Comparison of near‐infrared fluorescent deoxyglucose probes with different dyes for tumor diagnosis in vivo

Glucose plays a central role in the cellular energy metabolism. Malignant tumors exhibit an elevated rate of glycolysis over normal tissues. In this study, two near‐infrared fluorescent dyes, Cypate and ICG‐Der‐02, with different water solubility, were conjugated to 2‐amino‐2‐deoxy‐d ‐glucose (2DG) to form Cypate‐2DG and ICG‐Der‐02‐2DG, respectively, for NIR fluorescent imaging of tumors in nude mice. The clear routes and tumor targeting abilities of the two NIR fluorescent 2DG probes were compared. Results showed that ICG‐Der‐02‐2DG with higher hydrophilicity was cleared faster by kidneys than the more lipophilic Cypate‐2DG. Cypate‐2DG had slower but stronger tumor targeting ability compared with ICG‐Der‐02‐2DG. To investigate the correlation between the targeting ability of the probe and the glucose transporter (GLUT1) expression levels of cancer cells, the accumulation of Cypate‐2DG in tumors was assessed in MCF‐7/estradiol, U87MG, MCF‐7 and MDA‐MB‐435 tumor xenografts, which express different levels of GLUT1. The results show that both Cypate‐2DG and ICG‐Der‐02‐2DG possess tumor targeting ability on all the tumors examined, with a proportional correlation to GLUT1 expression. The findings demonstrate the broad applicability of these molecular probes for optical imaging of tumors and glucose‐related pathologies. Copyright © 2012 John Wiley & Sons, Ltd.     

A fast tumor‐targeting near‐infrared fluorescent probe based on bombesin analog for in vivo tumor imaging

Bombesin (BBN), an analog of gastrin‐releasing peptide (GRP), of which the receptors are over‐expressed on various tumor cells, is able to bind to GRP receptor specifically. In this study, a near‐infrared fluorescent dye (MPA) and polyethylene glycol (PEG) were conjugated to BBN analog to form BBN[7–14]–MPA and BBN[7–14]–SA–PEG–MPA. The successful synthesis of the two probes was proved by the characterization via sodium dodecylsulfate–polyacrylamide gel electrophoresis, infrared and optical spectra. Cellular uptakes studies indicated that BBN‐based probes were mediated by gastrin‐releasing peptide receptors (GRPR) on tumor cells and the PEG modified probe had higher affinity. The dynamic distribution and clearance investigations showed that the BBN‐based probes were eliminated by the liver–kidney pathway. Furthermore, both of the BBN‐based probes displayed tumor‐targeting ability in GRPR over‐expressed tumor‐bearing mice. The PEG modified probe exhibited faster and higher tumor targeting capability than BBN[7–14]–MPA. The results implied that BBN[7–14]–SA–PEG–MPA could act as an effective fluorescence probe for tumor imaging. Copyright © 2014 John Wiley & Sons, Ltd.     

Optical imaging detection of microscopic mammary cancer in ErbB‐2 transgenic mice through the DA364 probe binding αvβ3 integrins

Despite spontaneous tumor growth in genetically engineered mice being one of the most recognized tools for the in vivo evaluation of novel diagnostic and therapeutic anticancer compounds, monitoring early stage lesions in live animals is a goal that has yet to be achieved. A large number of targets for the molecular imaging of various diseases have been identified and many imaging technologies, including optical techniques are emerging. One of the most commonly exploited targets in tumor imaging is α_vβ₃ integrin, which plays an important role in the expansion, invasiveness and metastatic capability of a number of cancers, including breast cancer. The aim of this study was to set up an optical imaging method for the early detection of autochthonous mammary cancer in female BALB/c mice transgenic for the rat‐ErbB‐2 oncogene (BALB‐neuT). We show that DA364, a near‐infrared fluorescence arginine–glycine–aspartic acid cyclic probe, was taken up by neoplastic mammary glands and that its uptake increased with cancer progression. By contrast, the nonaccumulation of DA364 in the healthy mammary glands of virgin and lactating wild‐type mice suggests that the probe specifically targets breast cancers. Comparisons of optical imaging with whole‐mount and histological findings showed that DA364 allows the noninvasive visualization of atypical hyperplasia and microscopic foci of in situ carcinoma 2 months before mammary lesions become detectable by palpation. Moreover, DA364 was successfully used to monitor the outcome of anticancer vaccination. Therefore, it can be considered a promising early detection tool in near‐infrared noninvasive optical imaging for the early diagnosis of breast cancer. Copyright © 2013 John Wiley & Sons, Ltd.     

A new optical imaging probe targeting αVβ3 integrin in glioblastoma xenografts

α_Vβ₃ Integrins are a widely recognized target for in vivo molecular imaging of pathological conditions such as inflammation, cancer and rheumatoid arthritis. We have evaluated the sensitivity of a new, near‐infrared fluorescence (NIRF), RGD cyclic probe (DA364) in noninvasive detection of α_Vβ₃ integrin‐overexpressing tumors. DA364's binding affinity for α_Vβ₃ integrin was first evaluated in vitro. Human α_Vβ₃ integrin‐positive, U‐87 MG glioblastoma cells were then xenografted in nude mice, and DA364 was injected intravenously (i.v.) to evaluate its in vivo distribution, specificity and sensitivity in comparison with a commercially available probe. DA364 bound α_Vβ₃ integrin on U‐87 MG cells with high affinity and specificity, both in vitro and in vivo. This binding specificity was corroborated by the strong inhibition of its tumor uptake induced by nonfluorescent, cyclic‐RGD peptides. Ex vivo analysis showed that DA364 accumulated at the tumor site, whereas very low levels were detected in liver and spleen. In conclusion, DA364 allows sensitive and specific detection of transplantable glioblastoma by NIRF imaging, and is thus a promising candidate for the elaboration of imaging and therapeutic probes for α_Vβ₃ integrin‐overexpressing tumors. Copyright © 2011 John Wiley & Sons, Ltd.     

Effect of near‐infrared rays on female menstrual pain in Korea

Most Korean women who experience menstrual pain have reported taking pain medicine and making use of complementary alternative therapies. However, because some interventions may cause side effects, more effective pain‐relieving measures need to be identified. This study using a non‐equivalent group design, evaluated the effects of near‐infrared rays on dysmenorrhea among Korean women. The experimental group wore a near‐infrared ray abdominal belt for the duration of one menstrual cycle until the end of the menstrual period, while the control group used hot packs. The level of menstrual pain, menstrual pain duration, and pain medicine use were measured. The menstrual pain, average menstrual pain duration, and use of analgesics were reduced in the near‐infrared rays group. The results of this study indicate that the near‐infrared ray LED belt was effective in reducing menstrual pain, menstrual pain duration compared to the use of analgesics in Korean women with dysmenorrhea. Therefore, near‐infrared rays may be used to relieve menstrual pain and improve the quality of life of women with dysmenorrhea in Korea.     

Collagen‐mimetic peptides mediate flow‐dependent thrombus formation by high‐ or low‐affinity binding of integrin α2β1 and glycoprotein VI

Summary. Background: Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen‐derived triple‐helical peptides have identified the GXX’GER motif as an adhesive ligand for platelet integrin α2β1, and (GPO)_n as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). Objective: The potency was investigated of triple‐helical peptides, consisting of GXX’GER sequences within (GPO)_n or (GPP)_n motifs, to support flow‐dependent thrombus formation. Results: At a high‐shear rate, immobilized peptides containing both the high‐affinity α2β1‐binding motif GFOGER and the (GPO)_n motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co‐immobilized VWF was needed for thrombus formation. The (GPO)_n but not the (GPP)_n sequence induced GPVI‐dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low‐affinity (GASGER, GAOGER) α2β1‐binding motifs formed procoagulant thrombi only if both (GPO)_n and VWF were present. At a low‐shear rate, immobilized peptides with high‐ or low‐affinity α2β1‐binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)_n. Conclusions: Triple‐helical peptides with specific receptor‐binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high‐shear rate, either GPIb or high‐affinity (but not low‐affinity) GXX’GER mediates GPVI‐dependent formation of procoagulant thrombi. By extension, high‐affinity binding for α2β1 can control the overall platelet‐adhesive activity of native collagens.     

Optimizing quantitative in vivo fluorescence imaging with near‐infrared quantum dots

Quantum dots (QDs) are fluorescent nanoparticles with broad excitation and narrow, wavelength‐tunable emission spectra. They are used extensively for in vitro fluorescence imaging studies and more recently for in vivo small animal and pre‐clinical studies. To date there has been little concern about the selection of QD size (and thus emission wavelength peak) and excitation wavelengths, as they have little relevance to the results of in vitro studies. In vivo imaging, however, poses additional constraints, such as the scattering and absorption by tissue, which may influence the signal intensity at the body surface. Here, we demonstrate that longer‐wavelength excitation and emission yield less quantization error in measured relative fluorescence intensity, using three near‐infrared QDs (QD655, QD705 and QD800) applied to in vivo lymphatic imaging, and a range of excitation wavelengths from the blue to the red. Statistically significant differences in quantization error were observed between nearly all pairs of excitation wavelengths (445–490, 503–555, 575–605, 615–665 and 671–705 nm). Similarly, quantization error decreased with longer emission wavelengths (655, 705 and 800 nm). Light absorbance and scattering were demonstrated to be more potent factors than absorbance efficiency of QDs in producing quantization error in the measured fluorescence intensity. As a result, while wavelengths can be adjusted for qualitative experiments, the longest possible wavelengths should be used if quantification is desired during QD imaging experiments. Copyright © 2010 John Wiley & Sons, Ltd.     

In vivo near‐infrared fluorescence targeting of T cells: comparison of nanobodies and conventional monoclonal antibodies

The large size of conventional antibodies impedes tissue penetration and renal elimination, resulting in suboptimal in vivo targeting. Here we assess the utility of nanobodies and nanobody‐Fc‐fusion proteins as alternatives to monoclonal antibodies as theranostics, using T cell ADP–ribosyltransferase 2 (ART2) as a model antigen for specific targeting of lymph nodes. ART2‐specific monovalent nanobody s + 16a (17 kDa), a bivalent Fc‐fusion protein of s + 16a (s + 16‐mFc, 82 kDa), and conventional antibody Nika102 (150 kDa) were labeled with AlexaFluor680. In vitro binding and inhibitory properties of the three AF680 conjugates were assessed by flow cytometry. For in vivo imaging experiments, AF680 conjugates were intravenously injected in mice lacking (KO) or overexpressing (TG) ART2. We monitored circulating and excreted AF680 conjugates in plasma and urine and performed in vivo near‐infrared fluorescence imaging. Nanobody s + 16a⁶⁸⁰ and s + 16mFc⁶⁸⁰ labeled and inhibited ART2 on T cells in lymph nodes within 10 min. In contrast, mAb Nika102⁶⁸⁰ required 2 h for maximal labeling without inhibition of ART2. In vivo imaging revealed specific labeling of ART2‐positive lymph nodes but not of ART2‐negative lymph nodes with all AF680 conjugates. Even though bivalent s + 16mFc⁶⁸⁰ showed the highest labeling efficiency in vitro, the best lymph node imaging in vivo was achieved with monovalent nanobody s + 16a⁶⁸⁰, since renal elimination of unbound s + 16a⁶⁸⁰ significantly reduced background signals. Our results indicate that small single‐domain nanobodies are best suited for short‐term uses, such as noninvasive imaging, whereas larger nanobody‐Fc‐fusion proteins are better suited for long‐term uses, such as therapy of inflammation and tumors. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparative study of alpha‐ and beta‐pinene effect on PTZ‐induced convulsions in mice

Convulsions occur in response to a loss of balance between excitatory and inhibitory neurotransmitters, and the treatment for this condition consists in restore such lost balance. Many anticonvulsant drugs present side effects which may limit their use. This fact has stimulated the search for new sources of treatment from aromatic plants. Many monoterpenes commonly present in essential oils are known because of their anticonvulsant properties. The anticonvulsant effect of α‐ and β‐pinene, two structural isomers, is still little studied. Thus, the present work evaluated the anticonvulsant effect of α‐ and β‐pinene in pentylenetetrazole‐induced convulsions model. Initially, the oral LD₅₀ for α‐ and β‐pinene was estimated. Following the oral administration, a mild sedation was observed and no deaths were recorded; the LD₅₀ estimated for both monoterpenes was greater than 2 000 mg/kg, p.o. Further, animals were orally treated with α‐pinene (100, 200 and 400 mg/kg), β‐pinene (100, 200 and 400 mg/kg) and the equimolar mixture of α‐ and β‐pinene (400 mg/kg) and subjected to the pentylenetetrazole‐induced convulsions model. In this model, only the dose of 400 mg/kg of the compounds was able to significantly decrease the seizure intensity. The latency of first convulsion was significantly increased by the mixture of α‐ and β‐pinene (400 mg/kg). In addition, β‐pinene and the mixture of the two monoterpenes, both at a dose of 400 mg/kg, significantly increased the time of death of animals. The treatment with β‐pinene and the equimolar mixture of the two monoterpenes significantly reduced hippocampal nitrite level and striatal content of dopamine (DA) and norepinephrine (NE). Taken together, the results suggest that α‐pinene appears to be devoid of anticonvulsant action. This fact, however, seems to be dependent on the chemical structure of the compound, since pretreatment with the β‐pinene increased the time of death pf PTZ‐treated mice, which seems to depend on the ability of the compound to reduce nitrite concentration and NE and DA content, during the pentylenetetrazole‐induced seizure.     

Anti‐inflammatory effect of 4‐methylcyclopentadecanone in rats submitted to ischemic stroke

This study aimed to investigate the anti‐inflammatory effect of 4‐methylcyclopentadecanone (4‐MCPC) in rats suffering from a cerebral ischemia/reperfusion (I/R) injury. In this study, the focal cerebral ischemia in rats was induced by middle cerebral artery occlusion (MCAO) for 2 h, and the rats were treated with 4‐MCPC (8 mg/kg) just 0.5 h before reperfusion. The ischemic infarct volume was recorded 24 h after the MCAO. In addition, myeloperoxidase (MPO) activity and TNF‐α and IL‐1β levels in the ischemic cerebral cortex were determined by ELISA, while nuclear translocation of NF‐κB p65 subunit and expression of p‐IκBα were investigated by Western blotting. Our results showed that 4‐MCPC treatment decreased infarct volume significantly, compared with I/R group (16.8%±7.5% vs. 39.7%±10.9%); it reduced MPO activity (0.43 ± 0.10 vs. 1.00 ± 0.51 U/g) and expression levels of TNF‐α (18.90 ± 3.65 vs. 35.87 ± 4.87 ng/g) and IL‐1β (1.68 ± 0.23 vs. 2.67 ± 0.38 ng/g) in ischemic brain tissues of rats. Further study revealed that 4‐MCPC treatment markedly reduced nuclear translocation of NF‐κB p65 subunit and expression of p‐IκBα in ischemic cerebral cortex. Taken together, our results suggest that 4‐MCPC protects against cerebral I/R injury and displays anti‐inflammatory actions through inhibition of the NF‐κB signal pathway.     

Effects of variation in the human α2A‐ and α2C‐adrenoceptor genes on cognitive tasks and pain perception

Background: The mechanisms underlying interindividual variability in pain perception and cognitive responses are undefined but highly heritable. α_2C‐ and α_2A‐adrenergic receptors regulate noradrenergic activity and are important mediators of pain perception and analgesia. We hypothesized that common genetic variants in these genes, particularly the ADRA2C 322–325 deletion variant, affect pain perception or cognitive responses. Methods: We studied 73 healthy subjects (37 Caucasians and 36 African–Americans) aged 25.4 ± 4.6 years. Pain response to a cold pressor test was measured using a 10 cm visual analog scale and again on the next day, after three infusions of the selective α₂‐agonist dexmedetomidine. Standardized cognitive tests were administered at baseline and after each infusion. The contribution of ADRA2C deletion genotype, dexmedetomidine concentration, and other covariates to pain perception and cognitive responses was determined using multiple linear regression models. Secondary analysis examined the effects of ADRA2A and other ADRA2C variants on pain perception. Results: ADRA2C Del homozygotes had higher pain scores in response to cold at baseline (6.3 ± 1.8 cm) and after dexmedetomidine (5.6 ± 2.2 cm) than insertion allele carriers (4.6 ± 2.1 cm [baseline] and 3.8 ± 1.9 cm [after dexmedetomidine]; adjusted P‐values = 0.019 and 0.004, respectively). Cognitive responses were unrelated to ADRA2C Ins/Del genotype. None of the other ADRA2A and ADRA2C variants was significantly related to cold pain sensitivity before dexmedetomidine; after dexmedetomidine, ADRA2A rs1800038 was marginally associated (P = 0.03). Conclusion: The common ADRA2C del322–325 variant affected pain perception before and after dexmedetomidine but did not affect other cognitive responses, suggesting that it contributes to interindividual variability in pain perception.     

CD40 ligand shedding is regulated by interaction between matrix metalloproteinase‐2 and platelet integrin αIIbβ3

Summary. Background: CD40 ligand (CD40L, CD154) in the circulatory system is mainly contained in platelets, and surface‐expressed CD40L on activated platelets is subsequently cleaved by proteolytic activity to generate soluble CD40L (sCD40L). However, the enzyme responsible for the shedding of CD40L in activated platelets has not been clearly identified yet. We have recently found that molecular interaction of matrix metalloproteinase‐2 (MMP‐2) with integrin α_IIbβ₃ is required for the enhancement of platelet activation. Objectives: To elucidate the biochemical mechanism of MMP‐2‐associated sCD40L release. Methods: Localization of MMP‐2 and CD40L in platelets was analyzed by flow cytometry and fluorescence microscopy. The release of sCD40L from activated platelets was measured by enzyme‐linked immunosorbent assay. MMP‐2 binding to α_IIbβ₃ was analyzed by immunoprecipitation and western blotting. Recombinant hemopexin‐like domain and MMP‐2‐specific inhibitor were used to characterize the nature of MMP‐2 binding and catalytic activity. Results: It was revealed that interaction of MMP‐2 with α_IIbβ₃ is required for effective production of sCD40L in activated human platelets. Platelet activation and release of sCD40L were significantly affected by inhibition of platelet‐derived MMP‐2 activity or by inhibition of binding between the enzyme and the integrin. It was also found in platelet‐rich plasma that MMP‐2 activity is responsible for generating sCD40L. Conclusions: The results presented here strongly suggest that MMP‐2 interacts with α_IIbβ₃ to regulate the shedding of CD40L exposed on the surfaces of activated human platelets.