Eric L. Kaijzel

Optical Image-guided Surgery—Where Do We Stand?

In cancer surgery, intra-operative assessment of the tumor-free margin, which is critical for the prognosis of the patient, relies on the visual appearance and palpation of the tumor. Optical imaging techniques provide real-time visualization of the tumor, warranting intra-operative image-guided surgery. Within this field, imaging in the near-infrared light spectrum offers two essential advantages: increased tissue penetration of light and an increased signal-to-background-ratio of contrast agents. In this article, we review the various techniques, contrast agents, and camera systems that are currently used for image-guided surgery. Furthermore, we provide an overview of the wide range of molecular contrast agents targeting specific hallmarks of cancer and we describe perspectives on its future use in cancer surgery.

Nasal vaccination with N-trimethyl chitosan and PLGA based nanoparticles: Nanoparticle characteristics determine quality and strength of the antibody response in mice against the encapsulated antigen

Nasal vaccination is a promising, needle-free alternative to classical vaccination. Nanoparticulate delivery systems have been reported to overcome the poor immunogenicity of nasally administered soluble antigens, but the characteristics of the ideal particle are unknown. This study correlates differences in physicochemical characteristics of nanoparticles to their adjuvant effect, using ovalbumin (OVA)-loaded poly(lactic-co-glycolic acid) nanoparticles (PLGA NP), N-trimethyl chitosan (TMC) based NP (TMC NP) and TMC-coated PLGA NP (PLGA/TMC NP). PLGA NP and PLGA/TMC NP were prepared by emulsification/solvent extraction and TMC NP by ionic complexation. The NP were characterized physicochemically. Their toxicity and interaction with and stimulation of monocyte derived dendritic cells (DC) were tested in vitro. Furthermore, the residence time and the immunogenicity (serum IgG titers and secretory IgA levels in nasal washes) of the nasally applied OVA formulations were assessed in Balb/c mice. All NP were similar in size, whereas only PLGA NP carried a negative zeta potential. The NP were non-toxic to isolated nasal epithelium. Only TMC NP increased the nasal residence time of OVA compared to OVA administered in PBS and induced DC maturation. After i.m. administration all NP systems induced higher IgG titers than OVA alone, PLGA NP and TMC NP being superior to PLGA/TMC NP. Nasal immunization with the slow antigen releasing particles, PLGA NP and PLGA/TMC NP, did not induce detectable antibody titers. In contrast, nasal immunization with the positively charged, fast antigen releasing TMC NP led to high serum antibody titers and sIgA levels. In conclusion, particle charge and antigen release pattern of OVA-loaded NP has to be adapted to the intended route of administration. For nasal vaccination, TMC NP, releasing their content within several hours, being mucoadhesive and stimulating the maturation of DC, were superior to PLGA NP and PLGA/TMC NP which lacked some or all of these characteristics.

Molecular imaging of tumor angiogenesis using αvβ3-integrin targeted multimodal quantum dots

Molecular imaging of angiogenesis is urgently needed for diagnostic purposes such as early detection, monitoring of (angiostatic) therapy and individualized therapy. Multimodality molecular imaging is a promising and refined technique to study tumor angiogenesis, which has so far been largely unexplored due to the lack of suitable multimodal contrast agents. Here, we report on the application of a novel αvβ3-specific quantum dot-based nanoparticle, which has been optimized for both optical and magnetic resonance detection of tumor angiogenesis. Upon intravenous injection of RGD-pQDs in tumor-bearing mice, intravital microscopy allowed the detection of angiogenically activated endothelium at cellular resolution with a small scanning window and limited penetration depth, while magnetic resonance imaging was used to visualize angiogenesis at anatomical resolution throughout the entire tumor. Fluorescence imaging allowed whole-body investigation of angiogenic activity. Using these quantum dots and the aforementioned imaging modalities, the angiogenic tumor vasculature was readily detected with the highest angiogenic activity occurring in the periphery of the tumor. This nanoparticle may be employed for multimodality imaging of a variety of diseases that are accompanied by activation of endothelial cells. Furthermore, the current technology might be developed for molecular imaging of other pathophysiological processes.

Whole-Body Optical Imaging in Animal Models to Assess Cancer Development and Progression

Different optical-based imaging models were used to investigate tumor progression and metastasis with particular emphasis on metastasis to bone and bone marrow. We describe how optical imaging can be used to follow important processes in tumor development and treatment response, including angiogenesis, apoptosis, and proteolysis. Finally, we discuss the translation of one optical imaging modality, near-IR fluorescence, from animal validation studies to applications in the clinic related to cancer management.

Allele-specific quantification of tumor necrosis factor alpha (TNF) transcription and the role of promoter polymorphisms in rheumatoid arthritis patients and healthy individuals.

Interindividual variation in the expression of tumor necrosis factor α (TNF) suggests the existence of functionally distinct TNF alleles that could play a role in susceptibility to TNF associated diseases such as rheumatoid arthritis (RA). To determine whether differential expression of TNF alleles exists, the relative contribution of TNF alleles in total TNF RNA production in peripheral blood mononuclear cells (PBMC) of healthy individuals and synovial tissue of RA patients was analyzed. By using a Tai I restriction fragment length polymorphism (RFLP) located at position +489 in the first intron of the gene, the relative contribution of each allele in precursor transcript production in heterozygous individuals could be measured. By means of this method we studied whether differences exist between TNF alleles in TNF pre-mRNA production. The relative contribution of TNF alleles to the non-spliced RNA pool was measured in PBMC of healthy individuals which were stimulated with LPS, PMA and anti-CD3 and anti-CD28 monoclonal antibodies for different time periods. Moreover, synovial biopsy material of RA patients was analyzed. The results of this study do not reveal a difference in the contribution of distinct TNF alleles in TNF pre-mRNA production upon in vitro and physiological stimulation conditions in healthy individuals and RA patients. Since some of the individuals whose PBMC were tested were also heterozygous for either −308, −1031, −863, −857 TNF promoter/enhancer single nucleotide polymorphisms (SNPs), the data argue against functional relevance of these TNF promoter/enhancer SNPs in the regulation of transcription. In conclusion, the data do not provide evidence for the existence of transcriptionally distinct TNF alleles to explain interindividual variation in TNF expression.

Atlas-based whole-body segmentation of mice from low-contrast Micro-CT data

This paper presents a fully automated method for atlas-based whole-body segmentation in non-contrast-enhanced Micro-CT data of mice. The position and posture of mice in such studies may vary to a large extent, complicating data comparison in cross-sectional and follow-up studies. Moreover, Micro-CT typically yields only poor soft-tissue contrast for abdominal organs. To overcome these challenges, we propose a method that divides the problem into an atlas constrained registration based on high-contrast organs in Micro-CT (skeleton, lungs and skin), and a soft tissue approximation step for low-contrast organs. We first present a modification of the MOBY mouse atlas (Segars et al., 2004) by partitioning the skeleton into individual bones, by adding anatomically realistic joint types and by defining a hierarchical atlas tree description. The individual bones as well as the lungs of this adapted MOBY atlas are then registered one by one traversing the model tree hierarchy. To this end, we employ the Iterative Closest Point method and constrain the Degrees of Freedom of the local registration, dependent on the joint type and motion range. This atlas-based strategy renders the method highly robust to exceptionally large postural differences among scans and to moderate pathological bone deformations. The skin of the torso is registered by employing a novel method for matching distributions of geodesic distances locally, constrained by the registered skeleton. Because of the absence of image contrast between abdominal organs, they are interpolated from the atlas to the subject domain using Thin-Plate-Spline approximation, defined by correspondences on the already established registration of high-contrast structures (bones, lungs and skin). We extensively evaluate the proposed registration method, using 26 non-contrast-enhanced Micro-CT datasets of mice, and the skin registration and organ interpolation, using contrast-enhanced Micro-CT datasets of 15 mice. The posture and shape varied significantly among the animals and the data was acquired in vivo. After registration, the mean Euclidean distance was less than two voxel dimensions for the skeleton and the lungs respectively and less than one voxel dimension for the skin. Dice coefficients of volume overlap between manually segmented and interpolated skeleton and organs vary between 0.47+/-0.08 for the kidneys and 0.73+/-0.04 for the brain. These experiments demonstrate the methods effectiveness for overcoming exceptionally large variations in posture, yielding acceptable approximation accuracy even in the absence of soft-tissue contrast in in vivo Micro-CT data without requiring user initialization.

Association of the TNF +489 polymorphism with susceptibility and radiographic damage in rheumatoid arthritis

Multiple genetic factors contribute to susceptibility to rheumatoid arthritis (RA). The extent of variability in disease presentation in RA may be related to genetic heterogeneity. In this study we investigated the association of the TNF gene polymorphism at position +489 with susceptibility to and severity of RA. Analysis of the frequency of the +489 A and G alleles in a group of 293 consecutive RA patients and 138 healthy controls revealed a significant decrease of the A allele. The +489 GA patients had a 3.9 times decreased chance of having erosive disease than +489 GG patients. These results were confirmed in a prospective study using a cohort of 112 patients who were followed for 12 years. The progression rate of the erosion score over 12 years expressed as Sharp score for X-rays of hands and feet was 3.4 per year for the GA-genotyped patients and 12.1 for the GG-genotyped patients. These associations were independent of rheumatoid factor and HLA-shared epitope positivity. In conclusion, these data suggest that the intron TNF +489 polymorphism is associated with susceptibility to and disease severity of RA independently of HLA-shared epitope-positive alleles.

Sensitive Dual Color In Vivo Bioluminescence Imaging Using a New Red Codon Optimized Firefly Luciferase and a Green Click Beetle Luciferase

Background Despite a plethora of bioluminescent reporter genes being cloned and used for cell assays and molecular imaging purposes, the simultaneous monitoring of multiple events in small animals is still challenging. This is partly attributable to the lack of optimization of cell reporter gene expression as well as too much spectral overlap of the color-coupled reporter genes. A new red emitting codon-optimized luciferase reporter gene mutant of Photinus pyralis, Ppy RE8, has been developed and used in combination with the green click beetle luciferase, CBG99. Principal Findings Human embryonic kidney cells (HEK293) were transfected with vectors that expressed red Ppy RE8 and green CBG99 luciferases. Populations of red and green emitting cells were mixed in different ratios. After addition of the shared single substrate, D-luciferin, bioluminescent (BL) signals were imaged with an ultrasensitive cooled CCD camera using a series of band pass filters (20 nm). Spectral unmixing algorithms were applied to the images where good separation of signals was observed. Furthermore, HEK293 cells that expressed the two luciferases were injected at different depth in the animals. Spectrally-separate images and quantification of the dual BL signals in a mixed population of cells was achieved when cells were either injected subcutaneously or directly into the prostate. Significance We report here the re-engineering of different luciferase genes for in vitro and in vivo dual color imaging applications to address the technical issues of using dual luciferases for imaging. In respect to previously used dual assays, our study demonstrated enhanced sensitivity combined with spatially separate BL spectral emissions using a suitable spectral unmixing algorithm. This new D-luciferin-dependent reporter gene couplet opens up the possibility in the future for more accurate quantitative gene expression studies in vivo by simultaneously monitoring two events in real time.

Polymorphism within the tumor necrosis factor α (TNF) promoter region in patients with ankylosing spondylitis

In addition to HLA-B27, other genetic factors are thought to be involved in the pathogenesis of ankylosing spondylitis (AS). Because of the location of the TNF gene in the vicinity of the HLA-B locus, and the prominent role in inflammation of its product, we investigated the association between AS and two G to A transition polymorphisms located at position -238 and -376 in the promoter region of the TNF gene. The distribution of the TNF alleles was determined in 86 HLA-B27+ AS patients and 163 healthy controls. From the 86 AS patients, 33 suffered from acute anterior uveitis (AAU). No significant difference for the TNF-376 polymorphism in AS and healthy controls was observed. The frequency of the TNF-238A allele in HLA-B27+ AS patients was significantly decreased compared to random controls (p = 0.021). However, the frequency of the TNF-238A allele in HLA-B27+ AS patients was not significantly different from that observed in HLA-B27+ healthy individuals (p = 0.6). Assessment of association showed that the TNF-238G allele is in linkage disequilibrium with the HLA-B27 allele (delta = 0.053; P = 0.008). Therefore, we conclude that the association between TNF-238G and AS is secondary to the HLA-B27 gene and that TNF-238 and-TNF-376 alleles are not likely to be involved in the susceptibility to AS.

Role of trimethylated chitosan (TMC) in nasal residence time, local distribution and toxicity of an intranasal influenza vaccine

The nose is a promising immunization site and intranasal (i.n.) vaccination studies with whole inactivated influenza virus (WIV) adjuvanted with N,N,N-trimethylchitosan (TMC-WIV) have shown promising results. In this study, the influence of TMC on the i.n. delivery of WIV was studied in mice by comparing the nasal residence time and the specific location in the nasal cavity of WIV and TMC-WIV. Additionally, the local toxicity profile of the WIV formulations was assessed. In vivo fluorescence imaging was used to study the nasal residence time and the fate of the bulk vaccine in mice that received vaccines fluorescently labeled with IRDye800CW. An immunohistochemical (IHC) staining method for nasal cross-sections was developed to visualize the antigen in the nasal cavity. Therefore, mice were sacrificed at different time points after vaccination with various vaccine formulations and nasal cross-sections were made. The local toxicity was assessed using hematoxylin and eosin staining for the nasal cross-sections. No significant differences in the nasal residence time between WIV and TMC-WIV were observed. However, IHC revealed a striking difference in the location and distribution of WIV in the nasal cavity. When formulated as plain WIV, positive staining was mainly found in the nasal cavity, presumably in mucus blobs. TMC-coated WIV, on the other hand, was mostly present as a thin layer on the epithelial surfaces of the naso- and maxilloturbinates. This difference in staining pattern correlates with the observed differences in immunogenicity of these two vaccines and indicates that TMC-WIV results in a much closer interaction of WIV with the epithelial surfaces than WIV alone, potentially leading to enhanced uptake and induction of immune responses. This study further shows that both WIV and TMC-WIV formulations induce minimal local toxicity. Taken altogether, these results provide more insight in the mode of action and safety of TMC and justify further research to develop TMC-adjuvanted nasal vaccines.