury. Expression of PDGFR-α in myofibroblasts supports these findings.
doi:10.1016/j.exer.2009.03.017
PMCID: PMC2720438  PMID: 19344713
Myofibroblasts; α-SMA (alpha – smooth muscle actin); PDGFR-α; wound healing; photorefractive keratectomy (PRK
16.  Corneal myofibroblast viability: Opposing effects of IL-1 and TGF β1 
Experimental eye research  2009;89(2):152-158.
The purpose of this study was to test the effect of corneal epithelial scrape on myofibroblasts associated with haze and elucidate the effect of interleukin-1 and transforming growth factor beta-1 on corneal stromal myofibroblasts viability and death in vitro. Corneal epithelial scrape was performed in rabbit eyes with severe haze at one month after -9 diopter photorefractive keratectomy. Corneas were processed for immunocytochemistry for myofibroblast marker α-smooth muscle actin (α-SMA) and the TUNEL assay to detect apoptosis. Rabbit corneal fibroblasts were cultured with 2 ng/ml of transforming growth factor-β1 (TGF β1) to induce myofibroblast differentiation confirmed by monitoring α-SMA expression. Fluorescence-based TUNEL assay was performed to analyze the apoptotic response of myofibroblasts to IL-1α or IL-1β in the presence or absence of TGFβ1. Dose response experiments were performed after withdrawal of TGFβ1 and exposure to 1, 5, or 10 ng/ml of IL-1α or IL-1β for 1 hour. Subsequent experiments were performed with myofibroblasts exposed to 5 ng/ml IL-1α or IL-1β in conjunction with 0, 1, 5, or 10 ng/ml TGFβ1. Corneal epithelial scrape with a scalpel blade produced myofibroblast apoptosis. Exposure to TGF β1 in vitro resulted in greater than 99% transformation of corneal fibroblasts to α-SMA+ myofibroblasts. There was a statistically significant dose dependent increase in the percentage of TUNEL+ cells with either IL-1α or IL-1β initiated at concentrations as low as 1 ng/ml. For example, after withdrawal of TGFβ1, the % TUNEL+ cells at 1 hour after exposure to IL-1α increased significantly with increasing concentration (0 ng/ml, 2.4±0.8 [S.E.M.] %; 1 ng/ml, 15.4±1.8%; 5 ng/ml, 47.4±3.9%; or 10 ng/ml, 70.3±3.2%). Similar results were obtained with IL-1β. The differences between the means of apoptotic myofibroblasts for the different concentrations of cytokine for either IL-1α or IL-1β were significantly different (ANOVA p<0.001). When myofibroblasts were exposed to 5 ng/ml IL-1α or IL-1β, the % TUNEL+ cells at 1 hour were reduced in a significant dose dependent manner when TGF β1 at a concentration of 5 ng/ml or 10 ng/ml was present in the medium (ANOVA p<0.01). IL-1α or IL-1β triggers the death of myofibroblasts in vitro and TGF β1 reduces the IL-1 effect on cell death. TGF β1 and IL-1 have opposing effects on myofibroblast viability and likely interact to modulate haze generation after corneal injury.
doi:10.1016/j.exer.2009.03.001
PMCID: PMC2713809  PMID: 19285499
17.  Dynamics of the expression of intermediate filaments vimentin and desmin during myofibroblast differentiation after corneal injury 
Experimental eye research  2009;89(2):133-139.
Previous studies have suggested that abnormal corneal wound healing in patients after photorefractive keratectomy (PRK) is associated with the appearance of myofibroblasts in the stroma between two and four weeks after surgery. The purpose of this study was to examine potential myofibroblast progenitor cells that might express other filament markers prior to completion of the differentiation pathway that yields α-smooth muscle actin (SMA)-expressing myofibroblasts associated with haze localized beneath the epithelial basement membrane after PRK. Twenty-four female rabbits that had -9 diopter PRK were sacrificed at 1 week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were collected, frozen at -80 °C, and analyzed by immunocytochemistry using anti-vimentin, anti-desmin, and anti-SMA antibodies. Double immunostaining was performed for the co-localization of SMA with vimentin or desmin with SMA. An increase in vimentin expression in stromal cells is noted as early as 1 week after PRK in the rabbit cornea. As the healing response continues at two or three weeks after surgery, many stromal cells expressing vimentin also begin to express desmin and SMA. By 4 weeks after the surgery most, if not all, myofibroblasts express vimentin, desmin and SMA. Generalized least squares regression analysis showed that there was strong evidence that each of the marker groups differed in expression over time compared to the other two (p < 0.01). Intermediate filaments - vimentin and desmin co-exist in myofibroblasts along with SMA and may play an important role in corneal remodeling after photorefractive keratectomy. The earliest precursors of myofibroblasts destined to express SMA and desmin are detectible by staining for vimentin at 1 week after surgery.
doi:10.1016/j.exer.2009.02.022
PMCID: PMC2716066  PMID: 19285070
photorefractive keratectomy (PRK); myofibroblasts; α-SMA; vimentin and desmin; wound healing
18.  Corneal stroma PDGF blockade and myofibroblast development 
Experimental eye research  2008;88(5):960-965.
Myofibroblast development and haze generation in the corneal stroma is mediated by cytokines, including transforming growth factor beta (TGF β), and possibly other cytokines. This study examined the effects of stromal PDGF-β blockade on the development of myofibroblasts in response to -9.0 diopter photorefractive keratectomy in the rabbit. Rabbits that had haze generating photorefractive keratectomy (PRK, for 9 diopters of myopia) in one eye were divided into three different groups: stromal application of plasmid pCMV.PDGFRB.23KDEL expressing a subunit of PDGF receptor b (domains 2-3, which bind PDGF-B), stromal application of empty plasmid pCMV, or stromal application of balanced salt solution (BSS). The plasmids (at a concentration 1000 ng/μl) or BSS was applied to the exposed stroma immediately after surgery and every 24 hours for 4-5 days until the epithelium healed. The group treated with pCMV.PDGFRB.23KDEL showed lower αSMA+ myofibroblast density in the anterior stroma compared to either control group (P≤ 0.001). Although there was also lower corneal haze at the slit lamp at one month after surgery, the difference in haze after PDGF-B blockade was not statistically significant compared to either control group. Stromal PDGF-B blockade during the early postoperative period following PRK decreases stromal αSMA+ myofibroblast generation. PDGF is an important modulator of myofibroblast development in the cornea.
doi:10.1016/j.exer.2008.12.006
PMCID: PMC2674136  PMID: 19133260
Corneal stroma; myofibroblasts; platelet derived growth factor; platelet derived growth factor beta receptor (PDGFR-β); wound healing; soluble cytokine receptors
19.  Effect of Femtosecond Laser Energy Level on Corneal Stromal Cell Death and Inflammation 
PURPOSE
To analyze the effects of variations in femtosecond laser energy level on corneal stromal cell death and inflammatory cell influx following flap creation in a rabbit model.
METHODS
Eighteen rabbits were stratified in three different groups according to level of energy applied for flap creation (six animals per group). Three different energy levels were chosen for both the lamellar and side cut: 2.7 μJ (high energy), 1.6 μJ (intermediate energy), and 0.5 μJ (low energy) with a 60 KHz, model II, femtosecond laser (IntraLase). The opposite eye of each rabbit served as a control. At the 24-hour time point after surgery, all rabbits were euthanized and the corneoscleral rims were analyzed for the levels of cell death and inflammatory cell influx with the terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunocytochemistry for monocyte marker CD11b, respectively.
RESULTS
The high energy group (31.9±7.1 [standard error of mean (SEM) 2.9]) had significantly more TUNEL-positive cells in the central flap compared to the intermediate (22.2±1.9 [SEM 0.8], P=.004), low (17.9±4.0 [SEM 1.6], P≤.001), and control eye (0.06±0.02 [SEM 0.009], P≤.001) groups. The intermediate and low energy groups also had significantly more TUNEL-positive cells than the control groups (P≤.001). The difference between the intermediate and low energy levels was not significant (P=.56). The mean for CD11b-positive cells/400× field at the flap edge was 26.2±29.3 (SEM 12.0), 5.8±4.1 (SEM 1.7), 1.7±4.1 (SEM 1.7), and 0.0±0.0 (SEM 0.0) for high energy, intermediate energy, low energy, and control groups, respectively. Only the intermediate energy group showed statistically more inflammatory cells than control eyes (P=.015), most likely due to variability between eyes.
CONCLUSIONS
Higher energy levels trigger greater cell death when the femtosecond laser is used to create corneal flaps. Greater corneal inflammatory cell infiltration is observed with higher femtosecond laser energy levels.
doi:10.3928/1081597X-20090917-08
PMCID: PMC2769018  PMID: 19835327

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