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In view of the ethno medicinal use of Enhydra fluctuans for the treatment of kidney stones; the present study aimed to elucidate the molecular mechanisms involved in the amelioration of nephrolithiasis through a network pharmacology approach. The phytoconstituents were queried in DIGEP-Pred to identify the regulated proteins. The modulated proteins were then enriched in the STRING to predict the protein-protein interactions and the probably regulated pathways were traced in the Kyoto Encyclopedia of Genes and Genomes. Further, the network was constructed using Cytoscape ver 3.5.1. Results showed that β-carotene was found to be regulating maximum targets i.e. 26. In addition, 63 proteins were triggered by the components in which the vitamin D receptor was targeted by the maximum phytoconstituents i.e. 16. The enrichment analysis identified the regulation of 67 pathways in which fluid shear stress and atherosclerosis-associated pathways (KEGG entry hsa05418) regulated ten genes. Further, protein kinase C-α was traced in 23 different pathways. In addition, the majority of the regulated genes were identified from the extracellular space via the modulation of 43 genes. Also, nuclear receptor activity had the maximum molecular function via the regulation of 7 genes. Likewise, the response to organic substance was predicted to trigger the top genes i.e. 43. In contrast, Stigmasterol, Baicalein-7-o-glucoside, and Kauran-16-ol were found to have a high affinity to bind with the VDR receptor confirmed by the molecular modelling and the dynamics. Hence, the study elucidated the probable molecular mechanisms of E. fluctuans in managing nephrolithiasis and identified the lead molecules, their targets, and possible pathways. Communicated by Ramaswamy H. Sarma

Supplementary Data from FDA Approval Summary: Belumosudil for Adult and Pediatric Patients 12 Years and Older with Chronic GvHD after Two or More Prior Lines of Systemic Therapy

On July 16, 2021, the FDA approved belumosudil, a kinase inhibitor, for adult and pediatric patients 12 years and older with chronic GvHD (cGvHD) after failure of at least two prior lines of systemic therapy. Approval was based on the results of Study KD025–213, which included 65 patients with cGvHD treated with belumosudil 200 mg daily in an open-label, single-arm cohort. Efficacy was determined by the overall response rate (ORR) through Cycle 7 Day 1, which included complete response (CR) or partial response (PR) according to the 2014 NIH consensus criteria, and durability of response. The ORR through Cycle 7 Day 1 was 75% [95% confidence interval (CI), 63–85]; 6% of patients achieved a CR, and 69% achieved a PR. The median duration of response was 1.9 months (95% CI, 1.2–2.9), and 62% (95% CI, 46–74) of responding patients remained alive without new systemic therapy for at least 12 months from response. The common adverse reactions were infections, asthenia, nausea, diarrhea, dyspnea, cough, edema, hemorrhage, abdominal pain, musculoskeletal pain, headache, phosphate decreased, gamma-glutamyl transferase increased, lymphocytes decreased, and hypertension. Additional study is warranted to confirm safety with long-term use.

Major depressive disorders (MDD) pose major health burdens globally. Currently available medications have their limitations due to serious adverse effects, long latency periods as well as resistance. Considering the highly complicated pathological nature of this disorder, it has been suggested that multitarget drugs or multi-target-directed ligands (MTDLs) may provide long-term therapeutic solutions for the treatment of MDD. In the current review, recent lead design and lead modification strategies have been covered. Important investigations reported in the last ten years (2013–2022) for the preclinical development of MTDLs (through synthetic medicinal chemistry and biological evaluation) for the treatment of MDD were discussed as case studies to focus on the recent design strategies. The discussions are categorized on the basis of pharmacological targets. Based on these important case studies, the challenges involved in different design strategies were discussed in detail. Even though large variations were observed in the selection of pharmacological targets, some potential biological targets (NMDA, melatonin receptors) are required to be explored extensively for the design of MTDLs. Similarly, apart from structure activity relationship (SAR), in silico techniques such as multitasking cheminformatic modeling, molecular dynamics simulation and virtual screening should be exploited to a greater extent.

Purpose To image colon-expressed alternatively spliced D domain of tenascin C in preclinical colitis models using near infrared (NIR)-labeled targeted molecular imaging agents. Procedures. A human IgG1 with nanomolar binding affinity specific to the alternatively spliced D domain of tenascin C was generated. Immunohistochemistry identified disease-specific expression of this extracellular matrix protein in the colon of mice given dextran sulfate sodium in the drinking water. The antibody reagent was labeled with the NIR fluorophore IRDye 800CW via amine chemistry and intravenously dosed to evaluate in vivo targeting specificity. Increasing doses of imaging agent were given to estimate the saturating dose. Results The NIR-labeled proteins successfully targeted colonic lesions in a murine model of colitis. Co-administration of a molar excess competing unlabeled dose reduced normalized uptake in diseased colon by > 70%. Near infrared ex vivo images of colon resected from diseased animals showed saturation at doses exceeding 1 nmol and was confirmed with additional quantitative ex vivo biodistribution. Cellular-level specificity and protein stability were assessed via microscopy. Conclusions Our imaging data suggest the alternatively spliced D domain of tenascin C is a promising target for delivery-based applications in inflammatory bowel diseases.

Intestinal permeability and neutrophil activity are closely linked to inflammatory bowel disease (IBD) pathophysiology. Here we discuss two techniques for assessing permeability and neutrophil activity in mouse IBD models using near infrared (NIR) detection. To address the limitation of visible light readouts—namely high background—IRDye 800CW was used to enable rapid, non-terminal measurements of intestinal permeability. The increased sensitivity of NIR readouts for colon permeability is shown using dextran sulfate sodium (DSS) and anti-CD40 murine colitis models in response to interleukin-22 immunoglobulin Fc (IL22Fc) fusion protein and anti-p40 monoclonal antibody treatments, respectively. In addition to enhanced permeability, elevated levels of neutrophil elastase (NE) have been reported in inflamed colonic mucosal tissue. Activatable NIR fluorescent probes have been extensively used for disease activity evaluation in oncologic animal models, and we demonstrate their translatability using a NE-activatable reagent to evaluate inflammation in DSS mice. Confocal laser endomicroscopy (CLE) and tissue imaging allow visualization of spatial NE activity throughout diseased colon as well as changes in disease severity from IL22Fc treatment. Our findings with the 800CW dye and the NE probe highlight the ease of their implementation in preclinical IBD research.

On July 16, 2021, the FDA approved belumosudil, a kinase inhibitor, for adult and pediatric patients 12 years and older with chronic GvHD (cGvHD) after failure of at least two prior lines of systemic therapy. Approval was based on the results of Study KD025–213, which included 65 patients with cGvHD treated with belumosudil 200 mg daily in an open-label, single-arm cohort. Efficacy was determined by the overall response rate (ORR) through Cycle 7 Day 1, which included complete response (CR) or partial response (PR) according to the 2014 NIH consensus criteria, and durability of response. The ORR through Cycle 7 Day 1 was 75% [95% confidence interval (CI), 63–85]; 6% of patients achieved a CR, and 69% achieved a PR. The median duration of response was 1.9 months (95% CI, 1.2–2.9), and 62% (95% CI, 46–74) of responding patients remained alive without new systemic therapy for at least 12 months from response. The common adverse reactions were infections, asthenia, nausea, diarrhea, dyspnea, cough, edema, hemorrhage, abdominal pain, musculoskeletal pain, headache, phosphate decreased, gamma-glutamyl transferase increased, lymphocytes decreased, and hypertension. Additional study is warranted to confirm safety with long-term use.

Purpose To image colon-expressed alternatively spliced D domain of tenascin C in preclinical colitis models using near infrared (NIR) labeled targeted molecular imaging agents.Methods Human IgG and scFv fusion proteins specific to the alternatively spliced D domain of tenascin C were generated. Immunohistochemistry identified disease-specific expression of this extracellular matrix in mouse colitis models. Proteins were labeled with the NIR fluorophore IRDye 800CW via amine chemistry and intravenously dosed to evaluate targeting specificity in preclinical rodent and primate colitis models.Results The NIR labeled proteins successfully targeted colonic lesions in a murine model of colitis and appeared as distinct punctate spots. Co-administration of a blocking dose reduced the whole colon standardized uptake of the fluorescent dose >7-fold in mouse models. Estimates suggest local expression at >100 nM in diseased mouse colon. Macroscopic targeting specificity was not observed in diseased primate colon. Cellular level specificity was assessed via microscopy and immunohistochemistry.Conclusion Our imaging data suggest the alternatively spliced D domain of tenascin C is a promising target for delivery-based applications in inflammatory bowel diseases.

To assess the ability of monoclonal antibodies (mAbs) specific for fibronectin extra-domain A (FnEDA) to target diseased tissues of mouse collagen induced arthritis (mCIA) models. To explore the parameters of the targeting exhibited by anti-FnEDA mAbs including timing and location.Targeting capabilities of anti-FnEDA mAbs were demonstrated by biodistribution study where i.v. injected antibodies were detected by conjugated near-infrared (NIR) fluorophore,Anti-FnEDA mAbs show specific targeting to diseased paws of mCIA animal. The targeting was focused on inflamed synovium which is consistent with FnEDA expression profile in both mCIA and human RA tissues. Anti-FnEDA mAbs accumulated in diseased tissue at pharmacologically relevant concentrations, the targeting was sustained for up to 14 days and FnEDA was able to support targeting of multiple doses of anti-FnEDA mAbs given 5 days apart.FnEDA is specifically upregulated in the inflamed tissues of mCIA. Antibodies specific for FnEDA can be useful as molecular delivery vehicles for disease specific targeting of payloads to inflamed joint tissue.

While immune checkpoint blockade leads to potent antitumor efficacy, it also leads to immune-related adverse events in cancer patients. These toxicities stem from systemic immune activation resulting in inflammation of multiple organs, including the gastrointestinal tract, lung, and endocrine organs. We developed a dual variable domain immunoglobulin of anti-CTLA4 antibody (anti-CTLA4 DVD, where CTLA4 is defined as cytotoxic T lymphocyte–associated antigen-4) possessing an outer tumor-specific antigen-binding site engineered to shield the inner anti-CTLA4–binding domain. Upon reaching the tumor, the outer domain was cleaved by membrane type-serine protease 1 (MT-SP1) present in the tumor microenvironment, leading to enhanced localization of CTLA4 blockade. Anti-CTLA4 DVD markedly reduced multiorgan immune toxicity by preserving tissue-resident Tregs in Rag 1(–/–) mice that received naive donor CD4(+) T cells from WT C57BL/6j mice. Moreover, anti-CTLA4 DVD induced potent antitumor effects by decreasing tumor-infiltrating Tregs and increasing the infiltration of antigen-specific CD8(+) T lymphocytes in TRAMP-C2–bearing C57BL/6j mice. Treg depletion was mediated through the antibody-dependent cellular cytotoxicity (ADCC) mechanism, as anti-CTLA4 without the FcγR-binding portion (anti-CTLA4 DANA) spared Tregs, preventing treatment-induced toxicities. In summary, our results demonstrate an approach to anti-CTLA4 blockade that depletes tumor-infiltrating, but not tissue-resident, Tregs, preserving antitumor effects while minimizing toxicity. Thus, our tumor-conditional anti-CTLA4 DVD provides an avenue for uncoupling antitumor efficacy from immunotherapy-induced toxicities.

Lymphangioleiomyomatosis (LAM) is a rare disease in women. Patients with LAM develop metastatic smooth-muscle cell adenomas within the lungs, resulting in reduced pulmonary function. LAM cells contain mutations in tuberous sclerosis genes (TSC1orTSC2), leading to up-regulation of mTORC1 activity and elevated proliferation. The origin of LAM cells remains unknown; however, inactivation ofTsc2gene in the mouse uterus resulted in myometrial tumors exhibiting LAM features, and approximately 50% of animals developed metastatic myometrial lung tumors. This suggests that LAM tumors might originate from the uterine myometrium, possibly explaining the overwhelming prevalence of LAM in female. Here, we demonstrate that mouseTsc2-null myometrial tumors exhibit nearly all the features of LAM, including mTORC1/S6K activation, as well as expression of melanocytic markers and matrix metalloproteinases (MMPs). Estrogen ablation reduces S6K signaling and results inTsc2-null myometrial tumor regression. Thus, even without TSC2, estradiol is required to maintain tumors and mTORC1/S6K signaling. Additionally, we find that MMP-2 and -9, as well as neutrophil elastase (NE), are overexpressed inTsc2-null myometrial tumors in an estrogen-dependent fashion.In vivofluorescent imaging using MMP- or NE-sensitive optical biomarkers confirms that protease activity is specific to myometrial tumors. Similar to LAM cells, uterineTsc2-null myometrial cells also overexpress melanocytic markers in an estrogen-dependent fashion. Finally, we identify glycoprotein NMB (GPNMB) as a melanocytic marker up-regulated inTsc2-null mouse uteri and human LAM samples. Our data highlight the potential importance of estradiol in LAM cells, suggesting that anti-estrogen therapy may be a treatment modality. Furthermore, proteases and GPNMB might be useful LAM biomarkers.

During drug development, tissue samples serve as indicators of disease activity and pharmacodynamic responses. Reliable non-invasive measures of drug target engagement will facilitate identification of promising new treatments. Here we develop and validate a novel bi-transgenic mouse model of myotonic dystrophy type 1 (DM1) in which expression of either DsRed or GFP is determined by alternative splicing of an upstream minigene that is mis-regulated in DM1. Using a novel in vivo fluorescence spectroscopy system, we show that quantitation of the DsRed/GFP ratio provides an accurate estimation of splicing outcomes in muscle tissue of live mice that nearly doubles throughput over conventional fluorescence imaging techniques. Serial in vivo spectroscopy measurements in mice treated with a C16 fatty acid ligand conjugated antisense (LICA) oligonucleotide reveal a dose-dependent therapeutic response within seven days, confirm a several-week duration of action, and demonstrate a two-fold greater target engagement as compared to the unconjugated parent oligonucleotide., Myotonic dystrophy type 1 (DM1) is associated with aberrant transcript splicing. Here, the authors develop a transgenic mouse model expressing a bi-chromatic reporter system that allows non-invasive monitoring of splicing of a transcript altered in DM1 in vivo, and show that it allows for evaluation of the therapeutic response to treatment with antisense oligonucleotides.

Because of the heterogeneous nature of articular cartilage tissue, penetration of potential therapeutic molecules for osteoarthritis (OA) through the articular surface (AS) is complex, with many factors that affect transport of these solutes within the tissue. Therefore, the goal of this study is to investigate how the size of antibody (Ab) variants, as well as application of cyclic mechanical loading, affects solute transport within healthy cartilage tissue. Penetration of fluorescently tagged solutes was quantified using confocal microscopy. For all the solutes tested, fluorescence curves were obtained through the articular surface. On average, diffusivities for the solutes of sizes 200 kDa, 150 kDa, 50 kDa, and 25 kDa were 3.3, 3.4, 5.1, and 6.0 μm2/s from 0 to 100 μm from the articular surface. Diffusivities went up to a maximum of 16.5, 18.5, 20.5, and 23.4 μm2/s for the 200 kDa, 150 kDa, 50 kDa, and 25 kDa molecules, respectively, from 225 to 325 μm from the surface. Overall, the effect of loading was very significant, with maximal transport enhancement for each solute ranging from 2.2 to 3.4-fold near 275 μm. Ultimately, solutes of this size do not diffuse uniformly nor are convected uniformly, through the depth of the cartilage tissue. This research potentially holds great clinical significance to discover ways of further optimizing transport into cartilage and leads to effective antibody-based treatments for OA.

We report the use of optical imaging strategies to noninvasively examine photosensitizer distribution and physiological and host responses to 2-[1-hexyloxyethyl]-2 devinyl pyropheophorbide-a (HPPH)-mediated photodynamic therapy (PDT) of EMT6 tumors established in the ears of BALB/c mice. 24 h following intravenous (IV) administration of 1 μmol kg(-1) HPPH, wide-field fluorescence imaging reveals tumor selectivity with an approximately 2-3-fold differential between tumor and adjacent normal tissue. Confocal microscopy demonstrates a relatively homogeneous intratumor HPPH distribution. Labeling of host cells using fluorophore-conjugated antibodies allowed the visualization of Gr1(+)/CD11b(+) leukocytes and major histocompatibility complex class II (MHC-II)(+) cells in vivo. Imaging of the treated site at different time-points following irradiation shows significant and rapid increases in Gr1(+) cells in response to therapy. The maximum accumulation of Gr1(+) cells is found at 24 h post-irradiation, followed by a decrease at the 48 h time-point. Using IV-injected FITC-conjugated dextran as a fluorescent perfusion marker, we imaged tissue perfusion at different times post-irradiation and found that the reduced Gr1(+ )cell density at 48 h correlated strongly with functional damage to the vasculature as reported via decreased perfusion status. Dual color confocal imaging experiments demonstrates that about 90% of the anti-Gr1 cell population co-localized with anti-CD11b labeling, thus indicating that majority of the Gr1-labeled cells were neutrophils. At 24 h post-PDT, an approximately 2-fold increase in MHC-II+ cells relative to untreated control is also observed. Co-localization analysis reveals an increase in the fraction of Gr1(+) cells expressing MHC-II, suggesting that HPPH-PDT is stimulating neutrophils to express an antigen-presenting phenotype.

Background and Objective We investigated the relationship among heat shock protein 70 (hsp70) promoter activation, extracellular HSP70 protein levels, and tumor cure in an animal model of meso-tetrahydroxyphenyl chlorin (mTHPC; Foscan®)-mediated photodynamic therapy (PDT). Materials and Methods Using Western blot analysis, we compared HSP70 protein levels in control and PDT-treated EMT6 cells with the amplitude of hsp70-promoter driven green fluorescent protein (GFP) expression in identically treated, stably transfected hsp70-GFP/EMT6 cells. A clonogenic survival assay was performed to assess the relationship among promoter activation, HSP70 levels, and cell survival in vitro. Tumor growth studies with this transfected cell line were performed to examine responses to fluences from 0.1 to 10 J cm−2, which ranged from sub-curative to curative. In vivo stereofluorescence and confocal fluorescence imaging were used to assess the temporal kinetics in hsp70 activation in tumors subjected to these fluences and the intratumor spatial correlation between hsp70 induction and extracellular levels of HSP70, respectively. Results Maximum GFP expression and HSP protein levels in cells were observed at PDT doses that corresponded to 30% cell survival. The relative changes in GFP and HSP70 protein accumulation as analyzed using Western immunoblots agreed very well, thereby confirming the validity of fluorescent reporter assessment of gene expression in our studies. In vivo imaging revealed that hsp70 promoter-driven GFP expression and accumulation of extracellular HSP70 in PDT-treated tumors subjected to non-curative doses exhibit minimal spatial correlation. There is a strong correlation between mTHPC-PDT doses that result in long-term tumor cure and those that cause high levels of surface exposed or extracellularly released HSP70s. Conclusion Treatment conditions that induce strong promoter activation do not correspond to tumor cure. PDT doses that result in long-term tumor growth control also produce significant accumulation of extracellular HSP70. Lasers Surg. Med. 43:632–643, 2011. © 2011 Wiley-Liss, Inc.

Background and Objective The fungus Candida albicans commonly causes mucosal and cutaneous infections in patients with impaired immunity. We investigated the effectiveness of the photosensitizer meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP-1363) in the photodynamic treatment (PDT) of C. albicans infection in vitro and its selectivity in an animal model. Materials and Methods The efficacy of TMP-1363 in PDT of C. albicans in vitro was compared to that of methylene blue (MB) using a colony forming unit (CFU) assay. In vivo infection in the mouse was established by inoculation of C. albicans yeast in the intradermal space of the ear pinna. Two days post-infection, 0.3 mg ml−1 TMP-1363 was administered topically. Thirty minutes after TMP-1363 application, the ears were irradiated at 514 nm using a fluence of 90 J cm−2 delivered at an irradiance of 50 mW cm−2. The ears were excised 2 hours post-irradiation, homogenized, and the organism burden was determined by a CFU assay. In vivo wide field and confocal fluorescence imaging assessed the localization of the photosensitizer in relationship to C. albicans. Results Photosensitization with TMP-1363 resulted in a greater than three-log increase in killing of C. albicans in vitro compared to MB. In vivo fluorescence imaging demonstrated a high degree of selective labeling of C. albicans by TMP-1363. PDT of infection using TMP-1363 resulted in a significant reduction in CFU/ear relative to untreated controls. Infected ears subjected to PDT displayed complete healing over time with no observable damage to the pinna. Conclusion Our in vitro and in vivo findings support TMP-1363-mediated PDT as a viable therapeutic approach for the PDT of candidiasis. Lasers Surg. Med. 43:324–332, 2011. © 2011 Wiley-Liss, Inc.

We evaluated intratumor (IT) versus intravenous (IV) administration of the photosensitizer Pc 4 with respect to tumor photosensitizer concentration, specificity, and responses to irradiation. BALB/c mice bearing intradermal EMT6 tumors were given 0.3 mg/kg Pc 4 injected IT or IV through the tail vein. Photosensitizer concentration was evaluated by chloroform extraction and localization assessed by fluorescence imaging and spectroscopy in vivo. Tumors were irradiated at 667 nm, 50 mW/cm(2), and 100 J/cm(2). Cures were defined as no palpable tumor 90 days after irradiation. Tumor Pc 4 concentrations 1 hour after IT administration were 35,000-fold higher than measured 24 hours after IV administration (0.112 vs 0.317 x 10(-5)microg Pc 4/mg tumor). Exquisite tumor selectivity was observed 1 hour after IT injection. Fluorescence imaging of freshly sectioned tumors revealed no regions devoid of sensitizer at this time point, with pixel intensities in a midline section within a factor of 3 of the peak intensity. For identical photosensitizer doses, IT administration significantly improved tumor responses to irradiation, with more than 70% of tumors cured with IT-Pc 4-PDT. In this model, IT-Pc 4 administration provides improved tumor control, greater selectivity, and opportunity for a short drug-light interval.

Background and Objective In a recently completed pilot clinical study at Roswell Park Cancer Institute, patients with superficial basal cell carcinoma (sBCC) received topical application of 20% 5-aminolevulinic acid (ALA) and were irradiated with 633 nm light at 10–150 mW cm−2. Protoporphyrin IX (PpIX) photobleaching in the lesion and the adjacent perilesion normal margin was monitored by fluorescence spectroscopy. In most cases, the rate of bleaching slowed as treatment progressed, leaving a fraction of the PpIX unbleached despite sustained irradiation. To account for this feature, we hypothesized a decrease in blood flow during ALA-photodynamic therapy (PDT) that reduced the rate of oxygen transported to the tissue and therefore attenuated the photobleaching process. We have performed a detailed analysis of this hypothesis. Study Design/Materials and Methods We used a comprehensive, previously published mathematical model to simulate the effects of therapy-induced blood flow reduction on the measured PpIX photobleaching. This mathematical model of PDT in vivo incorporates a singlet-oxygen-mediated photobleaching mechanism, dynamic unloading of oxygen from hemoglobin, and provides for blood flow velocity changes. It permits simulation of the in vivo photobleaching of PpIX in this patient population over the full range of irradiances and fluences. Results The results suggest that the physiological equivalent of discrete blood flow reductions is necessary to simulate successfully the features of the bleaching data over the entire treatment fluence regime. Furthermore, the magnitude of the blood flow changes in the normal tissue margin and lesion for a wide range of irradiances is consistent with a nitric-oxide-mediated mechanism of vasoconstriction. Conclusion A detailed numerical study using a comprehensive PDT dosimetry model is consistent with the hypothesis that the observed trends in the in vivo PpIX photobleaching data from patients may be explained on the basis of therapy-induced blood flow reductions at specific fluences. Lasers Surg. Med. 41:686–696, 2009. © 2009 Wiley-Liss, Inc.

Cummings, R. J., Mitra, S., Foster, T. H. and Lord, E. M. Migration of Skin Dendritic Cells in Response to Radiation Exposure. Radiat. Res. 171, 687–697 (2009). We describe an imaging assay that monitors the migration of two unique subsets of immune dendritic cells (DC), interstitial dendritic cells (iDC) and Langerhans cells (LC), found in the dermal and epidermal layers of skin, respectively. Using this assay, we study responses of these cells to ionizing radiation. Results obtained using whole-mount histology and fluorescence microscopy suggest that ionizing radiation triggered the migration of both major histocompatibility complex (MHC) class II+ iDC and Langerin+ LC in a dose- and time-dependent manner. Migration appeared to be limited by local administration of recombinant IL-12, a potent immunostimulatory cytokine known to induce DNA repair. Those findings were extended to an in vivo model by injecting fluorescently conjugated anti-MHC class II antibodies intradermally into the ears of liv...

The interaction of sucrose and starch with bacterial glucosyltransferases and human salivary amylase may enhance the pathogenic potential of Streptococcus mutans within biofilms by influencing the structural organization of the extracellular matrix and modulating the expression of genes involved in exopolysaccharide synthesis and specific sugar transport and two-component systems.

Mucosal infections caused by the pathogenic fungus Candida are a significant infectious disease problem and are often difficult to eradicate because of the high frequency of resistance to conventional antifungal agents. Photodynamic treatment (PDT) offers an attractive therapeutic alternative. Previous studies demonstrated that filamentous forms and biofilms of Candida albicans were sensitive to PDT using Photofrin as a photosensitizer. However, early stationary phase yeast forms of C. albicans and Candida glabrata were not adversely affected by treatment. We report that the cationic porphyrin photosensitizer meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP-1363) is effective in PDT against yeast forms of C. albicans and C. glabrata. Respiratory-deficient (RD) strains of C. albicans and C. glabrata display a pleiotropic resistance pattern, including resistance to members of the azole family of antifungals, the salivary antimicrobial peptides histatins and other types of toxic stresses. In contrast to this pattern, RD mutants of both C. albicans and C. glabrata were significantly more sensitive to PDT compared to parental strains. These data suggest that intact mitochondrial function may provide a basal level of anti-oxidant defense against PDT-induced phototoxicity in Candida, and reveals pathways of resistance to oxidative stress that can potentially be targeted to increase the efficacy of PDT against this pathogenic fungus.

Full details of the total syntheses of five members of the eupomatilone family of lignans are reported.

We report the influence of fluence rate on the photobleaching and cell survival in Colo 26 multicell spheroids photosensitized by meta-tetra-(hydroxyphenyl)chlorin (mTHPC). Photosensitizer degradation and therapeutic efficacy increased dramatically and progressively when the fluence rate was reduced over the range from 90 to 5 mW cm−2. These experimental results were compared to a mathematical model of photobleaching based on self-sensitized singlet oxygen reactions with the photosensitizer ground state. This model incorporates photophysical parameters obtained from microelectrode measurements of oxygen depletion at the surface of mTHPC-sensitized spheroids and was refined by including the inhomogeneous distribution of mTHPC in spheroids and oxygen depletion in the bulk medium. Since the model is consistent with the experimental data we conclude that the fluence rate dependence of the cell survival and of mTHPC photobleaching is due to photochemical oxygen consumption and a predominantly singlet ...

Cellular responses to photodynamic therapy (PDT) include induction of heat shock proteins (HSP). We examined meso-tetrahydroxyphenyl chlorin (mTHPC) PDT–mediated HSP activation in EMT6 cells stably transfected with a plasmid containing the gene for green fluorescent protein (GFP) driven by an hsp70 promoter. mTHPC incubation induced concentration-dependent GFP expression. Irradiation of cells exposed to a sensitizer concentration that induced a slight increase in GFP and no loss of cell viability resulted in fluence-dependent GFP accumulation. In response to drug only and to PDT, GFP levels increased to a maximum of four- to five-fold above control levels with increasing drug or fluence and then decreased at higher doses. A trypan blue–exclusion assay confirmed that decreased GFP levels in both cases were due to a loss of cell viability. For initial evaluation in vivo, HSP70/GFP–transfected EMT6 tumors were grown in BALB/c mice and subjected to mTHPC-PDT with a fluence of 1 J/cm2. Six hours after...

Meso-tetra-hydroxyphenyl-chlorin (mTHPC) is one of the most potent photosensitizers currently available for clinical photodynamic therapy (PDT). However the reason or reasons for its high photodynamic efficacy remain(s) unresolved. To investigate the PDT efficacy of mTHPC vs Photofrin we use the knowledge of photophysical parameters extracted from the analysis of oxygen electrode measurements in spheroids to compute and compare their respective singlet oxygen (1O2) dose depositions. The electrode measurements indirectly report the bleaching kinetics of mTHPC and indicate that its photobleaching mechanism is consistent with 1O2-mediated reactions. mTHPC's photodegradation via 1O2 reactions is confirmed by a more direct evaluation of the spatially resolved fluorescence in confocal sections of intact spheroids during irradiation. The PDT efficacy comparisons establish that mTHPC's enhanced potency may be accounted for completely on the basis of its ability to sequester tightly in cells and its photophysical properties, in particular its higher extinction coefficient at a redshifted wavelength. We extend the efficacy comparison to include the influence of hemoglobin absorption of PDT treatment light and show that incorporating the influence of wavelength-dependent light attenuation in tissue further contributes to significantly higher efficacy for mTHPC- vs Photofrin-PDT.

We have developed a comprehensive theoretical model for rigorously describing the spatial and temporal dynamics of oxygen ( O 2 3 ) consumption and transport and microscopic photodynamic dose deposition during photodynamic therapy(PDT)in vivo. Previously published models have been improved by considering perfused vessels as a time-dependent O 2 3 source and linking the O 2 3 concentration in the vessel to that within the tissue through the Hill equation. The time-dependent photochemical O 2 3 consumption rate incorporates sensitizer photobleaching effects and an experimentally determined initially nonuniform photosensitizer distribution. The axial transport of O 2 3 is provided for in the capillaries and in the surrounding tissue. A self-sensitized singlet oxygen ( O 2 1 ) -mediated bleaching mechanism and the measured, initially nonuniform distribution of meso-tetrahydroxyphenyl chlorin at 3 h after intravascular administration were used to demonstrate the capabilities of the model. Time-evolved distributions of O 2 3 concentration were obtained by numerically solving two-dimensional diffusion-with-reaction equations both in the capillary and the adjacent tissue. Using experimentally established physiological and photophysical parameters, the mathematical model allows computation of the dynamic variation of hemoglobin- O 2 3 saturation ( SO 2 ) within the vessels, irreversible sensitizer degradation due to photobleaching, and the microscopic distributions of O 2 3 , sensitizer concentration, and O 2 1 dose deposition under various irradiation conditions. The simulations reveal severe axial gradients in O 2 3 and in photodynamic dose deposition in response to a wide range of clinically relevant treatment parameters. Thus, unlike former Krogh cylinder-based models, which assume a constant O 2 3 concentration at the vessel, this new model identifies conditions in which O 2 3 depletion and minimal deposition of reacting O 2 1 exist near the end of axial segments of vessels and shows that treatment-limiting O 2 3 depletion is induced at fluence rates as low as 10 mW cm − 2 . These calculations also demonstrate that intercapillary heterogeneity of photosensitizer contributes significantly to the distribution of photodynamic dose. This more rigorous mathematical model enables comparison with experimentally observable, volume-averaged quantities such as SO 2 and the loss of sensitizer fluorescence through bleaching that have not been included in previous analyses. Further, it establishes some of the intrinsic limitations of such measurements. Specifically, our simulations demonstrate that tissue measurements of SO 2 and of photobleaching are necessarily insensitive to microscopic heterogeneity of photodynamic dose deposition and are sensitive to intercapillary spacing. Because prior knowledge of intercapillary distances in tumors is generally unavailable, these measurements must be interpreted with caution. We anticipate that this model will make useful dosimetry predictions that should inform optimal treatment conditions and improve current clinical protocols.

In this review we focus on the widely used Suzuki–Miyaura reaction and show that Pd/C is an efficient catalyst for carbon–carbon bond formation. The advantages and limitations of Pd/C are presented through selected examples. Mechanistic as well as practical aspects are also discussed. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

Treatment of mucocutaneous and cutaneous Candida albicans infections with photosensitizing agents and light, termed photodynamic therapy (PDT), offers an alternative to conventional treatments. Initial studies using the clinically approved photosensitizer Photofrin demonstrated the susceptibility of C. albicans to its photodynamic effects. In the present study, we have further refined parameters for Photofrin-mediated photodynamic action against C. albicans and examined whether mechanisms commonly used by microorganisms to subvert either antimicrobial oxidative defenses or antimicrobial therapy, including biofilm formation, were operative. In buffer and defined medium, germ tubes preloaded with Photofrin retained their photosensitivity for up to 2 hours, indicating the absence of degradation or export of Photofrin by the organism. The addition of serum resulted in a gradual loss of photosensitivity over 2 hours. In contrast to an adaptive response by germ tubes to oxidative stress by hydrogen peroxide, there was no adaptive response to singlet oxygen-mediated stress by photodynamic action. C. albicans biofilms were sensitive to Photofrin-mediated phototoxicity in a dose-dependent manner. Finally, the metabolic activity of C. albicans biofilms following photodynamic insult was significantly lower than that of biofilms treated with amphotericin B for the same time period. These results demonstrate that several of the mechanisms microorganisms use to subvert either antimicrobial oxidative defenses or antimicrobial therapy are apparently not operative during Photofrin-mediated photodynamic treatment of C. albicans . These observations provide support and rationale for the continued investigation of PDT as an adjunctive, or possibly alternative, mode of therapy against cutaneous and mucocutaneous candidiasis.

We report results of experiments that evaluated the influence of oxygenation on the penetration of red light in tissue, with particular emphasis on 630 and 650 nm laser wavelengths commonly used in photodynamic therapy (PDT) of solid tumours. Direct measurements in tissue-simulating phantoms comprised of intact human erythrocytes suspended in a scattering emulsion demonstrated significant enhancements in fluence rate at depths of 0.5-2 cm from the irradiated surface when the cells were fully oxygenated versus fully deoxygenated. The 630 and 650 nm fluence rates at depth in the homogeneous phantoms continued to increase when examined over a continuous range of oxygen partial pressures from 0 to 160 Torr. When considered as a function of haemoglobin oxygen saturation, the largest increases in fluence rate were observed as the saturation increased beyond 70%. Dramatic increases in optical fluence rate were measured at the base of 1-cm-thick subcutaneous EMT6 mammary carcinomas in vivo when the tumour-bearing mouse was subjected to carbogen through a nose cone. These results indicate that improved tumour oxygenation is important in PDT not only for the maintenance of the oxygen-dependent photochemistry but, through the effects reported here, may also enable more efficient treatment of thicker lesions.

The development of renin inhibitors with favorable oral pharmacokinetic profiles has been a longstanding challenge for the pharmaceutical industry. As part of our work to identify inhibitors of BACE1, we have previously developed iminopyrimidinones as a novel pharmacophore for aspartyl protease inhibition. In this letter we describe how we modified substitution around this pharmacophore to develop a potent, selective and orally active renin inhibitor.

Studies investigating the interaction of Candida with a single host cell type such as phagocytes or epithelial cells are typically performed in vitro. The advantages of in vitro studies include the ability to compare multiple conditions in one experiment, the ability to perform thorough biochemical and immunologic analyses, the relatively short duration of experiments, lower experimental costs, and the ability to reduce the use of laboratory animals. The major disadvantage of in vitro experiments is that it is not possible to fully replicate the myriad of cellular and extracellular signals provided by the host environment. Several animal models of candidiasis are regularly used, including models of oropharyngeal, vaginal, and disseminated candidiasis. Information about the interaction of specific host cell types with Candida organisms in vivo can sometimes be obtained through conventional histology or immunohistochemistry, but these analyses require sacrifice of the animal and thus are often limited to a single time point during the course of infection. Several fluorescent proteins have recently been codon optimized for expression in yeast. The authors suspect that any strain of Candida that has relatively bright fluorescence could be used in this model. For imaging, an inverted confocal microscope with an enclosed stage incubator chamber and generic stage insert is used. Recently, a stage insert was constructed allowing the use of an Olympus FV1000 laser scanning confocal microscope at the confocal microscopy core facility. Images were successfully obtained using both 10x, 0.4to 0.45-numerical-aperture and 20x, 0.75-NA dry objective lenses.

We describe the design and implementation of an inverted laser scanning confocal fluorescence microscope utilizing the commercial Nikon Diaphot TMD platform. An external confocal scanner was retrofitted through the video side port of the Diaphot. With 10×, 0.5 NA dry and 60×, 1.4 NA oil immersion objectives, the depth discrimination is 5.8 μm and 0.8 μm, respectively, as determined by derivatives of fluorescence edge responses measured in liquid samples of rhodamine 6G dissolved in DMSO. We present sample edge response curves and representative confocal fluorescence images of tumor cells in monolayer culture.

Synthesis of protoporphyrin IX (PpIX) in intact murine mammary cancer cell spheroids is reported from optical sections obtained using a laser scanning confocal fluorescence microscope. EMT6 spheroids 275–350 μ m in diameter were incubated in 0.1–15 mM aminolevulinic acid (ALA) or 0.001–2 mM ALA-hexylester (h-ALA) to test the ability of both pro-drugs to diffuse into the spheroids and induce PpIX production. Spheroids incubated with ALA show significant fluorescence nonuniformity for all concentrations, with the outermost cells exhibiting greater porphyrin fluorescence. Comparable levels of fluorescence throughout the optical section are achieved with approximately 100-fold lower h-ALA concentrations, indicating that the interior cells maintain esterase activity and porphyrin synthesis and that h-ALA diffuses efficiently to the spheroid interior. Fluorescence gradients are less pronounced with h-ALA incubation, in part because of apparent saturation of esterase activity in the spheroid perimeter. Proliferating (Ki67 positive) and quiescent cell populations exhibit remarkably different h-ALA concentration dependencies. The incubation concentration resulting in maximum fluorescence with ALA is 10 mM, while the optimal concentration for h-ALA is 200-fold lower at 0.05 mM. Exceeding these optimal concentrations for both pro-drugs leads to an overall loss of fluorescence. © 2001 Cancer Research Campaign http://www.bjcancer.com

Recently, we published an improved mathematical model of photodynamic therapy (PDT) dose deposition on length scales corresponding to intercapillary distances.1 This model describes the spatial and temporal dynamics of oxygen (O23) consumption and transport and microscopic singlet oxygen (O21) dose deposition during PDT treatment. It also enables simulation of volume-averaged quantities like hemoglobin oxygen saturation (SO2) and photosensitizer fluorescence photobleaching, which are accessible experimentally. In a subsequent modeling study of the kinetics of the recovery of SO2 following the interruption of PDT irradiation, we were troubled by what appeared to be physically unreasonable simulation results. The origin of these erroneous results was a sign error in one of the terms buried deep in the original code, which had the effect of creating a tumor microenvironment that was more hypoxic that we had specified as an initial condition. We emphasize that in our previously published article on this model, there were no errors in what was described, but the plots presented in that article were quantitatively affected by this sign error. Specifically, this error was in the numerator of Eq. (2) of Wang et al.,1 which was correct in the text; the error was only in the code. In this erratum, we show the revised figures generated by using the same photophysical and physiological parameters described previously.1 We also use this occasion to describe a few relatively minor improvements to the model. The oxygen transport equation in the capillary is revised slightly from the original and is now written (1+S)∂Ccap∂t=Dcap[1r∂∂r(r∂Ccap∂r)]+Dcap∂2Ccap∂z2−V(1+S)∂Ccap∂z,0⩽r⩽Rc, (1) where S=CsatnC50nCcapn−1(C50n+Ccapn)2. (2) Here, we include a new term, (1+S), on the left-hand side of the transport equation for the capillary [Eq. 1], which more correctly provides for the dynamic unloading of O23 from hemoglobin.2 Further, the revised code uses Michaelis–Menten kinetics to describe the rate of metabolic O23 consumption not only in the calculation of the time-dependent state, as was done previously, but also in the calculation of the steady-state with axial diffusion. The equation for the tissue region remains as previously described ∂Ctiss∂t=Dtiss[1r∂∂r(r∂Ctiss∂r)]+Dtiss∂2Ctiss∂z2−Γ,Rc⩽r⩽b. (3) The definitions and the values of the parameters were previously described. Using the corrected code and implementing the above modifications to the model, we repeated the simulations for the case of a 130 μm intercapillary spacing. Figure ​Figure11 shows the revised computed spatial distributions of O23 concentration, [O23](r,z), for (a) 0 J cm−2 and mTHPC-PDT conducted at irradiances of (b) 10 mW cm−2 for a fluence of 50 J cm−2, (c) 100 mW cm−2 for a fluence of 3 J cm−2, and (d) for a fluence of 50 J cm−2, assuming a nonuniform initial sensitizer distribution 3 h after i.v. injection. The anoxic regions shown in Figs. ​Figs.3b,3b, ​,3c,3c, ​,3d3d of our original article,1 which do not appear in these corrected results, were a result of the sign error in the code. Figure 1 Calculated axial and radial distributions of the O23 concentration [O23](r,z) for (a) 0 J cm−2 and mTHPC-PDT conducted at irradiances of (b) 10 mW cm−2 for a fluence of 50 J cm−2, (c) 100 mW cm−2 for a fluence of 3 J cm ... Figure 3 Computed spatial distributions of O21 dose, [O21](r,z), deposited during mTHPC-PDT at irradiances of 10 and 100 mW cm−2 for a fluence of 50 J cm−2, assuming initially uniform [(a1) and (a2)] and nonuniform [(b1), and (b2)] sensitizer distributions. ... Revised numerical calculations of volume-averaged hemoglobin O23 saturation, ⟨SO2⟩, within the capillary versus irradiation time, the normalized, volume-averaged ground state sensitizer concentration, ⟨[S0]⟩, versus fluence, and the volume-averaged O21 dose, ⟨[O21]⟩, versus fluence for two fluence rates, 10 and 100 mW cm−2, assuming nonuniform initial sensitizer distribution are shown in Figs. ​Figs.2a,2a, ​,2b,2b, ​,2c,2c, respectively. The initial decrease in ⟨SO2⟩ in Fig. ​Fig.2a2a is not as significant as shown in Fig. 4(b) of Wang et al.1 Compared to Figs. 5(a) and 7(a) of our previous article, the rates of photobleaching and dose deposition for the 10 and 100 mW cm−2 cases in Figs. 2(b) and 2(c) are more rapid, and for a given fluence, the differences in the extent of sen-sitizer degradation and dose deposition between these two fluence rate cases are smaller. The differences between these and our previous results originate from greater oxygen availability in the tumor region after correcting the sign error. Figure 2 (a) Computed ⟨SO2⟩ within the capillary vs irradiation time (s), (b) computed normalized ⟨[S0]⟩ vs fluence (J cm−2), and (c) computed ⟨[O21]⟩ vs fluence (J cm−2) for two fluence rates, 10 ... Figure ​Figure33 shows the computed spatial distributions of O21 dose, [O21](r,z), deposited during mTHPC-PDT at irradiances of 10 and 100 mW cm−2 for a fluence of 50 J cm−2, assuming initially uniform [(a1) and (a2)] and nonuniform [(b1), and (b2)] sensitizer distributions. Compared to our previously published Fig. 8,1 the axial gradients in [O21] are now less severe. The corrected MATLAB code is available, and interested readers should contact the authors for the updated version.

Agriculture has a significant role in economy of the most of the developing countries. A significant amount of crops are damaged in every year due to fungi, fungus, bacteria, Phytoplasmas, bad weather etc. Grapes are one of the most widely grown fruit crops in the world with significant plantings in India. Grapes are used in the production of wine, brandy, or non-fermented drinks and are eaten fresh or dried as raisins. Sometimes grape plants are affected by downy mildew, a serious fungal disease. Therefore, farmers try to detect the stage of the disease in plant at an early stage so that they can take necessary steps in order to prevent the disease from spreading to others parts of the fields. This article presents a novel technique for detection of downy mildew disease present in the grape leaves based on fuzzy importance factor. The proposed technique uses some digital image processing operations and fuzzy set theory concept. We experimented on thirty one diseased and non-diseased images and got 87.09% success. The experimental results reveal that the proposed technique can effectively detect the present of downy mildew disease in the grape leaves.

The inflammatory response elicited by photodynamic therapy (PDT) represents a complex and dynamic process characterized by elements of the innate immune system.1,2 A hallmark of this host response is the massive recruitment of leukocytes, a significant fraction of which are neutrophils, to the treated tumor site.3,4 This phenomenon has been observed with several different photosensitizers, including the US FDA approved Photofrin.5 In our own work using in vivo imaging, we have recently demonstrated that PDT with the phthalocyanine photosensitizer, Pc4, or with 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH), induces a significant increase in Gr1+ cells in irradiated versus control EMT6 mouse mammary carcinoma tumors.6,7 Several animal studies have also investigated the importance of intratumor neutrophil accumulation to the long-term curative outcome of PDT using selective depletion or inactivation of neutrophils and have demonstrated that the depletion of these effector cells results in a decrease of the PDT-mediated tumor cure rate.3,8,9 Neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G are three serine proteases stored in large quantities in neutrophil cytoplasmic azurophilic granules. These proteases are externalized in an active form during neutrophil activation at inflammatory sites, and recent studies have started to elucidate their contribution to the immune response. In this context, NE has received particular attention.10,11 Although NE has been implicated in a variety of diseases due to its capacity to degrade the extracellular matrix, new findings provide compelling evidence that NE contributes considerably to the regulation of host functions.12 A recent study reported that NE is involved in polymorphonuclear leukocyte-mediated host protection in a mouse model of Pseudomonas aeruginosa-induced pneumonia.13 The study also demonstrated that extracellularly active NE has the capacity to induce mRNA expression of the proinflammatory cytokines tumor necrosis factor α (TNF-α), macrophage inflammatory protein-2, and interleukin-6 (IL-6). The growing recognition of the role of proteinases and proteolytic cascades in both the growth and metastasis of tumors has led to the development not only of therapeutic strategies using proteinase inhibitors but also of methods to detect and image tumors in vivo via tumor-associated proteolytic activities.14–16 These selective protease probes belong to a family of imaging agents that are optically invisible or at least display reduced fluorescence when administered intravenously until activated by enzymatic cleavage at the disease site. This allows localization of fluorescence signal in areas of enzyme activity with low background in tissue regions that do not activate the probes. Thus, increased signal levels report enhanced proteolytic activity. Recently, several of these activatable imaging probes have been developed. For example, commercially available protease activatable probes targeting matrix metalloproteinases (MMPs) (MMPSense®, Perkin Elmer, Hopkinton, MA) have been widely used to illustrate enzyme activity in tumors and in other non-neoplastic diseases, such as stroke and arthritis.17–19 In light of recent findings that NE mediates the upregulation of proinflammatory cytokines13 and based on the knowledge that the local inflammatory response to PDT is also characterized by increased expression of several cytokines, including IL-1β, TNF-α and IL-6,20 we performed an imaging study to characterize the NE enzyme activity in PDT-treated tumors using a newly developed NE-selective near infrared activatable optical probe, neutrophil elastase 680 FAST (NE680). We evaluated if PDT conditions that induce significant neutrophil accumulation in treated tumor sites alter local NE protease activity. We report that in vivo imaging results are consistent with significantly increased NE activity in PDT-treated tumors, and the extent of probe accumulation correlates with the magnitude of intratumor neutrophil influx associated with different treatment protocols.

We measured the optical properties of freshly excised kidneys with renal parenchymal tumors to assess the feasibility of photodynamic therapy (PDT) in these patients. Kidneys were collected from 16 patients during surgical nephrectomies. Spatially resolved, white light, steady-state diffuse reflectance measurements were per- formed on normal and neoplastic tissue identified by a pathologist. Reflectance data were fit using a radiative trans- port model to obtain absorption (μa) and transport scattering coefficients (μ 0 s), which define a characteristic light propagation distance, δ. Monte Carlo (MC) simulations of light propagation from cylindrical diffusing fibers were run using the optical properties extracted from each of the kidneys. Interpretable spectra were obtained from 14 kidneys. Optical properties of human renal cancers exhibit significant inter-lesion heterogeneity. For all diagnoses, however, there is a trend toward increased light penetration at longer wavelengths. For renal cell carcinomas (RCC), mean values of δ increase from 1.28 to 2.78 mm as the PDT treatment wavelength is increased from 630 to 780 nm. MC simulations of light propagation from interstitial optical fibers show that fluence distribution in tumors is significantly improved at 780 versus 630 nm. Our results support the feasibility of PDT in selected renal cancer patients, especially with photosensitizers activated at longer wavelengths. © 2012 Society of Photo-Optical Instrumentation