3D wide-field multispectral photoacoustic imaging of human melanomas in vivo: a pilot study.

BACKGROUND: The Breslow depth is an important parameter to determine the excision margin and prognosis of melanoma. However, it is difficult to accurately determine the actual Breslow depth before surgery using the existing ocular micrometer and biopsy technique. OBJECTIVES: To evaluate the use of 3D wide-field multispectral photoacoustic imaging to non-invasively measure depth and outline the boundary of melanomas for optimal surgical margin selection. METHODS: Six melanoma patients were examined in vivo using the 3D multispectral photoacoustic imaging system. For five cases of melanomas (one in situ, three nodular, and one acral lentiginous type melanoma), the spectrally unmixed photoacoustic depths were calculated and compared against histopathological depths. RESULTS: Spectrally unmixed photoacoustic depths and histopathological depths match well within a mean absolute error of 0.36 mm. In particular, the measured minimum and maximum depths in the in situ and nodular type of melanoma were 0.6 and 9.1 mm, respectively. In the 3D photoacoustic image of one metastatic melanoma, feeding vessels were visualized in the melanoma, suggesting the neovascularization around the tumour. CONCLUSIONS: The 3D multispectral photoacoustic imaging not only provides well-measured depth and sizes of various types of melanomas, it also visualizes the metastatic type of melanoma. Obtaining accurate depth and boundary information of melanoma before surgery would play a useful role in the complete excision of melanoma during surgery.

Combination treatment of copanlisib and gemcitabine in relapsed/refractory PTCL (COSMOS): an open-label phase I/II trial.

BACKGROUND: Current treatment options for peripheral T-cell lymphomas (PTCLs) in the relapsed/refractory setting are limited and demonstrate modest response rates with rare achievement of complete response (CR). PATIENTS AND METHODS: This phase I/II study (NCT03052933) investigated the safety and efficacy of copanlisib, a phosphatidylinositol 3-kinase-α/-δ inhibitor, in combination with gemcitabine in 28 patients with relapsed/refractory PTCL. Patients received escalating doses of intravenous copanlisib on days 1, 8, and 15, administered concomitantly with fixed-dose gemcitabine (1000 mg/m2 on days 1 and 8) in 28-day cycles. RESULTS: Dose-limiting toxicity was not observed in the dose-escalation phase and 60 mg copanlisib was selected for phase II evaluation. Twenty-five patients were enrolled in phase II of the study. Frequent grade ≥3 adverse events (AEs) included transient hyperglycemia (57%), neutropenia (45%), thrombocytopenia, (37%), and transient hypertension (19%). However, AEs were manageable, and none were fatal. The overall response rate was 72% with a CR rate of 32%. Median duration of response was 8.2 months, progression-free survival was 6.9 months, and median overall survival was not reached. Combination treatment produced a greater CR rate in patients with angioimmunoblastic T-cell lymphoma than those with PTCL-not otherwise specified (55.6% versus 15.4%, respectively, P = 0.074) and progression-free survival was significantly longer (13.0 versus 5.1 months, respectively, P = 0.024). In an exploratory gene mutation analysis of 24 tumor samples, TSC2 mutation was present in 25% of patients and occurred exclusively in responders. CONCLUSION: The combination of copanlisib and gemcitabine is a safe and effective treatment option in relapsed/refractory PTCLs and represents an important new option for therapy in this rare group of patients.

Elevated cerebrospinal fluid tumour necrosis factor is associated with acute and long-term neurocognitive impairment in cerebral malaria.

Systemic tumour necrosis factor-α (TNF-α) may contribute to the pathogenesis of cerebral malaria (CM) by promoting endothelial activation and parasite sequestration. However, less is known about the role of central nervous system (CNS) TNF-α in CM. We assessed plasma (n=249) and cerebrospinal fluid (CSF) (n=167) TNF-α levels in Ugandan children with CM, plasma TNF-α in Ugandan community control children (n=198) and CSF TNF-α in North American control children who had recovered from leukaemia (n=13). Plasma and CSF TNF-α were measured by magnetic bead assay. We compared plasma and CSF TNF-α levels in children with CM to mortality, acute and chronic neurologic deficits and long-term neurocognitive impairment. Plasma and CSF TNF-α levels were higher in CM than control children (P<.0001 for both). CSF TNF-α levels were higher in children who had neurologic deficits at discharge or 6-month follow-up (P≤.05 for both). Elevated CSF but not plasma TNF-α was associated with longer coma duration (Spearman's rho .18, P=.02) and deficits in overall cognition in children 5 years and older (ß coefficient -.74, 95% CI -1.35 to -0.13, P=.02). The study findings suggest that CNS TNF-α may be involved in the development of acute and chronic neurologic and cognitive sequelae in children with CM.

A leukotriene B4 receptor-2 is associated with paclitaxel resistance in MCF-7/DOX breast cancer cells.

BACKGROUND: Breast cancer is the most common malignancy in women. Although chemotherapeutic agents, such as paclitaxel, are effective treatments for the majority of breast cancer patients, recurrence is frequent and often leads to death. Thus, there is an urgent need to identify novel therapeutic targets that sensitise tumour cells to existing chemotherapy agents. METHODS: The levels of leukotriene B4 receptor-2 (BLT2) in multidrug-resistant MCF-7/DOX cells were determined using quantitative PCR and FACS analysis. The potential role of BLT2 in the paclitaxel resistance of MCF-7/DOX cells was assessed using a pharmacological inhibitor and small interfering RNA knockdown, and the BLT2-associated resistance mechanism was assessed. RESULTS: The expression levels of BLT2 were markedly upregulated in MCF-7/DOX cells. The inhibition of BLT2 by pre-treatment with LY255283 or siBLT2 knockdown significantly sensitised MCF-7/DOX cells to paclitaxel and induced significant levels of apoptotic death, suggesting that BLT2 mediates paclitaxel resistance. We also demonstrated that BLT2-induced paclitaxel resistance was associated with the upregulation of P-glycoprotein. Finally, co-treatment with a BLT2 inhibitor and paclitaxel markedly reduced tumour growth in an MCF-7/DOX in vivo model. CONCLUSION: Together, our results demonstrate that BLT2 is a novel therapeutic target that sensitises drug-resistant breast cancer cells to paclitaxel.

Differences in presentation of younger and older systemic sclerosis patients in clinical trials.

OBJECTIVES: To compare the characteristics of younger and older subjects with diffuse cutaneous systemic sclerosis (SSc) entering clinical trials. METHODS: Subjects were participants in three randomised interventional trials that shared relative uniformity of demographics and disease characteristics. Only subjects with diffuse cutaneous systemic sclerosis were evaluated. To maximise possible differences, the lowest (age<38 years) and highest quartiles (age>53 years) were used, and a total of 264 diffuse cutaneous SSc (dcSSc) subjects were identified. For the comparison between the two age groups, generalised linear mixed or linear models with adjustment for population norms, demographics and medications were employed to assess differences attributable to subject age. RESULTS: After adjustment for population norms and study effects, differences in diastolic blood pressure, alkaline phosphatase, AST, and creatinine phosphokinase (CK) were found between the two age groups. After further adjustment for demographics, disease duration and medications, older SSc patients still had significantly higher alkaline phosphatase (11 U/L higher), and lower CK (76 U/L lower) than younger patients (p<0.003 for all). All other variables were not significantly different in the two age groups. CONCLUSIONS: Clinical baseline differences exist between younger and older patients with SSc. However, after adjustment for population norms and potential confounders, including medications, only differences in alkaline phosphatise (only 11U/L) and CK (76 U/L) remain. Overall, older patients with SSc in clinical trials seem to be more similar to younger patients than was previously thought.

Systemic sclerosis and bilateral lung transplantation: a single centre experience.

Lung involvement is the leading cause of death in systemic sclerosis (SSc), but lung transplantation (LT) for systemic disease remains controversial. Our objective was to comprehensively evaluate post-LT outcomes for SSc compared to idiopathic pulmonary fibrosis (IPF). We retrospectively evaluated bilateral LT recipients (LTRs) with SSc or IPF at our centre between January 1, 2003 and December 31, 2007. The primary end-point was all-cause mortality at 1 yr post-LT. Secondary end-points included assessments of acute rejection (AR), pulmonary function, infection and chronic rejection. 14 patients with SSc and 38 patients with IPF underwent LT. Apart from a younger SSc cohort (53.2 versus 58.8 yrs; p = 0.02), the two groups were well matched. 1-yr all-cause mortality was no different between SSc (6.6%) and IPF (13.1%) groups, after adjusting for age (p = 0.62). Rates of (AR) ≥2 were significantly increased for the SSc compared with the IPF group (hazard ratio (HR) 2.91; p = 0.007). Other end-points, including chronic rejection, infection and pulmonary function, showed no differences. SSc LTRs experience similar survival 1 yr post-LT when compared to IPF. AR rates may be significantly higher in the SSc group. Longer follow-up is necessary to determine the effects of gastrointestinal dysfunction and AR on late allograft function in SSc LTR.

Advanced treatment for taste and odour control in drinking water: case study of a pilot scale plant in Seoul, Korea.

Taste and odour problems of tap water in Seoul are attributed to 2-methylisoborneol (2-MIB) and trans-1,10-dimethyl-trans-9-decalol (geosmin), which are the result of metabolism of algae and chlorine for disinfection. This study was carried out to measure 2-MIB and geosmin in the raw water from the Han River, to investigate removal efficiency of GAC and BAC integrated with post-ozonation, and to minimise and quantify the required chlorine concentration as a final disinfectant through the candidate process.

Minimally important difference in diffuse systemic sclerosis: results from the D-penicillamine study.

OBJECTIVE: To estimate minimally important differences (MIDs) in scores for the modified Rodnan Skin Score (mRSS) and Health Assessment Questionnaire-Disability Index (HAQ-DI) in a clinical trial on diffuse systemic sclerosis (SSc). PARTICIPANTS AND METHODS: 134 people participated in a 2-year, double-blind, randomised clinical trial comparing efficacy of low-dose and high-dose D-penicillamine in diffuse SSc. At 6, 12, 18 and 24 months, the investigator was asked to rate the change in the patient's health since entering the study: markedly worsened, moderately worsened, slightly worsened, unchanged, slightly improved, moderately improved or markedly improved. Patients who were rated as slightly improved were defined as the minimally changed subgroup and compared with patients rated as moderately or markedly improved. RESULTS: The MID estimates for the mRSS improvement ranged from 3.2 to 5.3 (0.40-0.66 effect size) and for the HAQ-DI from 0.10 to 0.14 (0.15-0.21 effect size). Patients who were rated to improve more than slightly were found to improve by 6.9-14.2 (0.86-1.77 effect size) on the mRSS and 0.21-0.55 (0.32-0.83 effect size) on the HAQ-DI score. CONCLUSION: MID estimates are provided for improvement in the mRSS and HAQ-DI scores, which can help in interpreting clinical trials on patients with SSc and be used for sample size calculation for future clinical trials on diffuse SSc.

The role of nitric oxide in ocular surface diseases.

For the first time, the current series of studies provide a possible pathophysiologic mechanism of NO-induced ocular surface disease. NO is present in tear and aqueous humor and is suspected of having an important physiological role in maintaining normal homeostasis of the ocular surface. NO concentrations are higher in aqueous humor compared to tears, though some variability exists between different species. When inflammation was induced by PTK wounding or LPS, three forms of NOS expression were seen in corneal cells. Each isoform of NOS was expressed uniquely according to the specific location of inflammation. When concentrations of NO peaked, the levels of iNOS were markedly increased in fibroblasts and inflammatory cells. The correlation between NO and inflammation was confirmed by treatment with NOS inhibitor, which abrogated the amount of both NO and inflammation. The tissue damage by NO was measured by nitrotyrosine formation. Damage was detected mainly in inflammatory cells, especially those localized in and around the limbal vessel. It is likely that expression of iNOS in limbal fibroblasts has other roles related to survival of limbal stem cells and fibroblasts as well. Because the main source of NO are fibroblasts, we were able to determine the effect of various concentrations of NO on cell viability using a fibroblast culture system. Cell viability increased in dose dependent manner from 10 microM to 500 microM of the NO generator SNAP, but decreased at concentrations above 1000 microM, suggesting that the in vivo mechanism of cell death was indirect, through specific biologic pathways. Therefore, the pathophysiological mechanism of NO action is bimodal with a toxicological component in ocular surface diseases. Furthermore, its concentration and interaction with other oxygen mediators appear to vary depending on the degree of inflammation.

Non-lipid effects of statin on hypercholesterolemic patients established to have coronary artery disease who remained hypercholesterolemic while eating a step-II diet.

BACKGROUND: Results of clinical trials of statin therapy demonstrate that an improvement in incidence of cardiovascular end points and coronary stenosis can be achieved. The beneficial effects of statins on clinical events may involve nonlipid mechanisms that affect endothelial function, such as inflammatory responses, formation of thrombi, and stabilization of plaque. OBJECTIVE: To investigate levels of serologic markers, which may be useful surrogates for activity of vascular disease after administration of statin. METHODS: We administered 20-40 mg simvastatin daily for 14 weeks to 13 patients established to have coronary artery disease who remained hypercholesterolemic during step-II diet therapy. RESULTS: Administration of simvastatin significantly lowered lipoprotein levels and the low: high-density lipoprotein cholesterol level ratio and apolipoprotein B:A-I level ratio compared with pretreatment values (P < 0.01). Administration of simvastatin significantly lowered plasma levels of matrix metalloproteinase-9 (MMP-9) and monocyte chemoattractant protein-I [33+/-46 and 13+/-19%, respectively (P = 0.027 and 0.020, respectively)]. Furthermore, administration of simvastatin tended to lower plasma levels of plasminogen activator inhibitor type-1 and tumor necrosis factor-alpha [by 20+/-44 and 13+/-29%, respectively (P= 0.066 and 0.110, respectively)]. There were significant inverse correlations between pretreatment levels of MMP-9 and the degree of change in those levels after administration of simvastatin (r = -0.714, P= 0.005). However, there was no significant correlation between levels of lipoprotein and levels of MMP-9, monocyte chemoattractant protein-I, and plasminogen activator inhibitor type-1 during administration of simvastatin. CONCLUSIONS: Our current data support the hypothesis that nonlipid mechanisms elicited by administration of simvastatin contribute to the decrease in incidence of cardiovascular events and explain the early clinical benefit observed in clinical trials, independent of changes in levels of lipoprotein.

Efficacy of heparin-coated stent in early setting of acute myocardial infarction.

Primary stenting has been reported to be superior to balloon percutaneous transluminal coronary angioplasty (PTCA) in acute myocardial infarction (AMI) for recurrent ischemia, target lesion revascularization, and restenosis. However, concerns about early reocclusion or thrombosis after stenting in the very thrombotic environment of acute myocardial infarction still remain. Therefore, postprocedural short-term heparin or GpII(b)/III(a) receptor blockades has been used. The aim of our study was to evaluate the safety, feasibility, and long-term efficacy of heparin-coated stent in the early setting of AMI without postprocedural heparin or GpII(b)/III(a) receptor blockade infusion. We studied 102 consecutive patients presenting to cardiac catheterization laboratory < or = 6 hr from the onset of chest pain. No patients who were implanted with heparin-coated stents received heparin or GpII(b)/III(a) receptor blockade infusion after the procedures, not even patients who showed an angiographically large thrombus burden before stenting. Patients were evaluated for clinical endpoints at 30 days and 6 months. Coronary angiography was required for all patients at 2 weeks and 6 months after the procedure. Angiographic and procedural successes were 100% and 98%, respectively. Two patients (2%) died of heart failure without evidence of reocclusion of stented vessel during the hospitalization and 4 (4%) additional patients died of refractory heart failure within the first 6 months. Major bleeding complication occurred in one patient (1%). Recurrent myocardial infarction developed in one patient at 4 months. Early angiographic follow up at 2 weeks was performed in 88% of all patients, none of whom showed thrombotic stent occlusion. Six-month angiographic follow-up was completed in 71%(64/91) of eligible patients and binary restenosis was present in 17.2% of stented vessels. Eight(8%) patients underwent repeat PTCA. Cardiac event-free survival rate at 6 months was 86.3%. This study demonstrates that heparin-coated stents are safe in the early setting of acute myocardial infarction and no additional heparin infusion after stenting is necessary, which may reduce bleeding complications. Angiographic restenosis rate compares favorably to the binary restenosis rate from other studies with uncoated stents.

The role of nitric oxide in ocular surface diseases.

The role of nitric oxide (NO) in ocular surface diseases remains unknown. We investigated the conditions leading to increase NO generation in tears and the main sources of ocular surface tissue. We evaluated the possibility of a dual action (cell survival or cell death) depending on the amount of NO. The concentration of nitrite plus nitrate, the stable end-product of NO, was measured in the tears of various ocular surface diseases. We also examined the main source of nitric oxide synthase (NOS) using immunohistochemical staining & Western blot analysis. When cultured human corneal fibroblasts were treated with NO producing donor with or without serum, the viability of cells was studied. We found that sources of NO in ocular surface tissue primarily included corneal epithelium, fibroblasts, endothelium and inflammatory cells. Three forms of NOS (eNOS, bNOS, & iNOS) were expressed in experimentally induced inflammation. Cell death by NO revealed TUNEL positive staining, however in the EM finding, this NO specific cell death was an atypical necrosis showing perinuclear large vacuolization and mitochondrial swelling. In the fibroblasts culture system, the NO donor (SNAP, S-nitroso-N-acetyl-D, L-penicillamine) prevented the death of corneal fibroblasts caused by serum deprivation in a dose dependent manner up to 500 m SNAP, although a higher dose decreased cell viability. This study suggested that NO might act as a double-edged sword in ocular surface disease depending on the degree of inflammatory condition related with NO concentration.

Binding of equine infectious anemia virus rev to an exon splicing enhancer mediates alternative splicing and nuclear export of viral mRNAs.

In addition to facilitating the nuclear export of incompletely spliced viral mRNAs, equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of the tat/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for Rev binding and transport of incompletely spliced RNA. The presence of a purine-rich exon splicing enhancer (ESE) was required for exon 3 recognition, and the addition of Rev inhibited exon 3 splicing. Glutathione-S-transferase (GST)-Rev bound to probes containing the ESE, and mutation of GAA repeats to GCA within the ESE inhibited both exon 3 recognition in RNA splicing experiments and GST-Rev binding in vitro. These results suggest that Rev regulates alternative splicing by binding at or near the ESE to block SR protein-ESE interactions. A 57-nucleotide sequence containing the ESE was sufficient to mediate Rev-dependent nuclear export of incompletely spliced RNAs. Rev export activity was significantly inhibited by mutation of the ESE or by trans-complementation with SF2/ASF. These results indicate that the ESE functions as a Rev-responsive element and demonstrate that EIAV Rev mediates exon 3 exclusion through protein-RNA interactions required for efficient export of incompletely spliced viral RNAs.

Alterations of Fas (Apo-1/CD95) gene in non-small cell lung cancer.

Fas (Apo-1/CD95) is a cell-surface receptor involved in cell death signaling. The key role of the Fas system in negative growth regulation has been studied mostly within the immune system, and somatic mutations of Fas gene in cancer patients have been described solely in lymphoid-lineage malignancies. However, many non-lymphoid tumor cells have been found to be resistant to Fas-mediated apoptosis, which suggests that Fas mutations, one of the possible mechanisms for Fas-resistance, may be involved in the pathogenesis of non-lymphoid malignancies as well. In this study, we have analysed the entire coding region and all splice sites of the Fas gene for the detection of the gene mutations in 65 human non-small cell lung cancers by polymerase chain reaction, single strand conformation polymorphism and DNA sequencing. Overall, five tumors (7.7%) were found to have the Fas mutations, which were all missense mutations. Four of the five mutations identified were located in the cytoplasmic region (death domain) known to be involved in the transduction of an apoptotic signal and one mutation was located in the transmembrane domain. This is the first report on the Fas gene mutations in non-lymphoid malignancies, and the data presented here suggests that alterations of the Fas gene might lead to the loss of its apoptotic function and contribute to the pathogenesis of some human lung cancers.

Practical synthesis of Taxol side chain.

Practical large scale synthesis of N-benzoyl-(2R,3S)-phenylisoserine methyl ester of the Taxol side chain has been attained from the coupling of chiral imine of N-[(S)-methylbenzyl]benzaldimine with (Z)-alpha-methoxy trimethylsilyl ketene acetal followed by the sequential reactions of lactamization, demethylation, methanolysis and N-benzoylation.