Orotracheal manganese‐enhanced MRI (MEMRI): An effective approach for lung tumor detection

Lung cancer is a primary cause of cancer deaths worldwide. Timely detection of this pathology is necessary to delay or interrupt lung cancer progression, ultimately resulting in a possible better prognosis for the patient. In this context, magnetic resonance imaging (MRI) is especially promising. Ultra‐short echo time (UTE) MRI sequences, in combination with gadolinium‐based contrast agents, have indeed shown to be especially adapted to the detection of lung neoplastic lesions at submillimeter precision. Manganese‐enhanced MRI (MEMRI) increasingly appears to be a possible effective alternative to gadolinium‐enhanced MRI. In this work, we investigated whether low‐dose MEMRI can effectively target non‐small‐cell lung cancer in rodents, whilst minimizing the potential toxic effect of manganese. Both systemic and orotracheal administration modalities allowed the identification of tumors of submillimeter size, as confirmed by bioluminescence imaging and histology. Equivalent tumor signal enhancements and contrast‐to‐noise ratios were observed with orotracheal administration using 20 times lower doses compared with the more conventional systemic route. This finding is of crucial importance as it supports the observation that higher performances of contrast agents can be obtained using an orotracheal administration route when targeting lung diseases. As a consequence, lower concentrations of contrast media can be employed, reducing the dose and potential safety issues. The non‐detectable accumulation of ionic manganese in the brain and liver following orotracheal administration observed in vivo is extremely encouraging with regard to the safety of the orotracheal protocol with low‐dose Mn²⁺ administration. To our knowledge, this is the first time that a study has clearly allowed the high‐precision detection of lung tumor and its contours via the synergic employment of a strongly T₁‐weighted MRI UTE sequence and ionic manganese, an inexpensive contrast agent. Overall, these results support the growing interest in drug and contrast agent delivery via the airways to target and diagnose several diseases of the lungs.     

Manganese‐enhanced MRI (MEMRI) via topical loading of Mn2+ significantly impairs mouse visual acuity: a comparison with intravitreal injection

Manganese‐enhanced MRI (MEMRI) with topical loading of MnCl₂ provides optic nerve enhancement comparable to that seen by intravitreal injection. However, the impact of this novel and non‐invasive Mn²⁺ loading method on visual function requires further assessments. The objective of this study is to determine the optimal topical Mn²⁺ loading dosage for MEMRI and to assess visual function after MnCl₂ loading. Intravitreal administration was performed to compare the two approaches of MnCl₂ loading. Twenty‐four hours after topical loading of 0, 0.5, 0.75, and 1 M MnCl₂, T₁‐weighted, T2‐weighted, diffusion tensor imaging and visual acuity (VA) assessments were performed to determine the best topical loading dosage for MEMRI measurements and to assess the integrity of retinas and optic nerves. Mice were perfusion fixed immediately after in vivo experiments for hematoxylin and eosin and immunohistochemistry staining. Topical loading of 1 M MnCl₂ damaged the retinal photoreceptor layer with no detectable damage to retina ganglion cell layers or prechiasmatic optic nerves. For the topical loading, 0.75 M MnCl₂ was required to see sufficient enhancement of the optic nerve. At this concentration the visual function was significantly affected, followed by a slow recovery. Intravitreal injection (0.25 μL of 0.2 M MnCl₂) slightly affected VA, with full recovery a day later. To conclude, intravitreal MnCl₂injection provides more reproducible results with less adverse side‐effects than topical loading. Copyright © 2014 John Wiley & Sons, Ltd.     

Cancer metabolism in a snapshot: MRS(I)

The contribution of MRS(I) to the in vivo evaluation of cancer‐metabolism‐derived metrics, mostly since 2016, is reviewed here. Increased carbon consumption by tumour cells, which are highly glycolytic, is now being sampled by ¹³C magnetic resonance spectroscopic imaging (MRSI) following the injection of hyperpolarized [1‐¹³C] pyruvate (Pyr). Hot‐spots of, mostly, increased lactate dehydrogenase activity or flow between Pyr and lactate (Lac) have been seen with cancer progression in prostate (preclinical and in humans), brain and pancreas (both preclinical) tumours. Therapy response is usually signalled by decreased Lac/Pyr ¹³C‐labelled ratio with respect to untreated or non‐responding tumour. For therapeutic agents inducing tumour hypoxia, the ¹³C‐labelled Lac/bicarbonate ratio may be a better metric than the Lac/Pyr ratio. ³¹P MRSI may sample intracellular pH changes from brain tumours (acidification upon antiangiogenic treatment, basification at fast proliferation and relapse). The steady state tumour metabolome pattern is still in use for cancer evaluation. Metrics used for this range from quantification of single oncometabolites (such as 2‐hydroxyglutarate in mutant IDH1 glial brain tumours) to selected metabolite ratios (such as total choline to N‐acetylaspartate (plain ratio or CNI index)) or the whole ¹H MRSI(I) pattern through pattern recognition analysis. These approaches have been applied to address different questions such as tumour subtype definition, following/predicting the response to therapy or defining better resection or radiosurgery limits.     

The pH sensitivity of APT‐CEST using phosphorus spectroscopy as a reference method

The pH value is a potential physiological marker for clinical diagnosis as it is altered in pathologies such as tumors. While intracellular pH can be measured noninvasively via phosphorus spectroscopy (³¹P MRSI), Amide Proton Transfer‐Chemical Exchange Saturation Transfer (APT‐CEST) MRI has been suggested as an alternative method for pH quantification. To assess the suitability of APT‐CEST contrast for pH quantification, two approaches (magnetization transfer ratio asymmetry [MTR_asym] and Lorentzian difference analysis [LDA]) for analyzing the Z‐spectrum have been correlated with pH values obtained by ³¹P MRSI. Fourteen patients with glioblastoma and 12 healthy controls were included. In contrast to MTR_asym, the LDA is modeling the direct water saturation and the semi‐solid magnetization transfer, allowing a separate evaluation of the aliphatic nuclear Overhauser effect and the APT‐CEST. The results of our study show that the pH values obtained by ³¹P MRSI correspond well with both methods describing the APT‐CEST contrast. Two‐sample t‐test showed significant differences in MTR_asym, LDA and pH obtained by ³¹P MRSI for regions of interest in glioblastoma, contralateral control areas and normal appearing white matter (P < 0.001). A slightly improved correlation between the amide signal and pH was found after performing LDA (r = 0.78) compared with MTR_asym (r = 0.70). While both methods can be used to monitor pH changes, the LDA approach appears to be better suited.     

Denaturing high-performance liquid chromatography (DHPLC) as a reliable high-throughput prescreening method for aberrant promoter methylation in cancer

Aberrant promoter hypermethylation of CpG dinucleotides is a frequent and significant mechanism of tumor suppressor gene (TSG) silencing in cancer. As increasing numbers of downregulated putative TSGs are emerging from large-scale expression profiling studies, high-throughput techniques are needed to screen for hypermethylation. DHPLC has been established as a reliable, highly sensitive technique for mutation analysis. In this study, the use of DHPLC as a prescreening method for the identification of CpG methylation was developed by analyzing DNA samples with different, well-characterized methylation patterns of the CDKN2A/p16 promoter. Bisulfite treatment of genomic DNA was followed by PCR-amplification of unmethylated as well as methylated CDKN2A/p16 promoter sequences. PCR products were denatured and renatured, permitting the formation of heteroduplex DNA detectable by DHPLC. Methylation of all CpG-sites results in a single peak (homoduplex) with a shift in retention time, whereas partial methylation can be recognized by additional signals representing diverse heteroduplex structures. After method development, 35 DNA samples from primary bladder and breast carcinomas were analyzed in a blinded fashion, revealing complete or partial methylation of the p16 promoter in eight cases and a heterozygous mutation in one case. In conclusion, DHPLC is a highly sensitive and convenient method for methylation screening.     

Visualization of phosphatidylcholine (16:0/16:0) in type II alveolar epithelial cells in the human lung using imaging mass spectrometry

Imaging mass spectrometry (MS) is an emerging technique that can detect numerous biomolecular distributions in a non‐targeting manner. In the present study, we applied a mass imaging modality, mass microscopy, to human lung tissue and identified several molecules including surfactant constituents in a specific structure of the lung alveoli. Four peaks were identified using imaging MS, and the ion at m/z 772.5, in particular, was localized at some spots in the alveolar walls. Using an MS/MS analysis, the ion was identified as phosphatidylcholine (PC)(16:0/16:0), which is the main component of lung surfactant. In a larger magnification of the lung specimen, PC (16:0/16:0) was distributed in a mottled fashion in a section of the lung. Importantly, the distribution of PC (16:0/16:0) was identical to that of anti‐SLC34A2 antibody immunoreactivity, which is known to be a specific marker of type II alveolar epithelial cells, in the same section. Our experience suggests that imaging MS has excellent potential in human pathology research.     

A rapid and reliable PCR method for genotyping the ABO blood group. II: A2 and O2 alleles

PCR permits direct genotyping of individuals at the ABO locus. Several methods have been reported for genotyping ABO that rely on differentiating the A, B, and O alleles at specific base substitutions. However, the O allele as defined by serology comprises at least two alleles (O¹ and O²) at the molecular level, and most current ABO genotyping methods only take into account the O¹ allele. Determining the presence of the O² allele is critical, as this not-infrequent allele would be mistyped as an A or a B allele by standard PCR typing methods. Furthermore, none of the methods to date distinguish between the A¹ and A² alleles, even though 10% of all white persons are blood group A². We have developed a method for genotyping the ABO locus that takes the O² and A² alleles into account. Typing for A² and O² by diagnostic restriction enzyme digestion is a sensitive, nonradioactive assay that provides a convenient method useful for forensic and paternity testing and for clarifying anomalous serological results. © 1996 Wiley-Liss, Inc.     

Focus on the glycerophosphocholine pathway in choline phospholipid metabolism of cancer

Activated choline metabolism is a hallmark of carcinogenesis and tumor progression, which leads to elevated levels of phosphocholine and glycerophosphocholine in all types of cancer tested so far. Magnetic resonance spectroscopy applications have played a key role in detecting these elevated choline phospholipid metabolites. To date, the majority of cancer‐related studies have focused on phosphocholine and the Kennedy pathway, which constitutes the biosynthesis pathway for membrane phosphatidylcholine. Fewer and more recent studies have reported on the importance of glycerophosphocholine in cancer. In this review article, we summarize the recent literature on glycerophosphocholine metabolism with respect to its cancer biology and its detection by magnetic resonance spectroscopy applications.     

卵巢肿瘤患者外周血NK细胞受体NKG2A、NKG2D及NK活性检测的临床意义

通过研究卵巢癌及良性卵巢肿瘤患者外周血NK细胞表面受体的表达情况及NK活性的变化,分析探讨宿主NK细胞受体与肿瘤免疫逃逸的关系及其临床价值。分离受检者外周血单个核细胞,应用MTT法检测NK细胞的细胞毒活性,流式细胞术检测NK细胞受体NKG2D和NKG2A的表达,并结合临床病理因素作比较分析。结果显示,与良性卵巢肿瘤组和正常组相比,卵巢癌患者外周血NK细胞的细胞毒活性降低,NK细胞表面NKG2D的表达水平降低,而NKG2A的表达水平明显升高,其变化与卵巢癌的病情进展有关。此结果表明,卵巢癌患者机体NK细胞杀伤活性下降,NKG2D与NKG2A二者之间的平衡表达可能对NK细胞的功能状态起着重要的调节作用。     

Diagnosis of the sentinel lymph node in breast cancer: a reproducible molecular method: a multicentric Spanish study

Bernet L, Cano R, Martinez M, Dueñas B, Matias‐Guiu X, Morell L, Palacios J, Rezola R, Robles‐Frias M, Ruiz I, Velasco A, Vieites B, Sevilla F, Torro J, Medrano J & Ballester B
(2011) Histopathology  58 , 863–869
Diagnosis of the sentinel lymph node in breast cancer: a reproducible molecular method: a multicentric Spanish study Aims: Standardization of the sentinel node (SN) as a diagnostic tool has not yet been achieved, because the protocol for histopathological study is highly variable between centres. We compared the results of a new method with conventional histological tests and evaluated its feasibility for intra‐operative evaluation, and propose it as a method to standardize the sentinel node evaluation procedure. Methods and results: Trial 1 included 181 cases; in parallel, 2‐mm‐thick sections of the SN were processed alternately for histological analysis and for the one‐step nucleic acid amplification (OSNA) procedure. A final concordance of 99.45% was observed in the first trial of our study. For trial 2, the timing of every procedural step was recorded in an electronic database in order to discern the time spent for each step, the total SN evaluation time and to identify areas of improvement. In the second trial, after a learning period and feedback on data recorded, we spent a mean of 31 min for the entire SN evaluation procedure. Conclusion: Our multi‐centric trial using the OSNA assay for sentinel node evaluation in breast cancer demonstrates that this is a highly sensitive, specific and reproducible technique that allows for standardization of the SN diagnostic procedure, a necessary, and until now unresolved, issue.     

Magnetic resonance spectroscopy (MRS) in the evaluation of pediatric brain tumors, Part II: Clinical analysis.

Over a 1-year period (1994-1995), 75 children with brain neoplasms were evaluated with a new automated magnetic resonance spectroscopy (MRS) software package called Proton Brain Exam/Single-Voxel (PROBE/SV) to determine the efficacy of this modality in children. The children ranged in age from newborn to 17 years and were comprised of 30 girls and 45 boys. The types of brain neoplasms consisted of 45 astrocytomas, 4 medulloblastomas, 2 ependymomas, 3 craniopharyngiomas, 3 germinomas, 1 pineoblastoma, 2 teratomas, 1 choroid plexus papilloma, 4 meningiomas, 2 astroblastomas, 3 rhabdoids, and 5 metastases from primary brain neoplasms. All children underwent magnetic resonance imaging (MRI) at the same setting as the MRS examination. The MRS examination was performed with the stimulated echo acquisition mode (STEAM) pulse sequence in all children, and occasionally the point resolved spectroscopy (PRESS) sequence also was used. Qualitative spectra were obtained in all children, and at times quantification data also were obtained. We found that our spectra over the brain neoplasms were consistent with the MRS findings of brain neoplasms in the literature. There was markedly elevated choline with markedly decreased or absent N-acetylasparate and at times elevated lactate and lipid peaks. In children with meningiomas, there was also an elevated alanine peak. We found MRS to be extremely useful in 1) characterizing a brain mass as a neoplasm, 2) differentiating radiation necrosis and radiation-induced meningiomas from the recurrent primary tumor, 3) following treatment response of the primary neoplasm, 4) differentiating residual or recurrent primary neoplasm from postsurgical changes, and 5) identifying inactive neoplasms or neoplasms in remission.     

Multifunctional hollow CaF2:Yb3+/Er3+/Mn2+-poly(2-Aminoethyl methacrylate) microspheres for Pt(IV) pro-drug delivery and tri-modal imaging

Combining the multi-modal medical imaging with cancer therapy in one single system has attracted the great interests for theranostic purpose. In this paper, CaF₂:Yb³⁺/Er³⁺/Mn²⁺-poly(2-Aminoethyl methacrylate) (UCHNs-PAMA) hybrid microspheres were successfully fabricated. The synthetic route to the nanocomposite based on a facile hydrothermal method for fabrication of hollow upconversion (UC) nanospheres at first and then post-filling the PAMA interiorly through photo-initiated polymerization. The UCHNs showed orange fluorescence under 980 nm near infrared (NIR) laser excitation, which provided the upconverting luminescence (UCL) imaging modality. Meanwhile, the presence of functional Mn²⁺ and Yb³⁺ offered the enhanced T₁-weighted magnetic resonance (MR) and computed tomography (CT) imaging, respectively. Thanks to introducing amine groups-containing PAMA inside the hollow nanospheres, the Pt(IV) pro-drug, c,c,t-Pt(NH₃)₂Cl₂(OOCCH₂CH₂COOH)₂ (DSP), can be conveniently bonded on the polymer network to construct a nanoscale anti-cancer drug carrier. The UCHNs-PAMA-Pt(IV) nanocomposite shows effective inhibition for Hela cell line via MTT assay. In contrast, Pt(IV) pro-drug and UCHNs-PAMA microspheres behave little cytotoxicity to Hela cells. This should be attributed the fact that the anti-cancer ability can be recovered only when Pt(IV) pro-drug was reduced to Pt(II)-drug in cellular environment. Furthermore, the in vivo experiments on small mice also confirm that the hybrid microspheres have relatively low toxic side effects and high tumor inhibition rate. These findings show that the multifunctional hybrid microspheres have potential to be used as UCL/MR/CT tri-modal imaging contrast agent and anti-cancer drug carriers.     

Ultrashort echo time spectroscopic imaging (UTESI): an efficient method for quantifying bound and free water

Biological tissues usually contain distinct water compartments with different transverse relaxation times. In this study, two‐dimensional, multi‐slice, ultrashort echo time spectroscopic imaging (UTESI) was used with bi‐component analysis to detect bound and free water components in musculoskeletal tissues. Feasibility studies were performed using numerical simulation. Imaging was performed on bovine cortical bone, human cadaveric menisci and the Achilles' tendons of volunteers. The simulation study demonstrated that UTESI, together with bi‐component analysis, could reliably quantify both T₂* and fractions of the short and long T₂* components. The in vitro and in vivo studies each took less than 14 min. The bound water components showed a short T₂* of ~0.3 ms for bovine bone, ~1.8 ms for meniscus and ~0.6 ms for the Achilles' tendon. The free water components showed about an order of magnitude longer T₂* values, with ~2 ms for bovine bone, ~14 ms for meniscus and ~8 ms for the Achilles' tendon. Bound water fractions of up to ~76% for bovine bone, 50% for meniscus and ~75% for the Achilles' tendon were measured. The corresponding free water components were up to ~24% for bovine bone, 50% for meniscus and ~25% for the Achilles' tendon by volume. These results demonstrate that UTESI, combined with bi‐component analysis, can quantify the bound and free water components in musculoskeletal tissues in clinically realistic times. Copyright © 2011 John Wiley & Sons, Ltd.     

Spondyloepiphyseal dysplasia in a cape town family: Linkage with the gene for type II collagen (COL2A1)

A moderately severe form of autosomal dominant (AD) spondyloepiphyseal dysplasia (SED) has been documented in 14 individuals in 3 generations of a family in Cape Town, South Africa. Affected persons had a short trunk; radiographic investigations indicated that skeletal involvement was worst in the hips and spine. Linkage studies with restriction fragment length polymorphisms (RFLPs) associated with the COL2A1 gene and the phenotype yielded a maximal LOD score of 4.51 at theta = 0.00. This result suggests that the structural locus for type II collagen is primarily involved in the pathogenesis of this form of SED. © 1992 Wiley-Liss, Inc.     

Calcium and cAMP signaling induced by gamma-hydroxybutyrate receptor(s) stimulation in NCB-20 neurons

The NCB-20 neurohybridoma cells differentiated with dibutyryl-cyclic-AMP represent an interesting model to study several components of the gamma-hydroxybutyrate (GHB) system in brain. In particular, an active Na⁺-dependent uptake and a depolarization-evoked release of GHB is expressed by these cells, together with high affinity specific binding sites for this substance. However, only little is known about cellular mechanisms following GHB receptor(s) stimulation in these neurons. Electrophysiological data indicate that GHB can differently affect Ca²⁺ currents. L-type calcium channels were typically inhibited by GHB when NCB-20 cells were depolarized. In contrast, when NCB-20 cells were at resting potential, GHB induced a specific Ca²⁺ entry through T-type calcium channels. In this study, we investigated the effect induced on cytosolic free Ca²⁺ level and cAMP production by GHB receptor(s) stimulated with micromolar concentrations of GHB or structural analogues of GHB. Ca²⁺ movements studied by cellular imaging were dose-dependently increased but disappeared for GHB concentrations >25 μM. In addition, nanomolar doses of GHB inhibited forskolin-stimulated adenylate cyclase. This effect was also rapidly desensitized at higher GHB concentrations. Acting as an antagonist, NCS-382 decreased GHB receptor(s) mediated cAMP and calcium signals. The agonist NCS-356 mimicked GHB effects which were not affected by the GABA_B receptor antagonist CGP-55-845. Our results reveal the occurrence of Ca²⁺-dependent adenylate cyclase inhibition in NCB-20 neurons after GHB receptor(s) stimulation by GHB concentrations <50 μM. Above this dose, GHB effects were inactivated. In addition, at GHB concentrations exceeding 50 μM, GTP-γS binding was also reduced, confirming the desensitization of GHB receptor(s). Taken together, these results support the existence in NCB-20 neurons of GHB receptors belonging to GPCR family that may recruit various G protein subtypes.     

Preliminary criteria for the definition of allergic rhinitis: a systematic evaluation of clinical parameters in a disease cohort (II)

BACKGROUND: This study is a development of the work done in 'Preliminary criteria for the definition of allergic rhinitis: a systematic evaluation of clinical parameters in a disease cohort (I)'. OBJECTIVE: We sought to develop criteria which could be routinely used to define allergic rhinitis. METHODS: A total of 47 allergic rhinitis and 23 normal subjects were evaluated with a detailed questionnaire and history, physical examination, serum total immunoglobulin (Ig) E, skin prick tests and serum EAST's. RESULTS: Based on this data, we developed a preliminary scoring system by which a reliable diagnosis of allergic rhinitis can be made, which is based on the prevalence adjusted strength of association delta ( partial differential) rank values described in this report. Cumulative frequency histograms were constructed for allergic rhinitis and normal subjects in a variety of configurations. CONCLUSION: A simple scoring system which can be used to diagnose allergic rhinitis on a routine basis was developed in this study.     

Contaminating cells alter gene signatures in whole organ versus laser capture microdissected tumors: a comparison of experimental breast cancers and their lymph node metastases

Genome-wide expression profiling has expedited our molecular understanding of the different subtypes of breast cancers, as well as defined the differences among genes expressed in primary tumors and their metastases. Laser-capture microdissection (LCM) coupled to gene expression analysis allows us to understand how specific cell types contribute to the total cancer gene expression signature. Expression profiling was used to define genes that contribute to breast cancer spread into and/or growth within draining lymph nodes (LN). Whole tumor xenografts and their matched whole LN metastases were compared to LCM captured cancer cells from the same tumors and matched LN metastases. One-thousand nine-hundred thirty genes were identified by the whole organ method alone, and 1,281 genes by the LCM method alone. However, less than 1% (30 genes) of genes that changed between tumors and LN metastases were common to both methods. Several of these genes have previously been implicated in cancer aggressiveness. Our data show that whole-organ and LCM based gene expression profiling yield distinctly different lists of metastasis-promoting genes. Contamination of the tumor cells, and cross reactivity of mouse RNA to human-specific chips may explain these differences, and suggests that LCM-derived data may be more accurate.